Nutritional Bioavailability

of Manganes

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 In Nutritional Bioavailability of Manganese; Kies, C.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
ACS SYMPOSIUM SERIES 354

Nutritional Bioavailability
of Manganese
Constance Kies, EDITOR
University of Nebraska

Developed from a symposium sponsored

by the Division of Agricultural and Food Chemistry
at the 192nd Meeting
of the American Chemical Society,
Anaheim, California,
September 7-12, 1986

American Chemical Society, Washington, D C 1987

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
Library of Congress Cataloging-in-Publication Data
Nutritional bioavailability of manganese.
(ACS symposium series, ISSN 0097-6156; 354)
"Developed from a symposium sponsored by the
Division of Agricultural and Food Chemistry at the
192nd Meeting of the American Chemical Society,
Anaheim, California, September 7-12, 1986."
Includes bibliographies and indexes.
1. Manganese—Bioavailability—Congresses.
2. Manganese in human nutrition—Congresses.
I. Kies, Constance, 1934- .
Chemical Society. Division of
Chemistry. III. American Chemical Society. Meeting
(192nd: 1986: Anaheim, Calif.)
[DNLM:1.Biological availability—congresses.
2. Manganese—metabolism—congresses.
3. Nutrition—congresses. QU 130 N976 1986]
QP535.M6N88 1987 612'.3924 87-19553
ISBN 0-8412-1433-6

Copyright © 1987
American Chemical Society
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In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 ACS Symposium Series
M . Joan Comstock, Series Editor

1987 Advisory Board

Harvey W. Blanch Vincent D. McGinniss
University of California—Berkeley Battelle Columbus Laboratories

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Clemson University J. T. Baker Chemical Company

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Nabisco Brands, Inc. Exxon Chemical Company

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Martin L. Gorbaty C. M. Roland

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Rudolph J. Marcus Douglas B. Walters

Consultant, Computers & National Institute of
Chemistry Research Environmental Health

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Foreword
The ACS SYMPOSIUM SERIES was founded in 1974 to provide a
medium for publishing symposia quickly in book form. The
format of the Series parallels that of the continuing ADVANCES
IN CHEMISTRY SERIES except that, in order to save time, the
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In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Preface

^N^ANGANESE NUTRITION in plants and animals has long been a topic

of practical and esoteric research interest. However, only recently has it
become a hot topic among investigators whose concerns are centered
directly or indirectly around the nutritional and general physiological health
of humans. At the symposium on which this book is based, information
presented ranged from basic theoretical biochemistry to applied nutrition
based on laboratory-controlle
research models.
The chapters in this book particularly emphasize the dietary and
nondietary factors that apparently influence the absorption and use of
manganese and, thus, the bioavailability and bioutilization of manganese.
Updated papers that were presented at the symposium are included in this
volume; several other chapters that were solicited by the editor give more
complete coverage of the topic. Increased knowledge of the involvement of
manganese in metabolic functions of the living organism has contributed to
the current excitement about manganese research. Also contributing to this
excitement is the development of instrumentation that allows for greater
accuracy and ease in analysis of manganese and of organic compounds
containing manganese or dependent on nutritional adequacy of manganese.
This volume is meant to present current information on the bioavail-
ability of manganese, to share enthusiasm of the participants with other
investigators who have a current or possible future interest in manganese,
and to alert scientists and practitioners to this field of investigation.
I would like to acknowledge the help and hard work of Donna Hahn
in organizing the symposium and this book.

CONSTANCE KIES
University of Nebraska
Lincoln, NE 68583

May 7, 1987

ix

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Chapter 1

Manganese Bioavailability Overview

Constance Kies
Department of Human Nutrition and Food Service Management,
University of Nebraska, Lincoln, NE 68583

Manganese is recognized as an essential nutrient for

humans and animals; however, in excessive amounts
manganese is also a toxic substance. Manganese has
been defined as being a component of several enzymes
and an activato
although other mineral
stitute for manganese in many but not all of these
metabolic processes. Since nutritional adequacy
of manganese is necessary for the enzyme manganese
superoxide dismutase, research dealing with this
enzyme is of current interest. Only one case of
frank manganese deficiency in the human has been
identified and publicized in the literature.
However, animal studies indicate that manganese
deficiencies affect bone, brain and reproductive
systems. Manganese absorption from the gastro-
intestinal tract is thought to be poor but its
absorption is thought to be at least in part
governed by its valence state. In the United
States recent research suggests that mean mangan-
ese intakes of women are considerably lower than
are the NRC Estimated Safe and Adequate Daily
Dietary Intakes while those of formula fed infants
are greater than the NRC ESADDI listings for this
age group. Depending upon manganese source and
availability of manganese from these sources,
manganese adequacy or toxicity might be a problem
for these segments of the American population.

Manganese has been found t o be an e s s e n t i a l n u t r i e n t f o r t h e human

as w e l l as f o r many o t h e r l i v i n g o r g a n i s m s ; however, i n e x c e s s i v e
amounts, i t i s a l s o a t o x i c m a t e r i a l ( 1 - 6 ) . D e f i c i e n c y symptoms
f o r manganese i n s e v e r a l s p e c i e s have been c r e a t e d and manganese
m e t a b o l i c r o l e s have, a t l e a s t i n p a r t , been d e f i n e d (5,7-15). So
c a l l e d "normal" manganese c o n c e n t r a t i o n s i n b l o o d have been e s t a b -
l i s h e d (1,10,16). K i n e t i c s o f manganese uptake and m e t a b o l i s m by

0097-6156/87/0354-0001 $06.00/0
© 1987 American Chemical Society

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
2 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

t i s s u e s under c o n d i t i o n s o f manganese t o x i c i t y and d e f i c i e n c y have

been reviewed by Keen and L o n n e r d a l i n t h i s volume.

Manganese Enzymes Systems

Manganese i s a component o f p y r u v a t e c a r b o x y l a s e and m i t o c h o n d r i a l

s u p e r o x i d e dismutase and a c t i v a t e s a number o f enzymes i n c l u d i n g
p h o s p h a t a s e s , k i n a s e s , d e c a r b o x y l a s e s , and g l y c o s y l t r a n s f e r a s e s t h a t
are i n v o l v e d i n t h e s y n t h e s i s o f p o l y s a c c h a r i d e s and g l y c o p r o t e i n s
(12,17,18). I t i s a s s o c i a t e d w i t h the s y n t h e s i s o f p r o t e i n , DNA,
and RNA w i t h c a r t i l e g e m u c o p o l y s a c c h a r i d e s y n t h e s i s (5,19). Since
i s has a l s o been shown t o be i n v o l v e d i n amino a c i d t r a n s p o r t and
c a t a b o l i s m , t h i s s u g g e s t s t h a t i n c r e a s e d p r o t e i n i n t a k e might i n -
c r e a s e t h e need f o r manganese i n t h e t i s s u e s ( 5 ) . R e c e n t l y , i n v e s -
t i g a t i o n s o f f a c t o r s a f f e c t i n g a c t i v i t y l e v e l s and f u n c t i o n s o f man-
ganese s u p e r o x i d e dismutase have been p a r t i c u l a r l y a c t i v e .
Superoxide dismutases
branes from l i p i d p e r o x i d a t i o n
forms, one c o n t a i n i n g manganese and t h e o t h e r c o n t a i n i n g copper and
z i n c ( 2 0 ) . A r e l a t i o n s h i p between manganese consumption and super-
o x i d e dismutase a c t i v i t y has been found. H e p a t i c c o n c e n t r a t i o n s o f
manganese and h e p a t i c manganese s u p e r o x i d e dismutase a c t i v i t i e s were
h i g h e r but h e p a t i c c o p p e r - z i n c d i s m u t a s e l e v e l s were lower i n e t h a n o l
fed monkeys than i n c o n t r o l monkeys ( 2 1 ) .
When d i f f e r e n t groups o f w e a n l i n g r a t s were f e d r a t i o n s con-
t a i n i n g graded l e v e l s o f manganese r a n g i n g from 0.2 t o 29.7 mg/kg o f
r a t i o n , s i g n i f i c a n t c o r r e l a t i o n s between d i e t a r y manganese l e v e l s
w i t h h e a r t and k i d n e y manganese s u p e r o x i d e dismutase enzyme a c t i v i -
t i e s , l i v e r a r g i n a s e and plasma a l k a l i n e phosphatase a c t i v i t i e s and
h e a r t , k i d n e y , l i v e r and plasma manganese c o n c e n t r a t i o n s were found;
however, no changes o c c u r r e d i n t h e a c t i v i t i e s o f c o p p e r - c o n t a i n i n g
superoxide dismutase o r g l u t a t h i o n e peroxidase i n the t i s s u e analyzed
(22).
I n manganese d e f i c i e n c y i n t h e c h i c k e n , manganese i s r e p l a c e d
w i t h magnesium so t h a t no l o s s o f p y r u v a t e c a r b o x y l a s e o c c u r s ( 2 3 ) .
DeRosa e t a l . (18) v e r i f i e d a d e c r e a s e i n manganese s u p e r o x i d e d i s -
mutase a c t i v i t y i n manganese d e f i c i e n t r a t s and mice. Zidenberg-
Cherr e t a l . (17) r e p o r t e d t h a t l i p i d p e r o x i d a t i o n i n c r e a s e d t o a
g r e a t e r e x t e n t i n m a n g a n e s e - d e f i c i e n t than i n m a n g a n e s e - s u f f i c i e n t
r a t s . C o n c u r r e n t l y manganese s u p e r o x i d e dismutase a c t i v i t y i n c r e a s e d
to a much l e s s e r e x t e n t i n t h e d e f i c i e n t animals (17) • Hence,
m i t o c h o n d r i a l membrane damage found i n manganese d e f i c i e n t a n i m a l s
might be due t o i n c r e a s e d f r e e r a d i c a l p r o d u c t i o n due t o depressed
manganese s u p e r o x i d e dismutase a c t i v i t y .

Manganese D e f i c i e n c y Symptoms

Manganese d e f i c i e n c y symptoms i n animals a f f e c t t h r e e systems - bone,

r e p r o d u c t i v e and b r a i n (6,24). Impaired growth, s k e l e t a l abnormal-
i t i e s , depressed r e p r o d u c t i v e f u n c t i o n and a t a x i a i n newborn appear
to be s i m i l a r manganese symptoms i n a l l s p e c i e s s t u d i e d .
The s i n g l e r e p o r t e d case o f f r a n k manganese d e f i c i e n c y i n humans
a r o s e u n i n t e n t i o n a l l y i n an a d u l t male p a r t i c i p a t i n g i n a v i t a m i n K
d e f i c i e n c y study ( 2 5 ) . I n a d v e r t e n t l y manganese was o m i t t e d from t h e
p u r i f i e d diet mixture. The s i g n s and symptoms o f w e i g h t l o s s ,

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
1. KIES Manganese Bioavailability Overview 3

t r a n s i e n t d e r m a t i t i s , nausea, slow growth and r e d d e n i n g o f the h a i r

and b e a r d , h y p o c h o l e s t e r o l e m i a and depressed v i t a m i n K depending
c l o t t i n g f a c t o r s d i d not respond t o v i t a m i n K t h e r a p y but were
c o r r e c t e d by the a d m i n i s t r a t i o n of manganese.
Because o f the b l o o d l o w e r i n g c h o l e s t e r o l e f f e c t s o f a mangan-
ese d e f i c i e n c y , involvement o f manganese i n l i p i d m e t a b o l i s m has
been a t o p i c o f r e s e a r c h i n t e r e s t a s reviewed by Johnson and K i e s i n
t h i s volume.

Manganese A b s o r p t i o n and E x c r e t i o n

Manganese i s o n l y p o o r l y absorbed from the i n t e s t i n a l t r a c t ; how-

e v e r , a b s o r p t i o n o c c u r s i n t o mucosa c e l l s throughout the s m a l l i n -
t e s t i n e s (13,26). E x c r e t i o n of manganese through b i l e and pan-
c r e a t i c j u i c e i s a p p a r e n t l y more i m p o r t a n t than a b s o r p t i o n i n main-
tenance of manganese h o m e o s t a s i s a l t h o u g h young a n i m a l s seem t o l a c k
the a b i l i t y t o e x c r e t e manganese
i n the +2 v a l e n c e s t a t
same a b s o r p t i o n s i t e s (27) . Mechanisms of manganese uptake and
r e t e n t i o n both i n e x p e r i m e n t a l a n i m a l s and humans a r e d i s c u s s e d i n
the c h a p t e r by Keen et a l . Three o f the many d e t e r m i n a n t s o f man-
ganese a b s o r p t i o n and r e t e n t i o n a r e : 1) d e v e l o p m e n t a l s t a t u s ; 2)
d i e t a r y c o n s t i t u e n t s ; and 3) membrane t r a n s l o c a t i o n of the element.
However, much o f the i n f o r m a t i o n on manganese a b s o r p t i o n has been
o b t a i n e d from a n i m a l s t u d i e s w i t h the a s s u m p t i o n made t h a t t h e s e
mechanisms a l s o a p p l y t o humans.
Other n u t r i e n t s have been found t o i n f l u e n c e the a b s o r p t i o n o f
manganese. Manganese a b s o r p t i o n has been found t o be a s s o c i a t e d
w i t h h i g h i n t a k e s o f d i e t a r y c a l c i u m a s d i s c u s s e d by McDermott and
K i e s i n t h i s volume. The p o s s i b l e r e l a t i o n s h i p s o f c a l c i u m , man-
ganese and bone h e a l t h may be o f importance i n the o c c u r r e n c e o f
o s t e o p o r o s i s as d i s c u s s e d by S t r a u s s and Saltman i n another c h a p t e r .
S i n c e i r o n and manganese compete f o r b i n d i n g s i t e s i n the i n t e s t i n e s
i t i s not s u r p r i s i n g t h a t d i e t a r y i r o n a p p a r e n t l y i n h i b i t s manganese
u t i l i z e d a s d i s c u s s e d i n the c h a p t e r by Gruden.

D i e t a r y Manganese Needs and I n t a k e s o f Humans

Because o f a l a c k of i n f o r m a t i o n on manganese c o n t e n t s o f f o o d s ,
manganese i n t a k e s a r e not u s u a l l y i n c l u d e d i n n u t r i e n t i n t a k e
s u r v e y s . However, the s e v e r a l s u r v e y s w h i c h have been done i n the
U n i t e d S t a t e s and i n the U n i t e d Kingdom have y i e l d e d somewhat s i m i l a r
r e s u l t s r e g a r d i n g u s u a l manganese i n t a k e s of human a d u l t p o p u l a t i o n s .
Schroeder et a l . (6) e s t i m a t e d manganese i n t a k e s t o be between 2.2
and 8.8 mg/day; Wenlock e t a l . (28) e s t i m a t e d mean i n t a k e s t o be 4.6
mg/day and W a s l i e n (29) found i n t a k e s t o range from 0.9 t o 7.0 mg/
day. U s i n g a n a l y z e d , model U.S. d i e t s , P e n n i n g t o n et a l . (30) found
a d u l t i n t a k e s o f manganese t o range from 3.52 t o 3.67 mg/day d u r i n g
the 1977-1982 time p e r i o d . A t the U n i v e r s i t y of N e b r a s k a , manganese
i n t a k e s o f young c o l l e g e women consuming s e l f - s e l e c t e d d i e t s ranged
from 0.8 t o 5.2 mg/day w i t h a mean o f 1.28 mg/day a s e s t i m a t e d from
one week d i e t a r y d i a r i e s and from a n a l y s e s o f manganese c o n t e n t s o f
feces (31).
An i n t e r e s t i n g r e c e n t paper (32) i n c l u d e d not o n l y a n a l y z e d
manganese c o n t e n t s o f a l a r g e number o f foods based on an e x c e l l e n t

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
4 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

s a m p l i n g p r o c e d u r e b u t a l s o i n c l u d e d e s t i m a t e d mean manganese l e v e l s
of e i g h t age-sex group d i e t s compared w i t h t h e NRC E s t i m a t e d Safe
and Adequate D a i l y D i e t a r y I n t a k e s (ESADDI). The mean i n t a k e o f 1.10
mg manganese/day f o r 6-11 month i n f a n t s s l i g h t l y exceeded t h e ESADDI
and t h a t f o r t h e two-year o l d c h i l d was on t h e v e r y h i g h end o f t h e
s c a l e (a mean i n t a k e o f 1.47 r e l a t e d t o a ESADDI s c a l e o f 1.00-
1.50). F o r a l l o f t h e male g r o u p , t h e mean manganese i n t a k e s were
f
w i t h i n t h e range l i m i t s o f t h e ESADDI s b u t were toward t h e lower end
of t h e s c a l e . F o r a l l female g r o u p s , t h e mean manganese i n t a k e s were
1 T
c o n s i d e r a b l y below t h e ESADDI s• S i n c e t h e ESADDI s f o r men and
women a r e t h e same, t h e low i n t a k e s o f manganese o f women i n compar-
1
i s o n t o t h e ESADDI s a r e i n p a r t due t o t h e lower food i n t a k e s o f
women due t o l o w e r c a l o r i c needs. However, t h e s e d a t a do suggest
t h a t manganese i n t a k e s f o r many Americans may be s e r i o u s l y l o w .

Manganese Content o f M i l k and I n f a n t Formulas

E s t i m a t i o n o f manganese
t i c u l a r r e s e a r c h i n t e n s i t y . W h i l e e s t i m a t i o n s o f manganese content
o f human m i l k v a r y , t h e r e i s g e n e r a l agreement t h a t t h e manganese
content o f human m i l k i s s u b s t a n t i a l l y lower than t h a t o f cow's m i l k
( 3 3 ) . Manganese i n cow's m i l k i s combined w i t h d i f f e r e n t and s m a l l e r
p r o t e i n m o l e c u l e s t h a n i s t h a t o f human m i l k (34-36). Degree o f
a b s o r p t i o n may be d i f f e r e n t depending upon l i g a n b i n d i n g i n t h i s m i l k
and may be d i f f e r e n t than t h a t o f f r e e manganese from s u p p l e m e n t a l
manganese s a l t s .
T h i s has c r e a t e d something o f a dilemma f o r p r o d u c e r s o f i n f a n t
f o r m u l a s f o r b o t t l e f e e d i n g w h i c h has l e d t o a d i v e r s i t y o f l e v e l s
o f manganese content i n t h e s e p r o d u c t s . S i n c e human m i l k , cow's
m i l k and/or f o r m u l a a r e t h e p r i n c i p a l foods consumed by i n f a n t s ,
manganese i n t a k e s a r e determined t o a l a r g e e x t e n t by t h e q u a n t i t a -
t i v e manganese c o n t e n t s o f t h e s e s u b s t a n c e s . B r e a s t m i l k - f e d
i n f a n t s , t h e r e f o r e , have l o w e r i n t a k e s o f manganese t h a n those f e d
cow's m i l k f o r m u l a s b u t whether o r n o t t h e manganese i s e q u a l l y
a v a i l a b l e i s unknown. I n t h i s book, L o n n e r d a l e t a l . p r e s e n t e v i -
dence t h a t manganese r e t e n t i o n from m i l k and from m i l k f o r m u l a s i s
h i g h w h i l e t h a t from soy f o r m u l a s i s much l o w e r .

Manganese C o n t e n t s o f Foods

Foods v a r y i n t h e i r c o n t e n t s o f manganese (32,37,38). Comparisons

o f manganese c o n t e n t s o f d i f f e r e n t foods a r e g i v e n i n T a b l e I .
P l a n t o r i g i n foods such as t e a , whole g r a i n c e r e a l s , some dark green
l e a f y v e g e t a b l e s , and n u t s c o n t a i n h i g h amounts o f manganese. How-
e v e r , t h e s e p r o d u c t s o f t e n c o n c u r r e n t l y c o n t a i n h i g h amounts o f
t a n n i n s , o x a l a t e s , p h y t a t e s and f i b e r . These d i e t a r y c o n s t i t u e n t s
have been found t o i n h i b i t t h e a b s o r p t i o n o f o t h e r m i n e r a l s ; hence,
might have a n e g a t i v e e f f e c t on manganese a b s o r p t i o n .
A n i m a l o r i g i n p r o d u c t s such as eggs, m i l k , f i s h , r e d meats and
p o u l t r y c o n t a i n low amounts o f manganese. A b s o r p t i o n o f such
m i n e r a l s as i r o n , copper, phosphorus and c a l c i u m i s s u p e r i o r from
a n i m a l s p r o d u c t s t h a n from p l a n t - o r i g i n foods. As r e p o r t e d by K i e s
e t a l . ( i n t h i s b o o k ) , manganese a p p a r e n t l y i s b e t t e r absorbed by
humans from meals c o n t a i n i n g meat and f i s h t h a n from those c o n t a i n -
i n g p l a n t - p r o t e i n replacement p r o d u c t s . Because o f t h e low c o n t e n t

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
1. KIES Manganese Bioavailability Overview 5

Table I . Comparative Manganese Contents o f Foods

1
Food Item mg Mn/100 g F o o d

M i l k and m i l k p r o d u c t s
Cheese, American p r o c e s s e d 0.0464
Meat, p o u l t r y , f i s h , eggs
B e e f , ground, cooked 0.019
B e e f , chuck, o v e n - r o a s t e d 0.025
Chicken, oven-roasted 0.046
Frankfurters 0.024
Fish f i l l e t 0.058
Egg, s o f t b o i l e d 0.037
Legumes and n u t s
Pork and beans, canned
Peanut b u t t e r 1.32
G r a i n s and g r a i n p r o d u c t s
R i c e , w h i t e , e n r i c h e d , cooked 0.807
White b r e a d , e n r i c h e d 0.376
Corn bread 0.315
Biscuits 0.244
Whole wheat bread 1.120
Fruits
Peaches, canned 0.017
A p p l e s a u c e , canned 0.014
F r u i t c o c k t a i l , canned 0.122
P e a r s , canned trace
C h e r r i e s , sweet 0.085
Vegetables
Coleslaw with dressing 0.093
Cauliflower 0.102
French f r i e s 0.150
Mashed p o t a t o e s 0.075
Boiled potatoes 0.083
S p i n a c h , canned o r f r o z e n 0.501
Mixed d i s h e s
B e e f - v e g e t a b l e stew 0.101
P i z z a , cheeze 0.259
C h i l i con c a r n e 0.140
C h i c k e n noodle c a s s e r o l e 0.140
V e g e t a b l e beef soup 0.181
Desserts
Y e l l o w cake 0.390
Pumpkin p i e 0.620
Gelatin dessert 0.005
^ h e s e v a l u e s were r e c a l c u l a t e d from t h o s e r e p o r t e d by Tack (50) and
a r e i n r e a s o n a b l y good agreement w i t h t h o s e r e p o r t e d by P e n n i n g t o n
e t a l . (32) and Gormican e t a l . ( 3 8 ) .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
6 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

o f manganese i n meat and f i s h , t h i s i m p l i e s manganese a b s o r p t i o n and

r e t e n t i o n from these p r o d u c t s i s e x c e l l e n t and i m p l i e s t h a t these
a n i m a l o r i g i n p r o d u c t s enhance u t i l i z a t i o n o f manganese s u p p l i e d by
p l a n t - o r i g i n p r o d u c t s as w e l l .

Methods o f E v a l u a t i o n o f Manganese S t a t u s

Most s t u d i e s on f a c t o r s a f f e c t i n g manganese needs o f human s u b j e c t s

have employed t h e manganese b a l a n c e t e c h n i q u e d u r i n g s h o r t - t e r m
f e e d i n g p e r i o d s r a n g i n g from f i v e t o 14 days. However, measurements
of o t h e r t i s s u e , b l o o d o r u r i n e components might be o f v a l u e i n
assessment o f n u t r i t i o n a l s t a t u s o f humans o r a n i m a l s . F o r example,
c h i l d r e n w i t h l e a r n i n g d i s a b i l i t i e s have been r e p o r t e d t o have h i g h
h a i r manganese l e v e l s ( 3 9 ) , e l e v a t e d b l o o d serum l e v e l s o f mangan-
ese have been r e p o r t e d i n such d i s e a s e s t a t e s as c o n g e s t i v e h e a r t
f a i l u r e ( 4 0 ) , i n f e c t i o n s (40) and A l z h e i m e r - l i k e d i s e a s e s (41) and
e l e v a t e d manganese c o n c e n t r a t i o n
i n d i v i d u a l s w i t h e x c e s s manganes
a r t h r i t i s (44) and i r o n - d e f i c i e n c y anemia ( 4 5 ) . Whole b l o o d mangan
ese l e v e l s o f many but n o t a l l a d u l t s w i t h c o n v u l s i v e d i s o r d e r s
have been found t o be lower than i n normal c o n t r o l s ( 4 6 ) .

B a s i s f o r Manganese "Safe I n t a k e L e v e l s "

The Food and N u t r i t i o n B o a r d , N a t i o n a l Research C o u n c i l , N a t i o n a l

Academy o f S c i e n c e s (47) has l i s t e d s a f e i n t a k e l e v e l s o f manganese
a l t h o u g h i n f o r m a t i o n was, a t t h e time o f t h e 1980 l i s t i n g , t o o
f r a g m e n t a r y f o r exact recommendations t o be made. These manganese
" s a f e i n t a k e l e v e l " l i s t i n g s were as f o l l o w s : 2.5-5.0 mg/day f o r
a d u l t s , 0.7-1.0 mg/day f o r i n f a n t s and 1.0-5.0 mg/day f o r t o d d l e r s .
I n l a b o r a t o r y c o n t r o l l e d s t u d i e s , p o s i t i v e manganese b a l a n c e s
( c a l c u l a t e d from the f o r m u l a : manganese b a l a n c e = d i e t a r y manganese
- f e c a l manganese - u r i n e manganese) have been observed when s u b j e c t s
were m a i n t a i n e d a t 2.5 mg manganese/day o r h i g h e r but n e g a t i v e b a l -
ances o c c u r r e d when s u b j e c t s were f e d 0.7 mg manganese/day ( 4 8 ) . An
e x t e n s i v e r e v i e w o f t h e l i t e r a t u r e r e l a t i v e t o human s t u d i e s on
manganese r e q u i r e m e n t s o f humans i s p r e s e n t e d by F r e e l a n d - G r a v e s e t
a l . i n t h i s volume. A f a c t o r i a l method f o r e s t i m a t i o n o f manganese
r e q u i r e m e n t s o f humans i s g i v e n by these a u t h o r s .
I n r e c e n t s t u d i e s r e p o r t e d by Rao and Rao ( 4 9 ) , I n d i a n men
r e q u i r e d 3.72 mg manganese/day t o m a i n t a i n manganese b a l a n c e . In
s t u d i e s conducted a t t h e U n i v e r s i t y o f N e b r a s k a , American a d u l t
s u b j e c t s a l s o f a i l e d t o be i n manganese b a l a n c e when manganese i n t a k e
was m a i n t a i n e d a t 2.5 mg/day ( 3 1 ) .
C u r r e n t e s t i m a t i o n o f d i e t a r y manganese adequacy may be t o o
low, p a r t i c u l a r l y i f d i e t s c o n t a i n s u b s t a n t i a l amounts o f f i b e r o r
a r e based l a r g e l y on p l a n t p r o d u c t s . However, f o r meat c o n t a i n i n g
d i e t s , t h e c u r r e n t e s t i m a t e d l e v e l s o f adequacy may be q u i t e ade-
quate.

Conclusion

W h i l e manganese n u t r i t i o n a l s t a t u s i s n o t c u r r e n t l y r e c o g n i z e d as a
problem i n t h e U n i t e d S t a t e s o r i n t h e r e s t o f t h e w o r l d , c e r t a i n
groups may have l e s s than o p t i m a l manganese n u t r i t i o n a l s t a t u s

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
1. KIES Manganese Bioavailability Overview 7

because o f lower i n t a k e s than p r e v i o u s l y s u s p e c t e d from s e l f -

s e l e c t e d d i e t s . There i s a need f o r e s t a b l i s h m e n t o f manganese
r e q u i r e m e n t s f o r a l l age/sex groups consuming d i e t based on c u r r e n t
or recommended food p a t t e r n s . F u r t h e r m o r e , t h e f e a s a b i l i t y o f
e x p r e s s i n g manganese c o n t e n t s o f foods on t h e b a s i s o f b i o l o g i c a l l y
a v a i l a b l e manganese c o n t e n t r a t h e r than on c h e m i c a l l a b o r a t o r y v a l u e s
deserves c o n s i d e r a t i o n .

Acknowledgment s

P u b l i s h e d as U n i v e r s i t y o f Nebraska A g r i c u l t u r a l Research D i v i s i o n
J o u r n a l A r t i c l e S e r i e s No. 8062a. Supported by Nebraska A g r i c u l t u r a l
Research D i v i s i o n P r o j e c t 91-031 and USDA, CSRS R e g i o n a l Research
P r o j e c t W-143.

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1980, 110, 795.
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20. Lonnerdal, B.; Keen, C.L. and Hurley, L.S. FEBS Lett. 1979,
68, 1024.
21. Keen, C.L.; Tamura, T.; Lonnerdal, B.; Hurley, L.S. and Halsted,
C.H. Am. J . Clin. Nutr. 1985, 41, 929.

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ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
8 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

22. Paynter, D.I. J. Nutr. 1979, 110, 437.

23. Scrutton, M.C.; Utter, M.F. and Mildvan, A.S. J. Biol. Chem.
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25. Doisy, E.A., Jr. In Trace Substances in Environmental Health;
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1983, 27, 488.
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Biol. Med. 1961, 107, 734.
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Med. 1966, 67, 836.
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44. Cotzias, G.C.; Papavasilious, P.S.; Hughes, E.R.; Tang, L . ;
and Borg, D.C. J. Clin. Invest. 1968, 49, 992.
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1969, 19, 100.
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Y.Y. and Aronson, R.B. Neurology 1979, 29, 1466.
47. Food and Nutrition Board. Recommended Dietary Allowances; 9th
Rev. Ed.; National Academy of Sciences: Washington, DC, 1980.
48. McLeod, B.E. and Robinson, M.R. Br. J . Nutr. 1972, 27, 221.
49. Rao, C.N. and Rao, B.S.N. Nutr. Reports Int. 1982, 26, 1113.
50. Tack, K. 1984. M.S. Thesis, University of Nebraska-Lincoln.
RECEIVED July 21, 1987

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Chapter 2

Manganese Uptake and Retention

Experimental Animal and Human Studies
1,2 1,3 1 4
Bo Lönnerdal , Carl L. Keen , J. G. Bell , and B. Sandström
1
Department of Nutrition, University of California—Davis, Davis, CA 95616
2
Laboratory for Energy-Related Health Research, University of California—Davis,
Davis, CA 95616
3
Department of Internal Medicine, University of California—Davis, Davis, CA 95616
4
Department of Clinical Nutrition, University of Gothenburg, Gothenburg, Sweden

Retention of dietary manganese is very high during

the neonatal period
decreases considerably
decreased uptake and increased excretion of absorbed
Mn via bile. Studies on brush border membranes from
suckling rat small intestine demonstrate two compo-
nents involved in the uptake of Mn, one saturable
with limited capacity and one non-saturable, indi-
cating passive uptake above a certain level of Mn.
Although mucosal factors strongly affect Mn absorp-
tion, dietary factors also influence its uptake. In
early life, Mn absorption from human milk and cow's
milk formula is high compared to soy formula. These
differences are also observed at later stages in
life, although the differences are less pronounced.
Age, Mn intake and dietary factors affect Mn absorp-
tion and retention and need to be considered when
establishing requirements.

At present our knowledge concerning the uptake and retention of Mn

in humans and experimental animals i s limited (1). The convention-
a l balance technique has serious l i m i t a t i o n s for studying the
absorption and retention of Mn due to the low retention of the
element from any single meal and the slow turnover of the mineral
in the body. There have been a few limited studjLgs on the absorp-
t i o n and excretion of Mn using the radioisotope Mn; yet the
mechanisms of Mn absorption are not well understood. I t i s
believed that absorption of Mn occurs throughout the length of the
small intestine (2). The e f f i c i e n c y of Mn absorption i s r e l a t i v e l y
low, and i t i s not thought to be under homeostatic c o n t r o l . For
the adult human, i t has been reported that approximately 3-4% of
dietary Mn i s absorbed (3). High levels of dietary calcium,
phosphorus, and phytate have been shown to increase the require-
ments for Mn i n several species, possibly by adsorption of Mn i n

0097-6156/87/0354-0009$06.00/0
© 1987 American Chemical Society

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
10 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

the i n t e s t i n a l t r a c t r e s u l t i n g i n a reduction of soluble element

which can be absorbed (4).
The neonatal period poses several p o t e n t i a l problems with
regard to Mn n u t r i t i o n . In contrast to other trace elements such
as iron, zinc and copper, Mn stores are not thought to be accrued
during f e t a l l i f e (5); therefore, the infant may be dependent on an
adequate supply of Mn during early postnatal l i f e and consequently
p a r t i c u l a r l y susceptible to Mn deficiency since the concentration
of t h i s element i n milk i s low. On the other hand, Mn absorption
i s high during early l i f e and therefore high dietary levels of Mn
may lead to excess retention. That infant formulas can contain
high levels of Mn was shown by Lonnerdal et a l . (6) and Stastny et
a l . (7). There have been reports correlating high h a i r Mn levels
to learning d i s a b i l i t i e s (8,9) i n which the authors suggested
excessive Mn intake during early l i f e resulting i n neurological
disturbances. We have therefore found i t important to assess the
b i o a v a i l a b i l i t y of Mn fro
responsible for the uptak

Manganese Absorption at D i f f e r e n t Ages

Several trace elements are known to be absorbed to a higher extent

i n the newborn than i n adult l i f e . Preliminary studies by Mena C3)
indicated that infants, p a r t i c u l a r l y premature i n f a n t s , retain a
higher proportion of Mn than adults. This i s i n agreement with
studies i n experimental animals. Using everted i n t e s t i n a l sacs
from rats of various ages, Kirchgessner et a l . (10) showed that Mn
absorption decreased with increasing age. Whole body and tissue Mn
uptake studies by Gruden (11) also showed high retention of Mn i n
a r t i f i c i a l l y reared rat pups up to the age of 17 days, while these
values decrease s u b s t a n t i a l l y between days jL^ and 21. In our
studies using suckling rats intubated with Mn after fasting (12),
we also found a precipitous drop i n Mn absorption but i t occurred
between day 15 and day 16 (Fig. 1). These differences may be
explained by differences i n experimental design or that, whatever
the mechanism behind the lower absorption, the a r t i f i c i a l feeding
(cow milk) caused a delay of the change i n Mn absorption. In rats
15 days of age or younger, about 80% of the Mn was retained 24 h
post-intubation, while by day 18 only about 30% was retained. In a
subsequent study, Raghib et a l . (13) reported that Mn absorption
was lower at day 13 than at day 11 and that carcass values ( i n -
cluding l i v e r ) were 17% and 30%, respectively. There are several
possible reasons for the difference i n t h e i r results and ours.
F i r s t , they used a time period of 3 h post-intubation while we used
24 h retention values. I t i s obvious from t h e i r paper that the
stomach, the i n t e s t i n a l contents and the i n t e s t i n a l wall s t i l l
contained considerable quantities of radioisotope (and milk). The
fate of t h i s Mn i s , of course, not known, and may represent absorb-
able Mn that i s "bioavailable." In our study, stomachs and intes-
t i n a l perfusates were very low i n r a d i o a c t i v i t y , demonstrating
complete passage of the d i e t . Second, we added the Mn i n the small
i n t e s t i n a l wall to the carcass value, as we believe that t h i s
represents a pool of Mn that i s available to the body. The lower
values at day 13 reported by Raghib et a l . (13) may be indicative

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
2. L O N N E R D A L E T A L . Manganese Uptake and Retention 11

100-

80-

60
>
0
<
1 40

20

1 1 1 1 1 1 1 1 1
12 14 16 18 20
A G E (days)

F i g u r e 1. Whole body minus stomach, i n t e s t i n a l p e r f u s a t e , and

54
cecum-colon r e t e n t i o n of M n i n young r a t s 24 h p o s t i n t u b a t i o n
(n = 2-7 per p o i n t ; t o t a l n = 30; v a l u e s a r e means + SEM).
(Reproduced w i t h p e r m i s s i o n from Ref. 12. C o p y r i g h t 1986 L a n c a s t e r
Press.)

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
12 NUTRITIONAL BIOAVAILABILITY O F MANGANESE

of a slower transfer of Mn across the mucosa at that age, but may

not necessarily mean that net transfer (retention) i s lower. In
our studies, we found no difference i n net Mn retention for day 11
and day 14 pups, although we found a higher retention i n brain with
time at day 11 than at day 14, demonstrating d i f f e r e n t tissue
retention patterns at various ages (Fig. 2).
The mechanism behind the high retention of Mn at young age i s
not e n t i r e l y known. I t has been suggested that immature b i l i a r y
function at young age may be responsible since the major pathway of
Mn excretion i s v i a b i l e (14). Thus, while many trace elements are
absorbed to a high extent during neonatal l i f e , the capacity of the
body to r i d i t s e l f of p o t e n t i a l l y excessive doses of Mn has not yet
been developed. Some i n d i r e c t support for t h i s has been obtained
by balance studies on term and preterm infants (15). These authors
found a p o s i t i v e Mn balance that increased with the l e v e l of
dietary Mn given and they also found higher Mn retention i n pre-
mature (physiologically
balance was often negative
marily excreted v i a pancreatic or mucosal secretions, could be
eliminated from the body i n spite of high zinc absorption i n
premature infants. Further support for the high retention of Mn i n
early l i f e i s given by the study of Zlotkin and Buchanan (16) who
showed high accumulation of Mn i n premature infants given t o t a l
parenteral n u t r i t i o n .
Another p o t e n t i a l explanation for the lower Mn absorption
observed after day 15 of l i f e , i s the p o s s i b i l i t y of the infant
rats starting to nibble on the s o l i d food, from which Mn a v a i l a b i l -
i t y can be assumed to be lower than from rat milk. However, i n our
study we did not see any v i s u a l signs of s o l i d d i e t i n the gastro-
i n t e s t i n a l t r a c t of day 16-18 pups. In addition, we obtained
similar results when pups were separated from t h e i r dams and no
food was available for the pups post-intubation. Gruden {11) also
found these developmental changes when using a r t i f i c i a l feeding
with cow's milk and no introduction of s o l i d food. Therefore, i t
appears that the changes are induced by changes i n g a s t r o i n t e s t i n a l
physiology rather than by dietary changes.

Mechanisms Behind Manganese Uptake and Retention

In order to explain the high uptake of Mn i n early neonatal l i f e ,

we have studied Mn uptake by brush border membrane vesicles pre-
pared from rats of various ages (17). This method, which i s based
on the selective p u r i f i c a t i o n of brush border membranes and t h e i r
propensity to form v e s i c l e s i n solution, allows us to study the
uptake k i n e t i c s of Mn uptake into the mucosal c e l l . Proper p u r i -
f i c a t i o n of the membrane can be ascertained by the use of marker
enzymes s p e c i f i c for the brush border membrane, f u n c t i o n a l i t y tests
and that the right side of the membrane i s out can be assessed by
glucose uptake studies. We found that Mn uptake by such v e s i c l e s
reached a plateau e a r l i e r ( i . e . , was more rapid) when they were
prepared from day 14 rats than from day 18 or 21 r a t s . Maximum
uptake capacity was also higher (about 3-fold) at day 14 than at
the older ages studied. A possible explanation for the higher rate
and capacity f o r Mn uptake at younger age i s a higher permeability

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
LONNERDAL ET AL. Manganese Uptake and Retention 13

3 5 7
DAYS POST-INTUBATION

F i g u r e 2. Uptake and r e t e n t i o n o f Mn 6 h , 3 o r 7 d p o s t -
intubation i n r a t pups i n t u b a t e d a t ages 11, 14 o r 18 d (n = 2-7
per p o i n t ; t o t a l n = 41; v a l u e s a r e means ± SEM). • , L i v e r ; # ,
carcass; • , brain.
(Reproduced w i t h p e r m i s s i o n from R e f . 12. C o p y r i g h t 1986 L a n c a s t e r
Press.)

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
14 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

of the brush border membrane at t h i s age. Similar findings have

e a r l i e r been made for sodium (18), calcium (19), magnesium (20) and
zinc (21). Another p o s s i b i l i t y i s co-transport with sodium, as a l l
our experiments were made i n the presence of a sodium gradient.
Our results demonstrate that more than one uptake process i s
operating. One appears to be a low capacity, saturable process,
which may involve a membrane receptor. The other, non-saturable
process, indicative of passive transport of the element, may occur
v i a d i f f u s i o n and may therefore allow very high uptake of Mn. That
transport across the membrane occurred and Mn was not bound to the
membrane was demonstrated by using d i f f e r e n t osmolarities induced
by cholamine chloride. These results are i n agreement with those
reported by Garcia-Aranda et a l . (22) who used older rats and an
i n t e s t i n a l perfusion technique. They suggested that the saturable
process occurs v i a an active transport mechanism. We found lower
uptake of Mn i n the presence of ATP and M g C l I t should be
2+

recognized, however, tha

shown previously that AT
v e s i c l e membranes (23,24). Therefore i t i s impossible with t h i s
experimental design to elucidate whether t h i s i s an ATP-dependent
process or not.
A recent study i n weanling rats by Weigand et a l . (25) showed
that at t h i s age, tissue Mn homeostasis can e f f e c t i v e l y be achieved
over a wide range of dietary Mn l e v e l s . A major l e v e l of control
appears to be by lowering the true absorption as expressed as
percent of Mn uptake; thus, with increasing dietary Mn, absorption
varied from 29% down to 2%. However, additional control was
exerted by increased endogenous loss of Mn (presumably v i a b i l e ) .
Therefore, i f Mn absorption i n infancy already i s high and the
excretion pathway not yet developed, homeostasis may not occur.

Dietary Factors Affecting Manganese Absorption

There have been r e l a t i v e l y few studies on Mn b i o a v a i l a b i l i t y from

various types of diets as well as from individual factors i n the
d i e t . However, to better understand the requirement of Mn i n
humans i t i s essential to obtain such information. While Mn d e f i -
ciency i n humans appears to be rare (see Chapter by Keen et a l . ) ,
our knowledge about the signs of human Mn deficiency as well as our
means to c l i n i c a l l y assess Mn status i s very limited. The physio-
l o g i c a l requirement of Mn, i . e . , the amount that must be absorbed
to balance the d a i l y excretion and retention i n growing subjects,
i s not known. The observed whole body turnover rate i n human
adults (a h a l f - l i f e of about 40 days) and available estimates of
t o t a l body Mn content (20 mg) (26) speaks for a d a i l y turnover of
about 0.25 mg. With a low degree of absorption, the dietary
requirement w i l l be much higher.
Balance studies i n young g i r l s have led to an estimated
dietary requirement of 1 mg Mn/day with a suggested allowance of
1.25 mg (27). Adolescent females that were consuming ordinary
foods which were t y p i c a l of t h e i r normal d i e t s , had a negative Mn
balance at an intake of 3 mg per day (28). Other studies have
shown that i n adults t h i s d a i l y intake leads to p o s i t i v e Mn
balance. While i t i s possible that adolescents have a higher

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
2. LONNERDAL ET AL. Manganese Uptake and Retention 15

requirement for Mn, i t was speculated that the high content of

calcium i n these diets may have affected Mn absorption. Balance
data obtained from young women i n New Zealand indicated that 2.5-
3.2 mg Mn/day i s required (29) and data from males i n India (30)
led investigators of that study to suggest a requirement of 3.7-4.1
mg/day. Guthrie and Robinson reported that a d a i l y intake of less
than 2 mg of Mn did not produce any signs of deficiency (31). A
recent balance study by Friedman et a l . (32) indicates a d a i l y
requirement of 2.11 mg Mn. However, as stated previously, our
knowledge of the manifestations of Mn deficiency i s limited.
Limited data are available about the effects of i n d i v i d u a l
dietary components on absorption, and consequently the requirement,
of Mn. Dietary protein and phosphorus levels (33), calcium l e v e l
(34) and the e f f e c t of a p a r t i a l substitution of soy protein for
meat (28) have been tested i n balance studies without any obvious
e f f e c t of Mn absorption or retention. However, since the main
route of excretion i s
technique i s probably no
factors that influence Mn absorption.
In contrast to the estimated dietary requirement of approxi-
mately 35 ug/kg/day i n adults, the requirement of infants has been
estimated to be 0.2-0.6 ug/kg/day (35). This difference i s l i k e l y
explained by the differences i n degree of absorption or i n excre-
t i o n rates and s u s c e p t i b i l i t y to Mn at d i f f e r e n t ages. Because of
the v u l n e r a b i l i t y of the newborn to deficiency and excess of Mn, we
have investigated Mn i n various infant d i e t s and the extent to
which i t i s absorbed. The concentration of Mn i n human milk i s
very low, 4-8 ug/L, as compared to 20-50 ug/L i n cow's milk and
50-1300 ug/L i n U.S. infant formula (6,36,37). Thus, Mn intake of
breast-fed infants w i l l be 0.5-0.9 ug/kg/day, while the intake of
formula-fed infants w i l l be considerably higher and highly variable
depending on the formula used.

1
Table 1. Manganese uptake from milks and formulas i n d 14 rat pups

54
Mn Total Total
Mn retention
concen- perfused small 24 h 2 Mn re-
Diet tration intestine liver retention tained
(ng/dose
(ug/ml) (%) (%) (%) fed)
Human milk 0.01 19.2±2.6 34.8±3. 9 81.5 4.1
Cow milk 0.04 21.1±4.7 35.8±3. 2 89.4 17.9
Cow milk formula 0.05 32.1±2.7 31.0±3. 3 77.4 19.4
Soy formula 0.30 16.6±1.9 26.8±4. 4 64.5 96.8
1. Adapted from Ref. 12.
2. Z = l i v e r , small i n t e s t i n e , brain, kidney, spleen, carcass.

Using our suckling r a t pup model, we have found that the

retention of Mn was 82% from human milk (Table 1); 89% from cow's
milk; 77% from cow's milk formula and 64% from soy formula (12).
Taking into account the varying levels of Mn of these d i e t s , the
amount of Mn retained per dose given was 4, 18, 19 and 97 ng,
respectively, for the four d i f f e r e n t d i e t s . I f these values are

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
16 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

relevant to humans, infants fed cow's milk formula may retain f i v e

times more and infants fed soy formula, 25 times more Mn than
breast-fed i n f a n t s . Lower b i o a v a i l a b i l i t y of Mn from soy formula
as compared to human milk, cow's milk and cow's milk formula has
also been demonstrated i n human adults (38). However, although a
somewhat lower absorption of Mn from soy formula, t h i s i s not
adequate to compensate for the considerably higher native Mn con-
tent of soy formula. Although i t could be speculated that the
higher concentration of Mn could cause the lower percentage absorp-
t i o n (isotope d i l u t i o n ) , we did not f i n d lower absorption of Mn
when the concentration of human milk was increased to 500 times i t
o r i g i n a l concentration or when cow's milk formula Mn concentration
was increased to 100 times i t s o r i g i n a l concentration (Table 2;
12).
54
Table 2. E f f e c t of manganes
1 , 2 f
in d 15 rats
54
Mn retention
Dose Small Total % 4

Diet Mn intestine Liver Brain retained

(mg/L) (% of dose)
Human milk (HM) 0.01 16.6±1.1 31 .311.6 1.810.08 78.612 .1
HM + 10 x native Mn 0.1 18.412.0 28 .512.8 1.910.1 79.915 .5
HM + 50 x native Mn 0.5 14.311.7 32 .111.1 2.110.1* 85.811 .1*
HM + 500 x native Mn 5.0 26.914.0* 26 .911.7 2.110.2 84.512 .2
1. Values are means 1 SEM.
2. S i g n i f i c a n t l y d i f f e r e n t from nonsupplemented d i e t s by Student's
t - t e s t : *P < 0.05.
3. Adapted from Ref. 12.
4. I = small i n t e s t i n e , l i v e r , kidney, spleen and carcass.

S i m i l a r l y , i n our human studies, percent Mn retention was similar

when a 50 ug dose was given and when a 2500 ug dose was given (39).
These data are i n agreement with a recent study by Weigand et a l .
(25) that show s u b s t a n t i a l l y increased true absorption (ug) when
dietary Mn i s increased. In order to assess the influence of
various components of infant diets on Mn uptake, we have i d e n t i f i e d
the compounds binding Mn i n human and cow's milk (40). F i r s t , by
using ultracentrifugation and u l t r a f i l t r a t i o n , we showed that
i n t r i n s i c Mn added to milks and infant formula d i s t r i b u t e d among
major milk fractions (fat, whey, casein) i n a pattern very s i m i l a r
to that observed for native (cold) Mn present i n these diets (Fig.
3). This strongly indicates that an e x t r i n s i c tag of Mn e q u i l i -
brates with the i n d i v i d u a l components binding Mn i n these d i e t s .
We subsequently used e x t r i n s i c a l l y labeled diets for further
fractionation by gel f i l t r a t i o n and immunoaffinity chromatography.
Mn i n human milk was found to be predominantly (about 70%) bound to
l a c t o f e r r i n , the major iron-binding protein i n human milk. Minor
amounts were bound to casein i n human milk and the milk f a t globule
membrane. I t i s therefore possible that changes i n l a c t o f e r r i n
concentration i n human milk may explain the developmental pattern

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
2. LONNERDAL ET AL. Manganese Uptake and Retention 17

Figure 3. D i s t r i b u t i o n of e x t r i n s i c and native manganese among

fractions of milks and formulas.
(Reproduced with permission from Ref. 40. Copyright 1985 American
Society for C l i n i c a l Nutrition.)

120 -

uJ 100 -

80 -

p 60 h
<
r'0.83
| 40 - p< 0.005
01 0 < 5 >
Li. n. O °

20 -

5 10 15 20 25
Fe:Mn RATIO IN THE INTESTINE

Figure 4. The relationship between iron/manganese r a t i o i n

mouse i n t e s t i n a l and l i v e r .
(Reproduced with permission from Ref. 45. Copyright 1984
Raven Press.)

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
18 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

for Mn observed during the l a c t a t i o n period (36). In early l a c t a -

t i o n , l a c t o f e r r i n and Mn concentrations are high and l a t e r i n
l a c t a t i o n they both decline. Towards the end of l a c t a t i o n , invo-
l u t i o n of the mammary gland causes the l a c t o f e r r i n concentration to
increase with a concomitant r i s e i n Mn concentration. It should be
recognized, however, that although l a c t o f e r r i n binds the major part
of Mn i n human milk, the l a c t o f e r r i n content of Mn as compared to
that of iron i s very low (about 1:2000). In contrast to human
milk, Mn i n cow's milk i s mostly bound to casein (> 60%) and very
l i t t l e i s bound to the f a t (< 2%). Similar to cow's milk, Mn i n
most infant formulas i s bound to casein or to insoluble material
(soy formula).
Some recent studies have assessed the e f f e c t of low molecular
weight ligands on Mn uptake. Garcia-Aranda et a l . (22) found a
p o s i t i v e e f f e c t of h i s t i d i n e and c i t r a t e on Mn uptake by rat intes-
t i n a l sacs. We did not f i n d a s i g n i f i c a n t e f f e c t of h i s t i d i n e on
Mn uptake i n our brush borde
possible that Mn uptake
studied) that a pronounced e f f e c t on Mn uptake w i l l not be ob
served. We found, however, a p o s i t i v e e f f e c t of ascorbic acid
added to the Mn solution, indicating either an e f f e c t as a reducing
agent or as a chelator, s i m i l a r to what i s found for iron.
The influence of iron status and dietary iron on Mn absorption
should also be recognized. An interaction between these two
elements was demonstrated already by Matrone et a l . (41) who showed
impaired hemoglobin synthesis i n p i g l e t s given high levels of Mn,
presumably due to decreased iron absorption. Conversely, Thomson
and Valberg (42) showed increased Mn absorption i n i r o n - d e f i c i e n t
r a t s . We have also shown dramatically increased Mn absorption (45%
vs. 9% i n controls) i n an i r o n - d e f i c i e n t woman (39). In rats the
absorption and retention of Mn i s r e l a t i v e l y high for foods low i n
iron such as milk. If milk i s supplemented with iron, the percen-
tage Mn absorbed i s reduced (43). A s i g n i f i c a n t l y lower concentra-
t i o n of Mn i n the l i v e r has been shown i n weanling mice fed i r o n -
supplemented milk than from those fed non-supplemented milk,
presumably r e f l e c t i n g lower absorption of Mn (44). Although the
functional significance of t h i s finding remains to be determined,
the l e v e l of iron supplementation was similar to that of i r o n -
f o r t i f i e d infant formulas. Therefore, iron supplementation may
have an unwanted side e f f e c t of compromised Mn status. On the
other hand, i n i r o n - d e f i c i e n t infants Mn absorption may be i n -
creased, possibly leading to excess Mn uptake. We have shown a
strong p o s i t i v e c o r r e l a t i o n between the r a t i o of Mn i n intestine
and l i v e r , indicating that the control of absorption at the mucosal
l e v e l w i l l determine l i v e r uptake (Figure 4) (45).
In conclusion, i t i s evident that further studies are needed
on Mn absorption and retention during infancy and i t s c o r r e l a t i o n
to dietary composition and iron status.

Literature Cited
1. Keen, C. L.; Lonnerdal, B.; Hurley, L. S. In Biochemistry of
the Essential Ultratrace Elements; Frieden, E., Ed.; Plenum:
New York, 1984, pp. 89-137.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
2. LÖNNERDAL ET AL. Manganese Uptake and Retention 19

2. Thomson, A. B. R.; Olatunbosun, D.; Valberg, L. S. J . Lab.

Clin. Med. 1971, 78, 643-55.
3. Mena, I. In Disorders of Mineral Metabolism; Bronner, F. and
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Neonate 1979; 36, 225-32.
6. Lonnerdal, B.; Keen, C. L . ; Ohtake, M.; Tamura, T. Am. J .
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8. Pihl, R. O.; Parkes, M. Science 1977, 198, 204-6.
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Element Metabolism in Man and Animals (TEMA-4); McC. Howell,
J., Gawthorne, J
Academy of Sciences
11. Gruden, N. Nutr. Rep. Int. 1984, 30, 553-7.
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395-402.
13. Raghib, M. H.; Chan, W.-Y.; Rennert, O. M. Br. J . Nutr. 1986,
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14. Miller, S. T.; Cotzias, G. C.; Evert, H. A. Am. J . Physiol.
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15. Dörner, Κ.; Sievers, Ε.; Dziadzka, S. In Human Lactation,
Vol. 3; Goldman, A. S., Atkinson, S. A. and Hanson, L. Α.,
Eds.; Plenum: New York, 1987; (in press).
16. Zlotkin, S. H.; Buchanan, Β. E. Biol. Trace Element Res.
1986, 9, 271-9.
17. Bell, J . G.; Keen, C. L.; Lonnerdal, B. Fed. Proc. 1987; 46,
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18. Ghishan, F. K.; Wilson, F. Am. J . Physiol. 1984, 248, G47-52.
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20. Meneely, R. L . ; Leeper, L . ; Ghishan, F. K. Pediatr. Res.
1982, 16, 295-8.
21. Ghishan, F. K.; Sobo,G. Pediatr. Res. 1983, 17, 148-51.
22. Garcia-Aranda, J . Α.; Wapnir, R. Α.; Lifshitz, F. J . Nutr.
1983, 113, 2601-7.
23. Menard, M. R.; Cousins, R. J . J . Nutr. 1983, 113, 1434-42.
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Res. 1986, 10, 265-79.
26. Schroeder, Η. Α.; Balassa, J. J.; Tipton, I. H. J . Chron.
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28. Greger, J . L . ; Balinger, P.; Abernathy, R. P.; Bennett, O. A.;
Peterson, T. Am. J . Clin. Nutr. 1978, 31, 117-21.
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In Nutritional Bioavailability of Manganese; Kies, C.;

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32. Friedman, B. J.; Freeland-Graves, J . H.; Bales, C. W.;

Behmardi, F.; Shorey-Kutschke, R. L.; Willis, R. A.; Crosby,
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Trace Element Metabolism in Man and Animals (TEMA-6); Hurley,
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1987 (in press).
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RECEIVED August 20, 1987

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Chapter 3

Dietary Manganese Toxicity and Deficiency

Effects on Cellular Manganese Metabolism
1,2 1,3 1,3
Carl L. Keen , Sheri Zidenberg-Cherr , and Bo Lönnerdal
1
Department of Nutrition, University of California—Davis, Davis, CA 95616
2
Department of Internal Medicine, University of California—Davis, Davis, CA 95616
3
Laboratory for Energy-Related Health Research, University of California—Davis,
Davis, CA 95616

The kinetics of Mn with regard to tissue uptake and

metabolism unde
ciency were investigated
a marked difference in Mn uptake among tissues.
Liver, kidney and pancreas Mn uptake is higher than
that by the brain, suggesting a difference in the
mechanisms underlying the cellular transport of Mn
into these tissues. Cellular metabolism of Mn also
differs among tissues; Mn taken up by the liver under
toxic conditions is initially distributed between a
protein with a MW of 80,000 and low MW ligands, while
in pancreatic tissue Mn is mainly associated with LMW
ligands. Data on the effects of Mn deficiency on Mn
uptake show that the uptake and cellular localization
of the element are not influenced by a deficiency
state, suggesting that the cellular uptake and
binding of Mn is not amplified under deficiency
conditions.

Manganese i s a metal which was already recognized during the time

of the ancient Roman Empire. I t s name i s believed to be derived
from the Greek word ]iayyoL\)e\a., which roughly translates as "magic"
(1). Whether t h i s derivation i s or i s not correct i t i s c e r t a i n l y
a meaningful designation i n view of the d i v e r s i t y of metabolic
functions ascribed to t h i s element (2^) and numerous abnormalities
to which either manganese deficiency or t o x i c i t y can give r i s e t o .
The e s s e n t i a l i t y of manganese (Mn) f o r animals was established
i n 1931 by Orent and McCollum (3) and Kemmerer and co-workers (4)
who demonstrated poor growth i n mice and abnormal reproduction i n
rats fed diets d e f i c i e n t i n the element. Today i t i s known that
under r i g i d l y controlled laboratory conditions, Mn deficiency
r e s u l t s i n a wide v a r i e t y of structural and metabolic defects.
That Mn deficiency could p o t e n t i a l l y be a problem i n humans was
f i r s t suggested by Doisy i n 1972 (5) (Table I ) .

0097-6156/87/0354-0021$06.00/0
© 1987 American Chemical Society

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
22 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

TABLE I. Reported Cases of Suspected Human Manganese Deficiency

Observation Authors
Accidental Mn deficiency i n
a male subject Doisy, 1972 (5)
E p i l e p t i c s have low blood Mn Papavasiliou et a l . , 1979 (6)
A l i n k between mseleni disease
and Mn deficiency Fincham et a l . , 1981 (8)
Low tissue Mn i n MSUD and PKU Hurry and Gibson, 1982 (9)
Mn deficiency and h i p d i s -
location i n Down's syndrome Barlow and Sylvester, 1983 (10)
Low h a i r Mn i n infants with
congenital malformations
and t h e i r mothers Saner et a l . , 1985 (11)
Low blood Mn i n non-head
injury e p i l e p t i c s C a r l et a l . , 1986 (13)
Low blood Mn i n osteoporosi
Experimental Mn deficienc
i n male subjects Friedman et a l
-1 1987 (14)

The subject described i n h i s report was a male volunteer who

developed Mn deficiency following i t s accidental omission from a
p u r i f i e d d i e t that was being used to investigate the e f f e c t s of
vitamin K deficiency. Signs that were associated with the feeding
of t h i s d i e t that are not normally considered consequences of
vitamin K deficiency included weight l o s s , reddening of h i s black
h a i r , reduced growth of h a i r and n a i l s , dermatitis and hypocholes-
terolemia. Unfortunately, i t i s not possible to determine with
certainty which of the above signs were actually due to Mn d e f i -
ciency, since the subject was given a "control" h o s p i t a l d i e t after
i t was recognized that the element had been l e f t out of the d i e t .
Thus the e f f e c t s of s e l e c t i v e l y adding Mn back to t h i s d i e t on the
expression of the above signs were not determined. Despite the
report of Doisy, for a considerable period of time there was l i t t l e
interest i n studying the role of Mn n u t r i t i o n i n human health, i n
part due to the perception that Mn deficiency i n humans would not
occur under natural conditions due to a low requirement for the
element coupled with i t s r e l a t i v e l y high concentration i n the d i e t .
Interest i n the p o s s i b i l i t y of human Mn deficiency increased
i n 1979 with the report of Papavasiliou and co-workers (6) that
some e p i l e p t i c s were characterized by lower than normal blood
concentrations of Mn. Based on the knowledge that Mn deficiency i n
experimental animals could r e s u l t i n increased s u s c e p t i b i l i t y to
electroshock and drug-induced seizures (1), these authors suggested
that Mn deficiency could be an e t i o l o g i c a l factor for epilepsy i n
some i n d i v i d u a l s .
Subsequent to the report of Papavasiliou et a l . , several
investigators have hypothesized a role for abnormal Mn metabolism
in a variety of diseases. In 1981, Fincham et a l . (8) suggested
that Mn deficiency may be an underlying factor i n the development
of mseleni j o i n t disease. In 1982 Hurry and Gibson (9) reported
that Mn deficiency could be a c h a r a c t e r i s t i c of some inborn errors
of metabolism such as maple syrup urine disease (MSUD) and phenyl-
ketonuria (PKU). Poor Mn status has also been suggested to be a

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
3. KEEN ET AL. Dietary Manganese Toxicity and Deficiency 23

problem i n adult Down's syndrome patients with secondary d i s l o c a -

t i o n of the h i p (10). Hip abnormalities ranged from severe e p i -
p h y s i t i s of the femoral head to mild subluxation of the j o i n t . The
authors speculated that the h i p dislocations observed i n the Down's
patients were related to low Mn d i e t s fed when the subjects were
infants. In 1985, Saner et a l . (11) suggested that Mn deficiency
may play a role as one p o t e n t i a l factor underlying intrauterine
malformations. This hypothesis was based on t h e i r observation that
infants with congenital malformations, and t h e i r mothers, were
characterized by low h a i r Mn concentrations compared to healthy
infants and t h e i r mothers. F i n a l l y , Strause and Saltman (12) have
obtained evidence suggesting that i n some individuals Mn deficiency
may be a factor i n the development of osteoporosis.
Evidence for the idea that Mn deficiency may be a cause,
rather than an e f f e c t , of some human pathologies has recently been
provided by C a r l et a l . (13) who reported that the low blood Mn
concentrations observe
of patients characterize
than i n the subgroup i n which the epilepsy was known to be the
r e s u l t of head injury. This observation suggests that the low Mn
concentrations are not a function of either seizure a c t i v i t y or
medication, as these two parameters were s i m i l a r i n the two patient
subgroups reported on. Thus, i t i s reasonable to suggest that Mn
deficiency may be a causative factor i n some e p i l e p t i c s .
Despite increasing recognition that Mn deficiency may be a
factor underlying several human pathologies, the metabolism of t h i s
element i s poorly understood. In our opinion, the recent report by
Friedman and co-workers (14) concerning the r e l a t i v e l y rapid
induction of Mn deficiency i n male subjects fed diets low i n t h i s
element further underscores the need to understand the metabolism
of t h i s element i n humans.
Manganese t o x i c i t y i s also known to represent a serious health
hazard to humans, with toxic intakes of the element (either through
the a i r or diet) r e s u l t i n g i n severe pathologies, p a r t i c u l a r l y of
the central nervous system (15-19). The f i r s t observation of Mn
t o x i c i t y i n humans was made by Couper i n 1837 (15) (Table I I ) , who
reported a p a r a l y t i c disease i n workers of a pyrolusite (Mn
dioxide) m i l l .

Table I I . Selected Cases of Human Manganese T o x i c i t y

Observation Authors
P a r a l y t i c disease i n workers
i n a pyrolusite m i l l Couper, 1837 (15)
Neurological disorders due to
Mn t o x i c i t y v i a contaminated
water Kawamura et a l . , 1941 (16)
Neurological disorders i n Mn Cotzias et a l . , 1968 (17)
mine workers Mena, 1981 (18)
Mn t o x i c i t y due to excessive
o r a l Mn supplements Banta and Markesbury, 1977 (19)
Pancreatitis due to Mn-
contaminated d i a l y s i s f l u i d Taylor and Price, 1982 (20)

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
24 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

In 1941 Kawamura and co-workers (16) described an outbreak of Mn

t o x i c i t y i n individuals who consumed water contaminated with Mn.
These individuals were characterized by neurological disorders and
a variety of soft tissue pathologies. However, Mn t o x i c i t y was not
considered as a serious health hazard u n t i l i t was recognized by
Cotzias and co-workers (17) that there was a high incidence of Mn
t o x i c i t y i n Mn mine workers i n C h i l e . I t has recently been e s t i -
mated that the incidence of Mn t o x i c i t y i n Mn mine workers i n Chile
i s from 1-4%, while i n India the incidence i s estimated to be as
high as 25% i n a work force that varies between 30,000 and 100,000
individuals (18). In i t s more severe forms Mn t o x i c i t y can r e s u l t
i n a syndrome characterized by severe p s y c h i a t r i c symptoms, i n -
cluding h y p e r i r r i t a b i l i t y , v i o l e n t acts and h a l l u c i n a t i o n s . The
t o x i c i t y r e s u l t s i n a permanent c r i p p l i n g neurological disorder of
the extrapyramidal system, the morphological lesions of which are
similar to the lesions of Parkinson's disease (17). While the
majority of reported case
exposed to excessive airborn
the report of Kawamura , y (19)
have reported a case of Mn t o x i c i t y occurring i n an i n d i v i d u a l who
consumed Mn supplements for several years. In addition, Taylor and
Price (20) have reported a case of p a n c r e a t i t i s i n a patient who
received excess Mn from contaminated d i a l y s i s f l u i d . Given the
above evidence of pathologies associated with abnormal Mn metabo-
lism, i t i s evident that we need to better understand the metabolic
handling of t h i s element under conditions of t o x i c i t y as well as
deficiency.

Manganese T o x i c i t y

Using the rat as a model, our research group has recently i n i t i a t e d

a series of investigations on the k i n e t i c s of Mn with regard to
tissue uptake and i t s e f f e c t on metabolism under conditions of Mn
t o x i c i t y . In our i n i t i a l work (21) rats were given a single i p
i n j e c t i o n of Mn at 2.5, 10 or 40 mg/kg BW. I t was observed that
there was a rapid dose-dependent r i s e i n tissue (plasma, l i v e r ,
brain, and kidney) Mn concentrations which was rapidly cleared.
Liver and kidney Mn uptake was much higher than that of brain,
suggesting a difference i n the mechanisms underlying the c e l l u l a r
transport and/or binding of Mn by these tissues (Figure 1). Gel
f i l t r a t i o n chromatography of l i v e r 10,000 g supernatants from rats
injected with the 10 mg/kg Mn dose showed that the majority of the
Mn was i n i t i a l l y associated with a protein with a molecular weight
of about 80,000, and also with a low molecular weight compound (<
5,000 daltons). Suzuki and Wada (22) have suggested that t h i s low
molecular weight f r a c t i o n represents Mn that i s being transferred
into the b i l e for excretion. We have t e n t a t i v e l y i d e n t i f i e d the
80,000 molecular weight protein as t r a n s f e r r i n . We suggest that
t h i s transferrin-bound Mn may r e f l e c t a pool of Mn that w i l l be
transported into plasma for subsequent transfer of the element to
extra-hepatic t i s s u e .
In addition to the e f f e c t s of an acute Mn load on c e l l u l a r Mn
metabolism, we observed a marked dose-dependent hyperglycemia i n
the injected rats over a two hour period. This hyperglycemia

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
3. KEEN ET AL. Dietary Manganese Toxicity and Deficiency 25

occurred with a marked drop i n plasma i n s u l i n concentration (21).

The observation of hyperglycemia following Mn i n j e c t i o n i s consis-
tent with reports that Mn can stimulate gluconeogenesis i n v i t r o
using rat l i v e r perfusion and l i v e r culture systems (23-25).
An a l t e r n a t i v e explanation i s that the r i s e i n blood glucose
i s the r e s u l t of a Mn-induced breakdown and mobilization of l i v e r
glycogen. To test t h i s idea we evaluated the e f f e c t s of Mn i n j e c -
t i o n on blood glucose i n rats that were fed, or fasted for 24 or 48
hours (26). I t was found that fasting attenuated the r i s e i n blood
glucose; thus, part of the hyperglycemic response of the Mn appears
to be due to a mobilization of l i v e r glycogen. However, an i n -
crease i n blood glucose was observed even i n the 48 hour fasted
rats (180 mg/dl i n Mn-injected rats versus 120 mg/dl i n c o n t r o l s ) ,
suggesting that processes other than glycogen breakdown were
contributing to the r i s e i n blood glucose. For t h i s reason we
tested the idea that the hyperglycemia may partly be due to an
e f f e c t of Mn on pancreati function i show i Figur 2
pancreatic Mn concentration
j e c t i o n . This increas
drop i n plasma i n s u l i n concentrations and a r i s e i n plasma glucose
concentrations. These observations suggest that changes i n
pancreatic Mn concentration may regulate i n s u l i n production.
Consistent with t h i s idea are the reports of Dean and Matthews (27)
that 2.5 mM Mn can block glucose-induced action potentials i n the
pancreatic B - c e l l ; of Hermansen and Iverson (28) that 50 mM Mn can
i n h i b i t i n s u l i n release i n the isolated perfused canine pancreas;
and of Rorsman et a l . (29) that i n v i t r o , glucose can stimulate
pancreatic Mn uptake. To further test the hypothesis that changes
in pancreatic Mn concentration may be regulatory with regard to
i n s u l i n output, we studied the e f f e c t s of adrenalectomy on the
Mn-induced hyperglycemia (30). This study was based on the idea
that the adrenalectomy would prevent the hyperglycemic response due
to a block i n catecholamine-induced glucagon release and glycogen
breakdown. Thus we reasoned that the glucose-induced r i s e i n
pancreatic Mn concentration should also be attenuated i n these
animals. Consistent with t h i s hypothesis, adrenalectomy blunted
Mn-induced r i s e s i n blood glucose and pancreatic Mn concentration
(Figure 3). In contrast to the pancreas, adrenalectomy had no
e f f e c t on hepatic uptake of Mn following i n j e c t i o n . Consistent
with our r e s u l t s , Hughes et a l . (31) have reported that long-term
adrenalectomy of normoglycemic rats does not a f f e c t hepatic Mn
concentrations. Thus the i n i t i a l uptake of Mn by the pancreas may
i n part be a homeostatic response of the pancreas to the hyper-
glycemic condition. An interesting speculation i s that under
conditions of Mn t o x i c i t y , pancreatic Mn uptake i s excessive and
results i n a subsequent i n h i b i t i o n of i n s u l i n release. I t should
be recognized that i n s u l i n i n turn may a f f e c t the metabolism of Mn,
as hepatic and kidney concentrations of the element are markedly
increased i n diabetic animals (32). While t h i s increase i n tissue
Mn concentration may be secondary to increased gluconeogenesis and
ureagenesis i n the diabetic animal, a d i r e c t e f f e c t of i n s u l i n on
tissue Mn uptake and/or release has not been ruled out (32).

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

TIME POST INJECTION (min)

Figure 1. Changes i n tissue Mn concentrations following

i n j e c t i o n with 2.5 ( A ) , 10 (•) or 40 ( O ) mg Mn/kg BW i n
Sprague-Dawley r a t s .
(Reproduced with permission from Ref. 21. Copyright 1984 Humana
Press.)

i i i 1 1
e e.5 t i.s 2
TIME CHOURS)

Figure 2. Changes i n pancreatic Mn ( A ) plasma i n s u l i n (•) and

glucose (•) concentrations following i n j e c t i o n of 40 mg Mn/kq
BW i n Sprague-Dawley rats.
(Reproduced with permission from Ref. 26. Copyright 1985 Elsevier.)

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
KEEN ET AL. Dietary Manganese Toxicity and Deficiency

F i g u r e 3. Changes i n t h e m o l e c u l a r l o c a l i z a t i o n o f Mn i n r a t
l i v e r and p a n c r e a s f o l l o w i n g i n j e c t i o n w i t h 10 mg Mn/kg BW.
C o n t r o l r a t s (#•—•) , a d r e n a l e c t o m i z e d r a t s ( O — O ) . Chromatog-
raphy on N a B H - t r e a t e d Sepharose CL-6B. Column s i z e 1.6 x 90
4

cm. B u f f e r 0.01 ammonium a c e t a t e , pH 6.5.

(Reproduced w i t h p e r m i s s i o n from Ref. 30. C o p y r i g h t 1986 Academic
Press.)

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
28 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

Manganese Deficiency

It i s important to point out that similar to Mn t o x i c i t y , Mn

deficiency can a f f e c t carbohydrate metabolism. Manganese d e f i -
ciency has been shown to r e s u l t i n reduced pancreatic Mn content
and i n s u l i n synthesis and release i n rats and guinea pigs (33-35).
When Mn-deficient animals are given a glucose load, a diabetic-type
of glucose tolerance curve i s observed (33,34). Thus, similar to
what i s observed for Mn t o x i c i t y , Mn deficiency has a profound
e f f e c t on pancreatic function, strongly supporting the hypothesis
that changes i n i n t r a c e l l u l a r Mn concentrations may be an important
mechanism of c e l l u l a r metabolic control i n the pancreatic c e l l .
For the above reasons, our research group has begun to inves-
tigate Mn uptake and tissue d i s t r i b u t i o n under conditions of
dietary adequacy and deficiency. In these studies we used both the
Sprague-Dawley and Wistar strains of r a t . These two strains were
chosen because we have foun
to dietary Mn deficienc
For example, neonatal mortality i n the offspring of Sprague-Dawley
dams fed Mn-deficient diets during pregnancy and l a c t a t i o n was
approximately 73%, while i n the offspring of Wistar dams fed
d e f i c i e n t diets for similar periods of time i t was 30%. S i m i l a r l y ,
the incidence of congenital ataxia, an expression of prenatal Mn
deficiency (38) , i s over 80% i n the surviving offspring of Mn-
d e f i c i e n t Sprague-Dawley dams, while ataxia i s not observed i n the
offspring of Wistar dams fed the d e f i c i e n t d i e t . A d i f f e r e n t i a l
response to Mn deficiency between the strains can also be observed
when control weanling rats are fed Mn-deficient diets for a period
of six weeks. Sprague-Dawley rats fed the d e f i c i e n t d i e t for t h i s
period of time are characterized by lower than normal l i v e r choles-
t e r o l concentrations (2.43 vs 2.09 mg/g for control and d e f i c i e n t
r a t s , respectively), while cholesterol concentrations are normal i n
the Wistar rats fed the d e f i c i e n t d i e t (2.45 vs 2.48 mg/g) (37).
One interpretation of the above results i s that the Wistar rat
has a better a b i l i t y to adapt to a Mn-deficient d i e t than does the
Sprague-Dawley. Thus we hypothesized that t h i s d i f f e r e n t i a l
response to Mn deficiency between the strains would allow us to
better i d e n t i f y metabolic responses to the consumption of Mn-
d e f i c i e n t d i e t s . In our i n i t i a l study, weanling Sprague-Dawley and
Wistar rats were fed diets containing either 45 or 1 ug Mn/g for 6
weeks. After t h i s period, the rats were fasted overnight and then
jlntubated with 1 g of the 1 ug Mn/g d i e t which was labeled with
Mn. The d i e t was given as a 50% s l u r r y made with deionized
water. Six hours a f t e r intubation the animals were k i l l e d and
tissues were c o l l e c t e d and counted.
Mean Mn concentrations were similar i n Sprague-Dawley and
Wistar rats fed the control d i e t (2.3 and 2.2 ug Mn/g wet weight,
respectively). Manganese concentrations were markedly lower i n the
rats fed the d e f i c i e n t diets compared to the controls. Liver Mn
concentrations were s l i g h t l y lower i n the d e f i c i e n t Sprague-Dawley
rats than i n the d e f i c i e n t Wistar rats (0.4 and 0.7 ug/g, respec-
tively) .
By 6 hours post-intubation the majority of the a c t i v i t y was
recovered i n the intestine and cecum (Figure 4). The only i n t e r n a l

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
3. KEEN ET AL. Dietary Manganese Toxicity and Deficiency 29

organ which retained a substantial amount of a c t i v i t y at t h i s time

was the l i v e r . Despite the marked difference i n l i v e r Mn concen-
t r a t i o n between the d e f i c i e n t and control rats, there was no
difference i n the amount of a c t i v i t y recovered i n t h i s tissue among
the d i f f e r e n t grgups (Figure 5). S i m i l a r l y , the subcellular
d i s t r i b u t i o n of Mn i n the tissues was not affected by d i e t or
s t r a i n (Figure 6).
In t h i s study the low count rate observed i n extrahepatic
tissue made i t d i f f i c u l t to characterize the e f f e c t of dietary
treatment on extrahepatic tissue Mn uptake. Therefore, a second
study was conducted i n which Sprague-Dawley rats which had been fed
£lje control or d e f i c i e n t d i e t for 6 weeks were injected i p with
Mn and then k i l l e d four hours l a t e r . As i s shown i n Figure 7,
s i g n i f i c a n t uptake of the isotope occurred i n pancreas, kidney,
l i v e r , heart and diaphragm i n decreasing order. There were no
differences i n organ d i s t r i b u t i o n of the element between the d e f i -
cient and control animals
data, the subcellular d i s t r i b u t i o
dietary treatment. I t i s interesting to note that t h i s statement
i s true even i n tissues such as l i v e r and pancreas which show a
considerable difference i n subcellular d i s t r i b u t i o n of the element
(Figure 8). Data obtained for the Wistar rat were similar to those
obtained for the Sprague-Dawley r a t .
In addition to similar tissue and subcellular d i s t r i b u t i o n of
the isotope between d e f i c i e n t and control rats, we have g^tained
preliminary evidence that 4 hours after i p i n j e c t i o n of Mn, the
molecular l o c a l i z a t i o n of the isotope i n the 10,000 g l i v e r super-
natant f r a c t i o n i s i d e n t i c a l i n two groups. Chromatograms for both
control and d e f i c i e n t rats show that a large proportion of the
isotope i s recovered i n the low molecular weight region, with a
sharp peak of a c t i v i t y occurring i n the 4-6000 molecular weight
range, while a second peak of a c t i v i t y i s associated with high
molecular weight proteins (39).
Taken together, the above data suggest that Mn deficiency does
not r e s u l t i n an increase i n Mn absorption or an amplification of
c e l l u l a r Mn uptake processes. To further test t h i s idea, we have
investigated Mn uptake using l i v e r , heart and pancreas tissue
s l i c e s obtained from Mn-deficient and control rats. Tissues were
collected from rats which had been fed either control or Mn-
d e f i c i e n t diets for 6 weeks after weaning. Incubations were done
as described by Brandt and Schramm (40). Although l i v e r Mn concen-
trations were markedly lower i n ^ e d e f i c i e n t rats compared to
controls, l i v e r s l i c e uptake of Mn was similar i n the two groups.
S i m i l a r l y , despite markedly lower heart and pancreas Mn concentra-
tions i n s l i c e s from the d e f i c i e n t animals compared to controls,
heart and pancreatic s l i c e uptake k i n e t i c s were similar i n the two
groups (39).
Overall, these data support the idea that c e l l u l a r uptake of
Mn i s not a component i n the regulation of c e l l u l a r Mn concentra-
tions. Thus there appears to be a poor homeostatic response of the
r a t to Mn deficiency with regard to i n t e s t i n a l , hepatic or extra-
hepatic c e l l u l a r uptake of the element. An i n a b i l i t y to compensate
for a low dietary intake of Mn, or low tissue concentrations of Mn,
by increased c e l l u l a r uptake of the element, i s consistent with the

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
30 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

100"

88"

68-

28- f

0-J—
C-SD C-W D-SD D-W

54.,
Figure 4. Mn d i s t r i b u t i o n i g i n t e s t i n e (m)
4 and colon ( UJD )
6 hours after intubation of a Mn-labeled meal i n control (C)
and Mn-deficient (D) Sprague-Dawley (SD) and Wistar (W) rats.

1 .5

1 .2-

0.9-

8.6'

8.3-

C-SD C-W D-SD D-W

54
gjgure 5. Liver retention of Mn 6 hours after intubation of a
Mn-labeled meal i n control (C) and Mn-deficient (D) Sprague-
Dawley (SD) and Wistar (W) r a t s .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
KEEN ET AL. Dietary Manganese Toxicity and Deficiency

188

C-SD C-W D-SD C-W

Figure 6. Nuclear ( • ^ ^ r mitochondrial ( a ) and cytosoljlg

( CCD ) d i s t r i b u t i o n of Mn 6 hours after intubation of a Mn-
labeled meal i n control (C) and Mn-deficient (D) Sprague-Dawley
(SD) and Wistar (W) r a t s .

PANCREAS KIDNEY LIVER HEART

54
Figure 7. Mn d i s t r i b u t i o n i n pancreas, kidney, l i v e r , heart
and diaphragm 4 hours a f t e r i p i n j e c t i o n with Mn i n control
( Hi ) and Mn-deficient (HD ) Sprague-Dawley r a t s .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
32 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

tea

88 -

LIVER PANCREAS PANCREAS

Figure 8. Liver and pancreas nuclear^ ( • ), mitochondrial ( C D )

and cytosolic (,DJ) ) d i s t r i b u t i o n of Mn 4 hours after i p
i n j e c t i o n with Mn i n control (left) and Mn-deficient (right)
Sprague-Dawley rats.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
3. KEEN E T AL. Dietary Manganese Toxicity and Deficiency 33

observation of recent investigators that Mn deficiency may be an

underlying factor i n several disease states.

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2. Keen, C. L . ; Lonnerdal, B.; Hurley, L. S. In Biochemistry of
the Essential Ultratrace Elements; Frieden, E . , Ed.; Plenum:
New York, 1984; pp 89-132.
3. Orent, E. R.; McCollum, E. V. J . Biol. Chem. 1931, 92,
651-78.
4. Kemmerer, A. R.; Elvehjem, C. A.; Hart, E. B. J . Biol. Chem.
1931, 92, 623-30.
5. Doisy, E . , Jr. In Trace Substances in Environmental Health;
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25. Rognstad, R. In Manganese in Metabolism and Enzyme Function;
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Academic: New York, 1986; pp 3-16.
RECEIVED August 20, 1987

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Chapter 4

Manganese Homeostasis in the Chick

1 2 2,3
L. Lee Southern, David H. Baker, and Kevin M. Halpin
1
Department of Animal Science, Louisiana Agricultural Experiment Station,
Louisiana State University Agricultural Center, Baton Rouge, LA 70803
2
Department of Animal Sciences, University of Illinois, Urbana, IL 61801

Manganese is a nutritionally important trace

element for chicks. Dietary energy and protein
sources contain very little bioavailable Mn, and
these feed ingredients reduce the biopotency of
inorganic Mn supplements
exerted primaril
reduced Mn absorption and is mediated by the fiber
and/or ash components of the feedstuffs. Gut ab-
sorption efficiencies are higher when a phytate-
-and fiber-free casein-dextrose diet is fed than
when a corn-soybean meal diet is fed. Dietary i n -
terrelationships exist between Mn and Co and be-
tween Mn and Fe. Cobalt increases Mn absorption
and may precipitate Mn toxicosis. Excess dietary
Mn reduces Fe u t i l i z a t i o n , but excess Fe does not
affect Mn utilization. Eimeria acervulina infec-
tion increases Mn absorption.

Manganese n u t r i t u r e o f t h e c h i c k has been a f e r t i l e area o f i n -

v e s t i g a t i o n f o r over 50 y e a r s . Wilgus and a s s o c i a t e s (1) i n
1936 r e p o r t e d t h a t Mn would prevent p e r o s i s i n c h i c k s . Numerous
r e s e a r c h p u b l i c a t i o n s have s i n c e expanded t h e knowledge o f Mn
homeostasis i n t h e c h i c k . The c h i c k i s unique w i t h regard t o
Mn. I t has a h i g h e r Mn requirement than i t ' s mammalian c o u n t e r -
p a r t , and t h e Mn a b s o r p t i o n e f f i c i e n c y i n c h i c k s i s thought t o
be l e s s than i n mammals ( 2 ) .
The purpose o f t h i s r e p o r t w i l l be t o review c u r r e n t a s p e c t s
o f Mn homeostasis i n t h e c h i c k . In p a r t i c u l a r , Mn a b s o r p t i o n

^Current address: National Dairy Council, 6300 North River Road, Rosemont, IL 60018

0097-6156/87/0354-0035$06.00/0
© 1987 American Chemical Society

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
36 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

e f f i c i e n c y , s t o r a g e , t u r n o v e r , i n t e r a c t i o n s with o t h e r m i c r o -
m i n e r a l s and with macrominerals, e f f e c t o f c o c c i d i a l i n f e c t i o n s ,
requirement and t o x i c i t y , and b i o a v a i l a b i l i t y o f Mn i n conven-
t i o n a l p o u l t r y feeds and i n i n o r g a n i c Mn supplements w i l l be
addressed.
B i o a v a i l a b i l i t y : F e e d s t u f f s and I n o r g a n i c Supplements
Conventional P o u l t r y F e e d s t u f f s . Manganese i s one o f o n l y two
o r t h r e e t r a c e elements t h a t must be supplemented t o c o n v e n t i o n -
al p o u l t r y d i e t s . T h i s requirement f o r supplementation n e c e s s i -
t a t e s knowledge o f Mn b i o a v a i l a b i l i t y i n p o u l t r y f e e d s t u f f s .
Davis and coworkers (3) r e p o r t e d t h a t soybean p r o t e i n reduced Mn
u t i l i z a t i o n , Holmes and Roberts (4) observed an i n c r e a s e d i n c i -
dence o f p e r o s i s i n c h i c k s f e d rapeseed meal and Seth and C l a n -
d i n i n (5) r e p o r t e d t h a t rapeseed meal n o t o n l y i n c r e a s e d t h e i n -
c i d e n c e o f p e r o s i s , but t h a t i t reduced t h p e r o s i s - p r e v e n t a t i v
efficacy of inorganic M
Baker (6) concluded t h a , soybea , ,
bran and r i c e bran reduced Mn d e p o s i t i o n i n bone, b i l e and pan-
creas (Mn r e s p o n s i v e t i s s u e s ) when added t o a c a s e i n - d e x t r o s e
d i e t c o n t a i n i n g 1000 ppm Mn. With t h e e x c e p t i o n o f r i c e bran,
these f e e d i n g r e d i e n t s a l s o reduced t i s s u e Mn c o n c e n t r a t i o n s
when t h e d i e t a r y supplemental l e v e l o f Mn was reduced t o 14 pom
(the Mn requirement o f c h i c k s consuming c a s e i n - d e x t r o s e d i e t s ) .
Rice bran p r o v i d e d b i o a v a i l a b l e Mn when t h e d i e t was a t o r below
the c h i c k ' s Mn requirement. These experiments were conducted
f o r two-week p e r i o d s and, although supplementation o f these f e e d
i n g r e d i e n t s t o t h e c a s e i n - d e x t r o s e d i e t appeared t o reduce Mn
a v a i l a b i l i t y , c h i c k g a i n and e f f i c i e n c y o f g a i n were n o t ad-
v e r s e l y a f f e c t e d . In a subsequent seven-week i n v e s t i g a t i o n ( 7 ) ,
f i s h meal, wheat bran and a corn-soybean meal mixture ( 5 7 % corn
and 43% soybean meal t o s i m u l a t e i n g r e d i e n t r a t i o s i n a 23%
crude p r o t e i n d i e t ) reduced c h i c k performance and t i s s u e Mn con-
c e n t r a t i o n when added t o t h e c a s e i n - d e x t r o s e d i e t c o n t a i n i n g 14
ppm Mn. Only f i s h meal had a d e t r i m e n t a l e f f e c t when added t o a
M n - d e f i c i e n t d i e t (7 ppm Mn). F i s h meal reduced g a i n , f e e d
e f f i c i e n c y and t i s s u e Mn c o n c e n t r a t i o n s , and i t a l s o i n c r e a s e d
the i n c i d e n c e and s e v e r i t y o f p e r o s i s . Wheat bran and t h e c o r n -
soybean meal m i x t u r e , however, i n c r e a s e d bone Mn c o n c e n t r a t i o n
when added t o t h e d i e t c o n t a i n i n g 7 ppm Mn. These r e s e a r c h e r s
concluded from t h e long-term i n v e s t i g a t i o n t h a t wheat bran and
the corn-soybean meal mixture p r o v i d e d b i o a v a i l a b l e Mn when t h e
c h i c k was t h r e a t e n e d with a Mn d e f i c i t , but not when t h e d i e t
c o n t a i n e d adequate ( o r m a r g i n a l l y adequate) Mn. F i s h meal, on
the o t h e r hand, c o n t a i n e d no b i o a v a i l a b l e Mn, r e g a r d l e s s o f Mn
c o n c e n t r a t i o n i n t h e d i e t . Thompson and Weber (8) conducted a
s i m i l a r i n v e s t i g a t i o n over a four-week p e r i o d u s i n g a soybean
meal-dextrose basal d i e t . They added c e l l u l o s e ( 6 % ) , wheat bran
( 1 2 % ) , corn bran ( 9 . 7 % ) , soy bran ( 9 . 7 % ) , o a t h u l l s (7.3%) and
r i c e bran (16.67%) t o t h e basal d i e t such t h a t f e e d i n g r e d i e n t
a d d i t i o n s s u p p l i e d a p p r o x i m a t e l y 6% d i e t a r y n e u t r a l d e t e r g e n t
f i b e r (NDF). These r e s e a r c h e r s concluded t h a t r i c e bran reduced

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
4. SOUTHERN ET AL. Manganese Homeostasis in the Chick

growth, f e e d e f f i c i e n c y and bone Mn c o n c e n t r a t i o n compared w i t h

the basal d i e t . The o t h e r " f i b e r " sources d i d not a d v e r s e l y
a f f e c t c h i c k performance o r bone and l i v e r Mn d e p o s i t i o n . These
r e s u l t s d i f f e r s l i g h t l y from those o f H a l p i n and Baker ( 6 - 7 ) ,
but i n v e s t i g a t i o n a l d i s s i m i l a r i t i e s such as f i b e r c o n t e n t and Mn
l e v e l i n the basal d i e t s probably account f o r the d i s c r e p a n c i e s .
H a l p i n and Baker's d i e t c o n t a i n e d known amounts o f i n o r g a n i c Mn
and was e s s e n t i a l l y d e v o i d o f f i b e r . Thompson and Weber's basal
d i e t , however, c o n t a i n e d 3.8% n e u t r a l d e t e r g e n t f i b e r from soy-
bean meal and 117 ppm Mn from a mixture o f both o r g a n i c and i n -
organic sources.
Although minor d i f f e r e n c e s are e v i d e n t i n the r e s e a r c h de-
s c r i b i n g Mn b i o a v a i l a b i l i t y , i t i s c l e a r t h a t most c o n v e n t i o n a l
p o u l t r y f e e d s t u f f s c o n t a i n very l i t t l e b i o a v a i l a b l e Mn, and i n
many c a s e s , they a c t u a l l y reduce a v a i l a b i l i t y o f i n o r g a n i c Mn
supplements.
The mechanism by whic
occurs was r e c e n t l y e v a l u a t e
f o r 14 days and then p l a c e d on d i e t s c o n t a i n i n g 14 ppm Mn with
o r without 10% wheat bran s u p p l e m e n t a t i o n . Chicks were k i l l e d
s e r i a l l y f o r two weeks f o l l o w i n g the p e r i o d o f Mn " l o a d i n g " .
T i s s u e Mn l e v e l s decreased with t i m e , but wheat bran d i d not
a f f e c t r a t e o f t i s s u e Mn d e p l e t i o n . Thus, wheat bran d i d not
a f f e c t homeostasis o f s t o r e d Mn. In another experiment ( 9 ) , 10%
d i e t a r y wheat bran was f e d to c h i c k s r e c e i v i n g v a r y i n g amounts
o f i n o r g a n i c Mn a d m i n i s t e r e d v i a i n t r a p e r i t o n e a l i n j e c t i o n .
Wheat bran e r r a t i c a l l y a l t e r e d t i s s u e Mn c o n c e n t r a t i o n s , but i t s
e f f e c t d i d not approach the magnitude and c o n s i s t e n c y o f t h a t
o c c u r r i n g with o r a l Mn i n g e s t i o n . The s l i g h t l o w e r i n g e f f e c t o f
d i e t a r y wheat bran on t i s s u e Mn d e p o s i t i o n i n c h i c k s r e c e i v i n g
Mn v i a i n t r a p e r i t o n e a l i n j e c t i o n p r o b a b l y r e s u l t e d from wheat
bran i n h i b i t i n g r e a b s o r p t i o n o f Mn o f b i l i a r y and p a n c r e a t i c
o r i g i n . I t would seem, t h e r e f o r e , t h a t the gut i s the primary
s i t e o f a c t i o n o f the adverse e f f e c t o f wheat bran on Mn homeo-
s t a s i s i n the c h i c k .
A subsequent experiment (9) was conducted to determine
which chemical f r a c t i o n i n wheat bran, f i s h meal, r i c e bran and
a corn-soybean meal mixture i s r e s p o n s i b l e f o r the reduced Mn
a b s o r p t i o n . The n e u t r a l d e t e r g e n t f i b e r f r a c t i o n i n both wheat
bran and the corn-soybean meal mixture reduced t i s s u e Mn con-
c e n t r a t i o n s t o the same e x t e n t as the i n t a c t f e e d i n g r e d i e n t s .
However, the ash component was r e s p o n s i b l e f o r the Mn-lowering
e f f e c t o f f i s h meal, and both the ash and n e u t r a l d e t e r g e n t
f i b e r f r a c t i o n s o f r i c e bran reduced t i s s u e Mn c o n c e n t r a t i o n s ,
but not to the e x t e n t o f i n t a c t r i c e bran. T h i s s t u d y , although
i n c o n c l u s i v e , i n d i c a t e s t h a t the f i b e r (NDF) component o f some
f e e d s t u f f s and the mineral (ash) component o f o t h e r s have a
n e g a t i v e impact on Mn homeostasis i n c h i c k s . Harmuth-Hoene and
Schelenz (10) have r e c e n t l y r e v e a l e d a number o f p o s s i b l e
mechanisms by which f i b e r c o u l d i n t e r f e r e with mineral absorp-
t i o n , van der Aar and a s s o c i a t e s (11) have a l s o r e p o r t e d t h a t
d i e t a r y f i b e r reduces mineral a v a i l a b i l i t y i n c h i c k s .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
38 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

Phytate has been s t u d i e d e x t e n s i v e l y with regard t o mineral

(mostly Zn and Ca) s t a t u s o f a n i m a l s , and i t has been shown t o
reduce whole-body Mn r e t e n t i o n i n r a t s (12). P h y t a t e , however,
i s n o t p r e s e n t i n t h e n e u t r a l d e t e r g e n t f i b e r o r i n t h e ash com-
ponent o f f e e d s t u f f s . T h e r e f o r e , phytate does not appear t o be
r e s p o n s i b l e f o r t h e r e d u c t i o n o f Mn uptake i n c h i c k s f e d c o r n ,
soybean meal, wheat bran o r f i s h meal ( 9 ) . That phytate nega-
t i v e l y impacts Mn n u t r i t u r e a l s o d i s a g r e e s with t h e r e s e a r c h o f
Reinhold e t a_h ( 1 3 ) , who r e p o r t e d t h a t f i b e r , and n o t p h y t a t e ,
was t h e primary f a c t o r d e t e r m i n i n g b i o a v a i l a b i l i t y o f d i v a l e n t
mineral elements i n breads.
I n o r g a n i c Mn Supplements. The b i o a v a i l a b i l i t y o f i n o r g a n i c Mn
supplements has been s t u d i e d l o n g e r and more e x t e n s i v e l y than
the b i o a v a i l a b i l i t y o f Mn i n f e e d i n g r e d i e n t s . As e a r l y as
1939, G a l l u p and N o r r i s (14) added 50 ppm Mn as M n C l . 4 H 0 , 2 2

MnS0 -4H 0, KMnO^, MnC0

lt 2

d i e t and r e p o r t e d t h a t t h
e f f i c a c i o u s i n p r e v e n t i n g p e r o s i s i n c h i c k s . T h e i r high l e v e l
o f Mn supplementation (50 ppm), however, may have p r e c l u d e d t h e
p o s s i b i l i t y o f d e t e c t i n g small d i f f e r e n c e s between t h e com-
pounds. Hennig and coworkers ( 1 5 ) , u s i n g uptake o f r a d i o l a b e l e d
Mn l a t e r r e p o r t e d t h a t MnCL was more a v a i l a b l e t o t h e c h i c k
2

than MnSO^ o r Mn0 . Watson e t a l . (16) i n 1970 d e s c r i b e d a

2

growth and t i s s u e Mn uptake tecTirTique f o r a s s e s s i n g Mn b i o a v a i l -

a b i l i t y o f i n o r g a n i c supplements. These r e s e a r c h e r s subsequent-
l y r e p o r t e d (17) t h a t d i f f e r e n c e s i n Mn b i o a v a i l a b i l i t y e x i s t e d
between d i f f e r e n t sources o f feedgrade Mn oxide and between f e e d -
grade and reagent-grade Mn c a r b o n a t e . Manganese o x i d e and Mn
c a r b o n a t e , depending on t h e s o u r c e , however, were as a v a i l a b l e
as M n S 0 ^ H 0 . In a d d i t i o n , these r e s e a r c h e r s r e p o r t e d t h a t bone
2

Mn c o n c e n t r a t i o n was more r e f l e c t i v e o f d i e t a r y Mn c o n t e n t than

growth o r bone ash p e r c e n t , and t h a t bone Mn c o n c e n t r a t i o n i n -
c r e a s e d i n c r e m e n t a l l y as d i e t a r y Mn c o n c e n t r a t i o n s i n c r e a s e d be-
tween 10 and 120 ppm. Southern and Baker (18) compared t h e b i o -
a v a i l a b i l i t y o f MnS0n.H 0, MnCL .4H 0, MnC0 and Mn0 by adding
2 2 2 3 2

excess Mn (3000, 4000 o r 5000 ppm) t o a corn-soybean meal d i e t

and then measuring t i s s u e Mn uptake, l i v e r Fe a c c u m u l a t i o n , blood
hemoglobin and h e m a t o c r i t , as w e l l as growth and f e e d e f f i c i e n c y .
From these d a t a , i t was concluded t h a t MnS0^.H 0, MnCL -4H 0 and 2 2 2

MnC0 were more e f f i c a c i o u s sources o f Mn than Mn0 , and t h a t

3 2

MnC0 was s l i g h t l y l e s s e f f i c a c i o u s than MnS0 .H 0 and MnCL -4H 0.

3 tt 2 2 2

Estimates o f b i o a v a i l a b i l i t y were made u s i n g t h e s l o p e - r a t i o t e c h -

nique. A s s i g n i n g a value o f 100% t o MnSO^.H-O, a v a i l a b i l i t y
e s t i m a t e s were 102, 77 and 29% f o r MnCL .4H 0, MnCO^ and Mn0 ,
2 2 2

r e s p e c t i v e l y . Black and a s s o c i a t e s (19; used a s i m i l a r e x p e r i -

mental d e s i g n (1000, 2000 o r 4000 ppm excess Mn) and determined
the b i o a v a i l a b i l i t y o f Mn i n MnO and MnC0 r e l a t i v e t o MnS0 .H 0.
3 I+ 2

B i o a v a i l a b i l i t y e s t i m a t e s , again u s i n g t h e s l o p e - r a t i o t e c h n i q u e ,
were 32 and 60% f o r MnC0 and MnO, r e s p e c t i v e l y . S i m i l a r l y ,
3

Henry e t al_. (20) r e p o r t e d t h a t t h e Mn i n MnO was 66% a v a i l a b l e

compare? with 100% f o r MnSO^.H-0. Manganese l e v e l s used i n t h i s
i n v e s t i g a t i o n were 40, 80 and 120 ppm added t o a corn-soybean
meal d i e t .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
4. SOUTHERN ET AL. Manganese Homeostasis in the Chick 39

T i s s u e Mn: Storage and Turnover

Storage, From the d i s c u s s i o n o f Mn b i o a v a i l a b i l i t y , i t i s ob-
v i o u s t h a t d i e t a r y Mn i n t a k e i s a c c u r a t e l y r e f l e c t e d i n t i s s u e
Mn c o n c e n t r a t i o n i n the c h i c k . T h i s i s not s u r p r i s i n g i n t h a t
most m i n e r a l s are d e p o s i t e d i n t o t a r g e t t i s s u e s to some degree.
What i s s u r p r i s i n g , however, i s the remarkable c o n s i s t e n c y w i t h
which increments o f d i e t a r y Mn, r e g a r d l e s s o f the c o n c e n t r a t i o n ,
are d e p o s i t e d i n t o c e r t a i n t i s s u e s . Southern and Baker (18,21)
r e p o r t e d l i n e a r i n c r e a s e s i n bone and g a l l b l a d d e r Mn c o n c e n t r a -
t i o n i n c h i c k s f e d 0, 1500 o r 3000 ppm Mn (21) and i n b i l e con-
c e n t r a t i o n i n c h i c k s f e d 3000, 4000 o r 5000 ppm Mn (18). S i m i -
l a r l y , Black and coworkers (22) r e p o r t e d t h a t d i e t a r y s u p p l e -
ments o f 0, 1000, 2000 and 3000 ppm Mn l i n e a r l y i n c r e a s e d Mn
c o n c e n t r a t i o n i n l i v e r , kidney, muscle, bone and plasma with
c o r r e l a t i o n c o e f f i c i e n t s ( r ) o f .832, .933, .642, .974 and .893,
r e s p e c t i v e l y . Black an
to account f o r l e n g t h o
weeks). Chicks were f e d 0, 1000, 2000 o r 3000 ppm Mn and k i l l e d
a t one, two o r t h r e e weeks a f t e r i n i t i a t i o n o f treatment d i e t s .
M u l t i p l e r e g r e s s i o n a n a l y s i s a c c o u n t i n g f o r time o f Mn f e e d i n g
and d i e t a r y Mn c o n c e n t r a t i o n r e v e a l e d l i n e a r i n c r e a s e s i n l i v e r
(.915), kidney (.980), pancreas (.931), muscle (.945), bone
(.987) and plasma (.969) Mn c o n c e n t r a t i o n s ( c o r r e l a t i o n s c o e f -
f i c i e n t s in parentheses).
T i s s u e Mn c o n c e n t r a t i o n s are not o n l y r e s p o n s i v e to high
d i e t a r y Mn l e v e l s , but a l s o t o d i e t a r y Mn l e v e l s near the c h i c k ' s
requirement. Watson and coworkers (16-17) r e p o r t e d t h a t bone Mn
responded l i n e a r l y to d i e t a r y supplements o f 0 t o 120 ppm Mn.
T h i s f i n d i n g was l a t e r confirmed by Southern and Baker (21) and
by Henry e t aj_. ( 2 0 ) , and the r e s p o n s i v e t i s s u e s were expanded
t o include~"bone and kidney. Remarkably, s l o p e ( t i s s u e Mn concen-
t r a t i o n r e g r e s s e d on Mn i n t a k e ) was the same between 0 and 100
ppm d i e t a r y Mn as t h a t o c c u r r i n g between 100 and 1000 ppm Mn.
Turnover and U t i l i z a t i o n . There i s l i t t l e doubt t h a t c h i c k s f e d
excess l e v e l s o f Mn d e p o s i t t h i s m i n e r a l i n v a r i o u s body
t i s s u e s . H a l p i n (24) i n v e s t i g a t e d the c a p a c i t y o f the c h i c k t o
remove t h i s Mn from s t o r e d d e p o s i t s and t o use i t f o r normal
b o d i l y f u n c t i o n s . Chicks were f e d e i t h e r 14 o r 2000 ppm Mn i n a
c a s e i n - d e x t r o s e d i e t f o r two weeks (8 t o 22 days p o s t h a t c h i n g ) .
A l l c h i c k s f e d 2000 ppm Mn were then switched t o 14 ppm Mn and
s e r i a l l y k i l l e d on days 0, 3, 7, 10 and 14 f o l l o w i n g the d i e t
s w i t c h . T i s s u e Mn c o n c e n t r a t i o n s are presented i n T a b l e I.
D e p l e t i o n o f t i s s u e Mn was c u r v i l i n e a r with time. L o g - t r a n s f o r -
mation o f the d a t a , however, r e v e a l e d a l i n e a r (P<.01) r e d u c t i o n
i n Mn c o n c e n t r a t i o n i n each o f the t i s s u e s . H a l f - l i f e (the num-
ber o f days r e q u i r e d t o a t t a i n o n e - h a l f o f the i n i t i a l t i s s u e Mn
c o n c e n t r a t i o n ) o f the t i s s u e Mn determined by r e g r e s s i o n a n a l y s i s
on the l o g - t r a n s f o r m e d data was 6.0, 7.3 and 1.1 days i n bone,
pancreas and b i l e , r e s p e c t i v e l y . Suso and Edwards (25) o b t a i n e d
a whole-body b i o l o g i c a l h a l f - l i f e o f 5 days i n c h i c k s a d m i n i s t e r e d
5^Mn o r a l l y . From these i n v e s t i g a t i o n s , i t i s c l e a r t h a t t i s s u e

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
40 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

1
Table I. T i s s u e Mn D e p l e t i o n

T i s s u e Mn C o n c e n t r a t i o n , yg/g dry t i s s u e
D e p l e t i o n Time
Days Bone Pancreas Bile

2
Experimental Chicks
0 49.5? 25.4?,
3
7
10
14 n.i
2
Control Chicks
0
14

Means w i t h i n a column not s h a r i n g a common s u p e r s c r i p t d i f f e r

(P<.05). E x p e r i m e n t a l c h i c k s were f e d 2000 ppm Mn d u r i n g the
14-day p r e t e s t p e r i o d and then switched to 14 ppm Mn d u r i n g the
14-day d e p l e t i o n p e r i o d . C o n t r o l c h i c k s were f e d 14 ppm Mn
throughout the 14-day p r e t e s t p e r i o d and the 14-day d e p l e t i o n
period.

s t o r e s o f Mn are not i n e r t and t h a t they r e p r e s e n t a l a b i l e pool

o f Mn f o r the c h i c k .
Subsequent r e s e a r c h a t the U n i v e r s i t y o f I l l i n o i s (D.E.
L a u r i n , K.M. H a l p i n and D.H. Baker, unpublished data) was con-
ducted to assess the c h i c k ' s a b i l i t y to u t i l i z e s t o r e d Mn.
Chick growth and t i s s u e Mn c o n c e n t r a t i o n were determined i n
c h i c k s f e d a c a s e i n - d e x t r o s e , M n - d e f i c i e n t d i e t a f t e r having
p r e v i o u s l y been f e d d i e t s c o n t a i n i n g 14, 140 or 1400 ppm Mn from
0 to 8 days p o s t h a t c h i n g . Chicks f e d p r e t e s t d i e t s c o n t a i n i n g
14, 140 or 1400 ppm Mn r e q u i r e d 14, 21 and 28 days o f consuming
the M n - d e f i c i e n t d i e t , r e s p e c t i v e l y , b e f o r e they e x h i b i t e d a Mn-
d e f i c i e n t growth d e p r e s s i o n . Moreover, i n c h i c k s k i l l e d a t the
f i r s t s i g n o f growth d e p r e s s i o n , bone and b i l e Mn c o n c e n t r a t i o n s
were n e a r l y i d e n t i c a l to those o f c h i c k s f e d the d i e t c o n t a i n i n g
14 ppm Mn throughout the study. Bone Mn c o n c e n t r a t i o n s dropped
from t h e i r day-8 l e v e l s o f 11, 28 and 167 yg/g ash to 3.0, 2.6
and 2.1 yg/g ash f o r c h i c k s f e d 14, 140 and 1400 ppm Mn d u r i n g
the f i r s t 8 days p o s t h a t c h i n g , r e s p e c t i v e l y . Bone Mn c o n c e n t r a -
t i o n s o f c h i c k s f e d the 14 ppm d i e t throughout the study were
11.6 and 3.0 ppm a t days 8 and 36, r e s p e c t i v e l y . These r e s u l t s
i n d i c a t e t h a t c h i c k s can u t i l i z e s t o r e d Mn to m a i n t a i n optimum
growth when p l a c e d on a M n - d e f i c i e n t d i e t and t h a t optimum
growth w i l l be maintained u n t i l body Mn s t o r e s are d e p l e t e d .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
4. S O U T H E R N ET AL. Manganese Homeostasis in the Chick

Absorption
A poor a b s o r p t i o n r a t e o f Mn was thought t o be one o f the r e a -
sons f o r the high Mn requirement i n p o u l t r y r e l a t i v e to mammals
(2). Suso and Edwards (25) f e d c h i c k s a corn-soybean meal d i e t
c o n t a i n i n g 79 ppm Mn o r t h i s d i e t supplemented w i t h 55 o r 110
5tf
ppm Mn from MnS0 -H 0. Chicks were dosed w i t h .2 u C i . o f Mn,
tf 2

and Mn a b s o r p t i o n e f f i c i e n c i e s o f 2.3, 2.1 and 1.5% were ob-

t a i n e d f o r the basal ( B ) , B+55 ppm Mn and B+110 ppm Mn d i e t s ,
r e s p e c t i v e l y . Turk and a s s o c i a t e s ( 2 6 ) , a l s o u s i n g a c o r n -
5l+
soybean meal d i e t (104 ppm Mn), but 2 u C i . o f Mn from M n C l , 2

r e p o r t e d a Mn a b s o r p t i o n e f f i c i e n c y o f .00819%. H a l p i n , Chausow
and Baker (27) compared Mn a b s o r p t i o n e f f i c i e n c i e s i n c h i c k s f e d
a c o n v e n t i o n a l p o u l t r y d i e t (corn-soybean meal, 30 ppm Mn, no
i n o r g a n i c Mn supplementation) o r a c a s e i n - d e x t r o s e d i e t e s s e n -
t i a l l y d e v o i d i n f i b e r and excess n u t r i e n t s . A b s o r p t i o n e f f i -
c i e n c i e s were determine
compare bone Mn d e p o s i t i o
ing increments o f Mn w i t h t h a t o f c h i c k s i n j e c t e d i n t r a p e r i
t o n e a l ^ with i n c r e a s i n g increments o f Mn. The a b s o r p t i o n e f f i -
c i e n c y o f Mn was 1.71% i n the corn-soybean meal d i e t and 2.40%
in the c a s e i n - d e x t r o s e d i e t . The e s t i m a t e o f 1.71% e f f i c i e n c y
i s lower than the e s t i m a t e o f Suso and Edwards ( 2 5 ) , but con-
s i d e r a b l y h i g h e r than the e s t i m a t e o f Turk (26). In a d d i t i o n ,
Suso and Edwards (25) r e p o r t e d a reduced r a t e o f Mn a b s o r p t i o n
as a r e s u l t o f i n o r g a n i c Mn s u p p l e m e n t a t i o n . T h i s i s c o n t r a r y
to the f a c t t h a t bone Mn i n c r e a s e s l i n e a r l y as d i e t a r y Mn i n t a k e
i n c r e a s e s over a wide range (see T i s s u e Mn: Storage and Turn-
o v e r ) . In f a c t , H a l p i n e t a l . (27) r e p o r t e d t h a t r e g r e s s i o n o f
bone Mn c o n c e n t r a t i o n on H ^ a y Mn i n t a k e s was l i n e a r , w i t h
2
c o e f f i c i e n t s o f d e t e r m i n a t i o n ( r ) r a n g i n g from .95-1.00.
The data on a b s o r p t i o n e f f i c i e n c y o f Mn are by no means
c o n s i s t e n t , but e f f i c i e n c y i s p r o b a b l y i n the range o f one to
t h r e e p e r c e n t depending on the type o f d i e t and on the presence
o r absence o f d i e t a r y Mn a n t a g o n i s t s (eg., ash o r f i b e r ) . T h i s
range o f a b s o r p t i o n e f f i c i e n c i e s i n the c h i c k i s not g r e a t l y
d i f f e r e n t from e s t i m a t e s o f one to f o u r p e r c e n t made i n mammals
(28-29). Thus, the c h i c k ' s high Mn requirement r e l a t i v e t o
mammals i s p r o b a b l y not t o t a l l y a r e s u l t o f i n e f f i c i e n t Mn ab-
s o r p t i o n . I n s t e a d , a v i a n s may e x p e r i e n c e a more r a p i d t u r n o v e r
of s t o r e d Mn than mammals, although more d e f i n i t i v e data are
needed to v e r i f y t h i s h y p o t h e s i s .
Interactions with Minerals
M i c r o m i n e r a l s . Manganese p r o b a b l y i n t e r a c t s to some e x t e n t with
many t r a c e elements depending on the c o n c e n t r a t i o n o f Mn and on
the c o n c e n t r a t i o n o f the o t h e r i n t e r a c t i v e element. Other
d i e t a r y c o n s t i t u e n t s p r o b a b l y p l a y a r o l e as w e l l . The d i s c u s -
s i o n h e r e , however, w i l l be l i m i t e d to the i n t e r a c t i o n o f Mn
with Co and Fe. Thomson and V a l b e r g (30) p e r f u s e d t e s t s o l u -
t i o n s o f Fe, Co and Mn i n t o open-ended duodenal loops o f r a t s
and r e p o r t e d t h a t Mn uptake was i n h i b i t e d by both Co and Fe, and

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
42 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

t h a t Mn i n h i b i t e d uptake o f these m i n e r a l s i n r e t u r n . In a
p r e l i m i n a r y i n v e s t i g a t i o n , H a l p i n and Baker (31) r e p o r t e d t h a t
Co d i d not i n h i b i t Mn uptake, but i n s t e a d , 1000 ppm d i e t a r y Co
i n c r e a s e d t i s s u e Mn d e p o s i t i o n . T h i s f i n d i n g was l a t e r con-
f i r m e d by Brown and Southern (32) and by H a l p i n (24). The i n -
v e s t i g a t i o n s by H a l p i n and Baker (31) and by Brown and Southern
(32) were conducted u s i n g a corn-soybean meal d i e t . I t was
o r i g i n a l l y thought t h a t Co i n c r e a s e d Mn uptake by c o m p e t i t i v e l y
b i n d i n g to d i e t a r y f a c t o r s t h a t might otherwise b i n d Mn. In the
r e p o r t by H a l p i n ( 2 4 ) , however, the p o s i t i v e e f f e c t o f Co on Mn
uptake was e v i d e n t even i n a c a s e i n - d e x t r o s e d i e t d e v o i d o f
f i b e r and c o n t a i n i n g a minimum o f excess n u t r i e n t s . I t would
t h e r e f o r e appear t h a t the Mn-binding p r o p e r t i e s o f the c o r n -
soybean meal d i e t were not completely r e s p o n s i b l e f o r the Co-
induced enhancement o f Mn uptake.
An i n t e r a c t i o n a l s o e x i s t s between Fe and Mn a t the gut
l e v e l i n r a t s ( 3 0 ) , an
t h a t excess supplementa
mals and c h i c k s (2,18,21). H a l p i n (24) r e c e n t l y i n v e s t i g a t e d
the mutual i n t e r r e l a t i o n s h i p between Mn and Fe i n c h i c k s . In a
c a s e i n - d e x t r o s e d i e t c o n t a i n i n g e i t h e r 14 o r 1014 ppm Mn, Fe
r a n g i n g from d e f i c i e n t to a 2000 ppm excess had l i t t l e or no
e f f e c t on c h i c k g a i n o r on t i s s u e Mn c o n c e n t r a t i o n . The 1000
ppm Mn a d d i t i o n , however, reduced hemoglobin and h e m a t o c r i t
l e v e l s when d i e t a r y Fe was a t o r below the c h i c k ' s requirement.
These data i n d i c a t e t h a t the i n t e r r e l a t i o n s h i p between Fe and Mn
i s u n i d i r e c t i o n a l i n c h i c k s ; i e . , excess Mn a f f e c t s Fe s t a t u s o f
c h i c k s , but excess Fe does not a f f e c t Mn s t a t u s o f c h i c k s .
T h e r e f o r e , t h i s i n t e r a c t i o n appears to be s i m i l a r to the u n i -
d i r e c t i o n a l i n t e r a c t i o n s between Fe and Zn (33) and between Zn
and Cu (34-35).
M a c r o m i n e r a l s . The e f f e c t o f Ca and P on Mn r v u t r i t u r e o f c h i c k s
has been an area o f i n v e s t i g a t i o n s i n c e Mn supplements were
f i r s t shown to prevent p e r o s i s i n c h i c k s . S c h a i b l e and Bandemer
(36) r e p o r t e d t h a t excesses o f bone meal o r o f C a ( P 0 ) reduced 3 t + 2

Mn a v a i l a b i l i t y and p r e c i p i t a t e d Mn d e f i c i e n c y . More r e c e n t l y ,
high d i e t a r y Ca (2 and 3%) was shown to reduce Mn u t i l i z a t i o n ;
the i n c i d e n c e o f p e r o s i s was i n c r e a s e d and t i s s u e Mn c o n c e n t r a -
t i o n s were decreased by excess Ca (37). Unpublished data from
the U n i v e r s i t y o f I l l i n o i s r e v e a l e d t h a t 3% CaC0 reduced c h i c k
3

g a i n , f e e d e f f i c i e n c y and b i l e Mn c o n c e n t r a t i o n , but i t had no

e f f e c t on bone Mn l e v e l . Moreover, the adverse e f f e c t s o f CaC0 3

were not a l l e v i a t e d by Mn supplementation. T h i s would i n d i c a t e

t h a t the adverse e f f e c t s o f CaC0 were not mediated by a Ca-
3

induced Mn d e f i c i e n c y . The e a r l y r e s e a r c h o f S c h a i b l e and

Bandemer (36) and the more r e c e n t i n v e s t i g a t i o n o f Smith and
K a b a i j a (37) were conducted u s i n g o y s t e r s h e l l f l o u r to vary Ca
l e v e l s . The r e s e a r c h a t the U n i v e r s i t y o f I l l i n o i s used l i m e -
stone t o i n c r e a s e Ca content o f the experimental d i e t s . D i s -
s i m i l a r i t i e s i n micromineral p r o f i l e s , or macromineral f o r t h a t
m a t t e r , o f the two i n g r e d i e n t s c o u l d account f o r these d i s p a r a t e
r e s u l t s . Nonetheless, more r e s e a r c h i s needed to a b s o l v e the
i n t e r a c t i o n between Ca and Mn.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
4. SOUTHERN ET AL. Manganese Homeostasis in the Chick

Interaction with C o c c i d i o s i s
C o c c i d i a l i n f e c t i o n s r e s u l t i n g from i n f e c t i o n s by E i m e r i a
a c e r v u l i n a (duodenal c o c c i d i o s i s ) have been shown t o a f f e c t
n u t r i e n t a b s o r p t i o n (21,26,35,38). Turk e t a l . ( 2 6 ) , u s i n g
r a d i o l a b e l e d Mn, r e p o r t e d t h a t e x p e r i m e n t a l TT a c e r v u l i n a i n f e c -
t i o n decreased Mn a b s o r p t i o n d u r i n g t h e acute phase o f i n f e c t i o n
(6 days p o s t i n o c u l a t i o n ) , but t h a t t h e i n f e c t i o n i n c r e a s e d Mn
a b s o r p t i o n d u r i n g t h e r e c o v e r y p e r i o d . Southern and Baker (32)
and Brown and Southern (21) r e p o r t e d t h a t c h r o n i c E. a c e r v u l i n a
i n f e c t i o n i n c r e a s e d Mn a b s o r p t i o n . Bone and b i l e Mn c o n c e n t r a -
t i o n s were i n c r e a s e d and s i g n s o f Mn t o x i c o s i s symptoms, as
a s s e s s e d h e m a t o l o g i c a l l y , were exacerbated by t h e i n f e c t i o n .
The r a t e o f bone Mn uptake was n e a r l y doubled, and t h e Mn
requirement o f c o c c i d i o s i s - i n f e c t e d c h i c k s was reduced. Thus,
L* a c e r v u l i n a i n f e c t i o n c l e a r l y i n c r e a s e d Mn a b s o r p t i o n and/or
retention.

Requirement and T o x i c i t y
Requirement. The Mn requirement o f c h i c k s f e d a c a s e i n - d e x t r o s e
d i e t low i n f i b e r , phytate and excess n u t r i e n t s i s 14 ppm (7,21).
The N a t i o n a l Research C o u n c i l (39) has s e t a requirement o f 60
ppm i n c o n v e n t i o n a l p o u l t r y d i e t s . Because o f t h e p o t e n t i a l
negative e f f e c t o f conventional f e e d s t u f f s (see B i o a v a i l a b i l i t y
S e c t i o n ) on Mn b i o a v a i l a b i l i t y , t h e e s t i m a t e o f 60 ppm seems
appropriate.
T o x i c i t y . The t o x i c i t y o f Mn f o r c h i c k s was r e c e n t l y reviewed
(40). 3 i g n s o f t o x i c o s i s have been shown t o vary depending on
type o f d i e t used, mineral composition o f t h e d i e t and on source
o f Mn. H e l l e r and Penquite (41) r e p o r t e d t h a t 4779 ppm Mn from
MnC0 reduced c h i c k growth and caused 52% m o r t a l i t y . More r e -
3

c e n t l y , s i m i l a r l e v e l s o f Mn were f e d t o c h i c k s with o n l y m i l d
anemia and a s l i g h t growth d e p r e s s i o n observed (18,19,22).
Without a d e f i n i t i v e Mn t o x i c i t y t r i a l , i t i s d i f f i c u l t t o com-
ment s p e c i f i c a l l y on Mn t o x i c o s i s i n c h i c k s . G e n e r a l l y though,
high l e v e l s o f Mn a r e t o l e r a t e d w e l l by c h i c k s , e s p e c i a l l y i n
conventional poultry d i e t s .
Literature Cited
1. Wilgus J r . , H. S.; Norris, L. C.; Heuser, G. F. Science
1936, 84, 252-53.
2. Underwood, E. J . Trace Elements in Human and Animal Nutri-
tion; Academic Press: New York, 1977; Chapter 7.
3. Davis, P. N . ; Norris, L. C.; Kratzer, F. H. J . Nutr. 1962,
77, 217-23.
4. Holmes, W. B.; Roberts, R. Poultry Sci. 1963, 42, 803-9.
5. Seth, P. C. C.; Clandinin, D. R. Poultry Sci. 1973, 52,
1158-60.
6. Halpin, K. M.; Baker, D. H. Poultry Sci. 1986, 65,
995-1003.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
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7. Halpin, K. M.; Baker, D. H. Poultry Sci. 1986, 65, 1371-74.

8. Thompson, S. A.; Weber, C. W. Poul try Sci. 1981, 60,
840-45.
9. Halpin, K. M.; Baker, D. H. Poultry Sci. 1986, 64
Supplement 1, 111 (Abstr.).
10. Harmuth-Hoene, A.; Schelenz, R. J. Nutr. 1980, 110,
1774-84.
11. van der Aar, P. J . ; Fahey, G. C., J r . ; Ricke, S. C.; Allen,
S. E.; Berger, L. L. J. Nutr. 1983, 113, 653-61.
12. Davis, N. T.; Nightingale, R. Br. J. Nutr. 1975, 34,
243-58.
13. Reinhold, J. G.; Ismail-Beigi, F.; Faradji, B. Nutr. Rep.
Int. 1975, 12, 75-85.
14. Gallup, W. D.; Norris, L. C. Poultry Sci. 1939, 18, 76-82.
15. Hennig, A.; Anke, M.; Jeroch, H.; Kaltwasser, W.; Weidner,
G.; Hoffman, G.; Diettrich, M.; Marcy, H. Biol. Abstr.
1976, 45, 75618.
16. Watson, L. T.; Ammerman
Poultry Sci. 1970, 49, 1548-54.
17. Watson, L. T.; Ammerman, C. B.; Miller, S. M.; Harms, R. H.
Poultry Sci. 1971, 50, 1693-1700.
18. Southern, L. L.; BaTcer, D. H. Poultry Sci. 1983, 62,
642-46.
19. Black, J. R.; Ammerman, C. B.; Henry, P. R.; Miles, R. D.
Poultry Sci. 1984, 63, 1999-2006.
20. Henry, P. R.; Ammerman, C. B.; Miles, R. D. Poultry Sci.
1986, 65, 983-86.
21. Southern, L. L.; Baker, D. H. J. Nutr. 1983, 113, 172-77.
22. BUck, J. R.; Ammerman, C. B.; Henry, P. R.; Miles, R. D.
Nutr. Rep. Int. 1984, 29, 807-14.
23. Black, J. R.; Ammerman, C. B.; Henry, P. R.; Miles, R. D.
Poultry Sci. 1985, 64, 688-93.
24. Halpin, K. M. Ph.D. Thesis, University of Illinois,
Illinois, 1985.
25. Suso, F. A.; Edwards, H. M., Jr. Poultry Sci. 1968, 48,
933-38.
26. Turk, D. E.; Gunji, D. S.; Molitoris, P. Poultry Sci. 1982,
61, 2430-34.
27. Halpin, K. M.; Chausow, D. G.; Baker, D. H. J. Nutr. 1986,
116, 1747-51.
28. Greenburg, D. M.; Copp, D. H.; Cuthbertson, E. M. J. Biol.
Chem. 1944, 147, 749-56.
29. Sansom, B. F.; Gibbons, S. N.; Dixon, A. M.; Russell, A.
M.; Symonds, H. W. In Nuclear Techniques in Animal
Production and Health; International Atomic Energy Agency;
Viena, 1976; p 179-189.
30. Thompson, A. B. R.; Valberg, L. S. Amer. J. Phys. 1972,
223, 1327-29.
31. Halpin, K. M.; Baker, D. H. Poultry Sci. 1984, 63, 109
(Abstr.).
32. Brown, D. R.; Southern, L. L. J. Nutr. 1985, 115, 347-51.
33. Bafundo, K. W.; Baker, D. H.; Fitzgerald, P. R. J. Nutr.
1984, 114, 1306-12.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
4. SOUTHERN ET AL. Manganese Homeostasis in the Chick
34. O'Dell, B. L.; Reeves, P. G.; Morgan, R. F. In Trace
Substances in Environmental Health; University of Missouri,
Columbia, 1976; p 411-21.
35. Southern, L. L.; Baker, D. H. J. Nutr. 1983, 113, 688-96.
36. Schaible, P. J.; Bandemer, S. L. Poultry Sci. 1942, 21,
8-14.
37. Smith, O. B.; Kabaija, E. Poultry Sci. 1985, 64, 1713-20.
38. Turk, D. E. Fed. Proc. 1974, 33, 106-11.
39. National Research Council; Nutrient Requirements of
Poultry; National Academy of Sciences; Washington, DC,
1984.
40. Mineral Tolerances of Domestic Animals; National Academy of
Sciences; Washington, DC, 1980; p 290-303.
41. Heller, V. G.; Penquite, R. Poultry Sci. 1937, 16, 243-46.
RECEIVED January 28, 1987

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Chapter 5

Role of Manganese in Bone Metabolism

Linda Strause and Paul Saltman
Department of Biology (B-022), University of California—San Diego,
La Jolla, CA 92093

The role of trace elements in general, and Mn(II) in par-

ticular, has been examined in the metabolism of calcified
tissue at several levels. The tissue distribution of
54
Mn orally administered to mice was followed. Specific
Mn(II) binding site
characterized usin
months, dietary regimens depleted in Mn or low Mn-low Cu
showed significantly lower bone density and higher serum
Ca than controls. The application of ectopic subcutane-
ous implants of bone powder and demineralized bone powder
into animals on control or Mn deplete diets demonstrated
that osteoblast activity, a measure of bone formation,
was impaired as was osteoclast activity, a parameter of
bone resorption. The multiple cellular effects of Mn
deficiency include: decreased bone resorption, production
of labile bone, and decreased synthesis of organic
matrix. The serum level of Mn in a group of osteoporotic
postmenopausal women was significantly lower than age-
-matched controls.

Our i n t e r e s t i n the r o l e o f t r a c e elements i n bone m e t a b o l i s m

developed i n a r a t h e r b i z a r r e f a s h i o n . We became i n t e r e s t e d i n t h e
o r t h o p e d i c problems o f a prominent p r o f e s s i o n a l b a s k e t b a l l player,
Bill Walton. S e v e r a l y e a r s ago he was p l a g u e d by f r e q u e n t broken
bones, p a i n s i n h i s j o i n t s and an i n a b i l i t y t o h e a l bone f r a c t u r e s .
We h y p o t h e s i z e d t h a t he might be d e f i c i e n t i n t r a c e elements as a
r e s u l t of h i s very l i m i t e d vegetarian d i e t . In cooperation with h i s
p h y s i c i a n , we were a b l e t o a n a l y z e Walton's serum. We found no
d e t e c t a b l e manganese (Mn). H i s serum c o n c e n t r a t i o n s o f copper (Cu)
and z i n c (Zn) were below normal v a l u e s . D i e t a r y s u p p l e m e n t a t i o n w i t h
t r a c e elements and c a l c i u m (Ca) was begun. Over a p e r i o d o f s e v e r a l
months h i s bones h e a l e d and he r e t u r n e d t o p r o f e s s i o n a l b a s k e t b a l l
( 1 , 2 ) . I n c o o p e r a t i o n w i t h s e v e r a l other orthopedic p h y s i c i a n s , we
analyzed serum from other p a t i e n t s w i t h slow bone h e a l i n g . Several
of these p a t i e n t s a l s o had a b n o r m a l l y low Zn, Cu and Mn l e v e l s .

0097-6156/87/0354-0046$06.00/0
© 1987 American Chemical Society

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
5. S T R A U S E A N D SALTMAN Role of Manganese in Bone Metabolism 47

I t comes as no great s u r p r i s e t h a t t r a c e elements may a f f e c t the

growth and development of bone. Trace element d e f i c i e n c e s p r o f o u n d l y
a l t e r bone m e t a b o l i s m i n a n i m a l s e i t h e r d i r e c t l y or i n d i r e c t l y ( 3 ) .
The absence of a t r a c e element i n the d i e t can l e a d t o i n e f f i c i e n t
f u n c t i o n i n g o f a s p e c i f i c enzyme or enzymes t h a t r e q u i r e the t r a n s i -
t i o n element as a c o f a c t o r . An example o f t h i s i s the r o l e of Cu and
i r o n (Fe) i n the c r o s s - l i n k i n g o f c o l l a g e n and e l a s t i n s ( 4 - 9 ) . The
p a r t i c i p a t i o n o f Mn i n the b i o s y n t h e s i s o f m u c o p o l y s a c c h a r i d e s (10-
12) i s another example. Zn d e f i c i e n c y causes a r e d u c t i o n i n osteo-
blastic activity, c o l l a g e n and c h o n d r o i t i n s u l f a t e s y n t h e s i s and
a l k a l i n e phosphatase a c t i v i t y (13-16).
H u r l e y and her c o l l a b o r a t o r s have s t u d i e d t h e p e r o s i s (faulty
tendons) induced by b o t h Mn and Zn d e f i c i e n c i e s (17-19). Previous
workers have d e s c r i b e d s k e l e t a l a b n o r m a l i t i e s i n c h i c k s and r a t s
including disproportionate growth of s k e l e t o n , bone r a r e f a c t i o n and
c h o n d r o d y s t r o p h y , as o v e r t m a n i f e s t a t i o n o f z i n c d e f i c i e n c y (20,21).
H u r l e y e t a l . , were a b l
produced by Mn was q u i t
of Zn. I n the case of Mn t h e r e was no a l t e r a t i o n i n the m i n e r a l i z a -
t i o n p r o c e s s e s measured by the dynamics o f r a d i o c a l c i u m movement. The
influence of trace elements on _in v i t r o t i s s u e c u l t u r e s o f c h i c k
o s t e o b l a s t s has been r e p o r t e d (22). Among the elements r e q u i r e d were
Fe, Cu, Zn and Mn.
G e n e t i c models o f t r a c e element i n s u f f i c i e n c y a r e known i n
a n i m a l s and humans. The m u t a t i o n i n mice c a l l e d p a l l i d produces con-
d i t i o n s s i m i l a r t o those observed i n p r e n a t a l Mn d e f i c i e n c y (17,23).
There i s an abnormal and d e l a y e d o s s i f i c a t i o n o f the o t i c c a p s u l e and
f a i l u r e o f the o t o l i t h s t o c a l c i f y . These c o n d i t i o n s can be r e v e r s e d
by the a p p l i c a t i o n of therapeutic amounts o f Mn t o the pregnant
female and i n t o the d i e t o f t h e o f f s p r i n g . Over t h e p a s t several
y e a r s we have sought t o u n d e r s t a n d more t h o r o u g h l y the m e t a b o l i c f a c -
t o r s w h i c h r e g u l a t e and c o n t r o l t r a c e element metabolism ( 2 4 ) . We
d e c i d e d t o combine and e x t e n d these i n t e r e s t s t o l e a r n more about t h e
mechanisms o f uptake and t r a n s p o r t of Mn and how t h a t t r a c e element
may be i n v o l v e d i n v a r i o u s a s p e c t s o f bone m e t a b o l i s m . I n t h i s r e v i e w
we t r a c e the p r o g r e s s we have made w i t h r e s p e c t t o t r a n s p o r t and d i s -
t r i b u t i o n o f Mn i n the mouse, the development o f a model o f o s t e o -
p e n i a i n the r a t induced by t r a c e element d e f i c i e n c i e s , an i n v e s t i g a -
t i o n o f the r o l e of Mn i n o s t e o c l a s t and o s t e o b l a s t a c t i v i t y i n t h e
r a t , and u l t i m a t e l y d e s c r i b e some new c l i n i c a l f i n d i n g s w h i c h indi-
cate t h a t Mn d e f i c i e n c y may have a s i g n i f i c a n t r o l e i n o s t e o p o r o s i s .

Studies o f Manganese Uptake and D i s t r i b u t i o n bv the Mouse

R e l a t i v e l y l i t t l e a t t e n t i o n has been p a i d t o the mechanisms by which

Mn i s absorbed from the d i e t ^ M o s t s t u d i e s have used i n t r a p e r i t o n e a l
or i n t r a v e n o u s i n j e c t i o n s o f M n ( I I ) s a l t s (25,26). I n v i t r o s t u d i e s
using i n t e s t i n a l segments suggested t h a t membrane t r a n s p o r t c a r r i e r s
might be i n v o l v e d , b u t a l s o t h a t c o m p l e x a t i o n o f the m e t a l i n f l u e n c e s
transport ( 2 7 ) . The h y d r o l y s i s o f the t r a n s i t i o n m e t a l ions s t r o n g l y
i n f l u e n c e s t h e i r a b i l i t y t o c r o s s v a r i o u s b i o l o g i c a l membranes. The
presence of low m o l e c u l a r w e i g h t o r g a n i c c h e l a t i n g agents b o t h of a
d i e t a r y o r i g i n o r s y n t h e t i c complexing agents w i t h s p e c i f i c chemical
a f f i n i t i e s c o u l d a l t e r b o t h the r a t e o f t r a n s p o r t and d i s t r i b u t i o n o f

American Chemical Society

Library
1155 16th St., N.w.
In Nutritional Bioavailability of Manganese; Kies, C.;
Washington, D.C. 20036
ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
48 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

the m e t a l i o n absorbed. U s i n g t h e technique of whole body c o u n t i n g t o

f o l l o w the r e t e n t i o n o f a s i n g l e dose o f r a d i o a c t i v e Mn a d m i n i s t e r e d
per o s , we i n t u b a t e d e i t h e r MnCl 2 or Mn-NTA (nitrilotriacetic
a c i d ) i n t o mice ( 2 8 ) . The c u m u l a t i v e r e t e n t i o n was f o l l o w e d f o r a
p e r i o d o f 10 days ( F i g u r e 1 ) . No s i g n i f i c a n t d i f f e r e n c e was seen w i t h
or w i t h o u t the c h e l a t i n g agent. I t i s i n t e r e s t i n g t o note t h a t l e s s
than 3% o f the t o t a l dose a d m i n i s t e r e d was r e t a i n e d a f t e r 10 days.

I i i i i ' ' • » ' «

1 2 3 4 5 6 7 8 9 10
DAYS AFTER ORAL ADMINISTRATION
OF RADIOMANGANESE
f i g u r e 1. P e r c e n t d a i l y r e t e n t i o n o f a s i n g l e o r a l dose of
MnCl (o) a t pH 2 and MnNTA (•) a t pH 9 ( 2 8 ) . Each datum
p o i n t f o r whole body r e t e n t i o n r e p r e s e n t s the average 25 indi-
v i d u a l a n i m a l s . * = p<0.01.(Reproduced w i t h p e r m i s s i o n from
r e f . 28. C o p y r i g h t 1985 The Humana P r e s s . )

Distribution of Mn i n v a r i o u s t i s s u e s was determined 10 days a f t e r

a d m i n i s t r a t i o n o f the i s o t o p e s . Animals were k i l l e d by CO^ a s p h y x i a -
t i o n . H e a r t , s p l e e n , femur, gastrocnemius muscle and p o r t i o n s o f the
liver and s m a l l i n t e s t i n e were removed and weighed. B l o o d was c o l -
l e c t e d by h e a r t p u n c t u r e . I n i t i a l l y i t appeared t h a t MnCl^ was more
effectively absorbed than Mn-NTA. However t h e e n t i r e d i f f e r e n c e
between t h e two forms a d m i n i s t e r e d c o u l d be accounted f o r by the
r a p i d and p e r s i s t e n t a d s o r p t i o n o f the Mn onto t h e t e e t h when f e d as
the i o n i c s a l t . When c o r r e c t e d f o r a d s o r p t i o n t o t e e t h , l e s s than 1%
of the MnCl^ was absorbed by the a n i m a l . The g r e a t e s t amount of
r a d i o a c t i v i t y was accumulated i n the muscle mass and l i v e r . Approxi-
mately 10% was found i n t h e bone.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
5. S T R A U S E A N D S A L T M A N Role of Manganese in Bone Metabolism 49

The r a p i d l o s s o f r a d i o i s o t o p e f o l l o w i n g a s i n g l e o r a l dose sug-

gests t h a t the i n t e s t i n e i s the major component i n the r e g u l a t i o n o f
Mn a b s o r p t i o n . Our f i n d i n g o f a v e r y low e f f i c i e n c y o f mucosa t o
serosa t r a n s f e r of Mn i s c o n s i s t e n t w i t h the s t u d i e s of Thompson and
V a l b e r g (29,30). They demonstrated t h a t a p p r o x i m a t e l y 25% of t h e Mn
taken up i n the mucosal c e l l s i s t r a n s f e r r e d t o the c a r c a s s .
We were i n t r i g u e d by the v e r y t i g h t b i n d i n g o f Mn t o the s u r f a c e
of the t e e t h ^ j n mice. A s e r i e s o f t e e t h from the lower jaw of mice
intubated with MnCl^ s o l u t i o n were removed and examined by e l e c t r o n
paramagnetic resonance. With the c o o p e r a t i o n of Dr. Dennis Chasteen
of the Department o f Chemistry a t the U n i v e r s i t y o f New Hampshire, we
concluded t h a t Bin appears t o s u b s t i t u t e f o r Ca w i t h i n t h e h y d r o x y -
apatite s t r u c t u r e of the t o o t h . I n such a s i t e , the Mn i s p r o b a b l y
c o o r d i n a t e d t o s i x phosphate groups. Mn i s n o t simply surface
adsorbed. I t seems t o p e n e t r a t e the t o o t h and i s t e n a c i o u s l y bound.
S i m i l a r studies w i t h chemically prepared hydroxyapatite crystals
exposed t o M n ( I I ) r e v e a l e
ment than the t o o t h ename
t o u n d e r s t a n d how b i n d i n g o f M n ( I I ) t o t e e t h and bone might be
e x p l o i t e d i n l i n k i n g v a r i o u s compounds t o the s u r f a c e of hard t i s s u e .

A Model of O s t e o p e n i a Produced i n the Rat bv D e f i c i e n c i e s

i n Mn and Cu.

We have developed a s e m i - s y n t h e t i c animal d i e t i n w h i c h the l e v e l s o f

t r a c e elements can be c a r e f u l l y c o n t r o l l e d ( 3 1 ) . We s e l e c t e d t h r e e
d i e t a r y c o n d i t i o n s : c o n t r o l (C) 66 ppm Mn and 5ppm Cu; low-Mn low-Cu
(L) 2.5 ppm Mn and 0.5 ppm Cu; and Mn-deplete (D) no added Mn and 5
ppm Cu. Female Sprague Dawley r a t s were randomly d i v i d e d a t weaning
i n t o t h e t h r e e d i e t a r y groups. Food and d i s t i l l e d water were p r o -
v i d e d ad l i b i t u m f o r the e n t i r e e x p e r i m e n t a l p e r i o d o f twelve months.
The w e i g h t g a i n o f a l l animals was a p p r o x i m a t e l y the same.
F i v e a n i m a l s from each group were k i l l e d by CO^ a s p h y x i a t i o n
after a 12-hour o v e r n i g h t p e r i o d o f s t a r v a t i o n . Animals were k i l l e d
at 2, 13, 26, and 52 weeks a f t e r the s t a r t of t h e d i e t a r y regimen.
Various t i s s u e s were sampled, immediately f r o z e n i n l i q u i d n i t r o g e n
and s t o r e d a t -80°C. Trace element d e t e r m i n a t i o n s were made on a l l
t i s s u e and s e r a f o l l o w i n g d i g e s t i o n i n c o n c e n t r a t e d n i t r i c a c i d u s i n g
h i g h - p r e s s u r e T e f l o n chambers ( 3 1 ) . Ca, Cu, and Zn were determined by
flame atomic a b s o r p t i o n spectrophotometry. Mn was measured u s i n g
e l e c t r o t h e r m a l atomic a b s o r p t i o n spectrophotometry. I s o l a t e d humeri
from e i g h t animals per d i e t a r y group were X-rayed.
M i n e r a l l e v e l s i n serum and bone from r a t s on t h e 3 d i f f e r e n t
d i e t s a r e shown i n T a b l e I . Serum Ca was s i g n i f i c a n t l y h i g h e r i n
d e f i c i e n t r a t s a t s i x months. Serum Cu and Mn were significantly
lower i n L and D r a t s r e s p e c t i v e l y . The m i n e r a l c o n c e n t r a t i o n s o f Ca
i n the femur as w e l l as Mn were a f f e c t e d by the M n - d e f i c i e n t diets.
Ca concentrations o f t h e femur was i n v e r s e l y c o r r e l a t e d w i t h the
serum Ca c o n c e n t r a t i o n s i n the L and D r a t s . R a d i o g r a p h i c observa-
t i o n s o f i s o l a t e d humeri i n d i c a t e d t h a t o s t e o p e n i c - l i k e l e s i o n s were
a s s o c i a t e d w i t h the L and D regimens ( 3 1 ) .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
50 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

T a b l e I . M i n e r a l L e v e l s i n Serum and Bone from Rats Fed

Three D i f f e r e n t D i e t s f o r 12 mo '

Diets
Measure N L D
Serum (6)
Ca, mg/L 103 + 15 a
119 + 16 a b
134 + 15 b

P, mg/L 39 + l a
63 ± 4* 50 + 3 C

Cu, mg/L 1.2 + 0.6 a

0.08 + 0.05° 1.2 + 0.3 a

Mn, mg/L 0.08 + 0.05 a

0.03 + 0.02 a
0.03 + 0.02 a

Zn, mg/L 1.0 + 0.3 a

1.1 + 0.3 a
1.2 ± 0.3 a

Femur (6)
Ca, mg/g 272 + 6 0 a
221 + 3 0 a b
180 16
+
b

P, mg/g 167 + 14° 156 + 9 a

155 + 2 0 a

Cu, ug/g 1.46 + 0.55 8

0.71 + 0.45 a
0.95 + 0.40 a

Mn, ug/g 2.70

Zn, ug/g 282

V a l u e s a r e means + SD f o r the number of r a t s p e r group i n

parentheses.
W i t h i n a row, means n o t s h a r i n g a common s u p e r s c r i p t a r e s i g n i f i -
c a n t l y d i f f e r e n t (P < 0.05) by Student's t w o - t a i l e d u n p a i r e d t - t e s t .

I n s u f f i c i e n t i n t a k e s o f Mn and Cu r e s u l t e d i n s i g n i f i c a n t abnor-
malities i n b o t h serum and bone m i n e r a l l e v e l s w i t h i n t w e l v e months.
Why a c h r o n i c d e f i c i e n c y o f t r a c e elements should r e s u l t i n condi-
t i o n s of osteopenia i s n o t a t p r e s e n t c l e a r . I t has been suggested
t h a t o s t e o p e n i a i s a s s o c i a t e d w i t h an i n c r e a s e d r a t e of bone resorp-
t i o n (33). Others have i m p l i c a t e d decreased bone f o r m a t i o n o r o s t e o b -
l a s t a c t i v i t y i n some forms o f o s t e o p o r o s i s (34). What i s o b v i o u s l y
at i s s u e i s a b a l a n c e between t h e r a t e of bone r e s o r p t i o n and t h a t of
bone s y n t h e s i s (35-37). How t h a t e q u i l i b r i u m dynamic i s a f f e c t e d by
t r a c e elements w i l l be d i s c u s s e d below.

The E f f e c t o f Mn and Cu D e f i c i e n c i e s on Osteo-Induct i o n and

R e s o r p t i o n o f Bone P a r t i c l e s i n R a t s

One of t h e most s e n s i t i v e b i o a s s a y s f o r osteoblast and o s t e o c l a s t

a c t i v i t i e s _in v i v o i s t h e use o f e c t o p i c models o f bone f o r m a t i o n and
bone m a t r i x resorption (38,39). D e v i t a l i z e d , d e m i n e r a l i z e d bone
powders (DBP) a r e s u b c u t a n e o u s l y i m p l a n t e d i n young r a t s . There i s a
p h e n o t y p i c c o n v e r s i o n o f c o n n e c t i v e c e l l t i s s u e mesenchyme i n t o c a r -
tilage. Subsequently t h i s c a r t i l a g e becomes c a l c i f i e d , v a s c u l a r i z e d
and bone i s d e p o s i t e d i n two weeks. I f m i n e r a l - c o n t a i n i n g bone p a r -
t i c l e s (BP) a r e implanted, a d i f f e r e n t phenomenon i s observed. Large
m u l t i n u c l e a t e d o s t e o c l a s t - l i k e c e l l s a r e r e c r u i t e d t o t h e s i t e of
implantation. There i s a complete r e s o r p t i o n o f the BP f o u r weeks
a f t e r i m p l a n t a t i o n . I n c o l l a b o r a t i o n w i t h Dr. J u l i e G l o w a c k i of the
H a r v a r d U n i v e r s i t y School of M e d i c i n e , we took advantage of these
p r o c e d u r e s and used i m p l a n t s o f normal DBP and BP i n t o r a t s t h a t had
been m a i n t a i n e d on t h e three e x p e r i m e n t a l d i e t s : C, L, and D ( 4 0 ) .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
5. STRAUSE AND SALTMAN Role of Manganese in Bone Metabolism 51

I s o g e n i c bone powder was p r e p a r e d from femurs, humeri and t i b i a

of normal a d u l t r a t s . The c l e a n d i a p h y s e s were e x t r a c t e d w i t h abso-
l u t e e t h a n o l f o l l o w e d by anhydrous e t h y l e t h e r . Bones were p u l v e r i z e d
in a l i q u i d n i t r o g e n i m p a c t i n g m i l l and s i e v e d t o p a r t i c l e s i z e s
between 75 t o 250 pm• The d e m i n e r a l i z e d powder was p r e p a r e d by
e x t r a c t i n g BP w i t h 0.5 M HC1 f o r t h r e e hours a t room temperature f o l -
lowed by e x t e n s i v e washes i n d i s t i l l e d water t o remove a l l a c i d and
m i n e r a l s . S e q u e n t i a l washes w i t h a b s o l u t e e t h a n o l and f i n a l l y w i t h
anhydrous e t h e r p r e p a r e d the dry powder. B i l a t e r a l subcutaneous pock-
ets i n t h e t h o r a c i c area i n a n e s t h e t i z e d r a t s were i m p l a n t e d w i t h
e i t h e r DBP or BP. Sets o f s i x p e l l e t s were h a r v e s t e d from animals
under each o f the d i e t a r y c o n d i t i o n s a t 2, 6 and 17 weeks f o l l o w i n g
i m p l a n t a t i o n o f the DBP. Implants o f the BP were h a r v e s t e d a t 2 and 4
weeks f o l l o w i n g i m p l a n t a t i o n .
P e l l e t s were removed and p r e p a r e d f o r h i s t o l o g i c a l examination
as p r e v i o u s l y d e s c r i b e d ( 3 7 ) . An a r b i t r a r y " i n d u c t i o n i n d e x " ,
d e f i n e d as t h e mean f r a c t i o n a
(IC) and induced bone ( I B )
of o s t e o b l a s t i c a c t i v i t y by the DBP. A summary o f t h e r e s u l t s o f
these experiments i s p r e s e n t e d i n Table I I . A t 6 and 17 weeks f o l -
l o w i n g i m p l a n t a t i o n of the DBP s i g n i f i c a n t bone f o r m a t i o n i s observed
in the C r a t s . No c a r t i l a g e was apparent i n t h e 2 week specimens
from the L r a t s . At 6 and 17 weeks some induced bone was p r e s e n t .
At no time throughout the course o f experiments d i d the DBP e l i c i t
f o r m a t i o n o f e i t h e r c a r t i l a g e o r bone i n the Mn-deplete r a t s .

T a b l e I I . I n d u c t i o n Index from Normal DBP Implanted i n t o Test

R a t s M a i n t a i n e d on E x p e r i m e n t a l D i e t s f o r 6 months (40)

DIET
Weeks A f t e r
Implantation CONTROL LOW DEPLETE
2 12.7UC) 0 0
6 41.5UB) 49.5UC+IB) 0
17 31.3UB) 20.0UB) 0

I n d u c t i o n Index i s t h e mean f r a c t i o n a l histomorphometric^ area

r e p r e s e n t e d by induced c a r t i l a g e (IC) o r induced bone (IB) x 10 .

Table I I I p r e s e n t s t h e r e s u l t s of BP i m p l a n t s i n t o t h e 3 d i f -
f e r e n t groups o f r a t s . The percentage o f i m p l a n t e d BP p a r t i c l e s
r e s o r b e d p e r m i c r o s c o p i c f i e l d was used as a measure of o s t e o c l a s t
induction. The decrease i n r e l a t i v e r e s o r p t i o n o f BP i n the L and D
r a t s i s apparent. D e f i c i e n c i e s i n Mn and Cu appear t o decrease
osteoclast a c t i v i t y .
The i m p l a n t a t i o n experiments c o r r e l a t e w e l l w i t h t h e o b s e r v a -
t i o n s f o r s k e l e t a l development under the t h r e e d i e t a r y c o n d i t i o n s .
The o s t e o p e n i a observed i n t h e r a t s r a i s e d f o r 12 months on the L or
D d i e t s c o u l d be a m a n i f e s t a t i o n o f a d i s e q u i l i b r i u m between the
r a t e s o f o s t e o c l a s t i c and o s t e o b l a s t i c a c t i v i t y . Both of these cel-
lular activities a r e i n f l u e n c e d by the t r a c e element s t a t u s o f the
a n i m a l . I f the o s t e o b l a s t i c a c t i v i t y were more s t r o n g l y i n h i b i t e d by

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
52 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

T a b l e I I I . R e s o r p t i o n o f Normal Bone P a r t i c l e s (BP) i n T e s t

a
R a t s M a i n t a i n e d on E x p e r i m e n t a l D i e t s (40)

BP %BP Relative
DIET N (Area/Field) Resorbed Resorption
CONTROL 5 4584 + 528 46.2 100%
LOW 6 7203 + 612 15.4 33%
DEPLETE 5 7451 + 482 12.4
a
Specimens h a r v e s t e d a t 14 d a f t e r i m p l a n t a t i o n o f BP i n t o r a t s main-
t a i n e d on d i e t s f o r 12 months.
S i g n i f i c a n t l y d i f f e r e n t (P<.01) from c o n t r o l group by S t u d e n t ' s
t w o - t a i l e d u n p a i r e d t - t e s t c o r r e c t e d f o r m u l t i p l e comparisons.

the d e f i c i e n c i e s than
would be expected.
I t has been shown t h a t M n - d e f i c i e n t hens and r a t s show d i s p r o -
portionate growth of s k e l e t o n s and u n d e r - g l y c o s y l a t i o n o f p r o t e o g l y -
cans (41,42). L y s y l o x i d a s e , a C u - r e q u i r i n g enzyme, i s e s s e n t i a l f o r
the c r o s s - l i n k i n g o f e l a s t i n and c o l l a g e n (5,43). We c a r r i e d out a
study i n w h i c h the e c t o p i c i m p l a n t s were p r e p a r e d from bones o f
animals r a i s e d on t h e d i f f e r e n t d i e t a r y regimens t o c h a r a c t e r i z e pos-
s i b l e changes i n t h e i r s t r u c t u r e and a c t i v i t y . Bone powders were
p r e p a r e d from r a t s r a i s e d f o r 12 months on one o f the three d i e t s : L
or D. The BP was i m p l a n t e d i n t o normal 2 8 - d a y - o l d r a t s . The l a b i l -
ity o f t h e BP's t o t h e m o b i l i z e d o s t e o c l a s t s was measured. P r e l i m -
i n a r y experiments i n d i c a t e t h a t BP's from the L and D r a t s were sig-
n i f i c a n t l y more l a b i l e than those from the C r a t s , 121%, 123%, and
100%, r e s p e c t i v e l y . At t h i s time we a r e unable t o determine whether
Mn and Cu d e f i c i e n c y y i e l d s a bone l e s s r e s i s t a n t t o o s t e o c l a s t
a t t a c k , o r t h a t more o s t e o c l a s t s a r e m o b i l i z e d .
We measured c e l l number and o s t e o n e c t i n concentration using
f e t a l c a l v a r i a l c u l t u r e s from c o n t r o l (C) and Mn-deplete (D) r a t s . We
h y p o t h e s i z e d t h a t Mn d e f i c i e n c y may r e s u l t i n t h e u n d e r - g l y c o s y l a t i o n
of o s t e o n e c t i n . P r e l i m i n a r y experiments showed an i n c r e a s e i n
o s t e o b l a s t c g l l number i n c u l t u r e s from C r a t s as compared t o D r a t s ,
0.677 x 10 and 0.229 x 10 , r e s p e c t i v e l y . O s t e o n e c t i n l e v e l was
l o w e r ^ i n C c u l t u r e s than i n D c u l t u r e s , 181 ng/10 c e l l s and 995
ng/10 c e l l s , r e s p e c t i v e l y . When Mn was added t o t h e megia o f b o t h C
and D c u l t u r e s , o s t e o n e c t i n l e v e l s i n c r e a s e d , 221 ng/10 c e l l s and
1027 ng/10 c e l l s , r e s p e c t i v e l y . I n p r i m a r y o s t e o b l a s t c u l t u r e s i t
i s speculated that i f c e l l s are stimulated t o d i v i d e , phenotypic
markers may a c t u a l l y d e c r e a s e . The h i g h e r l e v e l of o s t e o n e c t i n i n
the D c u l t u r e s may be due t o t h e slower growth r a t e o f these cells.
A greater number of c e l l s i n t h e d i f f e r e n t i a t i o n stage would r e s u l t
i n p r o p o r t i o n a l l y g r e a t e r s y n t h e s i s o f p r o t e i n s such as o s t e o n e c t i n .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
5. STRAUSE AND SALTMAN Role of Manganese in Bone Metabolism 53

P r o t e o g l y c a n s are a s m a l l but s i g n i f i c a n t component of the

m i n e r a l i z e d bone m a t r i x . P r o t e o g l y c a n c o n t e n t of humeral epiphyses
from r a t s r a i s e d f o r s i x months on our D d i e t was e s t i m a t e d by
measuring g l y c o s a m i n o g l y c a n (GAG) l e v e l s . P r e l i m i n a r y r e s u l t s showed
a much g r e a t e r GAG l e v e l i n the C r a t s t h a n i n D r a t s . The t o t a l
p r o t e o g l y c a n and b o n e - s p e c i f i c proteoglycan i n C r a t bones (2.19
ug/mg and 0.119 ug/mg, r e s p e c t i v e l y ) was g r e a t e r than t h a t i n the D
bones (1.43 ug/mg and 0.016 ug/mg, r e s p e c t i v e l y ) . We observed a
s h i f t toward lowered bone d e n s i t y and a change i n the m i n e r a l i z a t i o n
p r o f i l e of bone d i a p h y s e s i n r a t s r a i s e d on the L and D d i e t s f o r s i x
months. No changes were d e t e c t e d i n the c o n t r o l r a t s .

C l i n i c a l C o r r e l a t i o n s of O s t e o p o r o s i s w i t h Serum Manganese

I n c o l l a b o r a t i o n w i t h D r . J e a n - Y v e s R e g i n s t e r at the M e d i c a l School
of the U n i v e r s i t y of L i e g e , B e l g i u m , we measured C a , Cu, Mn and Zn
c o n c e n t r a t i o n s i n serum
p o r o t i c and age matched normal women. The o s t e o p o r o t i c p a t i e n t s were
s e l e c t e d by the presence of at l e a s t one v e r t e b r a l n o n - t r a u m a t i c
c r u s h f r a c t u r e . Normal s u b j e c t s were s e l e c t e d on the b a s i s of t h e i r
bone m i n e r a l c o n t e n t and bone m i n e r a l d e n s i t y as measured by dual-
phot o n - abs or ptiome t r y . There were s i g n i f i c a n t d i f f e r e n c e s between
the o s t e o p o r o t i c and normal women i n parameters of bone d e n s i t y .
Serum Mn was s i g n i f i c a n t l y lower i n o s t e o p o r o t i c s (Table I V ) . No
s i g n i f i c a n t d i f f e r e n c e s were found i n the f o l l o w i n g : bone Cu, Mn, Z n ,
o s t e o i d volume, o s t e o i d s u r f a c e , o s t e o c l a s t i c r e s o r p t i o n s u r f a c e ; or
serum Cu, Z n , bone G l a p r o t e i n and 1,25 d i h y d r o x y v i t a m i n D3. For
all s u b j e c t s the t r a c e elements (Cu, Bin and Fe) were w i t h i n the n o r -
mal r a n g e . We a r e not a b l e to draw a d e f i n i t i v e c o n c l u s i o n t h a t
serum Mn i s d i r e c t l y r e l a t e d t o the p a t h o g e n e s i s of o s t e o p o r o s i s .
However, low serum Mn may be r e l a t e d t o decreased bone mass as seen
i n e x p e r i m e n t a l a n i m a l s . I t i s n e c e s s a r y t o c a r r y out l o n g i t u d i n a l
s t u d i e s i n l a r g e p o p u l a t i o n s of h e a l t h l y post-menopausal women com-
p a r i n g the r a t e of bone l o s s t o v a r i a t i o n s i n serum c o n c e n t r a t i o n s of
these t r a c e e l e m e n t s .

Table I V . Bone and Serum V a l u e s i n O s t e o p o r o t i c and Normal Women

Normal Subjects Osteoporotic Patients

Bone C a l c i u m (mg/g) 149.7 + 17.3 113.7 28

T r a b e c u l a r Bone Volume (%) 23.4 + 5.9 12.6 ± i-l:

± A*
Bone M i n e r a l Content (g/cm^ 5.7 + 0.5 3.7 ± <>•*••
Bone M i n e r a l D e n s i t y (g/cm ) 1.0 + 0.3 0.7 ± 0 . 1 0

Serum Manganese (mg/L) 0.04 + 0.03 0.01 + 0.004

P < 0.05; P < 0.01; P < 0.001

There i s a great d e a l of b o t h p o p u l a r and p r o f e s s i o n a l interest
i n the e t i o l o g y , d i a g n o s i s , p r e v e n t i o n , and treatment of o s t e o -
porosis. The e x t e n t of t h i s d i s e a s e i n the U n i t e d S t a t e s i s a major
p u b l i c h e a l t h concern. No s i n g l e cause can be i d e n t i f i e d . Certainly
the i n f l u e n c e of hormones, d i e t a r y i n t a k e s of C a , f l u o r i d e and v i t a -
min D are s i g n i f i c a n t . Our r e s u l t s suggest t h a t i t may be prudent to
c o n s i d e r the p o s s i b i l i t y t h a t t r a c e element d e f i c i e n c i e s , particu-
l a r l y of Mn, may be of s i g n i f i c a n c e .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
54 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

Acknowledgments
This work was supported in part by the USPHS NIH Research Grant
AM-123 86 and a gift from the Proctor and Gamble Co.
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33. Nordin, B.E.C.; Aaron, J.; Sepped, R.; Crilly, R.G. Lancet
1981, II, 277-279.
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35. Riggs, B.L. Mineral and Electrolyte Metab. 1981, 5, 265-275.
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RECEIVED January 28, 1987

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Chapter 6

Enhanced Tissue Lipid Peroxidation

Mechanism Underlying Pathologies Associated
with Dietary Manganese Deficiency

Sheri Zidenberg-Cherr1,2
and C a r l L. Keen
1,3

Department of Nutrition, University of California—Davis, Davis, CA 95616

1

2
Laboratory for Energy-Related Health Research, University of California—Davis,
Davis, CA 95616
3
Department of Internal Medicine, University of California—Davis, Davis, CA 95616

While i t is recognize
pathological consequences
lesions have not been defined. One hypothesis is
that Mn deficiency results in a reduction in Mn
superoxide dismutase (MnSOD) activity with a sub-
sequent increase in tissue lipid peroxidation and
cellular damage. In support of this idea, Mn--
deficient animals are characterized by low liver
MnSOD activity and high levels of liver mitochondrial
lipid peroxidation. Ultrastructural studies showing
mitochondrial membrane abnormalities in liver from
Mn-deficient rats support the hypothesis that in-
creased tissue lipid peroxidation results in cellular
damage. Based on the above i t can be speculated that
Mn deficiency should increase the cytotoxicity of
environmental insults which increase the production
of superoxide ion radical. Consistent with this idea
is evidence that the metabolism of, and the physio-
logical response to, a number of free radical
inducers is affected by Mn status.

The essentiality of manganese (Mn) for animals was established in

1931 by Orent and McCollum (1) who reported that this element is
required for normal reproduction in the rat, and Kemmerer and
colleagues (2) who showed that it was necessary for normal growth
and reproduction in the mouse. Since then several investigators
have verified the critical need of this nutrient for normal devel-
opment (3). Manifestations of perinatal Mn deficiency in experi-
mental animals include neonatal death, impaired growth, skeletal
abnormalities, depressed reproductive function, congenital ataxia,
and defects in protein, carbohydrate and lipid metabolism.
Although it is evident that Mn is needed for several biological
functions, its precise biochemical roles have not been delineated.
Manganese is involved in numerous biochemical reactions both
as an integral part of metalloenzymes and as an enzyme activator.
0097-6156/87/0354-0056$06.00/0
© 1987 American Chemical Society

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
6. ZIDENBERG-CHERR AND KEEN Enhanced Tissue Lipid Peroxidation 57

A l t h o u g h t h e r e a r e numerous enzymes known t o be a c t i v a t e d by Mn,

few Mn metalloenzymes have been r e c o g n i z e d . With the exception o f
t h e g l y c o s y l t r a n s f e r a s e enzymes, t h e enzymes f o r which Mn i s a
c o f a c t o r are u s u a l l y non-sjj>ecifically a c t i v a t e d , with other d i - +

v a l e n t c a t i o n s such as Mg b e i n g a b l e t o t a k e t h e p l a c e o f Mn
(4,5).
Enzymes which c o n t a i n Mn i n c l u d e p y r u v a t e c a r b o x y l a s e , a r g i -
nase, and s u p e r o x i d e d i s m u t a s e . T h i s p a p e r w i l l f o c u s on t h e r o l e
o f Mn as a component o f Mn s u p e r o x i d e d i s m u t a s e (MnSOD) and t h e
f u n c t i o n a l s i g n i f i c a n c e o f a l t e r a t i o n s i n the a c t i v i t y of t h i s
enzyme.

F u n c t i o n o f Manganese S u p e r o x i d e Dismutase (MnSOD)

The m a j o r i t y o f t h e O^ reduced by a e r o b i c c e l l s i s c a r r i e d o u t
t h r o u g h t h e 4 e l e c t r o n r e d u c t i o n s by cytochrome o x i d a s e ( 6 ) , t h u s
preventing the r e l e a s e
m e d i a t e s such as 0 " , 2

these r e a c t i v e intermediates are generated during the r e d u c t i o n o f

0 2 t o H 0 ( 7 ) . In a d d i t i o n t o t h e r e d u c t i o n o f 0 i n t h e e l e c t r o n
2

t r a n s p o r t c h a i n , t h e a c t i v i t y o f a group o f c e l l u l a r enzymes which

are i n v o l v e d i n c a t a l y z i n g o x i d a t i o n r e a c t i o n s r e s u l t s i n u n i v a l e n t
r e d u c t i o n o f 0 t o 0 ' . These i n c l u d e x a n t h i n e o x i d a s e and p e r o x -
2

idases. As many o f t h e s e enzymes a r e l o c a t e d i n t h e m i t o c h o n d r i a ,

t h e s e o r g a n e l l e s can c o n t r i b u t e s u b s t a n t i a l amounts o f 0^' . I n
a d d i t i o n , t h e a u t o x i d a t i o n o f a l a r g e group o f compounds a l s o
c o n t r i b u t e s t o t h e O^" c o n c e n t r a t i o n i n l i v i n g systems. These
i n c l u d e c a t e c h o l a m i n e s , f l a v i n s and f e r r e d o x i n ( 8 ) .
The 0 f l u x i n a e r o b i c c e l l s appears t o have n e c e s s i t a t e d t h e
#
development o f SOD's w h i c h c a t a l y z e t h e d i s m u t a t i o n o f 0 2 to H 02 2

+ 0 » 2 Two t y p e s o f SOD's have been d e s c r i b e d i n mammalian c e l l s .

One c o n t a i n s Mn and i s l o c a l i z e d p r i m a r i l y i n t h e m i t o c h o n d r i a .
The o t h e r c o n t a i n s Cu and Zn and i s found p r i m a r i l y i n t h e c y t o s o l .
Manganese s u p e r o x i d e d i s m u t a s e i s o l a t e d from c h i c k e n , r a t and human
l i v e r has a m o l e c u l a r w e i g h t o f 80,000 and c o n t a i n s 4 s u b u n i t s o f
e q u a l s i z e , each c o n t a i n i n g one atom o f Mn ( 9 ) . I n c o n t r a s t t o
p y r u v a t e c a r b o x y l a s e and a r g i n a s e , t h e Mn i n r e s t i n g SOD i s i n t h e
t r i v a l e n t s t a t e . The c a t a l y t i c c y c l e o f t h i s enzyme i n v o l v e s r e -
d u c t i o n and t h e n r e o x i d a t i o n o f t h e m e t a l c e n t e r d u r i n g s u c c e s s i v e
e n c o u n t e r s w i t h oxygen. L i k e o t h e r enzymes o f t h e m i t o c h o n d r i a
which a r e s y n t h e s i z e d i n t h e c y t o p l a s m , MnSOD i s s y n t h e s i z e d
i n i t i a l l y as a h i g h e r m o l e c u l a r w e i g h t p r e c u r s o r p o l y p e p t i d e .
F i n a l p r o c e s s i n g t o t h e mature form presumably o c c u r s i n t h e m i t o -
chondria.
The h i g h c o n c e n t r a t i o n o f p o l y u n s a t u r a t e d f a t t y a c i d s i n
c e l l u l a r and s u b c e l l u l a r membranes makes them p a r t i c u l a r l y s u s c e p -
t i b l e t o f r e e r a d i c a l damage. I n a d d i t i o n , m i t o c h o n d r i a l membranes
c o n t a i n f l a v i n s as a p a r t o f t h e i r b a s i c s t r u c t u r e , p o t e n t i a l l y
contributing 0 2 r e s u l t i n g i n f r e e r a d i c a l damage. The p r o c e s s o f
u n c o n t r o l l e d l i p i d p e r o x i d a t i o n can r e s u l t i n t h e l o s s o f e s s e n t i a l
p o l y u n s a t u r a t e d f a t t y a c i d s , and t h e f o r m a t i o n o f t o x i c h y d r o p e r -
o x i d e s and o t h e r s e c o n d a r y p r o d u c t s . The l o s s o f e s s e n t i a l f a t t y
a c i d s may t h e n r e s u l t i n l o s s o f membrane i n t e g r i t y and l o s s o f
function. E x t e n s i v e o x i d a t i o n can a l s o l e a d t o r u p t u r e o f

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
58 NUTRITIONAL BIOAVAILABILITY O F MANGANESE

s u b c e l l u l a r membranes w i t h subsequent r e l e a s e o f l y s o s o m a l enzymes,

and i r r e v e r s i b l e damage t o t h e c e l l (10).

I n f l u e n c e o f D i e t a r y Manganese on MnSOD A c t i v i t y

A d i e t a r y d e f i c i e n c y o f Mn h a s been shown t o r e s u l t i n a r e d u c t i o n
o f MnSOD a c t i v i t y i n r a t s , mice, and c h i c k e n s . I n a d u l t mice f e d
d i e t s d e f i c i e n t i n Mn (1 ug Mn/g d i e t ) p r e n a t a l l y and p o s t n a t a l l y ,
the a c t i v i t y o f t h i s enzyme was s i g n i f i c a n t l y lower i n l i v e r ,
b r a i n , h e a r t , and l u n g than i n t i s s u e s o f a n i m a l s f e d c o n t r o l d i e t s
(45 ug Mn/g d i e t ) (11) . I n c h i c k e n s , t h e r e was lower a c t i v i t y o f
MnSOD i n l i v e r a f t e r o n l y 7 days o f f e e d i n g a M n - d e f i c i e n t d i e t (1
ug Mn/g d i e t ) t o h a t c h l i n g s compared t o c o n t r o l s . The a c t i v i t y o f
t h e enzyme was q u i c k l y e l e v a t e d t o normal by t h e f e e d i n g o f Mn-
adequate d i e t s . C o n c o m i t a n t w i t h t h e d e c l i n e i n a c t i v i t y o f MnSOD,
t h e a c t i v i t y o f c o p p e r - z i n c SOD (CuZnSOD) was i n c r e a s e d , s u g g e s t i n g
a compensatory r e s p o n s e
lower MnSOD a c t i v i t y . Paynte
i n h e a r t and k i d n e y MnSOD c a n a l s o o c c u r i n r a t s when t h e d e f i c i e n t
d i e t s a r e i n i t i a t e d a t weaning.

F u n c t i o n a l S i g n i f i c a n c e o f Low MnSOD A c t i v i t y

While t h e above shows t h a t t h e a c t i v i t y o f t h e enzyme c a n be

a f f e c t e d by d i e t a r y Mn i n t a k e , i t i s i m p o r t a n t t o d e t e r m i n e i f t h i s
reduction i n a c t i v i t y i sof physiological significance. An e x c e l -
l e n t model t o study t h i s q u e s t i o n i s t h e neonate. B i r t h and
weaning a r e c r i t i c a l p e r i o d s i n t h e l i f e o f a mammal; b o t h a r e
a s s o c i a t e d w i t h s e v e r a l pronounced enzyme changes. Immediately
a f t e r b i r t h , t h e neonate must depend on i t s g l y c o g e n r e s e r v e s f o r a
continuous supply o f glucose. L a t e r , g l u c o n e o g e n i c enzymes emerge,
and t h e a n i m a l i s c a p a b l e o f s y n t h e s i z i n g g l u c o s e from g l y c o l y t i c
p r o d u c t s and amino a c i d s (13) . There i s an i n c r e a s e i n o x i d a t i v e
m e t a b o l i s m d u r i n g t h i s p e r i o d ; thus an i n c r e a s e i n t h e p r o d u c t i o n
o f O* and i n c r e a s e d a c t i v i t y o f SOD might be e x p e c t e d , i n o r d e r t o
combat i t s e l e v a t e d p r o d u c t i o n . Consistent with t h i s idea are the
f i n d i n g s by Y o s h i o k a e t a l . (14) who r e p o r t e d t h a t SOD a c t i v i t y was
a t i t s l o w e s t l e v e l d u r i n g t h e f e t a l p e r i o d t h r o u g h day 5 o f p o s t -
n a t a l development i n t h e r a t . There was a r a p i d r i s e i n a c t i v i t y
from day 10 t h r o u g h day 20, r e a c h i n g l e v e l s t h a t were 88% o f t h o s e
o b s e r v e d i n a d u l t s . S i m i l a r r e s u l t s were r e p o r t e d by L a n k i n and
c o l l e a g u e s (15) and M a v e l l i e t a l . (16). I f the Mn-deficient
a n i m a l i s u n a b l e t o m a i n t a i n an adequate l e v e l o f e n z y m a t i c p r o t e c -
t i o n a g a i n s t o x y g e n - d e r i v e d r a d i c a l s , d e t r i m e n t a l consequences may
occur. F o r example, t h i s may e x p l a i n t h e h i g h i n c i d e n c e o f neo-
n a t a l m o r t a l i t y observed i n the M n - d e f i c i e n t animal. To t e s t t h i s
i d e a , we examined t h e changes t h a t o c c u r from b i r t h t o m a t u r i t y (d
60) i n t h e a c t i v i t y o f MnSOD and CuZnSOD i n s e v e r a l t i s s u e s o f
M n - s u f f i c i e n t and - d e f i c i e n t mice.
In t h i s s t u d y , t i s s u e Mn c o n c e n t r a t i o n s i n c r e a s e d w i t h age i n
b o t h groups; however, t h e i m p o s i t i o n o f a d i e t a r y Mn d e f i c i e n c y
r e s u l t e d i n l o w e r than normal l e v e l s o f Mn by day 60 i n a l l t i s s u e s
examined. The d e v e l o p m e n t a l p a t t e r n o f MnSOD a c t i v i t y p a r a l l e l e d
t h a t f o r Mn c o n c e n t r a t i o n (17).

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
6. ZIDENBERG-CHERR ANDKEEN Enhanced Tissue Lipid Peroxidation 59

L i v e r and k i d n e y c o n t a i n e d t h e g r e a t e s t amount o f MnSOD

a c t i v i t y i n mice, a p p r o x i m a t e l y 30% more t h a n b r a i n and h e a r t . I n
the mature a n i m a l , Mn d e f i c i e n c y a f f e c t e d MnSOD a c t i v i t y most
d r a m a t i c a l l y i n t h e l i v e r , w i t h s m a l l e r changes i n k i d n e y and
h e a r t , and a l m o s t no change i n t h e b r a i n .
An i n t r i g u i n g o b s e r v a t i o n i s t h a t MnSOD a c t i v i t y was r e t a i n e d
even w i t h t h e v e r y low amounts o f t i s s u e Mn i n d e f i c i e n t a n i m a l s .
These f i n d i n g s s u g g e s t t h e importance o f e n z y m a t i c p r o t e c t i o n
a g a i n s t f r e e r a d i c a l damage. I t i s i n t e r e s t i n g t h a t normal l e v e l s
o f MnSOD were m a i n t a i n e d i n b r a i n s from M n - d e f i c i e n t mice; we have
l i m i t e d d a t a t h a t i n d i c a t e t h a t Mn c o n c e n t r a t i o n i s lower t h a n
normal i n b r a i n s from M n - d e f i c i e n t mice. Perhaps o t h e r r o l e s o f Mn
i n t h i s t i s s u e a r e b e i n g compromised i n o r d e r t o p r o v i d e adequate
MnSOD a c t i v i t y .
To a s s e s s t h e f u n c t i o n a l s i g n i f i c a n c e o f lower t h a n normal
a c t i v i t y o f MnSOD, we measured h e p a t i c l i p i d p e r o x i d a t i o n and MnSOD
activity i nMn-sufficien
used r a t s i n o r d e r t o
the l i p i d p e r o x i d a t i o n s t u d i e s . S i m i l a r t o t h e f i n d i n g s i n mice,
a c t i v i t y o f l i v e r MnSOD i n c r e a s e d from b i r t h t h r o u g h 60 days o f
age. By day 60, MnSOD a c t i v i t y i n M n - d e f i c i e n t r a t s was h a l f t h a t
observed i n M n - s u f f i c i e n t r a t s (Figure 1). That t h i s d i f f e r e n c e i n
SOD a c t i v i t y i s o f s i g n i f i c a n c e i s s u g g e s t e d by t h e o b s e r v a t i o n
t h a t a t day 60, m i t o c h o n d r i a l l i p i d p e r o x i d a t i o n i n M n - d e f i c i e n t
r a t s , as a s s e s s e d by m e a s u r i n g TBA r e a c t i n g p r o d u c t s , was 3 t i m e s
t h a t o b s e r v e d i n M n - s u f f i c i e n t r a t s ( F i g u r e 2) (18). These
f i n d i n g s s u g g e s t e d t h a t t h e damage t o m i t o c h o n d r i a l membranes
p r e v i o u s l y o b s e r v e d i n M n - d e f i c i e n t a n i m a l s (1) was due i n p a r t t o
d e p r e s s e d MnSOD a c t i v i t y which r e s u l t e d i n i n c r e a s e d l i p i d p e r o x i -
d a t i o n from f r e e r a d i c a l s . To i n v e s t i g a t e t h i s p o s s i b i l i t y t h e
e f f e c t s o f Mn d e f i c i e n c y d u r i n g p r e n a t a l and p o s t n a t a l development
on m i t o c h o n d r i a l s t r u c t u r e i n t h e r a t were a s s e s s e d . Despite
s i g n i f i c a n t d i f f e r e n c e i n MnSOD a c t i v i t y , l i v e r s from M n - s u f f i c i e n t
and - d e f i c i e n t r a t s from day 3 t o day 60 e x h i b i t e d normal u l t r a -
s t r u c t u r e (19). However, a t 9 months o f age, l i v e r from t h r e e o f
the f o u r M n - d e f i c i e n t r a t s showed abnormal m i t o c h o n d r i a , whereas
t h o s e o f c o n t r o l r a t s had normal u l t r a s t r u c t u r e . I n t h e d e f i c i e n t
a n i m a l s , l a r g e v a c u o l e s were p r e s e n t i n t h e m a t r i x o f many m i t o -
chondria. The i n n e r and o u t e r m i t o c h o n d r i a l membranes were sepa-
r a t e d from each o t h e r , c r e a t i n g open s p a c e s . Similar abnormalities
have been o b s e r v e d i n l i v e r from p a t i e n t s w i t h W i l s o n ' s d i s e a s e
(20), d i s e a s e s o f m i t o c h o n d r i a l myopathy (21) , and A d r i a m y c i n
t r e a t m e n t (22). The u n d e r l y i n g mechanisms o f t h e s e changes a r e
unknown; however, e x c e s s i v e l i p i d p e r o x i d a t i o n h a s been s u g g e s t e d
as a c o n t r i b u t i n g f a c t o r . We s u g g e s t t h a t t h e m i t o c h o n d r i a l
a b n o r m a l i t i e s o b s e r v e d i n t h e 9 month o l d M n - d e f i c i e n t r a t s a r e a t
l e a s t i n p a r t t h e r e s u l t o f t h e lower MnSOD a c t i v i t y o c c u r r i n g a t
60 days o f age accompanied by e x c e s s i v e m i t o c h o n d r i a l l i p i d p e r o x i -
dation. S i n c e no s t r u c t u r a l a b n o r m a l i t i e s were a p p a r e n t e a r l i e r ,
t h e r e s u l t i n g m i t o c h o n d r i a l damage o b s e r v e d i n t h i s study may have
r e s u l t e d from numerous f a c t o r s c o n t r i b u t i n g t o s t r u c t u r a l damage
over a p e r i o d o f time. F o r example, l i p i d c o m p o s i t i o n o f t h e
membrane may have been a l t e r e d due t o e l e v a t e d l i p i d p e r o x i d a t i o n .
A d d i t i o n a l l y , Mn i s a c o f a c t o r f o r s e v e r a l enzymes f u n c t i o n i n g i n

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 NUTRITIONAL BIOAVAILABILITY O F MANGANESE

0 I i i i i ' \\' ' ' ' i i i

0 I 2 3 4 5 10 20 30 40 50 60 70
AGE (days)

F i g u r e 1. A g e - r e l a t e
Mn-sufficient ( )
shown a r e u n i t s / g l i v e r wet w e i g h t . Each p o i n t r e p r e s e n t s t h e
mean o f a t l e a s t t h r e e r a t s .

700 r

oI ' ' ' ' t> i i i i i i |

x

I 2 3 4 5 10 20 30 4 0 50 60 7 0
AGE (days)

F i g u r e 2. L i p i d p e r o x i d a t i o n as measured by TBA r e a c t i n g
p r o d u c t s (absorbance a t 532 nm) i n l i v e r m i t o c h o n d r i a f o r
Mn-sufficient ( ) and - d e f i c i e n t ( - - - ) r a t s . Isolated
m i t o c h o n d r i a were i n c u b a t e d i n T r i s - H C l b u f f e r , pH 7 . 4 , w i t h t h e
a d d i t i o n o f oxygen i n i t i a t o r s . Each p o i n t r e p r e s e n t s t h e mean
of a t l e a s t three r a t s .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
6. ZIDENBERG-CHERR A N DKEEN Enhanced Tissue Lipid Peroxidation 61

c h o l e s t e r o l s y n t h e s i s and f a t t y a c i d s y n t h e s i s ; t h u s a l t e r a t i o n s i n
t h e s y n t h e s i s o f t h e s e compounds c o u l d c o n t r i b u t e t o abnormal
membranes ( 1 ) .

Influence o f Manganese D e f i c i e n c y on Response t o F r e e Radical

Inducers

In a d d i t i o n t o d i e t , t h e a c t i v i t y o f MnSOD can be i n d u c e d under

c o n d i t i o n s which r e s u l t i n an i n c r e a s e d p r o d u c t i o n o f 0* s u c h as
e x p o s u r e t o h y p e r b a r i c oxygen. H y p e r o x i a i n d u c e s b o t h MnSOD and
c a t a l a s e a c t i v i t y i n pulmonary macrophages whether t h e c e l l s a r e
i n c u b a t e d i n v i t r o o r i f t h e a n i m a l s a r e exposed i n v i v o (23) .
S i m i l a r l y , ozone (0 ) i n h a l a t i o n has been shown t o i n c r e a s e t o t a l
l u n g CuZnSOD and MnSOD a c t i v i t y i n mice (24). Taken t o g e t h e r t h e s e
f i n d i n g s s u g g e s t t h a t an a n i m a l w i t h an i n a d e q u a t e d e f e n s e system
such as low MnSOD a c t i v i t y may be more s u s c e p t i b l e t o t h e d e l e t e r -
i o u s consequences o f suc
s t a t u s on t h e r e s p o n s e
- d e f i c i e n t mice were exposed t o O (1.21 ± 0.02 ppm) o r f i l t e r e d
a i r f o r 7 days (25). R e g a r d l e s s o f d i e t a r y t r e a t m e n t , O^ exposure
r e s u l t e d i n an i n c r e a s e i n l u n g wet w e i g h t (Table I ) .

TABLE I . E f f e c t o f Ozone Exposure on Mouse

Body and Lung Weight, and Lung SOD A c t i v i t y

Body wt Lung wt CuZnSOD MnSOD

(g) (g) (u/ (u/ (u/ (u/
g lung) lung) g lung) lung)
Mn-adequate
Air 25. .8±2, .4 0.,17±0..01 230±10 40±4 120±5 25±3
Ozone 26. ,7±0, .86 0.,22±0..02 190± 8 60±2 90±3 30±3
Mn-deficient
Air 30. .2±2, .6 0,,22±0..02 230±10 50±5 115±5 28±3
Ozone 31. .1±1, .7 0..35±0..02 220± 8 75±2 50±4 28±3

T h i s i n c r e a s e r e f l e c t s t h e edematous and i n f l a m m a t o r y r e s p o n s e o f
t h e l u n g t o O e x p o s u r e . N e i t h e r l u n g CuZnSOD n o r MnSOD a c t i v i t y
was a f f e c t e d by d i e t i n a i r - b r e a t h i n g g r o u p s . I n marked c o n t r a s t ,
exposure t o O r e s u l t e d i n an i n c r e a s e i n t o t a l l u n g SOD a c t i v i t y
i n t h e M n - s u f f i c i e n t group; t h i s i n c r e a s e was a f u n c t i o n o f h i g h e r
a c t i v i t i e s o f b o t h MnSOD and CuZnSOD i n t h e s e a n i m a l s . Exposure t o
O^ a l s o r e s u l t e d i n an i n c r e a s e i n t o t a l SOD a c t i v i t y i n Mn-
d e f i c i e n t mice; however, i n t h e s e a n i m a l s t h e i n c r e a s e o c c u r r e d as
a r e s u l t o f a s e l e c t i v e i n c r e a s e i n CuZnSOD a c t i v i t y .
These r e s u l t s show t h a t t h e t y p i c a l i n c r e a s e i n MnSOD a c t i v i t y
i n r e s p o n s e t o O exposure i s i m p a i r e d by d i e t a r y Mn d e f i c i e n c y .
The o b s e r v a t i o n t h a t t h e r e was a compensatory i n c r e a s e i n t h e
a c t i v i t y o f CuZnSOD i n t h e M n - d e f i c i e n t mice exposed t o O^ s u g g e s t s
t h a t t h e i n c r e a s e i n t h e a c t i v i t i e s o f t h i s enzyme i s i n p a r t
s u b s t r a t e - i n d u c e d , and s t r o n g l y s u p p o r t s t h e h y p o t h e s i s t h a t t h e
i n c r e a s e i n l u n g SOD a c t i v i t y i s an i m p o r t a n t r e s p o n s e t o 0 3

e x p o s u r e . Thus i f t h e n e t i n c r e a s e i n l u n g SOD a c t i v i t y i s l i m i t e d
by t h e n u t r i t i o n a l s t a t u s o f t h e a n i m a l , t h e n e x c e s s i v e l u n g damage
may o c c u r due t o f r e e r a d i c a l - i n i t i a t e d p e r o x i d a t i o n s .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
62 NUTRITIONAL BIOAVAILABILITY O F MANGANESE

S i m i l a r t o 0 , a l c o h o l i s t h o u g h t t o e x e r t some o f i t s t o x i c
e f f e c t s t h r o u g h t h e p r o d u c t i o n o f 0^ d u r i n g i t s m e t a b o l i s m . Con-
s i s t e n t with t h i s theory i s the observation that a l c o h o l feeding
can r e s u l t i n i n c r e a s e d SOD a c t i v i t y . I n r a t s (26) and p r i m a t e s
(27) e t h a n o l consumption r e s u l t e d i n i n c r e a s e d MnSOD a c t i v i t y .
In c o n t r a s t t o i n c r e a s e d MnSOD a c t i v i t y , d e c r e a s e s i n l i v e r
CuZnSOD a c t i v i t y were found w i t h c h r o n i c e t h a n o l f e e d i n g i n r a t s
and p r i m a t e s . A c u t e l o a d s o f e t h a n o l f e e d i n g have a l s o been
r e p o r t e d t o a f f e c t SOD l e v e l s . Mandal and co-workers (28) found
t h a t b r a i n SOD l e v e l s were d e c r e a s e d i n r a t s f o l l o w i n g acute
e t h a n o l f e e d i n g , and have s u g g e s t e d t h a t some o f t h e e f f e c t s o f
e t h a n o l on t h e nervous system a r e due t o t h e c y t o t o x i c e f f e c t s o f
superoxide r a d i c a l s . I n c o n t r a s t , V a l e n z u e l a and co-workers (29)
r e p o r t e d an i n c r e a s e i n l i v e r SOD l e v e l s i n r a t s g i v e n an a c u t e
feeding of ethanol. The d i f f e r e n c e between t h e r e s u l t s o f Mandal
and co-workers and V a l e n z u e l a and co-workers may be due t o d i f f e r -
ences i n t h e d i e t a r y s t a t u
o r t h e time sequence o
The above s u g g e s t s t h a t t h e i n c r e a s e i n t i s s u e MnSOD a c t i v i t y
w i t h e t h a n o l _ c o n s u m p t i o n r e f l e c t s a compensatory r e a c t i o n t o t h e
i n c r e a s e d 0* l o a d . T h e r e f o r e t h e a b i l i t y o f t h e a n i m a l t o i n -
c r e a s e t h e amount o f t h i s enzyme may d i c t a t e t h e e x t e n t o f t h e
p a t h o l o g y which c o u l d o c c u r due t o t h i s i n s u l t . To t e s t t h i s
h y p o t h e s i s , M n - s u f f i c i e n t and - d e f i c i e n t r a t s were g i v e n e i t h e r 20%
(w/v) e t h a n o l o r d e i o n i z e d water as t h e i r d r i n k i n g f l u i d (30).
There was no d i f f e r e n c e i n d a i l y c a l o r i c i n t a k e between t h e Mn-
s u f f i c i e n t and - d e f i c i e n t r a t s n o t r e c e i v i n g e t h a n o l . Both groups
consumed a p p r o x i m a t e l y 15 g o f p u r i f i e d d i e t a day, which i s
e q u i v a l e n t t o about 68 k c a l o f m e t a b o l i z a b l e energy p e r day. From
days 2-6, t h e d a i l y c a l o r i c i n t a k e o f t h e e t h a n o l - f e d r a t s
d e c r e a s e d t o 76 and 42% o f t h a t found i n t h e r a t s n o t f e d e t h a n o l
f o r M n - s u f f i c i e n t and - d e f i c i e n t r a t s , r e s p e c t i v e l y . E t h a n o l - f e d
r a t s were consuming 50% o f t h e i r c a l o r i e s from e t h a n o l and 50% from
the d i e t . A f t e r day 6, t h e e t h a n o l - f e d M n - s u f f i c i e n t r a t s i n -
c r e a s e d t h e i r c a l o r i c i n t a k e above t h a t o f t h e r a t s n o t f e d e t h a n o l
by e a t i n g more f o o d and by d r i n k i n g more e t h a n o l than d u r i n g t h e
f i r s t 6 d a y s . T h e i r c a l o r i c i n t a k e d u r i n g t h i s p e r i o d a v e r a g e d 90
k c a l / d a y , 50% from e t h a n o l and 50% from d i e t . In contrast, the
e t h a n o l - f e d M n - d e f i c i e n t r a t s c o n t i n u e d t o consume an average o f 30
k c a l / d a y , 65% from e t h a n o l and 35% from t h e i r f o o d .
The body w e i g h t changes o f t h e r a t s d u r i n g t h i s time were
c o n s i s t e n t w i t h t h e i r c a l o r i c i n t a k e . D u r i n g t h e f i r s t week o f
e t h a n o l f e e d i n g , M n - s u f f i c i e n t r a t s l o s t 15% o f t h e i r i n i t i a l body
weight. However, d u r i n g week 2 t h e s e r a t s g a i n e d an average o f 9
g, which b r o u g h t them t o about 90% o f t h e i r i n i t i a l body w e i g h t .
In c o n t r a s t , t h e e t h a n o l - f e d M n - d e f i c i e n t r a t s l o s t 20% o f t h e i r
i n i t i a l body w e i g h t d u r i n g week 1 and c o n t i n u e d t o l o s e w e i g h t
d u r i n g week 2. A f t e r 14 days o f e t h a n o l f e e d i n g , t h e e t h a n o l - f e d
M n - d e f i c i e n t r a t s were e x t r e m e l y l e t h a r g i c and i n p o o r c o n d i t i o n ,
w i t h pigment e n c r u s t a t i o n o f t h e f a c i a l and neck r e g i o n . A f t e r 14
days t h e body w e i g h t o f t h i s group o f r a t s was o n l y 65% o f t h e
i n i t i a l w e i g h t . The consumption o f e t h a n o l by M n - d e f i c i e n t r a t s
r e s u l t e d i n a t r e n d toward h i g h e r l e v e l s o f l i v e r Mn and l i v e r
MnSOD a c t i v i t y t h a n t h o s e o b s e r v e d i n d e f i c i e n t r a t s t h a t were n o t

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
6. ZIDENBERG-CHERR AND KEEN Enhanced Tissue Lipid Peroxidation 63

f e d e t h a n o l . However, t h e s e f i n d i n g s may be due t o t h e f a c t t h a t

l i v e r s i z e had d e c r e a s e d i n t h i s group. This finding i s consistent
w i t h t h o s e o f Barak e t a l . (31) who o b s e r v e d t h a t a l c o h o l i n c r e a s e s
t h e l e v e l o f h e p a t i c Mn i n normal r a t s , and D r e o s t i e t a l . ( 2 6 ) ,
who r e p o r t e d an i n c r e a s e i n c y a n i d e - i n s e n s i t i v e SOD a c t i v i t y a f t e r
e t h a n o l consumption i n normal r a t s .
Based on t h e l a c k o f s i g n i f i c a n t d i f f e r e n c e s among t h e f o u r
groups w i t h r e g a r d t o MnSOD a c t i v i t y i t i s u n l i k e l y t h a t t h e
t o x i c i t y p r o d u c e d by e t h a n o l i n M n - d e f i c i e n t r a t s was due s i m p l y t o
i n a d e q u a t e l e v e l s o f MnSOD, b u t r a t h e r r e p r e s e n t s an o v e r a l l
d i m i n i s h e d a b i l i t y o f t h e d e f i c i e n t animal t o respond t o t h i s
p a r t i c u l a r agent.
Another agent which we have used t o e v a l u a t e t h e i n f l u e n c e o f
Mn on s u p e r o x i d e m e t a b o l i s m i s t h e a n t i b i o t i c A d r i a m y c i n (ADR).
A d r i a m y c i n i s c o n s i d e r e d one o f t h e most p o t e n t drugs i n t h e f i e l d
o f chemotherapy, y e t i t s c l i n i c a l u s e f u l n e s s i s compromised by a
number o f s e r i o u s s i d e
toxicity. Adriamycin-induce
d e g e n e r a t i o n o f t h e c a r d i a c muscle; m i t o c h o n d r i a a r e e n l a r g e d and
t h e i n t r a c r i s t a l spaces a r e s u b s t a n t i a l l y extended.
We have e v a l u a t e d t h e b i o c h e m i c a l r e s p o n s e o f mice t o ADR
t r e a t m e n t when f e d e i t h e r M n - s u f f i c i e n t o r - d e f i c i e n t d i e t s . I n
a d d i t i o n we v a r i e d t h e l e v e l o f v i t a m i n E t o a s s e s s t h e i n f l u e n c e
o f a combined d e f i c i t o f d i e t a r y a n t i o x i d a n t s on ADR t o x i c i t y ( 3 3 ) .
A d r i a m y c i n i n j e c t i o n had no e f f e c t on l i v e r Mn c o n c e n t r a t i o n .
I n c o n t r a s t , l i v e r Fe c o n c e n t r a t i o n was i n f l u e n c e d by b o t h d i e t and
ADR i n j e c t i o n . A l t h o u g h t h e r e was a t r e n d towards h i g h e r t h a n
normal c o n c e n t r a t i o n s o f l i v e r Fe i n a l l groups t r e a t e d w i t h ADR,
o n l y t h o s e a n i m a l s f e d d i e t s low i n b o t h a n t i o x i d a n t s had s i g n i f i -
c a n t l y h i g h e r l e v e l s (254.8 ± 72 ug Fe/g l i v e r ) t h a n t h e i r s a l i n e -
i n j e c t e d c o n t r o l s (140.7 ± 44 ug Fe/g l i v e r ) .
H e a r t MnSOD a c t i v i t y i n t h e M n - d e f i c i e n t mice was a p p r o x i m a t e -
l y 50% t h a t o f M n - s u f f i c i e n t mice. A d r i a m y c i n i n j e c t i o n h a d no
e f f e c t on h e a r t MnSOD a c t i v i t y .
The TBA i n d e x was h i g h e s t i n t h o s e a n i m a l s f e d d i e t s low i n
v i t a m i n E and Mn; v a l u e s f o r t h i s group were a p p r o x i m a t e l y 2 - f o l d
h i g h e r t h a n t h o s e o b s e r v e d i n a n i m a l s f e d d i e t s which were n u t r i -
t i o n a l l y complete. A d r i a m y c i n d i d n o t i n f l u e n c e t h e TBA i n d e x i n
any d i e t a r y group.
R e s u l t s from t h i s s t u d y showed t h a t SOD a c t i v i t y was n o t
a f f e c t e d by a c u t e ADR t r e a t m e n t . A second f i n d i n g was t h a t a c u t e
ADR t o x i c i t y d i d n o t promote c a r d i a c l i p i d p e r o x i d a t i o n . However,
i t was o b s e r v e d t h a t m i t o c h o n d r i a l l i p i d p e r o x i d a t i o n was h i g h e s t
i n mice f e d d i e t s low i n b o t h a n t i o x i d a n t s . Ultrastructural
examination r e v e a l e d m i t o c h o n d r i a l a b n o r m a l i t i e s i n c a r d i a c t i s s u e
from A D R - t r e a t e d a n i m a l s ( F i g u r e s 3 and 4 ) . There were l a r g e
v a c u o l e s w i t h i n t h e m i t o c h o n d r i a and c o n d e n s a t i o n o f t h e i n n e r and
o u t e r membranes o f t h e m i t o c h o n d r i a . The u l t r a s t r u c t u r a l e f f e c t s
o f ADR t r e a t m e n t were most s e v e r e i n t h e low E , M n - d e f i c i e n t m i c e .
I t i s r e a s o n a b l e t o s u g g e s t t h a t a h i g h e r t h a n normal l e v e l o f
l i p i d p e r o x i d a t i o n may p r e d i s p o s e t h e a n i m a l t o t i s s u e damage from
ADR. C o n s i s t e n t w i t h t h i s c o n c e p t , Meyers e t a l . (34) have
r e p o r t e d t h a t p r e t r e a t m e n t w i t h s u p p l e m e n t a l v i t a m i n E c a n reduce
t h e t o x i c i t y o f ADR i n mice.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 NUTRITIONAL BIOAVAILABILITY O F MANGANESE

F i g u r e 3. H e a r t m i t o c h o n d r i a from a c o n t r o l mouse showing

normal u l t r a s t r u c t u r e (x 22,000).

F i g u r e 4. H e a r t m i t o c h o n d r i a from an ADR-treated mouse f e d a

d i e t low i n v i t a m i n E and Mn (x 22,000).

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
6. ZIDENBERG-CHERR AND KEEN Enhanced Tissue Lipid Peroxidation 65

Summary
In the above it is evident that a consequence of Mn deficiency can
be a profound reduction in MnSOD activity. Data from lipid peroxi-
dation studies strongly support the concept that this reduction is
of functional significance. In addition, the above findings
demonstrate the fact that environmental insults and drugs which
exert their toxic effects through the production of 0^ may exacer-
bate the effects of Mn deficiency. However, it is evident from the
work on ozone and ADR that the response of Mn-deficient animals to
free radical generators can vary. This suggests that the response
to such insults may be tissue specific and/or dependent on the
total amount of free radical generated.

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Schaffner, F . , Eds.; Grune and Stratton: New York, 1972; pp
511-525.
21. DiMauro, S.; Schotland, D. L . ; Bonilla, E.; Lee, C. P.;
Gambett, P.; Rowland, C. P. Arch. Neurol. 1973, 29, 170-9.
22. Pelikan, P. C.; Weisfeldt, M. L . ; Jacobus, W. E . ; Miceli,
M. V.; Bulkley, B. H.; Gerstenblith, G. J. Cardiovascular
Pharmacology 1986, 8, 1058-66.
23. Stevens, J . B.; Autor, A. P. Fed. Proc. 1980, 39, 3138-43.
24. Dubick, M. A.; Keen, C. L. Toxicol. Lett. 1983, 17, 355-60.
25. Dubick, M. A.; Zidenberg-Cherr, S.; Rucker, R. B.; Keen, C. L.
Fed. Proc. 1987, 46, 912.
26. Dreosti, I. E.; Record, I. R.; Buckley, R. A.; Manuel, S. J.;
Fraser, F. J . In Trace Element Metabolism in Man and Animals
(TEMA-4); Howell, J
Eds.; Griffin Press
617-620.
27. Keen, C. L . ; Tamura, T.; Lonnerdal, B.; Hurley, L. S.;
Halsted, C. H. Am. J . Clin. Nutr. 1982, 35, 836.
28. Mandal, P.; Ledig, M.; M'Paria, J. R. Pharmacol. Biochem.
Behav. 1980, 13, 175-82.
29. Valenzuela, A.; Fernandez, N.; Fernandez, B.; Ugarte, G.;
Vitela, L. A. FEBS Lett. 1980, 111, 11-13.
30. Zidenberg-Cherr, S.; Hurley, L. S.; Lonnerdal, B.; Keen, C. L.
J. Nutr. 1985, 115, 460-7.
31. Barak, A. J.; Beckenhauer, H. C.; Kerrigan, F. J . Gut 1967,
8, 454-7.
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Grotzinger, K.; Young, R. C. Science 1977, 197, 165-6.
RECEIVED August 20, 1987

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Chapter 7

Iron in Manganese Metabolism

N. Gruden

Institute for Medical Research and Occupational Health, M. Pijade 158, 41001 Zagreb,
Yugoslavia

The interaction of iron and manganese was studied on

intact white neonatal and weanling rats, and on the
everted intestinal segment of adult rats. The stim-
ulating effect of milk diet on manganese absorption
was eliminated by addition of iron to milk. This
inhibition of manganes
5 mg Fe/100 ml milk
of feeding on iron supplemented milk and disappeared
on the fourth day after withdrawing the supplementary
iron. Manganese retention in the intestinal wall was
far less affected by iron than its transport. The
competition between iron and manganese absorption is
is not yet developed in neonates but develops rather
abruptly in the third week of rat's life. To function
properly this competitive mechanism needs to be set
either by iron pretreatment or by a higher iron dose.

It has been recognized for a long time that there area large
number of interactions among trace elements with possible profound
metabolic consequences (1,2). The knowledge about the physio-
logical functions and optimum intake of trace elements should
therefore be considered in the light of interactions between them-
selves and with the other elements.
There are various mechanisms by which such interactions may
take place, like chemical association, competition for a binding
ligand-carrier, metabolic changes, membrane alteratios. The
result is usually that one elements inhibits the metabolic action
of another, but the two can also act sinergistically, causing a
effect greater than either element causes alone. The situation is
frequently rather complex, with an interactions between metals in a
chain of reactions C3). The subject has been appreciated by many
nutritionists. However, for different reasons, the knowledge
pertinent to humans has been developing rather slowly (4). Further
research may reveal that such interactions are of greater conse-
quence to human health than it is now generally acknowledged.
We focus here our attention to the action of iron on manganese
metabolism, i.e. on the two most studied essential trace elements.

0097-6156/87/0354-0067$06.00/0
© 1987 American Chemical Society

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
68 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

We s h a l l a l s o b r i e f l y t o u c h upon the e f f e c t of i r o n on cadmium-

manganese i n t e r a c t i o n .
The importance of manganese as an e s s e n t i a l d i e t a r y component
is well established. I t i s i n t e r e s t i n g to n o t e t h a t f o r 25 y e a r s
a f t e r i t s e s s e n t i a l i t y was r e c o g n i z e d , manganese has been t r e a t e d
as the t r a c e element of p u r e l y academic i m p o r t a n c e . Nowadays i t i s
a feed a d d i t i v e .
Manganese d e f i c i e n c y r e s u l t s i n a wide v a r i e t y of s t r u c t u r a l ,
p h y s i o l o g i c a l and b i o c h e m i c a l d e f e c t s , f o r i t has been i m p l i c a t e d
i n a number o f m e t a b o l i c and e n z y m a t i c p r o c e s s e s ( 5 - 1 5 ) . Hurley
has summarized the e v i d e n c e t h a t manganese i s e s s e n t i a l f o r normal
p r e n a t a l and n e o n a t a l development, w i t h d e f i c i e n c y r e s u l t i n g i n a
v a r i e t y of c o n g e n i t a l m a l f o r m a t i o n s ( 1 6 ) .
Manganese i s a l s o a t o x i c a g e n t , though i t can be r e g a r d e d as
one o f the l e a s t t o x i c t r a c e e l e m e n t s . A wide m a r g i n of s a f e t y
exists between i t s i n t a k e which i s e s s e n t i a l f o r the o r g a n i s m and
the c o n c e n t r a t i o n s a s s o c i a t e
Growth i s r a p i d an
number and c o m p o s i t i o n throughout the f i r s t y e a r o f l i f e . Optimal
n u t r i t i o n i s thus most c r i t i c a l i n t h i s e a r l y p e r i o d . M i l k - the
o n l y s o u r c e of f o o d f o r the o f f s p r i n g of a l l mammals i n the e a r l y
months of l i f e - cannot meet the demands of o p t i m a l growth l a t e r on
i n the f i r s t y e a r of l i f e . T h i s i s e s p e c i a l l y so w i t h the e s s e n t i a l
elements such as i r o n and manganese whose low c o n t e n t i n m i l k (18-
23) does not meet the needs of a f a s t growing o r g a n i s m (24-^27).
The e f f e c t o f m i l k upon i o n s a b s o r p t i o n from the i n t e s t i n a l
t r a c t has been s t u d i e d e x t e n s i v e l y (28-33). The h i g h e r a b s o r p t i o n
of i o n s i n the young than i n the a d u l t age (34-40) c o u l d be ex-
p l a i n e d on the one hand by changes o c c u r i n g i n the i n t e s t i n a l mem-
brane d u r i n g the p r o c e s s of a g i n g (41-45), or on the o t h e r hand,
may be due to m i l k d i e t which i s d e p r i v e d of s e v e r a l e s s e n t i a l
elements (28-33). Yet m i l k d i e t does not a f f e c t the m e t a b o l i s m of
a l l ions e q u a l l y . For i n s t a n c e , a s e v e n - d a y - l o n g m i l k d i e t which
n e i t h e r a l t e r s the t r a n s p o r t of c a l c i u m nor of l e a d i n s i x - w e e k - o l d
female a l b i n o r a t s (46), s i g n i f i c a n t l y i n c r e a s e s the manganese
t r a n s p o r t and r e t e n t i o n i n the duodenum of t h e s e a n i m a l s ( 4 7 ) . The
l a t t e r e f f e c t c o u l d be e x p l a i n e d n e a t l y by the low manganese c o n t e n t
of m i l k (48-52) were i t not t h a t m i l k f o r t i f i c a t i o n by manganese
enhanced even f u r t h e r , h i g h l y s i g n i f i c a n t l y , the manganese t r a n s f e r
and i n t e s t i n a l r e t e n t i o n ( 4 7 ) . Thus i t seems f a i r l y c o n c l u s i v e
t h a t i t i s not manganese d e f i c i e n c y i n m i l k which i s r e s p o n s i b l e f o r
an o v e r a l l enhancement of manganese t r a n s f e r i n t o and t h r o u g h the
duodenal w a l l i n m i l k - t r e a t e d a n i m a l s .
There i s a p o s s i b i l i t y t h a t some m i l k c o n s t i t u e n t s r e g u l a t e the
a b s o r p t i o n of i o n s i n the i n t e s t i n e . In s t u d y i n g manganese meta-
b o l i s m we t u r n e d t o the low i r o n c o n t e n t i n m i l k . I r o n has r e c e i v e d
great a t t e n t i o n i n p e d i a t r i c n u t r i t i o n . The c o n c e r n has been to
p r e v e n t the anemia caused by i r o n d e f i c i e n c y e a r l i e r o f t e n found i n
childhood. Wide m i l k consumption by i n f a n t s and young c h i l d r e n
makes t h i s food an a t t r a c t i v e v e h i c l e f o r i r o n f o r t i f i c a t i o n .
I r o n - e n r i c h e d p r o p r i e t a r y m i l k s u b s t i t u t e s can a d e q u a t e l y p r e v e n t
the anemia common t o i n f a n t s who s u b s i s t l a r g e l y on l o w - i r o n
mother's or cow's m i l k ( 5 3 ) .
Y e t , t h e r e i s i n s u f f i c i e n t knowledge about the b i o l o g i c a l
a v a i l a b i l i t y of t h i s element, and the o p t i m a l l e v e l s have not y e t

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
7. GRUDEN Iron in Manganese Metabolism 69

been adequately determined. There i s a r i s k of too high a l e v e l

with putatively diverse e f f e c t s of increasing the dietary iron
content (54-59). Further, since the amount of iron i n the diet and
the iron body state influence the absorption of some essential and
non-essential elements (60-71), the interaction between the minerals
thay may have harmful effects on the body i s always possible. In
view of a l l this we considered i t worthwhile to study the e f f e c t of
iron upon manganese metabolism, following thus the l i n e i n i t i a t e d
by some authors years ago (72-74).
A l l our experiments were performed on r a t s . Although "man i s
not a big r a t " (75) and extrapolation of experimental findings from
animals to humans i s generally d i f f i c u l t , according to Mahoney and
Hendricks (76) rats and humans respond q u a l i t a t i v e l y s i m i l a r l y to
many dietary and physiological factors known to influence iron
u t i l i z a t i o n . These authors have found iron absorption by rats to be
highly correlated with that i n humans - a rather important finding
for our (iron-manganes

Studies In V i t r o

The experimental animals were female albino r a t s , mostly f i v e weeks

old. Iron-manganese interaction was studied on the everted duo-
denal segment (77) where the transport and absorption of the two
metals per unit of time are higher than i n the more d i s t a l parts of
the intestine (78-82). Manganese-54 and iron-59 were used as
markers for their stable isotopes.

Manganese Transport. Our f i r s t experiments showed that when milk

was enriched with iron i n doses which equalized the d a i l y amount of
iron received with milk to that of the stock diet (10 mg Fe/100 ml),
the transduodenal transport of manganese became equal for animals
fed on these two d i f f e r e n t diets (47). In other words, the stimu-
l a t i v e effect of the o r i g i n a l , iron-deficient milk upon manganese
transport disappeared completely, confirming thus the competition
of iron and manganese at the expense of the l a t t e r (72-74). It
comes to one's mind, of course, that the tissue deprived of mangan-
ese through a milk diet w i l l u t i l i z e more of the offered manganese.
However, experiments with rats fed manganese-enriched milk resulted
in more than a doubled transfer of manganese-54 (47) , showing thus
that i t i s not the manganese deficiency i n milk which i s responsible
for the enhanced manganese-54 transfer i n milk-fed animals. More-
over, when both manganese and iron content of milk were raised to
the l e v e l i n the stock d i e t , the i n h i b i t o r y effects of iron upon
manganese was s t i l l dominant (47).
A l l t h i s indicates that there are some transport mechanisms
common to iron and manganese so that i n the presence of both ions
manganese w i l l be discriminated i n favor of iron. These findings
suggest also that the pretreatment ^n vivo with p l a i n or enriched
milk diet induces permeability changes i n the duodenal wall which
p e r s i s t at least long enough for the manganese transport to be
accomplished jln v i t r o .

Dose Dependency. When the animals were fed only cow's milk f o r t i -
f i e d with different doses of ferrous sulphate (0.60-19.0 mg Fe/100
ml) for three days before k i l l i n g ^ manganese transfer and i t s

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
70 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

duodenal r e t e n t i o n a l i k e f o l l o w e d the i r o n - d o s e dependency suggest-

i v e of a s a t u r a t i o n e f f e c t ( 8 3 ) . The i n h i b i t i o n had a m i l d l y ex-
p r e s s e d maximum around 2.5 mg Fe/100 ml m i l k w i t h a l e v e l i n g of
above 5 mg Fe/100 ml m i l k . The s i m p l e s t and most p l a u s i b l e
r a t i o n a l i z a t i o n of t h i s e f f e c t was ( a g a i n ) t h a t t h e r e i s a compe-
t i t i v e r e l a t i o n s h i p between i r o n and manganese f o r the c a r r i e r so
t h a t i r o n i s the p r e f e r r e d i o n w i t h a p r o b a b l y h i g h e r a f f i n i t y f o r
the b i n d i n g s i t e s w i t h i n the mucosa.
To what e x t e n t the mechanism r e g u l a t i n g manganese a b s o r p t i o n
i s dependent on the amount of i r o n i n the d i e t i s shown a l s o by the
f o l l o w i n g . Even a t a t e n - f o l d i n c r e a s e i n d i e t a r y manganese (from
5.6 t o 56.0 mg Mn/100 g f o o d ) , at two c o n c e n t r a t i o n s of d i e t a r y
i r o n (13 and 59 mg Fe/100 g f o o d ) , changes i n the duodenal t r a n s -
p o r t of manganese were o n l y m a r g i n a l l y s i g n i f i c a n t ( 8 4 ) . On the
c o n t r a r y , f o r the animals f e d p l a i n i r o n d e f i c i e n t m i l k the r e s u l t s
showed a marked dependence on manganese consumption, i . e . by
i n c r e a s i n g manganese c o n c e n t r a t i o
m l ) , transduodenal t r a n s f e
the a d d i t i o n of manganes mg/10
t a i n i n g 5 or 20 mg of i r o n i n 100 ml s l i g h t l y reduced or l e f t
unchanged the t r a n s f e r o f radiomanganese through the duodenal w a l l
( 8 5 ) . When the same amount of manganese was added t o m i l k which
had not been e n r i c h e d w i t h i r o n , manganese t r a n s f e r s i g n i f i c a n t l y
increased (47).
A p o s s i b l e r e a s o n why manganese a d d i t i o n t o m i l k and s t o c k
d i e t s g i v e s d i f f e r e n t r e s u l t s may w e l l be a d i f f e r e n c e s i n i r o n
c o n t e n t between the two d i e t s . In the case o f an i r o n d e f i c i e n t
d i e t ( m i l k ) , the ( r e g u l a t i o n o f ) manganese a b s o r p t i o n i s s e t o n l y
by the manganese l e v e l of the d i e t . W i t h 5 and 20 mg Fe/100 ml the
i r o n c o n t e n t i s w i t h i n i t s s a t u r a t i o n ("plateau") l e v e l ( 8 3 ) . I t
thus i n f l u e n c e s manganese m e t a b o l i s m s i m i l a r l y t o the s t o c k d i e t .
O b v i o u s l y , by i n c r e a s i n g the i r o n l e v e l i n m i l k above a t h r e s h o l d
(2.5 mg/100 mg, 83) the t r a n s f e r and i n t e s t i n a l r e t e n t i o n of man-
ganese become independent of b o t h the i r o n and manganese l e v e l s .

Time Dependence. As the i n i t i a l i r o n d e f i c i e n c y (by m i l k f e e d i n g )

s t i m u l a t e s i r o n a b s o r p t i o n (53,86-90)> w h i c h i n t u r n may a f f e c t
n e g a t i v e l y manganese a b s o r p t i o n (as d e s c r i b e d h e r e ) , the body i r o n
s t a t e must a l s o be t a k e n i n t o a c c o u n t . I t i s t h e r e f o r e u s e f u l t o
e s t a b l i s h data about the time f a c t o r , i . e . how l o n g the a n i m a l s can
be t r e a t e d w i t h i r o n supplemented m i l k b e f o r e an a l t e r a t i o n i n man-
ganese t r a n s p o r t i s observed and a l s o , how l o n g i t t a k e s f o r mangan-
ese t r a n s p o r t t o r e t u r n to normal once i r o n treatment has ceased.
The r e l e v a n t e x p e r i m e n t s showed t h a t the i n h i b i t i o n o f manganese
t r a n s f e r was p r e s e n t a f t e r one day of f e e d i n g on i r o n supplemented
(10 mg Fe/100 ml) m i l k . The i n h i b i t i o n l e v e l e d o f f a l r e a d y a f t e r
the second day of such f e e d i n g . The r e v e r s e e f f e c t - on w i t h d r a w i n g
the supplementary i r o n ( i . e . 0.05 mg Fe/100 ml i n pure m i l k ) - was
much s l o w e r , the i n c r e a s e i n manganese t r a n s p o r t h a v i n g become
n o t i c e a b l e a f t e r the f o u r t h day ( 9 1 ) .
The f a c t t h a t the onset o f i n h i b i t i o n of manganese t r a n s p o r t
and r e t e n t i o n by i r o n i s f a s t e r t h a n i t s d i s a p p e a r a n c e may a l s o be
due t o a h i g h e r a f f i n i t y of the c a r r i e r b i n d i n g s i t e s f o r i r o n t h a n
f o r manganese. Once f i l l e d up w i t h i r o n these s i t e s w i l l r e s i s t
i r o n d e f i c i e n c y f o r a l o n g e r time than they would need t o get

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
7. GRUDEN Iron in Manganese Metabolism 71

l o a d e d w i t h i r o n a f t e r an i r o n d e f i c i e n t s t a t e . As b o t h the appear-
ance and d i s a p p e a r a n c e of the i n h i b i t o r y e f f e c t o f i r o n t a k e s days,
i t i s l i k e l y t h a t the i n t e s t i n a l w a l l undergoes some r e v e r s i b l e
changes.
I r o n a b s o r p t i o n i s r e g u l a t e d by the c e l l s o f the i n t e s t i n a l
mucosa a c c o r d i n g to the body's need (68,87-89,90,92). In i r o n d e f -
i c i e n c y the e p i t h e l i a l i r o n content i s reduced and i t s uptake as
w e l l as t r a n s f e r t o the b l o o d i n c r e a s e d , w h i l e i n the l o a d e d s t a t e
the s i t u a t i o n i s o p p o s i t e . Owing to the c h e m i c a l s i m i l a r i t y o f i r o n
and manganese i t i s r e a s o n a b l e t o assume t h a t the i r o n b i n d i n g
m a t e r i a l i n the i n t e s t i n a l mucosal c e l l s can a l s o b i n d manganese,
e s p e c i a l l y i n the i r o n d e f i c i e n t s t a t e . A f t e r t h r e e days o f f e e d i n g
w i t h a n i r o n - p o o r m i l k , i r o n d e p o s i t s i n the i n t e s t i n a l w a l l are
m o s t l y used up by the body. As the b i n d i n g s i t e s i n v o l v e d i n the
t r a n s f e r of m e t a l s from the mucosa to the s e r o s a show a g r e a t e r
a f f i n i t y f o r i r o n than f o r manganese, the l a t t e r can r e p l a c e i r o n
o n l y i n the i r o n d e f i c i e n
On the w h o l e , manganes
l e s s a f f e c t e d by i r o n than i t s t r a n s p o r t (47,91), w h i c h s u g g e s t s
t h a t the b i n d i n g s i t e s f o r manganese (or i r o n ) t r a n s p o r t are not the
same as f o r t h e i r r e t e n t i o n i n the mucosa. In o t h e r wards, the
t r a n s p o r t b i n d i n g s i t e s are more s e n s i t i v e t o i r o n d e f i c i e n c y .
N o t h i n g , o f c o u r s e , can be s a i d about a c t u a l m o l e c u l a r d i f f e r e n c e s
between the two t y p e s of b i n d i n g s i t e s .
The e f f e c t o f i r o n upon manganese t r a n s f e r was shown to be
s i g n i f i c a n t l y h i g h e r i n the duodenum t h a n i n the jejunum or i l e u m .
As f o r a n i m a l s ' age and sex i t was observed t h a t i r o n e f f e c t upon
manganese t r a n s f e r and i n t e s t i n a l r e t e n t i o n was more pronounced i n
the young (6-week-old) than i n the o l d r a t s (16- and 26-week o l d ) ,
and i n female t h a n i n male r a t s . S u r p r i s i n g l y , the e f f e c t was more
dependent on sex t h a n on a n i m a l s ' age (Gruden, N., u n p u b l i s h e d d a t a ) .

I r o n T r a n s p o r t . To g a i n a d d i t i o n a l i n s i g h t i n t o the iron-manganese
i n t e r a c t i o n , the e x p e r i m e n t s were performed i n w h i c h the influence
of m i l k , e i t h e r pure o r f o r t i f i e d w i t h i r o n and/or manganese, on
i r o n t r a n s d u o d e n a l t r a n s p o r t was s t u d i e d ( 9 3 ) . The r e s u l t s
c o r r o b o r a t e d our former i n t e r p r e t a t i o n . Namely, compared w i t h the
s t a n d a r d d i e t as c o n t r o l , a t h r e e - d a y f e e d i n g w i t h cow's m i l k a l o n e
r e s u l t e d i n a two and a h a l f t i m e s h i g h e r t o t a l t r a n s d u o d e n a l
radioiron transport. The s t i m u l a t o r y e f f e c t o f m i l k was the same i f
manganese was added to m i l k (1.1 mg Mn/100 m l ) , but d i s a p p e a r e d
c o m p l e t e l y a f t e r the a d d i t i o n of 10 mg Fe/100 ml, a l o n e o r t o g e t h e r
w i t h 1.1 mg manganese/100 m l m i l k .
S i m u l t a n e o u s l y , t h e r e was no s i g n i f i c a n t change i n r a d i o i r o n
uptake i n the i n t e s t i n a l w a l l . T h i s c o n f i r m s the assumption t h a t
the b i n d i n g s i t e s f o r i r o n (and manganese) t r a n s p o r t are not the same
as f o r t h e i r r e t e n t i o n i n the mucosa. These p r o c e s s e s c o u l d be r e -
garded as independent so t h a t changes i n one need not necessarily
be accompanied by changes i n the o t h e r (73,74).
Under i d e n t i c a l e x p e r i m e n t a l c o n d i t i o n s i r o n d e f i c i e n c y d e f i -
n a t e l y s t i m u l a t e s much more the t r a n s d u o d e n a l t r a n s p o r t and i n t e s -
t i n a l uptake o f radiomanganese than of r a d i o i r o n (47,93). T h i s
c o u l d be e x p l a i n e d by the much more s t a b l e a b s o r p t i o n and o t h e r mech-
anisms i n the i n t e s t i n e f o r i r o n than f o r manganese. Whereas the
h o m e o s t a s i s o f i r o n i s m a i n t a i n e d a t the l e v e l o f the i n t e s t i n a l

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
72 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

tract (68,86,87,89,90,92), manganese homeostasis i s regulated (also)

at the excretion l e v e l no matter how much manganese has been absorbed
from the intestine (8,94-97).
The fact that manganese had no effect on iron transport could be
explained by different K values for iron and manganese (73). This
m

again suggests that the shared i n t e s t i n a l transport system shows less

a f f i n i t y for manganese than for iron and explains a greater i n h i b i -
tory effect of iron on manganese absorption than of manganese on
iron absorption. It may also be that there exists iron-binding s i t e s
which i f free from i r o n could be used to manganese transport and that
other manganese-binding s i t e s are present i n much greater abundance
but are used exclusively for manganese ions. The l a t t e r conclusion,
reached also for Forth and Rummel (98) i s strongly supported by the
fact that the enrichment of iron-poor milk with manganese enhances
the transfer and i n t e s t i n a l retention of radiomanganese but does
not affect iron transport (93).
Two conclusions whic
derived so f a r : 1) i n
increasing simultaneously iro and manganese content t
might be possible to diminish the r i s k of manganese deficiency and
2) milk does not seem to be the best means of a d d i t i o n a l n u t r i t i o n
in exposure to manganese.

Studies In Vivo

In view of l i t e r a t u r e data and our (in v i t r o ) results on i r o n -

manganese interaction, we considered i t worthwhile to study the e f f e c t
of iron on manganese metabolism i n very young rats when absorptive,
homeostatic and competitive mechanisms i n the i n t e s t i n a l tract are
not yet functioning properly or are i n a developing phase. We per-
formed experiments to see what role iron dose, duration of treatment
1
and animals age have i n iron-manganese competition at an early
period of rat's l i f e .
Five- to 21-day-old white rats were used. The animals were
placed into groups according to the amount of iron they had received
in iron supplemented cow's milk for either one ("non-pretreated") or
four days ("pretreated"). Iron doses were from 52 to 1000 yg Fe/ml
milk. Radioisotopes (Mn-54 or Fe-59) were always administered by
the a r t i f i c i a l "drop-by-drop" feeding procedure (99). The radio-
a c t i v i t y was determined i n selected organs ( l i v e r , kidney, spleen,
brain, femure, i n t e s t i n a l t r a c t , stomach) i n addition to measurements
of the whole body and carcass.

Sucklings. Although the i n h i b i t o r y e f f e c t of iron on manganese

absorption i s recognized (2,47,72-74,83,91,100), i t i s not known when
p r e c i s e l y i t sets i n . It seems that i n the neonatal, six-day-old
rats the competition between iron and manganese absorption i s not yet
developed. Namely, the addition for one (101) or four days (102) of
low, p h y s i o l o g i c a l amounts of iron to milk diet (50 or 100 yg Fe/ml)
did not appreciably decrease manganese absorption. Moreover, i n
neonatal rats treated with these iron doses for four days, manganese-
54 values i n the whole body (absorption), intestine, l i v e r and
kidneys were even greater by 10 to 52 percent than i n the controls
receiving p l a i n cow's milk (102). No interpretation can be offered
why and how the introduction of low iron doses increased

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
7. GRUDEN Iron in Manganese Metabolism 73

radiomanganese absorption at the same dietary manganese l e v e l for

a l l animal groups.
However, when a dose of 200 yg Fe/ml or higher (410-1000 yg Fe/
ml) was administered even on a single day, i t caused i n these s i x -
day-old rats a s i g n i f i c a n t and mainly dose-dependent diminution of
manganese absorption and organ uptake, with the exception of the
l i v e r (103). Our explanation i s that a) at that early age the lower
i r o n doses (below 200 yg Fe/ml) have no e f f e c t whatsoever on the
route(s) of manganese transport and b) that owing to lack of homeo-
s t a t i c iron regulation at that age (104) the large(r) amounts of
iron introduced (doses of 200, 410 and 1000 yg Fe/ml) can block the
manganese pathways. The l a t t e r conclusion i s corroborated by the
enhanced iron retention (104) and the diminished manganese one i n
the l i v e r and i n t e s t i n e (103). In other words, i n the absence of a
regulatory mechanism for iron absorption, i t s enhanced uptake owing
to high amounts of iron added to the milk diet eliminates manganese.
This i n turn implies tha
more prone to accept iro

Weanlings. In three-week-old rats i . e . , at an age when some h i s t o -

biochemical changes take place i n the i n t e s t i n a l tract (41,42,45,105,
106) the e f f e c t of iron on manganese metabolism i s somewhat d i f f e r e n t
from that i n neonatal animals. According to some authors (107) the
iron content of the weanling diet plays even a c r i t i c a l role i n
terminal maturation of rat small i n t e s t i n e . Anyhow, doses of 100 and
200 yg Fe/ml, administered on a single day enhanced s i g n i f i c a n t l y
(by 30-40 percent) radiomanganese absorption i n weanling r a t s , thus
influencing also manganese d i s t r i b u t i o n within t h e i r organism. At
higher iron concentrations (410 and 1000 yg Fe/ml), a s i g n i f i c a n t
drop (25-35 percent with respect to the controls fed with no addi-
t i o n a l iron i n milk) i n manganese absorption was observed (103). I t
thus appears that i n the weaning age manganese does not have to com-
pete with iron i f iron doses can be controlled by the homeostatic
mechanism. However, with the dose of 410 yg Fe/ml iron-manganese
competition sets i n abruptly, as (probably) the s t i l l large absolute
amounts of iron block the manganese pathways to an appreciable
degree.
It must be emphasized that the homeostatic regulation of iron
absorption i s e f f i c i e n t i n weaning but not i n neonatal rats (104).
In consequence, large amounts of iron prevent manganese transport i n
neonatals. The same i s true of the weanlings but at the higher iron
doses, when the homeostatic iron regulation cannot suppress iron
amounts below the threshold causing competition with manganese. I t
turns out that the dose of 410 yg Fe/ml milk i s above the threshold
irrespective of whether i t i s applied during a four-day treatment
(4 x 100 yg Fe/ml, 108), or given on a single day (103).
It i s concluded that the mechanisms which regulate i r o n -
manganese competition, although operating i n the intestine of wean-
l i n g r a t s , i n order to function properly need to be activated either
by iron doses above a certain threshold (103) or by some duration
(four days - i n our case, 108) of treatment with lower iron doses.

The Development of Iron-Manganese Competition. The competitive

effect of iron upon manganese absorption depends not only on the
duration of iron treatment (101,102,108) and on the iron dose

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
74 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

a d m i n i s t e r e d ( 1 0 3 ) , but a l s o , t o a g r e a t e x t e n t on t h e a n i m a l s age
(108). The q u e s t i o n remains whether t h i s e f f e c t i s t r i g g e r e d o f f
a b r u p t l y i n the r a t ' s weaning r:ge, o r whether i t i s d e v e l o p i n g g r a d -
u a l l y from t h e n e o n a t a l t i l l t h e weaning p e r i o d .
Experiments performed on 8,11,14 and 17-day-old a n i m a l s t o g e t h e r
w i t h t h e e a r l i e r r e s u l t s on f i v e - d a y - (102) and t h r e e - w e e k - o l d r a t s
(108) i n d i c a t e t h a t manganese a b s o r p t i o n does n o t s i g n i f i c a n t l y
d i f f e r between t h e c o n t r o l ( p l a i n m i l k d i e t ) and i r o n - t r e a t e d r a t s
(100 yg Fe/ml d i e t - d u r i n g f o u r days) u n t i l t h e 17th day o f age.
From t h e n on i t s h a r p l y d e c r e a s e s i n t h e i r o n - p r e t r e a t e d a n i m a l s t o
a t t a i n i n t h e t h i r d week as much as 50 p e r c e n t l o w e r v a l u e s t h a n i n
c o n t r o l a n i m a l s ( 1 0 9 ) . The abrupt d i m i n u t i o n o f manganese r e t e n t i o n
(as w e l l as t h a t o f i r o n - 5 9 i n i d e n t i c a l e x p e r i m e n t s , (110) i n i r o n -
p r e t r e a t e d a n i m a l s between t h e 17th and 2 1 s t day of l i f e i s l i k e l y
to be due t o t h e a l t e r a t i o n s t a k i n g p l a c e i n t h e i n t e s t i n a l t r a c t of
the r a t a t t h a t age. Having i n mind t h a t t h a t i s a l s o t h e time o f
weaning, one i s i m p e l l e
sumed by t h e a n i m a l s . However
mother's t o cow's m i l k and n o t t o t h e s t a n d a r d l a b o r a t o r y f o o d .
Thus, any major e f f e c t due t o a change o f food c o n s i s t e n c y can be
e l i m i n a t e d . I n a l l l i k e l i h o o d i t i s t h e h i s t o b i o c h e m i c a l change i n
t h e i n t e s t i n e t h a t f a c i l i t a t e s t h e onset o f iron-manganese i n t e r -
a c t i o n w i t h i n t h i s b r i e f p e r i o d of l i f e .
Summarizing, i t may be c o n c l u d e d t h a t t h e mechanisms r e g u l a t i n g
iron-manganese i n t e r a c t i o n become o p e r a t i v e i n t h e t h i r d week o f
the r a t ' s l i f e , i . e . , a t t h e same time when t h e r e g u l a t i o n o f i r o n
a b s o r p t i o n i s f u l l y a c t i v a t e d . E x p e r i m e n t a l e v i d e n c e suggests t h a t
the onset o f t h i s r e g u l a t i o n i s n o t provoked by a change from m i l k
to s o l i d f o o d .

I r o n and Cadmium-Manganese Interactions

The e f f e c t o f i r o n on cadmium-manganese i n t e r a c t i o n w i l l be b r i e f l y
d e a l t w i t h . The d a t a about t h e e f f e c t o f cadmium on manganese
m e t a b o l i s m a r e r a t h e r s c a n t y (78,111-114). N e v e r t h e l e s s , some d a t a
c l e a r l y show t h a t manganese t r a n s f e r through and i t s r e t e n t i o n i n
the r a t ' s duodenal w a l l a r e s i g n i f i c a n t l y depressed i n t h e p r e s e n c e
of cadmium ( 1 1 5 ) . By t h e s i m u l t a n e o u s a d d i t i o n of i r o n t o t h e
a n i m a l s ' m i l k d i e t t h e a c t i o n o f i r o n and cadmium upon manganese
a b s o r p t i o n becomes s y n e r g i s t i c . T h i s i s s u b s t a n t i a t e d by t h e o b s e r -
v a t i o n t h a t t h e a l r e a d y e x i s t i n g i n h i b i t o r y e f f e c t o f cadmium i s
enhanced by 10 t o 60 p e r c e n t i n t h e p r e s e n c e o f i r o n and, i n a d d i -
t i o n , t h a t i t becomes n o t i c e a b l e even a t such low doses o f cadmium
at which o t h e r w i s e t h e r e i s none (Gruden, N., P r o c . 5 t h I n t . Symp.
Trace Elem., Jena 1986, i n p r e s s ) .
A s a t u r a t i o n e f f e c t i s i n d i c a t e d i n t h a t i r o n enhances i d e n -
t i c a l l y t h e cadmium i n h i b i t o r y e f f e c t upon manganese a b s o r p t i o n
i r r e s p e c t i v e o f t h e cadmium dose. I n a d d i t i o n , t h e i r o n dose does
not a l t e r o n l y the a l r e a d y s t r o n g e f f e c t o f h i g h cadmium dose.
F u r t h e r m o r e , w i t h i n a span o f 5.0-15.0 mg Fe/100 ml i r o n has an
e q u a l e f f e c t upon cadmium a c t i o n , w h i l e below 2.5 mg Fe/100 ml i t
does n o t a l t e r t h e e f f e c t of cadmium a t a l l . This i s y e t another
i n d i c a t i o n o f i r o n s a t u r a t i o n a t a l e v e l above 5.0 mg Fe/ml m i l k ,
as o b s e r v e d e a r l i e r ( 8 3 ) .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
7. G R U D E N Iron in Manganese Metabolism 75

A plausible r a t i o n a l i z a t i o n would be that there i s a competition

for the transport route through the i n t e s t i n a l wall between cadmium
and manganese on the one side, and between iron and manganese on the
other. The competition, i . e . , absorption i n the i n t e s t i n a l t r a c t ,
depends upon the r e l a t i v e concentration of these ions and k i n e t i c s
of and a f f i n i t y for their i n t e r a c t i o n with the binding s i t e s i n the
i n t e s t i n a l mucosa.

Concluding Remarks

During the past f i f t e e n to twenty years observations have accumu-

lated to an extent which enables us to grasp the framework of a
complex relationship between two essential elements, iron and
manganese.
The competition between the two for the transport through the
i n t e s t i n a l wall, i . e . , absorption and their retention within the
various organs must be
homeostatic mechanism o
a l t e r a t i o n s of the tissues on the other. It has thus been e s t a b l i s h
ed that the iron-manganese competition i n the i n t e s t i n e at the
expense of manganese sets i n rather suddenly between the 17th and
21st day of the rats l i f e . The reversal of iron-manganese e f f e c t s
in neonatal to those i n weanlings i s not due to the change from
l i q u i d (milk) to s o l i d food, but (most probably) to the f u l l a c t i -
vation of the mechanism regulating iron absorption. The competition
is evident with iron doses that can be controlled by the homeostatic
mechanism, but the difference i n the homeostatic s i t e s for iron and
manganese must be taken into account i n r a t i o n a l i z i n g their i n t e r -
play.
The pretreatment in vivo with p l a i n or enriched milk diet i n -
duces changes i n the permeability of the i n t e s t i n a l wall which per-
s i s t at least long enough for manganese transport to be accomplished
in v i t r o . There i s obviously a competitive relationship between iron
and manganese for the c a r r i e r so that iron i s the preferred ion with
a probably higher a f f i n i t y for the binding s i t e s within the mucosa.
This also explains the fact that the i n h i b i t i o n of manganese trans-
port and retention evolves f a s t e r on iron a p p l i c a t i o n , than i t
disappears a f t e r iron withdrawal. As both the appearance and d i s -
appearance of the i n h i b i t o r y e f f e c t of iron take days, i t i s l i k e l y
that the i n t e s t i n a l wall undergoes some r e v e r s i b l e changes.
Manganese retention i n the i n t e s t i n a l wall i s far less affected
by iron than i t s transport, which suggests that the binding s i t e s
for manganese (or iron) transport are not the same as for their
retention i n the i n t e s t i n a l mucosa. In other words, the transport
binding s i t e s are more sensitive to iron deficiency, which means
that iron deficiency stimulates much more the transduodenal transport
of manganese (and iron) than their i n t e s t i n a l uptake.
From the p r a c t i c a l standpoint of p a r t i c u l a r importance i s that
i t i s not the manganese deficiency i n milk that i s responsible for
an o v e r a l l enhancement of manganese transfer into and through the
duodenal wall i n milk-treated animals. I t i s rather the a v a i l -
a b i l i t y to manganese of the i r o n - c a r r i e r s i t e s owing to the low iron
content of milk. Hence, milk does not seem to be the best means of
additional n u t r i t i o n i n exposure to manganese. On the other hand, i t
might be possible to diminish the r i s k of manganese deficiency i n

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
76 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

combating n e o n a t a l i r o n d e f i c i e n c y by i n c r e a s i n g s i m u l t a n e o u s l y i r o n
and manganese c o n t e n t i n m i l k .
I n t h e f u t u r e r e s e a r c h w i l l have t o be engaged i n e x p l a i n i n g a t
the m o l e c u l a r l e v e l why i r o n i n h i b i t s manganese t r a n s f e r and a b s o r p -
t i o n i n the i n t e s t i n a l t r a c t .

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ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
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In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
7. GRUDEN Iron in Manganese Metabolism 79

108. Gruden, N. Nutr. Reports Int. 1982, 25, 849.

109. Gruden, N. Nutr. Reports Int. 1984, 30, 553.
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RECEIVED April 29, 1987

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Chapter 8

Absorption Studies of Manganese from Milk Diets

in Suckling Rats
1-3 1 1-3
Wai-Yee Chan , M. HassanRaghib ,and Owen M. Rennert
1
Department of Pediatrics, University of Oklahoma Health Sciences Center,
Oklahoma City, OK 73190
2
Department of Biochemistry, University of Oklahoma Health Sciences Center,
Oklahoma City, OK 73190
3
Department of Molecular Biology, University of Oklahoma Health Sciences Center,
Oklahoma City, OK 73190

Absorption of Manganes
bovine milk and infan
even though the endogenous Mn content of these milk diets was
different. Studies showed that absorption of Mn from all milk
diets decreased with advancing age of the suckling rats. Using
suckling rats it was shown that the decline in Mn absorption
with age followed three stages with the changes occurring at 10
to 13 days and 20 days postnatal. A switching from duodenum
to jejunum as being the site of maximal Mn absorption with
advancing age of the suckling rat was also demonstrated by
both in vivo and in vitro experiments. Enhanced absorption of
Mn from bovine milk and infant formula rather than human milk
was demonstrated by in vitro studies.

Manganese is an essential mineral required for normal growth and development

(1,2). In the neonatal stage, the only dietary source of Mn for the infant is the
mother's milk or its substitutes such as bovine milk or infant formulas. The
relative bioavailability of most trace minerals in bovine milk and infant
formulas as compared to human milk is not known. Very often even though
deficiency might not have been reported in infants, an essential element is
added to the breast milk substitute because it is known to be important for
healthy growth of the infant. The Mn concentration of human milk is much
lower than that found in bovine milk and infant formula (3). However, absolute
quantity may not reflect the biologically effective level of Mn for absorption.
For example, the high Mn requirement in poultry might be the result of
inhibition of Mn absorption by high levels of calcium and phosphorus in the diet
(4). In rats low levels of calcium and iron in the diet were associated with
increased Mn absorption (4). Previous studies by Chan and associates using
everted small intestinal sacs from adult rats demonstrated a difference in the
bioavailability of Mn from human milk, bovine milk and infant formula (5). This
was not totally surprising since the bioavailability of several other essential
trace minerals has also been reported to be different in different types of milk.
One well investigated case is that of iron. Using extrinsically tagged milk, the
bioavailability of iron in human milk was found to be five to ten times greater
0097-6156/87/0354-0080506.00/0
© 1987 American Chemical Society

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
8. CHAN ET AL. Absorption Studies of Manganese from Milk Diets 81

than that in infant formula even though the latter had a significantly higher
iron content (6,7). Besides dietary components, the absorption of the mineral
could also be affected by the age of the suckling infant. A number of examples
9
were reported in the nutrition literature. A decrease in zinc (8), ^Nb (9),
^ ^ C d (10), lead, cadmium and mercury (11) absorption was reported to occur in
older rats when compared to younger animals.
Infant formulas are supplemented with Mn and other trace elements in the
form of inorganic salts. Whether these supplemented minerals have different
bioavailabilities from their counterparts found naturally in human milk or
bovine milk has not been extensively investigated. Chan and associates
previously reported that Mn was bound to different molecules in human milk,
bovine milk and infant formula (12). Inadequate or excessive intake of Mn has
severe effects especially during infancy (1,13). It is therefore important to
evaluate the bioavailability of Mn in different milk diets. To achieve this aim,
suckling rats and everted intestinal sacs derived from these animals were used
as systems to study the absorption of Mn from rat milk, human milk, bovine
milk and infant formula. Effec
from various milk diets wa
this article have been reported previously (14-17).

Materials and Methods

Animals. Sprague-Dawley female rats raised on regular chow (Way Rodent Blox
8604) were bred at the Animal Facility of the University of Oklahoma Health
Sciences Center. Females were mated with males of the same strain. Pups
were kept with their dams from birth to the time of the experiment. Pups were
separated from their dams and fasted for 14 hours before each experiment.

Milk. Human breast milk was obtained from volunteers and fresh raw bovine
milk was obtained from local farms. Raw milk was used instead of pasteurized
milk in order to preserve the Mn binding ligands in their natural form. The
infant formula used was regular Similac (Ross Laboratories). Fresh rat milk
was obtained from lactating female rats 8-12 days after parturition as
described previously (17).

In Vivo Manganese Absorption Studies, Eight to thirteen day old suckling rats
were used for these experiments. Milk samples (0.1 to 0.2 ml) were incubated
with carrier free ^ M n C l 2 (New England Nuclear) buffered with 0.1 M sodium
bicarbonate, pH 7.4 for 2 hours at room temperature. The final isotope
5
concentration of ^ M n was 5 pCi/ml milk. This method has been shown to label
endogenous Mn binding ligands successfully (12). To eliminate individual
variation to the maximum extent, only litters of a minimum of 8 pups were used
for an experiment. A t least 2 pups from the same litter were allocated to each
diet category in any given feeding experiment and each experiment was
repeated three or more times. Pups were fed by gastric intubation with 0.1 to
0.2 ml of extrinsically labeled milk as described previously (16). Amount of fed
radioactivity was quantitated by whole body counting of the animal for 1
minute in a gamma counter after each feeding. If the pups were too large to
allow whole body counting, the total amount of ^ M n fed was calculated from
the amount and specific activity of the milk sample fed. Absorption of ^ M n
was quantitated three hours after feeding by counting the digestive tract and
the carcass with and without the liver.

In Vitro Manganese Absorption Studies. Rats of 10, 13 and 15 days of age were

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
82 NUTRITIONAL BIOAVAILABILITY O F MANGANESE

fasted 12 to 14 hours before sacrificing. The small intestine was removed and
washed thoroughly with ice-cold physiological saline solution (0.9 % NaCl).
Segments of 2 to 2.5 cm of the duodenum or jejunum were everted as described
previously (18). After tying one end, the intestinal segment was filled with 50
to 100 pi of oxygenated modified Krebs-Ringer solution (135 mM NaCl, 5 mM
glucose and 10 mM Tris, pH 7.4). The everted intestinal sacs were incubated
for one hour at 37°C in 2.5 ml of either human milk, bovine milk or infant
formula containing 0.5 jiGi ^ M n / m l .

Analytical Methods. Manganese content of milk samples was measured in

triplicate by flameless atomic absorption spectrophotometry using a Perkin-
Elmer model 703 Atomic Absorption Spectrophotometer equipped with a HGA
500 graphite furnace and an AS-1 autosampler. Standards were run after every
third sample. Manganese-54 radioactivity of milk and tissue samples were
quantitated by using a Beckman 8000 deep-well gamma counter. Due to the
geometrical difference of a whole carcass and the liver in the deep-well, the
carcass counts of the smal
correction factors determine
was determined by the method of Lowry et al. (19).

Statistical Analysis. Testing for significant differences between groups was

done by analysis of variance according to the SAS User's Guide 1977 edition,
SAS Institute, Raleigh, N . C . The results of several experiments were combined
and the differences among the means of the various categories were subjected
to analysis of variance allowing for an unequal number of animals in each group
as described (17). A l l dependent variables were consistent with respect to the
presence or absence of the missing values. The probability of occurrence by
chance of p< 0.05 was accepted as significant.

Results and Discussion

In Vivo Manganese Absorption. Recovery of ^ M n in all experiments was above

98% (results not shown). No loss of the isotope was encountered during the
experimental period. Previous experiments established the optimum fasting
time prior to feeding to be 12 to 14 hours and the optimum assimilation time to
be 3 hours (16). With a similar protocol the whole body retention of ^ M n in 9
day old rat pups was found to be similar regardless of whether human milk,
bovine milk, infant formula or rat milk was fed. The results are presented in
Table L No statistically significant ( p < 0.05) difference existed among the
four milk diets. Similar observation was also reported recently by another
laboratory (20). In all cases examined, liver retained over 65 % of the absorbed
^ M n indicating that liver Mn might be as good an index for assessing Mn
absorption as Mn content of the gut-free carcass. This observation is not
surprising since both Cotzias et al. (21) and Papavasiliou et al. (22) have shown
that the liver is responsible for regulation of Mn homeostasis and Black et al.
(23) in a recent report have shown a linear increase in liver Mn in a
bioavailability study of Mn in the chicken.
Human milk contained relatively less Mn when compared to the other milk
diets examined. The endogenous Mn concentrations of human milk, bovine milk,
infant formula and rat milk used in the present studies as determined by
flameless atomic absorption spectrophotometry were 8 + 3, 30 + 5, 73 + 4, 148 +
18 Mg/liter respectively. It is possible that the higher concentrations of
endogenous Mn in infant formula and rat milk might compete with the added
n for transport during the experiments. However, the addition of non-

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
8. C H A N E T AL. Absorption Studies of Manganese from Milk Diets 83

radioactive Mn in the form of MnCl2 to both human and bovine milk to

concentrations up to 150 fig/litre had little or no effect on the absorption of
*^Mn by 10 day old rats from these two kinds of milk (results not shown). These
results suggested that at the level of carrier free ^ M n used in the experiments
described in this report absorption of Mn would be proportional to the
absorption of ^ M n .
5
Table I. Retention of *Mn from Various Milk
Diets by 9 Day Old Suckling Rats*

Mean ^ M n as % of Dose

Milk Diet N b Carcass w i t h L i v e r c

Liver Per Gm Liver

Human Milk 13 31 +7d 19 +6 46 + 12

Bovine Milk 12 28+6 19+7 42 + 12
Infant Formula 13
Rat Milk 15

a. Results of 5 different feeding experiments were combined for each of the

milk diets examined.
b. Number of pups.
c. Whole body minus stomach and digestive tract.
d. Means + standard deviations.

Effect of Age of Suckling Rats on Absorption of Milk Manganese In Vivo.

Retention of absorbed milk Mn in the carcass was affected by the age of the
suckling neonate. Results are presented in Figure 1. Each bar in the histogram
represents results obtained from at least 10 pups. Manganese-54 radioactivity
was expressed as % of dose. Both means and standard deviations of the means
are shown. There appeared to be three stages in the course of the decline of
Mn absorption with increasing age of the suckling rat. This change was
observed for all three milk diets (human milk, bovine milk and infant formula)
and appeared to be independent of the nature of the milk diet. The amount of
5^Mn retained by the carcass of the 10 day old and younger rats was
comparable with no significant difference (p < 0.05) regardless of the type of
milk diet fed. The decline in ^^Mn absorption with age before 10 day postnatal
was very small if existed. The first and milder drop occurred between 10 to 13
day postnatal. The difference between the amount of ^ M n in the carcass of 10
and 13 day old rats was significant (p < 0.05). The decrease in ^ M n absorption
into the carcass of 13 and 20 day old rats was gradual but steeper than the
change occurring in the first stage. There was a drastic decline in the retention
5
of ^ M n in the carcass of rats older than 20 days postnatal. For rats older than
20 days postnatal, Figure 1 only shows the data for 30 day old rats. Absorption
of ^ M n in 23 and 25 day old rats was similar to that in 30 day old rats. It is
interesting to notice that rats usually wean at around 20 days postnatal.
The difference in carcass ^ M n radioactivity between 10 and 20 day old
rats was approximately three fold. This difference in ^ M n absorption between
the two age groups was also reflected in the amount of ^ M n retained by the
intestinal tissues of these two groups. In this experiment eleven 10 day old rats
were fed infant formula labeled with 54,M The rats were sacrificed three
n#

hours after feeding. The intestine was removed and the lumen content flushed
out by washing the tissue several times with ice-cold physiological saline until a

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
84 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

40

8 9 10 11 13 15 20 30
Postnatal Age In Days

Figure 1. Effect of Age on the Incorporation of ^ M n into the Carcass

of Suckling Rats Fed Various Milk Diets

| 1 Human Milk; H B Bovine Milk; \ I Infant Formula

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
8. C H A N E T A L . Absorption Studies of Manganese from Milk Diets 85

oonstant reading was obtained when the tissue was counted after two
consecutive washes. The same experiment was repeated with six 20 day old
rats. The means and standard deviations of the means of ^^Mn radioactivity (as
% of dose) retained by the duodenum, jejunum, ileum and the large intestine for
the 10 day old rats were 17.1 + 3.5, 1.7 + 0.6, 0.5 + 0.2, and 0.4 + 0.2
respectively. Significantly less (p < 0.05) ^ M n was found in the duodenum of 20
day old rats (1.2 + 1.0) while the other segments of the intestine of rats of this
age group retained more ^ M n when compared with the 10 day old rats (2.4 +
IA for jejunum, 2.2 + 1.2 for ileum, and \A + 0.8 for large intestine). The
difference in duodenal tissue ^ M n radioactivity between rats of the two age
groups was even bigger when they were expressed as % of dose per gram of wet
tissue (11.8 + 6.1 for 10 day old rats and 0.51 + 0.24 for 20 day old rats, both
values were for the whole duodenum plus 1/3 of jejunum). In both the 10 and 20
day old rats more than 55 % of the duodenal tissue ^ M n radioactivity was
retained by the brush border cells (57.5 % for 10 day old rats and 55A% for 20
day old rats).
Not only the site of maxima
of milk in the stomach and
the suckling rat. Ten and 20 day old rats were fed 0.2 ml of human milk, bovine
milk or infant formula labeled with 5^Mn. Three hours after feeding, the rats
were sacrificed. The gastrointestinal tract was removed and the stomach,
small intestine and large intestine were counted separately before and after the
lumen contents were emptied. Regardless of the type of milk diet, passage of
milk from the stomach to the small intestine and from the small to the large
intestine was faster in 20 days old rats than in 10 day old rats. The relative
distribution of ^ M n among the stomach, small intestine and large intestine was
very similar for all three milk diets examined. For 10 day old rats at three
hours after feeding with infant formula the percent distribution of total
gastrointestinal tract ^ M n radioactivity among the stomach, small intestine
and large intestine was 32.4 + 4.2, 62.5 + 4.4, and 5.1 + 3.6 respectively (values
represent means + standard deviations for 8 rats). On the other hand in 20 day
old rats, the stomach only retained 11.9 + 2.4 % while the small intestine and
5
large intestine retained 75.8 + 6.1 % and 12.3 + 6.6 % of the total ^ M n
radioactivity in the gastrointestinal tract, respectively (Values represent means
+ standard deviations for 8 rats). Even though in 10 day old rats the percent
distribution of ^4,yj j n nfi hed tissue of the different sections of the
us

gastrointestinal tract at 3 hours after feeding did not vary with the nature of
the milk diets, detailed analysis of the tissue distribution of ^ M n among
various sections of the small intestine showed that duodenum always retained
the majority of tissue ^ M n (n.5 + 5.8 % for human milk, 8.2 + 4.9 % for bovine
milk and 13.5 + 3.5 % for infant formula, all value as % of dose). However, if
jejunal tissue ^ M n contents were compared, the jejunum of rats fed bovine
milk retained more % n (4.0 + 1.2 % out of 13.4 + 2.3 % of total administered
dose of 54|yj retained by intestinal tissue including duodenum, jejunum and
n

ileum) than rats fed either human milk (2.0 + 1.3 % out of 15.3 + 2.8 % for
total intestinal tissue) or infant formula (2.1 + 1.2 % out of 16.3 + 5.5 % for
total intestinal tissue).
The pronounced effect of age on the ability of the suckling rats to retain
dietary Mn observed in the present study has also been reported by others
(20,24-26). This developmental change in the absorption of Mn from milk could
be related to the change in the absorption mechanism in response to lactational
change in the constituents of milk or because of intestinal maturation.
Naturally, change in intestinal resecretion of absorbed Mn could also be a
factor (Weigand, E . ; Kirchgessner, M . Proc. 5th Int. Symp. Trace Element

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
86 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

Metab. Man and Animal, in press). Occurrence of developmental stage specific

binding ligand in the intestine for a particular nutrient has also been suggested
(27). Even though convincing examples are lacking, the present studies cannot
rule this out as a factor causing the observed change in Mn absorption with age
in suckling rats. A change in the absorption mechanism of Mn with age is
obviated by the observation that a larger amount of ^ M n was retained by the
jejunum and ileum in comparison to the duodenum in older (20 day old) suckling
rats than in younger ones. In adult rats the major Mn absorption site has also
been reported to be the jejunum by other researchers (28). This switching of
maximal Mn absorption site from duodenum to jejunum with age could be one of
the reasons for the decline of Mn absorption in older rats. The contribution
from the intestinal excretion of Mn due to bile also should not be neglected
(29). On the other hand, the observed change in the passage of milk through the
gastrointestinal tract might be the consequence of an age related change in the
stomach function of suckling rats.

Table II. Relativ

In In

Age Milk Ratio of Mean Serosal Fluid ^ M n %

a
in Days N Diet In Duodenal Sac/3ejunal Sac

Human Milk 1.37^

10 Bovine Milk 1.2*
Infant Formula 1.38

Human Milk 1.56

13 Bovine Milk 1.81
Infant Formula 0.97

Human Milk 1.86

15 Bovine Milk 0.65
4 Infant Formula 0.92

a. Number of animals in each group.

b. Values presented were ratio of the mean value of ^ M n radioactivity
recovered in the serosal fluid expressed as percent of dose per gm wet
tissue of the duodenal sac to that of the jejunal sac.

In Vitro Manganese Absorption. To further examine the relative importance of

duodenal and jejunal Mn absorption at different ages in rats, in vitro
experiments were performed with different segments of the intestine. To
account for the difference in tissue mass of the intestine at different ages, the
ratio of the percent of dose of ^ M n per gm wet tissue recovered in the serosal
fluid of the everted duodenal and jejunal sac was computed. The results are
presented in Table II. Duodenum played a more important role in ^ M n
absorption in younger rats (10 day old). This is true for all three milk diets
examined. Except in the case of human milk, there appeared to be a switching
of maximal Mn absorption site from the duodenum to jejunum with increasing
age of the suckling rat. This observation coincided with the observation made
in in vivo studies of Mn absorption with infant formula. The duodenum
appeared to remain the major absorptive site for Mn even in rats of 15 days of
age.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
8. CHAN ET AL. Absorption Studies of Manganese from Milk Diets 87

Table III. In Vitro Absorption Studies of Manganese

from Different Milk Diets

Age in Milk ^ M n as % of Dose/Gm Intestinal Sac

a
Days N Diet Serosal Fluid 0
Intestinal Tissue 0

4 Human Milk 23.7 + 10.0 C

295.6 + 75.7
10 4 Bovine Milk 56.6 + 21.6 332.3 + 44.2
4 Infant Formula 52.8 + 18.3 516.7 + 186.3

4 Human Milk 8.0 + 2.4 119.2 + 18.1

13 4 Bovine Milk 11.4 + 2.0 100.4 + 20.0
4 Infant Formula 11.4 ± - 2 3 121.1 + 24.6

Human Milk 6.2 + 2.0 63.6 + 16.3

15 4 Bovin
4 Infan

a. Number of animals in each group.

b. Obtained by combining the duodenal and jejunal sac results.
c. Means + standard deviations.

The effect of age of the animal on the transport of milk Mn across the
intestinal wall was also investigated with this in vitro system. Radioactivity
o f ^ M n recovered in the intestinal tissue and serosal fluid when different milk
diets were incubated with everted sacs derived from suckling rats of different
ages was expressed as percent of dose per gm of wet tissue in these two
compartments. The results are presented in Table III.
For all three milk diets tested the absorption of Mn, in terms of both
being transported to the serosal fluid and being taken up by the intestinal
tissue, was higher in younger rats. This agreed with the results obtained in in
vivo studies with the suckling rats. Further analysis of the data showed that
the uptake of ^ M n into intestinal tissue was higher from bovine milk and infant
formula than from human milk. This is true when the intestinal tissues were
derived from rats of 10 or 15 days of age. The transport of ^ M n to serosal
fluid across the intestinal sac was also significantly higher (p < 0.05) from
bovine milk and infant formula than from human milk regardless of the age of
the rats from which the intestinal tissues were derived with the exception of
formula with 15 day old rats. This significant difference in the transport and
uptake of Mn from bovine milk and human milk is consistent with the
observation previously reported using everted intestinal sacs from adult rats (5)
but is different from results of the in vivo absorption studies presented above
and a recent report from another laboratory (26). The cause of this discrepancy
between the results of the in vivo and in vitro experiments is not clear. One
possibility is that gastrointestinal processing of the milk diet plays an important
role in the absorption of Mn. This is implicated by the results of the study of
the passage of milk through the gastrointestinal tract. The role of intestinal
secretion of Mn also cannot be neglected. Naturally, there is also the
possibility that the mechanism of Mn absorption in the in vivo situation might
be altered in the in vitro system. This therefore poses an important question
concerning the validity of everted intestinal sacs for studying the absorption of
nutrients.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
88 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

Conclusion

The present study showed that the absorption of Mn from milk in suckling rats
is largely regulated by the age, or in other words, the developmental stage of
the animal. Even though rat milk, bovine milk and infant formula contained
higher amounts of Mn than human breast milk, absorption of Mn from these four
types of milk is comparable in suckling rats. These results suggest that
supplementation of infant formulas with excessive Mn would not guarantee an
increased supply of the mineral to the infant. This contention is supported by
reports that serum manganese levels were not higher in formula-fed infants
than in breast-fed infants (30,31).
A decline in absorption of Mn with advancing age of the suckling rats has
been shown. This change in Mn absorption appeared to be correlated with the
switching of the site of maximal absorption. In younger animals the duodenum
is more active in Mn absorption while in older animals the jejunum plays a more
important role. This switching of absorption site is also demonstrable using
everted intestinal sacs from rats of different ages
The nature of the mil
absorption of Mn by sucklin
studies did show a difference in uptake of Mn from different milk diets. This
suggests that other physiological factors regulating intestinal Mn absorption
overshadow the difference in the transport of Mn from the different milk diets
across the mucosal/serosal membranes.

Acknowledgments

We would like to thank the mothers of the L a Lecbe League of Oklahoma City
for providing us with some of the milk samples used in these studies. We would
also like to thank Ms. Greta Shepherd for typing the manuscript. This study was
supported by NIH grants HD 16730 and HD 21793 awarded to W.Y.C.

Literature Cited

1. Shaw, J.C.L. Am. J. Dis. Child. 1980, 13, 74-87.

2. Hurley, L.S. In Clinical, Biochemical and Nutritional Aspects of Trace
Elements; Prasad, A.S., Ed.; Alan R. Liss, Inc.: New York, 1982; p. 369.
3. Committee on Nutrition Pediatric Nutrition Handbook; American
Academy of Pediatrician: Evanston, 1979; p. 92.
4. Forbes, R.M.;Erdman, J.W., Jr. Ann. Rev. Nutr. 1983, 3, 213-231.
5. Chan, W.Y.; Bates, J.M., Jr.; Rennert, O.M.: Mahmood, A.S.; Torres-
-Pinedo, R. Life Sci. 1984, 35, 2415-2419.
6. Saarinen, U.M.;Siimes, M.A.; Dallman, P.R. J. Pediat. 1977, 41, 36-39.
7. McMillan, J.A.; Oski, F.A.; Lourie, G.; Tomarelli, R.M.; Landaw, S.A.
Pediatrics 1977, 60, 896-900.
8. Shah, B.G. In Progress in Clinical and Biological Research; Alan R. Liss,
Inc.: New York, 1981; Vol. 77, p. 199.
9. Mraz, F.R.; Eisele, G.R. Pediat. Res. 1977, 69, 591-593.
10. Sasser, L.B.; Jarboe, G.E. Toxicol. Appl. Pharmacol. 1977, 41, 423-431.
11. Kostial, K.; Robar, I.; Blanusa, M.; Landeka, M. Proc. Nutr. Soc. 1977, 38,
251-256.
12. Chan, W.Y.; Bates, J.M., Jr.; Rennert, P.M. J. Nutr 1982, 112, 642-651.
13. Collipp, P.J.: Chen, S.Y.; Martinsky, S. Ann. Nutr. Metab. 1983, 27, 488-
494.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
8. CHAN E T AL. Absorption Studies of Manganese from Milk Diets
89

14. Raghib, M.H.; Chan, W.Y.; Rennert, O.M. Fed. Proc. 1984, 43, 489.
15. Raghib, M.H.; Chan, W.Y.; Rennert, O.M. Fed. Proc. 1985, 44, 1850.
16. Raghib, M.H.; Chan, W.Y.; Rennert, O.M. Nutr. Rep. Int. 1985, 32, 1201-
1210.
17. Raghib, M.H.; Chan, W.Y.; Rennert, O.M. Brit. J. Nutr. 1986, 55, 49-58.
18. Wilson, T.H.; Wiseman, G. J. Physiol. 1954, 123, 116-125.
19. Lowry, O.H.; Rosenbrough, N.J.; Farr, A.L.; Randall, R.J. J. Biol. Chem.
1951, 193, 265-275.
20. Keen, C.L.; Bell, J.G.; Lonnerdal, B. J. Nutr. 1986, 116, 395-402.
21. Cotzias, G.C.; Papavasiliou, P.S. Nature (London) 1964, 201, 828-829.
22. Papavasiliou, P.S.; Miller, S.T.; Cotzias, G.C. Am. J. Physiol. 1966, 211,
211-216.
23. Black, J.R.; Ammerman, C.B.; Henry, P.R.; Miles, R.D. Nutr. Rep. Int.
1984, 29, 807-814.
24. Rehnberg, G.L.; Hein, J.F.; Carter, S.D.; Laskey, J.W. J. Toxicol. Environ.
Health, 1980, 6, 217-226.
25. Rehnberg, G.L.; Hein
Health, 1981, 7, 263-272
26. Gruden, N. Nutr. Rep. Int. 1984, 30, 553-557.
27. Duncan, J.R.; Hurley, L.S. Am. J. Physiol. 1978, 235, E556-559.
28. Garcia-Aranda, J.A; Wapnir, R.A.; Lifshitz, F. J. Nutr. 1983, 113, 2601-
2607.
29. Miller, S.T.; Cotzias, G.C.; Evert, H.A. Am. J. Physiol. 1975, 229, 1080-
1084.
30. Stastny, D.; Vogel, R.S.; Picciano, M.F. Am. J. Clin. Nutr. 1984, 39, 872-
878.
31. Craig, W.J. Nutr. Rep. Int. 1984, 30, 1003-1008.
RECEIVED January 28, 1987

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Chapter 9

Manganese Requirements of Humans

1
Jeanne H. Freeland-Graves, Connie W.Bales ,and Fares Behmardi
Division of Nutrition, University of Texas at Austin, Austin, TX 78712

The estimated safe and adequate daily dietary allowance for manganese (Mn)
in adults and adolescents may be too low. In the past, dietary intakes were
higher because whole grains were an essential component of the diet. But the
larger proportion of meats and refined foods in current diets may lead to low
dietary intakes of Mn. Since numerous studies have reported negative
balances of the mineral in adults and adolescents consuming conventional
foods, a higher dietar
mg/day is based on a

The human requirement for manganese (Mn) is inferred from its essentiality in
animals since gross deficiencies of the mineral have not been observed in free-living
populations. However, there are reports of experimentally-induced deficiencies in
humans and a number of cases of sub-optimal status.

Human Deficiencies

The first report of an experimentally-induced deficiency was by Doisy (1) who fed
two subjects a formula diet that was deficient in vitamin K. One of the subjects
developed a slight reddening of the hair, a scaly, transient dermatitis, depressed
vitamin K-dependent clotting factors, and hypocholesterolemia. The symptoms were
unresponsive to vitamin K but disappeared when a normal diet was resumed. When
Doisey recalculated his purified diet, he realized that he had inadvertentiy omitted the
manganese. The diet had contained a manganese level of only 0.34 mg/day and
apparently had produced a manganese deficiency.
The second report was part of a metabolic balance study conducted in our
laboratory (2). Seven males were fed a semi-purified diet containing 0.11 mg Mn/day
for 39 days. On the 35th day, five of the seven subjects developed a finely scaling,
minimally erythematous rash that primarily covered the upper torso, but also affected
the groin area and lower extremities of some of the subjects. The rash was diagnosed
as Miliaria crystallina, a condition in which sweat cannot be excreted through the
surface of the skin and results in small, clear blisters filled with fluid. This rash was
exacerbated during exercise when sweating occurred. After 2 days, the blisters broke

7
Current address: Center for the Study of Aging and Human Development, Box 3003,
Duke University, Durham, NC 27710

0097-6156/87/0354-0090$06.00/0
© 1987 American Chemical Society

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
9. FREELAND-GRAVES ET AL. Manganese Requirements of Humans 91

and the affected areas of the skin became dry and flaky. The skin cleared rapidly but
manganese supplementation began on the fifth day following the outbreak of the rash.
A possible explanation for the transient dermatitis observed in both studies could
be related to the requirement of manganese for the activity of glycosyltransferases
and/or prolidase, enzymes that are necessary to maintain the integrity of the dermis.
Glycosyltransfereases participate in the synthesis of glycosarninoglycans, compounds
which form the mucopolysaccharides of collagen in the skin (3). Prolidase is an
enzyme that catalyzes the breakdown of collagen in dermal fibroblasts. A genetic
deficiency of prolidase leads to a severe dermititis with chronic cutaneous ulcers (4).
The subjects in our study also developed hypocholesterolemia. The decline in
total plasma cholesterol during manganese depletion in both studies is presumably
related to the need for manganese at several sites in the synthesis of cholesterol (5).
This same study observed elevated levels of serum calcium, phosphorus, and
alkaline phosphatase activity in the subjects. These changes suggest that manganese
was being mobilized from stores in bone. In rats fed a manganese deficient diet for 12
months, increased concentrations of serum calcium and phosphorus have also been
observed. In addition, the
manganese and developed a
deficiency of manganese in humans would eventually lead to osteoporosis warrants
further investigation.

Sub-optimal Status

A number of cases of suboptimal status of manganese have been observed. Hurry

and Gibson (7) found children with phenylketonuria (PKU), galactosemia, and
methylmalonic acidemia had lower concentrations of hair manganese than their
age-matched controls, even though dietary intakes were comparable. Another study
of children with P K U and maple syrup urine diseases reported significantly lower
retentions of manganese compared to normal children (8). Lower retention and
absorption of manganese have also been found in patients with exocrine pancreatic
insufficiency (9). In epilectics, Tanaka (10) first reported significant decreases in
whole blood manganese in approximately one-third of children having convulsive
seizures compared to neurologically normal children. Low levels of blood manganese
have also been found in two studies of adult epileptics (11,12). Papavasiliou et al.
(11) reported that the low blood manganese was independent of the type of
anticonvulsive drugs taken and related to the frequency of seizures. Carl et al. (12)
did not confirm these correlations but did find higher whole blood manganese levels
in patients whose epilepsy was a result of trauma compared to those with no history
of trauma.
In contrast, elevated concentrations of whole blood manganese have been
reported in patients with active rheumatoid arthritis or hydralazine syndrome (13),
diseases that involve defects in collagen metabolism. The increased blood
concentrations of manganese observed in these patients were surprising since they
5 4
had slower body turnover rates of M n compared to controls. These investigators
suggested that these patients had a relative deficiency of manganese due to an altered
body distribution of the mineral. It should be noted that manganese supplementation
has been used with success in treating humans and animals with hydralazine disease
(14,15).

Balance Studies in Children and Adolescents

The first manganese balance study in humans was conducted in 1934 by Everson and
Daniels (16) who studied children ages 3 to 5 years (Table 1). The children were fed

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
92 NUTRITIONAL BIOAVAILABILITY O F MANGANESE

Table 1. Summary of manganese balance studies

Manganese
Subject Design Intake Excretion Balance Reference
Age Sex Days Diet Urinary Fecal
yr mg/d mg/d mg/d mg/d

3-5 M, 7 Mixed 1.84- 0.007- 1.67- +0.17- (16)

F 5.03 0.012 4.04 +0.97

6-10 F 30- Varied protein 2.10- 1.95- +0.15- (17)

56 4.84 - 3.55 +1.29

7-9 M 30 Varied calcium 2.13- 0.01- 1.97- +0.15- (18)

& protei

7-9 F 8 Varied calcium 1.91- 0.010- 1.87- +0.01- (19)

12 & nitrogen 2.06 0.011 2.05 +0.26

0.02 3 Breastfed 0.007 0.000 0.04 +0.03 (20)

0.25- M, 11 Mixed 1.15 a

0.02 0.97 +0.16 (8)
8.5 F

12-14 F 30 Varied protein 3.00 - 3.52 -0.5 (21)

&zinc

19-30 M 6-9 Whole wheat 4.2- 4.1- +0.1- (22)

22.5 - 10.9 +11.6

24-28 M 6 Fish 10.70 - 7.33 +3.37 (23)

;
24-28 M 6-9 Rice & fish 9.81 6.60 +3.21 (24)
Whole wheat 9.61 6.33 +3.28
Sago 0.71 1.76 -1.05

Adult F - White flour 2.45 0.08 2.35 +0.02 (25)

Whole wheat 8.67 0.06 8.36 +0.26

18-21 F 41 Varied protein 3.70 0.20 1.97 +1.54 (26)

20-29 M 35 Vegetarian 7.07 0.21 3.53 +3.34 (27)

23-25 M 347 Self-chosen 3.3 0.04 2.5 +0.80 (28)

347 5.5 0.05 3.0 +2.50
b
19-22 27 Mixed 2.78 0.01 2.45 +0.32 (29)

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
9. F R E E L A N D - G R A V E S E T AL. Manganese Requirements of Humans 93

Table 1. Continued

Manganese
Subject Design Intake Excretion Balance Reference
Age Sex Days Diet Urinary Fecal
yr mg/d mg/d mg/d mg/d

Adult M 21 Varied calcium 2.13- 0.02- 2.27- -0.15- b

(30)
& flouride 2.23 0.02 2.50 -0.28

19.2- M 12 Varied protein 3.00- 2.66- -0.10- b

(31)
25.6 & phosphorus 3.14 - 3.14 +0.45

Adult M 20 Varied tin 3.28 - - -0.13 b

(32)

20-24 M 11 Indian

b
Adult M 11 Indian foods 3.60- 0.01- 3.49- -0.30- (34)
8.37 0.04 6.54 +2.24
b
22-32 M 21 Mixed 13.9 14.2 -0.7 (35)
28 15.0 - 14.1 +1.1
b
Adult M 45 Dephytinized 4.1 - 4.8 -0.7 (36)
bran

Adult M,F 28 Mixed 3.00 - - -0.16 b

(37)
b
Adult M,F 7 Vegetarian 3.6- - -0.5- (38)
Self-selected 5.1 - -1.1

Adult M 7 Self-selected 4.28 - 4.98 -0.70 b

(39)

Adult F 7 Varied iron 3.89 3.45- -0.13- b

(40)
_ 4.02 +0.44
c b d
19-22 M 39 Semi-purified 0.11 0.002 c
0.12 -0.02 ' (2)
b e
5 1.53 0.004 c
0.66 c
+0.84 '
b e
5 2.55 0.004 c
1.51 c
+1.02 '
c
19-20 M 38 Mixed 1.21 0.001 c
1.24 -0.09 >f-g b

c
21 2.06 0.001 c
2.03 -0.02^
b
14 2.65 0.001 c
2.47 c
+0.14 >d
c b
21 2.89 0.004 c
2.86 -0.08-<*
c c
11 3.79 0.002 3.02 +0.85 ' b f

Calculated for a 20 kg child. "Analyzed by flame atomic absorption spectroscopy.

c
Analyzed by graphite furnace atomic absorption spectroscopy. "Includes 0.02 mg
e
integumental loss/day. Includes 0.03 mg integumental loss/ day. Includes 0.01 mg
integumental loss/ day. ^Submitted for publication.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
94 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

three levels of manganese, 1.84, 2.52, and 5.03 mg/day, for a one week period. On
these dietary levels, balances were determined to be 0.17, 0.50, and 0.97 mg Mn,
respectively.
Over 30 years passed before subsequent studies were conducted in children. In
1967, Engel et al. (17) collected data from girls, ages 6 to 10, fed diets that varied in
protein quantity and type. Positive manganese balances from 0.15 to 1.29 mg/day
were observed in diets that ranged from 2.10 to 4.84 Mn mg/day. The influence of
protein on balance could not be ascertained since the dietary manganese was
substantially increased by the substitution of plant protein for animal protein sources.
In their next study of pre-adolescent girls (18), the diets varied in calcium from 260 to
620 mg/day and in proteinfrom25 to 46 g/day. No significant effects of these dietary
manipulations on balance were observed. Consumption of diets that contained from
2.13 to 2.43 mg Mn/day produced positive balances of 0.15 to 0.18 mg Mn/day. In
their final study (19), the diets varied in calcium from 300 to 1300 mg/day and dietary
nitrogen was elevated from supplements of ammonium citrate or synthetic limiting
amino acids. Manganese balance was not affected except from the addition of
ammonium citrate in the absenc
balance was seen but it wa
supplement. Dietary intakes of 1.91 and 2.06 mg Mn/day produced positive balances
that ranged from 0.005 to 0.258 Mn/day.
The manganese balance of ten 6-day old breast-fed infants was measured for a
3-day period by Widdowson in 1969 (20). For a 3.5 kg infant, an intake of 7 |ig/day
produced a negative balance of 31 jig. Alexander et al. (8) studied the manganese
requirements of children, 3 months to 9 years, fed normal and synthetic diets. From
calculation of their data, a manganese intake of 1.15 mg/day in a 20 kg child would
produce a positive retention of 0.16 mg.
The only manganese balance study in adolescents was conducted by Greger et
al. (21) who fed diets containing 3.0 mg Mn/day for 30 days. The diets varied in the
levels of zinc (7.4 and 13.4 mg) and defatted soy protein and meat. These diets
produced negative balances in 13 out of 14 girls. The mean balance was -0.5 mg
Mn/day and was not affected by zinc levels or substitution of soy protein for meat.

Balance Studies in Adults

Basu and Malakar (22) were the first investigators to study manganese balance in
adults. Positive manganese balances, ranging from 0.1 to 11.6 mg/day, were found
when fecal excretion of three men was measured. However the dietary levels of
manganese were high, ranging from 4.2 to 22.5 mg/day, because of the inclusion of
whole wheat in some of the diets.
High intakes of manganese were also reported in two studies of Indian diets. In
the first study (23), a mean balance of 3.37 mg Mn/day was reported for subjects
consuming typical diets with a mean level of 10.7 mg Mn/day. In the second study
(24), dietary levels ranged from 6.42 to 13.81 mg Mn/day. Mean manganese
balances of 3.21 and 3.28 mg/day were found for rice-fish and whole wheat-based
diets that had contained 9.81 and 9.61 mg Mn, respectively. The type of fat that was
used in the preparation of the diets had no effect on manganese balance. Both
manganese intake (0.71 mg/day) and balance (-1.05 mg/day) decreased substantially
when a sago diet was fed. De (23) observed that manganese balance became more
and more positive as dietary levels of the mineral increased.
In 1941, Kent and McCance (25) measured balance in three subjects eating
mixed diets containing 1.72, 2.21, and 6.64 mg Mn/day. In the first 7-day period
measured, mean respective balances of -0.05, +0.16, and -0.39 mg Mn were found.
In two other subjects, substitution of white for brown flour decreased the manganese
intake from 8.67 to 2.45 mg/day and the daily balance from 0.26 to 0.02 mg.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
9. F R E E L A N D - G R A V E S E T AL. Manganese Requirements of Humans 95

The first study of college women was conducted by North et al. (26) who fed
conventional foods that varied in protein content from 37 to 76 g/day. The daily
intake of manganese averaged 3.7 mg/day and produced a mean positive balance of
1.54 mg Mn. Changing the daily protein level had no effect. In 1965, Lang et al. (27)
fed college men a diet containing an even higher intake of manganese, 7.07 mg/day.
This produced a higher positive balance, 3.34 mg. Substitution of vegetable protein
for skim milk powder did not influence manganese retention. The researchers
observed that variations in height and weight were responsible for some of the
variation in retention; however, when these variables were held constant, retention
rates still varied considerably between subjects.
The longest balance study measuring manganese was conducted by Tipton et al.
(28) who collected duplicate diets and all urinary and fecal excreta of two men for 347
days. The self-chosen diets of the two men contained an average of 3.3 and 5.5 mg
Mn/day and produced positive balances of 0.8 and 2.5 mg/day, respectively.
McLeod and Robinson (29) published the first manganese balance study in
adults in which atomic absorption spectrophotometry was the method of analysis. A
positive balance of 0.32 mg/da
diet containing 2.78 mg Mn
cream with tea and coffee, and two subjects, orange juice
A more nutritionally-adequate diet was fed by Spencer et al. (30) who fed eight
adult males diets that contained from 2.13 to 2.23 mg Mn/day. Respective negative
balances of -0.15 to -0.28 mg Mn/day were reported. Varying the level of calcium
from 200 to 1500 mg/day or administering 10 mg flouride had no influence on the
balance of manganese.
In 1980, Greger and Snedeker (31) fed eight adult males diets that contained
varying levels of protein (8.1 and 24.1 g nitrogen) and phosphorus (1,010 and 2,525
mg) for four 12-day periods. Both small negative and positive balances of -0.10 to
+0.45 mg resulted when diets contained between 3.00 to 3.14 mg Mn/day. Varying
the level of protein or phosphorus had no effect. The same laboratory found similar
results in a study (32) investigating the influence of low (0.1 mg) and high (50 mg)
levels of tin on mineral balance. A small mean negative balance of -0.13 mg Mn
occurredfroma mixed diet containing 3.28 mg Mn/day. The level of tin did not affect
manganese balance.
Rao and Rao determined manganese balances in two studies of typical Indian
diets. In the first study (33), diets representative of different geographical locations in
India were fed to six subjects for five 11-day periods. The diets contained from 5.4 to
17.5 mg Mn/day and produced positive balances of 0.1 to 3.1 mg Mn/day. A
legume-based vegetarian diet produced lower absorption rates of manganese
compared to those that included fish or meat. In the second study (34), a basal
vegetarian diet supplemented with regional foods was fed to five men for five 11-day
periods. Dietary intakes of 3.6 to 4.0 mg Mn/day produced negative balances of
-0.30 to 0.39 mg Mn. Increasing the dietary level from 4.31 to 8.37 mg/day led to
postive balances of 0.08 to 2.24 mg Mn. The reported rate of absorption for
manganese was 43%.
The absorption rate reported by Rao and Rao (34) is relatively high considering
the elevated concentrations of fiber and phytate that are usually present in vegetarian
diets. Schwartz et al. (35) determined manganese absorption rates of -2.0 to 7.6 %
for high fiber, high phytate diets. In this study, the fiber and phytate levels of the diet
were increased by the daily inclusion of four slices of whole wheat bread and three
bran muffins. Despite high dietary intakes of 13.9 and 15.0 mg Mn/day, balances of
-0.7 and 1.1 mg Mn were found. In another study in which 8.6 g dephytinized
wheat bran was added to the diet of six men for 45 days, a mean negative balance of
-0.7 mg Mn/day was found on an intake of 4.1 mg/day (36).
Patterson et al. (37) measured the manganese balance of 28 free-living adults

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
96 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

who provided duplicates of their self-selected diet for one week periods during the
four seasons of the year. The subjects were in negative balance (x = -0.16 mg) on a
daily intake of 3.00 mg/day during each season. However, the data were complicated
by an approximate 15% reduction in caloric intake during the collection week.
Apparently, the subjects decreased their normal quantity of food. This abrupt
lowering of caloric intake during collection periods was suggested as a probable
cause of the negative balances observed.
Two other studies measured manganese balances in duplicate 7-day composite
diets. The first study found that diets of 59 adult free-living vegetarians contained
3.6, 4.8, 4.3, and 5.1 mg Mn/day in female and male Asian vegetarians and female
and male American vegetarians, respectively (38). These intakes produced negative
balances of -0.5,-0.4, -1.0, and -1.1 mg Mn/day, respectively. The second study
measured duplicate plate and bulk liquid collections of 15 adult men for 7 days (39).
A negative balance of -0.7 mg Mn/day was seen on a daily intake of 4.28 mg. A
third study also measured manganese balance for a one week period (40). The diet
fed to 14 women contained 3.89 mg Mn/day either with and without a 20 mg iron
supplement. The addition of
positive 0.44 mg/day to a negativ

Balance Studies in Adults that Measured Integumental Losses

The only studies that have reported integumental losses of manganese have been done
in our laboratory. In the first study (2), the integumental losses of men fed a
semi-purified manganese deficient diet represented 13% of total body losses of
manganese. After repletion with inorganic manganese for 10 days, the integumental
losses decreased to 2.5% of total losses. This latter figure is similar to our second
study which found integumental losses to range from 0.4 to 1.2% in men fed a
conventional foods diet of varying manganese levels (Freeland-Graves et al. J. Nutr.
Submitted for publication.) The highest percentage of integumental losses, 1.2%,
was found with the lowest dietary level given. Thus integumental losses can be a
significant route of excretion for manganese if intakes fall to very low levels. This
suggests that the balances reported in studies that did not measure integumental losses
may be more positive than is truely the case since output was underestimated.
In our first study described above (2), a negative balance of -0.02 mg Mn was
found on a dietary level of 0.11 mg/day. This figure is small considering levels
reportedfrompast studies. However, the diet fed to the subjects was semi-purified,
not whole foods. It is believed that retention of the mineral was enhanced by
increased physiological needs caused by a manganese depletion from consumption of
such a low dietary level. Furthermore, the diet did not contain any phytates and
limited amounts of fiber.
In our second study, a diet of conventional foods was fed to five males, ages
19-20 years old, for 105 days. A baseline diet was supplemented with varying levels
of inorganic manganese so that the total intake was 1.21, 2.06, 2.65, 2.89, and 3.79
mg Mn/day. Manganese balances were -0.09, -0.02, 0.14, -0.08, and 0.85 mg/day
for these diets, respectively. Hair and nail losses have not been included in the
integumental measurements in our laboratory or others. But the contribution of these
losses to total obligatory losses has been calculated to be 0.74 |ig/day (2). This small
amount, 0.08% of total losses, would not significantly affect manganese balance.

Absorption and Retention

Studies of manganese requirements in humans are complicated by the fact that

excretion, rather than absorption, is believed to be the major regulator of homeostatic
control. Britton and Cotzias (41) fed diets containing varying levels of manganese to

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
9. F R E E L A N D - G R A V E S E T AL. Manganese Requirements of Humans 97

rats that had been injected with ^Mn and found that the rate of excretion of the labeled
tracer was linearly related to dietary concentrations of the mineral. However, other
studies suggest that absorption may also play a regulating role under certain
circumstances (42).
In balance studies, the re-excretion of absorbed manganese in the bile into the
feces makes it difficult to isolate unabsorbed from endogenous (re-excreted)
manganese. Thus, true absorption is difficult to determine. Furthermore, the
excretion of manganese is highly variable between subjects and is dependent on the
quantity given and the body status (43,44). The variability of manganese absorption
is shown in the study by Sandstrom et al. (44) in which individual rates of
manganese absorption ranged from 1-7 to 14.5% in 14 subjects. When repeated
several times, these rates were highly reproducible within the same individual.
Whether these differences were due to variations in body status or dietary intakes is
unknown.
54
When Thomson et al. (45) used an occulsive balloon to perfuse M n into the
duodenum and proximal jejunum of eight subjects, the rate of absorption averaged
27% over a 1 hour period
absorption, 3%, for orally
weeks. However these authors felt that the measurements may have underestimated
the amount actually absorbed since they could not account for the amount that had
been absorbed and re-excreted.
North et al. (26) reported a retention rate of 41% with a mean intake of 3.7 mg
Mn/day. This high retention rate was attributed to the fact that the young college
women may still have been growing or due to incomplete collections since
integumental losses were not measured. Lang et al. (27) found a similar retention
rate, 47%, in subjects consuming 7.07 mg Mn/day. McLeod and Robinson (29)
found a much lower rate of retention, 12% of intake, but were still puzzled why it
was so high. The retention reported by these researchers, 0.32 mg/day, would
approach the lower limit of total body stores of 12-20 mg (47) within 38 days. The
higher retentions observed in older studies would saturate body stores even sooner.
McLeod and Robinson suggested that failure to measure dermal and menstrual losses
along with overestimation of intake might be contributing factors to the high
retentions reported.
In contrast, Mena et al.(46) reported a 1.6% total body retention at 10 days
54
following ingestion of MnCl . The retention was extrapolated to decline to 0.21 %
2

of the ingested dose by 50 days. In children, the median retention rate for all studies
was 12.6%. This value is slightly higher than the 8% retention reported by Mena in
newborn receiving a 10 ug dose of MnC^ (48).

Factors Affecting the Human Requirement of Mn

A number of factors that may influence human requirements of manganese have been
investigated in the literature. These factors include the iron status and age of the
individual and dietary factors such as iron, calcium, phosphorus, phytates and fiber.
There are numerous studies that suggest a competitive interaction between
manganese and iron (42). Mena et al. (46) reported that anemic subjects absorbed
7.5% of ingested manganese compared to 3.0% for normal subjects. Thomson et al.
(45) also found that manganese absorption was increased in iron-deficient patients.
When iron was added to a manganese-containing duodenal perfusate, the absorption
of manganese was enhanced but the additional manganese was not retained. Kies et
al. (40) found that the addition of 20 mg iron supplment to the diet of adult women
caused negative manganese balance despite a dietary intake of 3.89 mg Mn/day.
Another human study involving an interaction between iron and manganese has

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
98 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

recently been conducted in our laboratory (49). The presence of 40 mg dose of

nonheme iron almost completely blocked the plasma uptake of a similar quantity of
inorganic manganese. No such effect was seen with the same dose of heme iron.
A similar competition between iron and manganese has also been observed in
rats. Gruden (50) found that iron added to the milk fed animals decreased tissue
levels of manganese because of reduced intestinal absorption. However, this
competition for absorption did not occur until the animal was old enough to develop
mechanisms that regulate iron absorption. Similar age effects have been reported in
humans. Mena (48) observed that the retention rates of manganese in premature
infants, newborn, and adults declined from 15.6% to 8% to 1.6%, respectively.
Overall, these studies suggest that both iron status and dietary form, as well as age,
may influence human requirements for manganese.
Antagonist interactions have also been reported to occur between manganese and
high dietary levels of calcium (Ca) and/or phosphorus. The relationship between
manganese and calcium is particularly of concern because of the recent popularity of
calcium supplements among women. Animal studies (51) have observed that a
combination of excessive calciu
manganese. However, Pond
either calcium or phosphorus o bone o tissue concentratio o manganese i swine.
Two human studies have reported increased, but not significant, losses of fecal
manganese in subjects when dietary levels of phosphorus were increased from 800 to
1,500 mg (30) and from 1,010 to 2,525 mg/day (31). In animals, elevated dietary
levels of phosphorus have been reported to increase manganese requirements (51);
however, the dietary level of calcium in this study was also high.
In a metabolic study of adolescent girls, Greger (31) suggested that the negative
balances from diets containing 3.1 mg Ca/day may have been due to the high calcium
level of the diet, 1,060 mg/day. However, Spencer et al. (30) found no effect of
varying levels of calcium (200 to 1500 mg/day) on manganese balance in adult males.
Similarly, Price et al. observed that manganese balance in preadolesent girls was
unaffected by varying calcium from 260 to 620 g on high and low protein intakes
(18) or by varying calcium from 300 to 1300 on high and low nitrogen intakes (19).
The exception was a slight enhancement of balance by the addition of ammonium
citrate on a low calcium intake; but this was negated by increasing dietary calcium.
In a study of five adults, our laboratory (Freeland-Graves et al., Trace Element
Metabolism in Man and Animals. In press.) found that an oral dose of 800 mg of
calcium significantly depressed the plasma uptake of 40 mg inorganic manganese.
Whether or not this antagonistic relationship exists in normal food or diets is unclear.
The impact of a high fiber and high phytate diet on manganese bioavailability
was illustrated in a metabolic balance study by Schwartz et al. (35). A mean negative
manganese balance of -0.07 mg/day occurred despite a dietary manganese level of
13.9 mg/day. Excretion of phytate phosphorus was found to be significantly related
to the excretion of manganese (r = 0.86). However, it is difficult to separate out the
effect of the phytates from the fiber since both were present in high quantities.
The negative influence of fiber on manganese bioavailability has been shown by
manganese tolerance tests in our laboratory (54). Both cellulose and pectin had an
inhibitory influence on the plasma uptake of the mineral. Furthermore, excretion of
manganese may also be affected by dietary fiber because of a report that the addition
of pectin to a human diet increased fecal bile acid excretion by 33% (55). Since bile is
the principle route of excretion for manganese (42), an increase in bile acid excretion
would presumably also increase manganese excretion. Thus, it appears that diets high
in either phytates or fiber or both may substantially increase the dietary requirement
for manganese.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
9. F R E E L A N D - G R A V E S E T AL. Manganese Requirements of Humans 99

Dietary Levels and Sources of Manganese

Direct measurements of manganese in composite diets have been found to be 0.88

and 1.78 mg for summer and winter hospital menus, respectively (56); 2.3 mg in
Japanese subjects (57); 2.7 mg (range of 0.8 to 7.1 mg) in New Zealand women
(58); 3.0 mg in middle class U.S. men and women (37); 3.1 mg in U.S. adolescent
girls (21); 3.2 mg in Canadian university meals (59); 3.3-5.5 mg in U. S. men (28);
4.6 mg in British diets (60); and 2.9 to 17.9 mg in Indian diets (61).
Gibson and Scythes (62) have reported both calculated intakes and direct
analysis of 24 hr food composites in 100 Canadian women. The analyzed values of
manganese were 121% higher than the calculated value of 3.1 mg/day. Although this
mean value is within the range suggested by the Food and Nutrition Board (63), the
intakes ranged from 0.7 to 10.8 mg/day and 37% had levels below the 2.5 mg level.
The high intakes in this study (5 mg/day) were associated with frequent consumption
of tea, a beverage which is high in manganese. Consumption of tea has also been
reported to provide nearly half the manganese in the British diet (60).
Other significant food
leafy green vegetables are fai
eggs, milk, sugar, and refined foods. A possible reason for some of the low
manganese intakes that have been reported may be consumption of diets that are high
in meat or refined foods. Kent and McCance (25) found that the dietary level of
manganese was 8.7 mg/day when two subjects consumed diets that contained
40-50% of their energy from whole wheat flour. The substitution of a more refined
white flour for the whole wheat reduced the manganese intake to 2.5 mg/day. Thus,
the heavy reliance of western diets on meat, milk, sugary and refined foods, as well
as fast foods, may lead to low dietary levels of manganese.

Recommended Allowance in Children and Adolescents

A daily manganese allowance of 1.25 mg/day for children, ages 6-10 years, was
suggested by Engel et al. (17) based on a regression equation that calculated a value
of 1.0 mg Mn needed for equilibrium. An additional allowance of 25% for growth
and unmeasured losses increased the level to 1.25 mg. Schlage (64) reported that the
average intakes were 1.4 mg/day for 3 to 5 year old children and 2.18 mg/day for
10-13 year olds. The current estimated safe and adequate daily dietary allowances for
children are based on these studies. These range from 1.0 to 1.5 mg for ages 1-3,1.5
to 2.0 mg for ages 4-6, and 2.0 to 3.0 mg for ages 7-10 (63). These values appear to
include an adequate margain of safety.
In contrast, the estimated safe and adequate daily allowances for adolesents is
2.5-5.0 mg/day. In the only study of metabolic balance of manganese in adolesents
(31), 13 of the 14 subjects were in negative balance while consuming diets containing
3.0 mg/day. Thus, the lower range of this recommendation for adolescents appears to
be too low. However, the lack of other studies makes it difficult to provide a new
recommendation.

Recommended Allowance in Adults

The current estimated safe and adequate allowance for manganese set by the Food
and Nutrition Board for adults is 2.5-5 mg/day (63). The lower range of this figure
was based on the study by McLeod and Robinson (29) in which equilibrium or
excretion of manganese occured whenever the intake was 2.5 mg/day or higher.
However, numerous studies (31-32, 34-40) have reported negative balances in
subjects consuming mixed diets containing manganese levels ranging from 3.0 to
13.9 mg/day. These negative balances suggest that the lower limit of 2.5 mg for
adults may also be too low.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
100 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

A combination of all the data reported for manganese balances for adults in
Table 1 is plotted against dietary intake in Fig. 1. Excluded were data determined by
methods other than atomic absorption spectrophotometry and that from diets that were
semi-purified or that contained more than 11 mg Mn/day. The regression equation
calculatedfromthese data is y = 0.185x - 0.657, in which y = balance and x = intake
(r = 0.64, p<0.01). The point of theoretical manganese equilibrium is 3.55 mg/day.
However, there are still a number of negative balances chaining above the theoretical
point (34-36, 38, 40). Since the estimated safe and adequate daily dietary intake for
manganese for adults is 2.5-5.0 mg (63), these negative balances suggest that the
lower end of this range is too low. Our suggestions are for a lower limit of 3.5
mg/day for males. However, it should be recognized that intakes of 5 mg/day may be
necessary to consistently acheive positive manganese balance if the diet is high in
phytates, fiber, calcium, or iron.
The upper range of the recommended dietary level is open to speculation since
toxicity has not been reported to occurfromconsumption of normal diets. In animals,
toxicity does not occur until dietary levels reach 500-2000 ug/g (65). There is one
report of an individual developin
minerals after 4-5 years (66)
who drank well water that was contaminated with manganese from buried battteries
(67). But the common cause of toxicity is industrial exposure via inhalation of fumes
or dust which leads to a psychiatric disorder resembling schizophreniza. This is
followed by a crippling neurological condition that is similar to Parkinson's disease
(65). Since the toxicity level of manganese is so high and natural diets have been
reported to contain up to 17.9 mg without any ill effects (60), our suggestion is to
increase the upper limit to 7 mg.
In 1940, Basu and Malakar (22) first recommended a manganese requirement
for adults to range from 3.7 to 5.5 mg/day, based on fecal excretions that were near
positive balances. In 1949, De (23) calculated a lower requirement, 2.74 mg, using
regression analysis of manganese balance. However, no diets were fed near this
level. More recently, Rao and Rao have estimated adult requirements to be 3.72 mg
(34) and 4.15 mg (33) per day based on consumption of Indian foods.
In our first metabolic study (2), mean obligatory losses of manganese were
found to be 295 p,g/day. Using these data, a minimal requirement for males, age
19-22, was calculated to be 0.74 mg Mn/day. This requirement is substantially less
than that found in previous studies. However, this requirement was based on subjects
consuming a semi-purified diet, not conventional foods. Furthermore, the subjects
were consuming a manganese-deficient diet so that the obligatory losses were
measured when the body was trying to conserve body stores of manganese. It seems
reasonable that the addition of dietary factors which reduce bioavailability of the
mineral coupled with a normal dietary intake of manganese would greatly increase the
requirement. In our second study based on a diet of conventional foods, we found
mean obligatory losses for the male subjects to be 392 |ig/day. This higher figure is
believed to be more characteristic of what occurs in everyday diets.
Currently, there are no recommendations for manganese requirements in
pregnant and lactating women. In lactating women, Vuori et al. (68) have calculated
that mothers would lose approximately 0.004 mg/day. This value is based on the
third month of lactation with the loss of 950 ml of milk/day that contains 4.25
ug/liter. This additional 0.004 mg would not increase the manganese requirement.
Information on additional needs of manganese during pregnancy is not available.
Clearly, further investigations of the manganese requirements of humans are needed.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
9. FREELAND-GRAVES E T AL. Manganese Requirements of Humans 101

2.0 -i a

Y-0.185x
1.5 •

1.0 •
a
«»
o
0.5 •
a
CI
0

« 00
8
9 1 .4 3 4 5 6 7 8 9 10 1

If
ej
-0.5- a

2
-1.0- D
a

-1.5- Manganese Intake mg/day

Figure 1. Manganese balance is plotted against dietary intake. The

studies represented are ref. 29, «>); ref. 30, (•); ref. 31, (•); ref. 32,
(A); ref. 33, (A); ref. 34, (•); ref. 36, (•); ref. 37, (•); ref. 38, (•); ref. 39
(*); and the present study (<$>).

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
102 NUTRITIONAL BIOAVAILABILITY O F MANGANESE

Is There a Problem in Meeting the Requirement ?

When 7-day diet composites were collected during the four seasons of the year,
Patterson et al. (37) found a mean intake of 3.0 mg Mn/day and a nutrient density of
1.6 mg Mn/1000 kcal. Based on this figure, a consumption of 1560 kcal would be
adequate to meet the lower limit of the suggested safe and adequate range (63).
However, in Fig. 1 the theoretical point of equilibrium is 3.55 mg and it appears that
approximately 5 mg is needed to consistently maintain positive balance. These would
require a daily consumption of 2250 and 3125 Kcal, respectively. Thus, it seems
plausible that some individuals may be at risk for being in negative manganese
balance.

Conclusion

In the past, there was more concern about toxicity of manganese from industrial
exposure than deficiencies. Also, dietary intakes were higher when whole grains
were an essential componen
foods in current diets may
numerous studies have reported negative manganese balances on conventional diets
and manganese deficiency has been implicated as producing osteoporotic bone
changes, it would seem prudent for individuals, particularly women, to increase their
dietary intake. A suggested range of intake is 3.5-7 mg; however this lower limit may
be insufficient to produce positive balance when special diets are consumed.

Acknowledgment

This work was supported in part by USDA grant #84-CRCR-1-1497.

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RECEIVED December 1, 1986

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Chapter 10

Manganese Metabolism in Epilepsy:

Normal or Abnormal?
1-4 5 1 5
G. F. Carl , Carl L. Keen , B. B. Gallagher , and L. S. Hurley
1
Department of Neurology, Medical College of Georgia, Augusta, GA 30912
2
Department of Medicine, Medical College of Georgia, Augusta, GA 30912
3
Department of Psychiatry, Medical College of Georgia, Augusta, GA 30912
4
Medical Research Service, Veterans Administration Medical Center,
Augusta, GA 30910
5
Department of Nutrition, University of California—Davis, Davis, CA 95616

There is a relationshi
that remains undefined
shown that manganese deficiency increases seizure
susceptibility, clinical work has established that mean
whole blood manganese concentration is significantly
lower in an epileptic population than it is in a control
population. One suggestion has been that the lower
blood manganese concentration in epileptics is due to
seizure activity. Alternatively, others have suggested a
possible genetic origin of the lower manganese levels in
a subgroup of epileptics. It has also been reported
that soft tissue manganese levels are responsive to
adrenal steroids, and it is known that some epileptics
with a temporal lobe focus exhibit abnormal
pituitary/adrenal control suggesting a possible hormonal
cause for the lower blood manganese levels.

In 1961 Hurley and coworkers (1) observed that congenitally ataxic

rats were more susceptible to seizures induced by electroshock than
were controls. This report followed closely a publication of
preliminary findings (2) i n d i c a t i n g that seizures induced i n rats by
hydralazine i n j e c t i o n could be prevented by p r i o r i n j e c t i o n of
manganese chloride. These preliminary findings were l a t e r confirmed
(3) and indeed i t was shown that i t was the manganese deficiency
that increased the seizure s u s c e p t i b i l i t y of the rats independent of
the ataxia (3).

The f i r s t report of abnormal manganese concentrations i n human

e p i l e p t i c s was published i n 1967 and indicated high serum manganese
concentrations i n e p i l e p t i c s compared to non-epileptic controls (4).
The authors were apparently unaware of the previous animal work that
demonstrated a l i n k between manganese deficiency and increased
s u s c e p t i b i l i t y to seizure. It was not u n t i l 1978 that Tanaka (5)
presented data i n d i c a t i n g that whole blood manganese concentrations
were lower i n e p i l e p t i c children than i n control children of similar
age. A year l a t e r Papavasiliou and coworkers (6) i n a study of 52
0097-6156/87/0354-0105S06.00/0
© 1987 American Chemical Society

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
106 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

adult e p i l e p t i c patients found that the patients had a mean whole

blood manganese concentration that was 76% that of 24 controls
(p < 0.002). They examined t h e i r data for correlations between
manganese levels and either serum anticonvulsant concentrations or
seizure frequency. While they found no relationship between blood
manganese and medication, seizure frequency did show a s i g n i f i c a n t
(p < 0.001) relationship to blood manganese concentration by
analysis of variance. These investigators also did a limited study
into the p o s s i b i l i t y that there i s a genetic lesion underlying the
lower blood manganese concentrations observed i n some e p i l e p t i c s .
In t h e i r study the whole blood manganese concentrations i n six non-
e p i l e p t i c r e l a t i v e s of e p i l e p t i c patients were normal r e l a t i v e to
those i n the control population. Unfortunately, these r e l a t i v e s
were not further i d e n t i f i e d , so that i t remains unknown to which
patients they were related or whether or not they were related to
the same or d i f f e r e n t patients. Interestingly, these investigators
also measured manganese
controls. Although the
hair was lower than tha ,
t i c a l l y s i g n i f i c a n t due to the v a r i a b i l i t y i n the h a i r manganese
measurements. In addition, they found no s i g n i f i c a n t correlation
between hair manganese and blood manganese concentrations.

In 1980 Hoffman (7) reported that serum manganese concentra-

tions were the same i n e p i l e p t i c s and non-epileptic controls i n
populations of both adults and children. This observation suggests
that the differences reported by Tanaka (5) and by Papavasiliou, et
a l . (6) i n whole blood were due to differences i n manganese concen-
trations i n the blood c e l l s , probably erythrocytes. Whether t h i s
apparent deficiency i n the erythrocytes of some e p i l e p t i c s trans-
lates into lower manganese concentrations i n other tissues of these
e p i l e p t i c s i s unknown.

To further examine the relationship between blood Mn concentra-

tion and seizure frequency, Papavasiliou and M i l l e r (8) injected
5
mice with **Mn at various times before and after inducing a seizure
by either maximal electroshock or pentylene tetrazole i n j e c t i o n .
They followed the d i s t r i b u t i o n of the radioactive manganese i n the
54
tissues of the mice and found that Mn injected immediately after
the siezure was taken up more readily by the l i v e r (67% increase)
and less readily by brain (53% decrease) and carcass (42% decrease)
blf
than Mn injected either into sham-seized mice or into mice before
induction of a seizure. They also found that chronic electroshock-
induced seizure a c t i v i t y i n mice (2/day, 6 day/wk, 3 wk) resulted i n
a decrease i n brain manganese concentration (16%) and an increase i n
l i v e r manganese concentration (67%) compared to sham-treated con-
t r o l s . These data were interpreted as indicating that the large
energy demand of the seizure causes a s h i f t of manganese, which i s
important i n energy metabolism, from other tissues (such as erythro-
cytes and brain) into the l i v e r . They argue that r e e q u i l i b r a t i o n of
the manganese takes time, and intervening seizures further s h i f t the
manganese toward the l i v e r . Consequently, whole blood manganese
concentration (as well as manganese concentration i n other non-
hepatic tissues) decreases as seizure frequency increases.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
10. C A R L E T AL. Manganese Metabolism in Epilepsy 107

A r e c e n t study from our l a b o r a t o r y u s i n g age, sex, r a c e and

w e i g h t matched c o n t r o l s w i t h b l i n d e d a n a l y s e s c o n f i r m e d p r e v i o u s
r e p o r t s o f l o w c o n c e n t r a t i o n s o f manganese i n whole b l o o d o f e p i l e p -
t i c s (11.8 ppb v s . 8.3 ppb,p < 0.001) ( 9 ) . We a l s o c o n f i r m e d t h e
l a c k o f a c o r r e l a t i o n between a n t i c o n v u l s a n t m e d i c a t i o n and whole
b l o o d manganese l e v e l s , e i t h e r as a f u n c t i o n o f t o t a l m e d i c a t i o n
( F i g u r e 1) o r i n d i v i d u a l a n t i c o n v u l s a n t s (Table I ) , but were u n a b l e
t o c o n f i r m c o r r e l a t i o n between s e i z u r e frequency and b l o o d manganese
c o n c e n t r a t i o n ( F i g u r e 2 ) . However, we d i d r e p o r t t h a t e p i l e p t i c s
w i t h s e i z u r e s o f unknown e t i o l o g y had s i g n i f i c a n t l y lower b l o o d
manganese c o n c e n t r a t i o n s than e p i l e p t i c s whose s e i z u r e s c o u l d
r e a s o n a b l y be a t t r i b u t e d t o i n j u r y o r d i s e a s e . We suggested t h a t a
subgroup o f e p i l e p t i c s may e x h i b i t low b l o o d manganese l e v e l s o f
g e n e t i c o r i g i n . We a l s o examined plasma z i n c and copper concen-
t r a t i o n s i n o u r p a t i e n t s and i n c o n t r o l s and found no d i f f e r e n c e s
( T a b l e I I ) i n d i c a t i n g t h a t t h e lower manganese l e v e l s a r e not a s i g n
of a g e n e r a l i z e d t r a c e
to t h e controversy i n v o l v i n
b l o o d o f e p i l e p t i c s w h i c h h a s been r e v i e w e d i n one o f t h e most
r e c e n t r e p o r t s on t h i s s u b j e c t ( 1 0 ) .

Table I . C o r r e l a t i o n Between Whole B l o o d Manganese

C o n c e n t r a t i o n and Plasma A n t i c o n v u l s a n t
Concentrations i n E p i l e p t i c P a t i e n t s

Correlation
Drug N Slope Intercept Coefficient
Phenytoin 23 +0.149 6.25 0.291
Phenobarbital 16 -0.012 7.40 -0.037
Primidone 13 +0.011 6.25 0.017
Carbamazepine 26 -0.024 8.72 -0.020
Valproate 9 +0.056 7.78 0.182

Manganese c o n c e n t r a t i o n s were measured by atomic a b s o r p t i o n s p e c t r o -

photometry and a n t i c o n v u l s a n t c o n c e n t r a t i o n s by EMIT (Syva, P a l o
A l t o . CA). Linear regression a n a l y s e s i n d i c a t e no s i g n i f i c a n t
c o r r e l a t i o n between manganese c o n c e n t r a t i o n and any a n t i c o n v u l s a n t
concentration.

I t i s i n t e r e s t i n g t o n o t e t h a t g l u c o c o r t i c o i d s have been shown

t o a f f e c t t h e d i s t r i b u t i o n o f manganese i n the body o f t h e mouse
(11,12). S e i z u r e s have a l s o been shown t o a f f e c t manganese d i s t r i -
b u t i o n i n mice ( 8 ) , and, i n t e m p o r a l l o b e e p i l e p t i c s , t h e c o n t r o l o f
t h e g l u c o c o r t i c o i d output from the a d r e n a l g l a n d i s a p p a r e n t l y
abnormal ( 1 3 ) . J u s t how these independent o b s e r v a t i o n s might be
r e l a t e d i s not c l e a r . When mice were t r e a t e d w i t h ACTH, C o r t i s o l o r
p r e d n i s o l o n e , manganese showed a r e d i s t r i b u t i o n from t h e l i v e r t o
t h e c a r c a s s (11,12). However, adrenalectomy d i d not have t h e
o p p o s i t e e f f e c t , u n l e s s e x t r e m e l y h i g h doses o f manganese were g i v e n
(12). But, a s d e s c r i b e d above, s e i z u r e s a p p a r e n t l y caused a r e d i s -
t r i b u t i o n o f manganese i n t h e o p p o s i t e d i r e c t i o n , from the c a r c a s s
t o t h e l i v e r ( 8 ) . T h i s would i n d i c a t e t h a t t h e s e i z u r e s do n o t

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
108 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

16.0-1

15.0-
14.0
13.0
12.0-
11.0
10.0-
9.0
8.0-
7.0-
6.0-
5.0
4.0
3.0-
2.0-
1.0-
0 -I 1 r-
3 4 5
Medication Index

F i g u r e 1. Whole b l o o d manganese l e v e l s i n e p i l e p t i c p a t i e n t s
v s . m e d i c a t i o n i n d e x . Whole b l o o d manganese was measured by
atomic a b s o r p t i o n spectrophotometry. The m e d i c a t i o n index i s a
composite measure o f a n t i c o n v u l s a n t c o n c e n t r a t i o n s i n plasma as
a r e l a t i o n s h i p o f each a n t i c o n v u l s a n t t o i t s t h e r a p e u t i c range.
The m e d i c a t i o n i n d e x i n c r e a s e s as a f u n c t i o n o f t h e r e l a t i v e
c o n c e n t r a t i o n o f each a n t i c o n v u l s a n t and as a sum o f a l l a n t i -
c o n v u l s a n t c o n c e n t r a t i o n s . There was no c o r r e l a t i o n between
whole b l o o d manganese and m e d i c a t i o n i n d e x ( r = -0.236.)

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
10. C A R L E T AL. Manganese Metabolism in Epilepsy 109

16.0-1
15.0-
14.0-
13.0-
12.0-
11.0-
10.0-
9.0-
8.0-
7.0-
6.0-

5.0-
AJ0-
3J0-

2.0-
1.0-
0-
4 5
Seizure Index
F i g u r e 2. Whole b l o o d manganese l e v e l s i n e p i l e p t i c p a t i e n t s
v s . s e i z u r e i n d e x . Whole b l o o d manganese was measured by a t o m i c
a b s o r p t i o n spectrophotometry. S e i z u r e index i s a l i n e a r i z e d
measure o f s e i z u r e frequency. There was no c o r r e l a t i o n between
b l o o d manganese c o n c e n t r a t i o n and s e i z u r e i n d e x ( r = -0.067).

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
110 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

Table I I . Comparison of Plasma Z i n c and Plasma Copper C o n c e n t r a -

t i o n s Between E p i l e p t i c s and Age, Sex, Race and
Height/Weight R a t i o Matched C o n t r o l s

Epileptics Controls
N = 44 N - 44
Copper 1.12±0.28 1.1310.37
(ug/ml)
Zinc 0.5910.15 0.6210.36
(ug/ml)

Z i n c and copper v a l u e s a r e e x p r e s s e d as mean 1 S.D. Z i n c and copper

were measured u s i n g flame atomic a b s o r p t i o n spectrophotometry. A
two t a i l e d t - t e s t was used t o compare the means f o r copper (p = 0.8)
and f o r z i n c (p = 0.6).

cause an i n c r e a s e i n g l u c o c o r t i c o i d s . However, i n p a t i e n t s whose

s e i z u r e s o r i g i n a t e i n the t e m p o r a l l o b e , t h e r e i s an i n c r e a s e d
s e c r e t i o n of b o t h ACTH and C o r t i s o l and the f o r m e r , a t l e a s t , does
not seem to be d r u g i n d u c e d ( 1 3 ) . I n a d d i t i o n , s u r g i c a l r e m o v a l of
the s e i z u r e f o c u s from the t e m p o r a l l o b e a l l o w s ACTH and C o r t i s o l
s e c r e t i o n t o r e t u r n t o normal whether o r not the s e i z u r e s a r e con-
trolled. I t i s unknown a t t h i s time what e f f e c t the s u r g i c a l
r e d u c t i o n of s e i z u r e s might have on the d i s t r i b u t i o n of manganese i n
t h e s e p a t i e n t s o r whether t h e r e i s any r e l a t i o n s h i p between g l u c o c o r -
t i c o i d s and manganese d i s t r i b u t i o n i n humans.

W h i l e a r e l a t i o n s h i p between e p i l e p s y and b l o o d manganese

c o n c e n t r a t i o n has been e s t a b l i s h e d , the n a t u r e of the r e l a t i o n s h i p
i s u n c l e a r . I t i s e v i d e n t t h a t c o n s i d e r a b l e work needs t o be done
t o examine the mechanisms i n v o l v e d i n t h i s r e l a t i o n s h i p . I n our
o p i n i o n , the e f f e c t s and e f f i c a c y of manganese s u p p l e m e n t a t i o n i n
e p i l e p t i c p a t i e n t s w i t h low b l o o d manganese need t o be i n v e s t i g a t e d .
Keen and coworkers (14,15) have shown t h a t i n r a t s whole b l o o d
manganese c o n c e n t r a t i o n i s a r e f l e c t i o n of the r e l a t i v e l e v e l s of
manganese i n s o f t t i s s u e s . I f t h i s i s t r u e i n e p i l e p t i c p a t i e n t s
w i t h low b l o o d manganese, t h e n low c o n c e n t r a t i o n s of manganese can
be e x p e c t e d i n the t i s s u e s of these p a t i e n t s . T h i s p o s s i b i l i t y
needs t o be i n v e s t i g a t e d .

I n t e r e s t i n g l y manganese t o x i c i t y i s m a n i f e s t e d i n i t i a l l y i n the
c e n t r a l nervous system, g e n e r a l l y w i t h the i n d u c t i o n of b e h a v i o r a l
changes o r P a r k i n s o n i a n - l i k e symptoms ( 1 6 ) . Whether t h e r e i s a
r e l a t i o n s h i p between t h i s o b s e r v a t i o n and the d e f i c i e n c y of manga-
nese i n some e p i l e p t i c s i s a m a t t e r f o r s p e c u l a t i o n , but the l i m b i c
system seems t o be i n v o l v e d i n b o t h the b e h a v i o r a l e f f e c t s of
manganese t o x i c i t y , and the m a n g a n e s e - d e f i c i e n c y - r e l a t e d epileptic
s e i z u r e s , a t l e a s t those o f the t e m p o r a l l o b e t h a t r e s u l t i n i n -
c r e a s e d ACTH and C o r t i s o l l e v e l s (13). However, the s t r u c t u r e s of
the t e m p o r a l l o b e a p p a r e n t l y have manganese c o n c e n t r a t i o n s not
s i g n i f i c a n t l y d i f f e r e n t from o t h e r p a r t s of the b r a i n ( 1 7 ) .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
10. C A R L E T AL. Manganese Metabolism in Epilepsy 111

One point that i s abundantly clear about the relationship

between manganese and epilepsy i s that much remains to be learned.

Acknowledgement s

The authors wish to express t h e i r gratitude for support from the

Department of N u t r i t i o n of the University of C a l i f o r n i a at Davis and
the Department of Neurology of the Medical College of Georgia and
the Medical Research Service of the Veterans Administration.

Literature Cited
1. Hurley, L. S.; Woolley, D. E.; Timiras, P. S. Proc. Soc.
Exptl. Biol. Med. 1961, 106, 343-46.
2. Comens, P. In Metal-Binding in Medicine; Seven, M. J.; John-
son, L. A., Eds., Lippincott: Philadelphia, 1960; p 312.
3. Hurley, L. S.; Woolley
Am. J. Physiol. 1963
4. Mindadse, A. A.; Tschikowani, T. I. Dtsch. Gesundheitsw. 1967,
22, 1746-48.
5. Tanaka, Y.; Presented at the American Chemical Society National
Meeting, Chicago, 1978.
6. Papavasiliou, P. S.; Kutt, H.; Miller, S. T.; Rosal, V.; Wang,
Y. Y.; Aronson, R. B. Neurology 1979, 29, 1466-73.
7. Hoffman, H. Klin. Wochenschr. 1980, 58, 157-58.
8. Papavasiliou, P. S.; Miller, S. T. Exptl. Neurol. 1983, 82,
223-36.
9. Carl, G. F.; Keen, C. L . ; Gallagher, B. B.; Clegg, M. S.;
Littleton, W. H.; Flannery, D. B.; Hurley, L.S. Neurology
1986, 36, 1584-87.
10. Taylor, A.; Glose, K. Human Toxicol., 1986, 5, 195-200.
11. Hughes, E. R.; Cotzias, G. C. Am. J. Physiol. 1961, 201,
1061-64.
12. Hughes, E. R.; Miller, S. T.; Cotzias, G. C. Am. J. Physiol.
1966, 211, 207-10.
13. Gallagher, B. B.; Murvin, A.; Flanigin, H. F.; King, D. W.;
Luney, D. Epilepsia 1984, 25, 683-89.
14. Keen, C L . ; Clegg, M. S.; Lonnerdal, B.; Hurley, L. S. N. Eng.
J. Med. 1983, 308, 1230.
15. Clegg, M. S.; Lonnerdal, B.; Hurley, L. S.; Keen, C. L. Anal.
Biochem. 1986, 157, 12-18.
16. Keen, C. L . ; Lonnerdal, B; Hurley, L. S. In Biochemistry of
the Essential Ultratrace Elements; Frieden, E . , Ed.; Plenum:
New York, 1984; pp 89-132.
17. Bonilla, E.; Salazar, E.; Villasmil, J. J.; Villalobos, R.
Neurochem. Res. 1982, 7, 221-27.
RECEIVED May 11, 1987

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Chapter 11

Plasma Uptake of Manganese

Influence of Dietary Factors
1
Connie W.Bales ,Jeanne H. Freeland-Graves, Pao-Hwa Lin, Jan M. Stone,
and Virginia Dougherty

Division of Nutrition, University of Texas at Austin, Austin, TX 78712

The influence of three dietary factors - pectin,

cellulose and phytates - on plasma uptake of
manganese (Mn) was studied in adults administered Mn
tolerance tests Plasm sample collected
hourly intervals
doses of severa
50 mg dose of elemental Mn was necessary to produce
consistent plasma responses and that manganese
chloride (MnCl ) was better absorbed than the sulfate
2

or acetate form. In fasting subjects, plasma Mn was

0.64, 1.29, 1.12, 0.95, and 0.75 ug/L at hours 0, 1,
2, 3, and 4, respectively, following a 40 mg Mn dose
provided as MnCl . Uptake of Mn chloride was marked-
2

ly reduced by 15 g high-methoxyl pectin, and to a

lesser extent by both 15 g alpha cellulose and 300 mg
sodium phytate.

Manganese i s an e s s e n t i a l nutrient f o r humans with a d a i l y estimated

adequate safe and d a i l y dietary intake of 2.5 to 5.0 mg (1). Yet
trace mineral n u t r i t u r e depends not only upon dietary intake, but
also upon a v a i l a b i l i t y f o r absorption. Currently, l i t t l e i s known
regarding the influence of dietary factors on the absorption of
manganese. Thus the intent of these studies was to (a) develop a
test that would r e a d i l y measure Mn b i o a v a i l a b i l i t y i n humans and (b)
u t i l i z e t h i s test to determine the influences of various dietary
factors on Mn b i o a v a i l a b i l i t y .

Absorption of Manganese i n Adults

Understanding of manganese b i o a v a i l a b i l i t y i s limited by a paucity

of information concerning the mechanism of i t s absorption i n
humans. Although the proportion of o r a l manganese which i s

'Current address: Center for the Study of Aging and Human Development, Box 3003,
Duke University, Durham, NC 27710

0097-6156/87/0354-0112$06.00/0
© 1987 American Chemical Society

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
11. B A L E S ET AL. Plasma Uptake of Manganese 113

absorbed from the gut i s reported to be as low as 3 to 4% (2), the

s c a r c i t y of data i n healthy subjects prevents clear d e f i n i t i o n of
the rate of manganese absorption. Using whole body counting of
MnCl , Mena et a l . (3) found a 3.0 ± 0.5% rate of absorption i n
11 subjects; however, manganese reabsorbed into the enterohepatic
c i r c u l a t i o n was not taken into account i n t h i s study. Thus the
rate of manganese absorption may have been underestimated. A much
higher absorption § J j e was reported by Thomson et a l . (4) who
r

examined uptake of MnCl^ i n segments of duodenum and jejunum

using a double-lumen tube. A mean absorption rate of 27 ± 3% was
found for the eight subjects measured.
Manganese appears to be absorbed throughout the small bowel,
although the exact s i t e of maximum uptake i n humans has not been
conclusively determined. Animal studies indicate that absorption
i s more rapid i n the duodenum and jejunum than the ileum (4,5) and
that manganese i s most l i k e l y absorbed i n the divalent form (6).
When Garcia-Aranda et a l
i n adult r a t s , the absorptiv
saturated, suggesting a transport mechanism with a strong a f f i n i t y
but a low capacity for the mineral.
B i l i a r y excretion provides the major route f o r manganese
homeostatic control (7-9); t h i s mechanism i s apparently e s s e n t i a l
when intakes are high (10). However, there i s evidence that
absorption can also play a role i n the manganese homeostasis i n
c a t t l e (11, 12) and rats (10,13).

Plasma uptake of manganese

A series of experiments were i n i t i a t e d to examine the e f f e c t s of

dietary f i b e r s on the plasma uptake of an o r a l dose of manganese i n
humans. Stable isotope techniques are the i d e a l methods for measur-
ing the i n t e s t i n a l absorption of minerals; however t h i s technique i s
impossible for manganese because of i t s monoisotopic nature (14).
Also, e t h i c a l considerations p r o h i b i t the use of radioactive forms
of manganese. Thus, a technique which was both r e l a t i v e l y easy to
conduct and safe to administer repeatedly to the same subject was
developed.
The manganese tolerance test measures the plasma uptake of
pharmacological doses of manganese. This test i s analogous to the
plasma tolerance test f o r zinc, which was described i n 1973 (15) and
has subsequently been used extensively i n this (16) and other
laboratories as a q u a l i t a t i v e indicator of absorption (17,18).
In manganese tolerance t e s t s , blood samples are collected at
fasting and at regular time intervals following administration of an
o r a l manganese load. In t h i s study, r e s u l t s of tolerance tests with
manganese alone were subsequently compared with plasma responses i n
the same subjects when the manganese dose was accompanied by various
dietary components.

Development of the Protocol. Reported concentrations of plasma

manganese vary considerably according to sample preparation and
method of a n a l y s i s . Although plasma l e v e l s as high as 34.30 ug/L
have been reported, concentrations i n the range of 0.5 to 1.2 ug/L
are generally considered most accurate (19). In t h i s study, fasting
l e v e l s of manganese were measured on several occasions i n the same

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
114 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

healthy adult subjects. Mean concentration of plasma manganese was

found to be 0.62 ± 0.18 ug/L. As shown i n Figure 1, inter-subject
v a r i a t i o n appears to be more prevalent than intra-subject variance.
S i m i l a r l y , Cotzias et a l . (20) reported s i g n i f i c a n t variance i n
plasma manganese among subjects but l i t t l e within-subject v a r i a t i o n .
However, s t a t i s t i c a l analysis of our data indicated no s i g n i f i c a n t
differences among or within i n d i v i d u a l s .
The i n i t i a l plasma uptake tests were used to select a r e a d i l y
absorbable s a l t form of manganese since differences i n b i o a v a i l -
a b i l i t y have been reported f o r various s a l t s of other minerals
(18,21). In animal studies, most reports have indicated that a
variety of chemical forms of manganese are equally well absorbed and
retained. Yet r e s u l t s have varied according to the type of s a l t
used and the c r i t e r i a used f o r assessing b i o a v a i l a b i l i t y (22-26).
Henning et a l . (27) found that MnCl was retained i n the bodies of
2

chicks to a greater extent than was either MnO^ or MnSO^. However,

when these two s a l t s wer fed i Souther d Bake (26)
found l i t t l e difference
study, three forms of manganes
chloride, s u l f a t e , and acetate s a l t s . In most subjects, response to
a 50 mg dose of the chloride s a l t was better than the sulfate or
acetate form (Figure 2). Thus MnCl^ was the s a l t used for a l l
subsequent tolerance t e s t s .
The manganese load i n the tolerance tests was administered i n
capsule form along with 200 ml of deionized d i s t i l l e d water.
Subjects were not allowed to eat or drink anything else during the
test period. Samples of plasma were collected after a 12-hour fast
and hourly for 4 hours postdose. Plasma samples were analyzed f o r
manganese content using graphite furnace atomic absorption spectro-
photometry (Friedman, et a l . , J . Nutr. In press.) Consecutive tests
i n the same subjects were separated by a minimum of 14 days, since
preliminary testing indicated no residual e f f e c t of the manganese
dose a f t e r t h i s time i n t e r v a l .
As expected, the plasma response was more consistent and
s i g n i f i c a n t l y higher when the o r a l dose was increased. Although
every e f f o r t was made to keep the manganese load as low as possible,
doses providing less than 40 mg elemental manganese as MnCl did not 0

produce consistent plasma responses i n a l l subjects. Thus the 40 mg

dose was chosen as the baseline load f o r most tests; i t was found to
give a consistent plasma response but less gastric discomfort than
the 50 mg dose.

Results of Baseline Tests. Figure 3 presents the e f f e c t on f a s t i n g

levels of manganese when the standard o r a l dose of 40 mg elemental
manganese (as 144 mg MnCl ) was ingested by 11 subjects who l a t e r
participated i n b i o a v a i l a b i l i t y t e s t i n g . The f i v e female and s i x
male subjects were of normal height f o r weight, with a mean age of
27 ± 4 years. These subjects were i n good health and had not used
supplements of manganese p r i o r to the study.
The heavy l i n e i n Figure 3 represents the mean curve (n=ll) f o r
the change i n plasma manganese and the other lines show the t y p i c a l
plasma responses of i n d i v i d u a l subjects (n-5). The mean increase i n
plasma manganese peaked at hour 1, at approximately 103% above
f a s t i n g . The mean concentrations of plasma manganese were 0.64,
1.29, 1.12, 0.94 and 0.75 ug/L at hours 0,1,2,3, and 4 postdose,

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
11. B A L E S E T AL. Plasma Uptake of Manganese 115

1.5-

•X 0.5-

TTT
(4)

Figure 1. Variation i n fasting concentrations of plasma manganese

within and among normal subjects (n=ll). Number i n parentheses
indicates the number of measurements made on the same i n d i v i d u a l .
Straight l i n e indicates mean value for the group.

4.5 T

• • Mn Chloride
3.5 fVS3 Mn Sulfate
pTZl Mn Acetate
3

? 1.5

Hours

Figure 2. Typical plasma response to 50 mg of manganese provided

in the chloride, sulfate, or acetate form.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
116 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

respectively. Although responses were variable among subjects,

plasma l e v e l s generally peaked at hours one or two postdose and
were close to f a s t i n g levels by hour 4. This rapid clearance of
manganese from blood has also been observed with injected doses
(12,28).

Influence of Dietary Factors on Plasma Uptake of Manganese

Once the test had been developed and standardized i n the subjects,
the influence of dietary f i b e r s on manganese uptake was studied.
Fiber was examined since d i e t s providing large amounts have been
suggested to play a preventative r o l e f o r a v a r i e t y of disease
states, including diabetes, and diseases of the large bowel and
cardiovascular system (29,30). However, f i b e r has also been r e -
ported to have a possible detrimental impact on the b i o a v a i l a b i l i t y
of some minerals including zinc (31-33), copper (31), calcium
(33,34), i r o n (35) and magnesiu
Only a l i m i t e d numbe
tionship of f i b e r and manganese. In 1986, Halpin and Baker (37) fed
casein-dextrose diets supplemented with wheat bran or a corn-soybean
meal mixture for 7 weeks and found that both diets impaired growth
and depressed tissue concentrations of Mn. In a study of human
subjects fed diets containing bran f i b e r and phytate, Schwartz et
a l . (38) found negative or only s l i g h t l y positive manganese
balances, despite manganese intakes (13.9-17.7 mg/day) that were
well above the recommended l e v e l s (1). Possible reasons for the
detrimental influence of f i b e r on mineral b i o a v a i l a b i l i t y may be the
formation of mineral-fiber chelates i n the g a s t r o i n t e s t i n a l t r a c t ,
d i l u t i o n with extra water, and/or decreased t r a n s i t time (39).
Since almost nothing i s known about the e f f e c t of i s o l a t e d
f i b e r s on the absorption of manganese and constituents of dietary
f i b e r vary both i n chemical and physical properties, the e f f e c t s of
two major types of f i b e r — c e l l u l o s e and pectin — were examined
using our protocol. The e f f e c t of phytate was also measured since
i t i s associated with high f i b e r foods and has been reported to
increase requirements for manganese (40)•

Cellulose. Fiber components d i f f e r i n t h e i r a b i l i t y to bind to b i l e

(41), the primary route of excretion of manganese (9). Cellulose,
the most abundant natural f i b e r , has been studied for i t s p o t e n t i a l
e f f e c t s on b i l e and l i p i d metabolism (42). It i s n a t u r a l l y present
i n high f i b e r d i e t s and i s also commonly added to many commercially-
produced food products. Previous studies have shown c e l l u l o s e to
decrease i n t e s t i n a l uptake of a v a r i e t y of minerals, including zinc,
phosphorus, calcium, magnesium, and i r o n (32,43,44). In many cases,
however, the e f f e c t of c e l l u l o s e has not been considered to be
a n t i - n u t r i t i o n a l (32) . It has been generally believed that c e l l u -
lose has a lower binding a f f i n i t y for minerals than other c o n s t i -
tuents of dietary f i b e r (33,45).
In order to determine the independent e f f e c t of c e l l u l o s e on
manganese b i o a v a i l a b i l i t y , manganese tolerance tests were admini-
stered with 15 g of alpha c e l l u l o s e (Sigma Co., Inc., St. Louis, MO)
to s i x human subjects. The c e l l u l o s e was given i n g e l a t i n capsules
i n addition to a 40 mg dose of manganese as MnCl and 200 ml of
deionized water. As seen i n Figure 4, the addition of c e l l u l o s e to

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
BALES ET AL. Plasma Uptake of Manganese

1.2 n

Hours after dot*

F i g u r e 3: Mean plasma uptake of 40 mg manganese as MnCl^ i s

shown as dark l i n e (n=10). The o t h e r f i v e curves illustrate
t y p i c a l manganese uptake i n i n d i v i d u a l s u b j e c t s .

Hours after dOM

F i g u r e 4: E f f e c t o f 15g c e l l u l o s e o r high-methoxy1 p e c t i n on
plasma uptake o f 40 mg manganese (n=6).

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
118 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

a manganese dose caused a reduction i n plasma uptake of manganese.

This tendency was noted throughout the t e s t , but was s i g n i f i c a n t
only at the second hour postdose.
I t i s possible that the effect of p u r i f i e d c e l l u l o s e on man-
ganese d i f f e r s from that of c e l l u l o s e provided from whole foods.
Ranhotra et a l . (46) found that products naturally high i n c e l l u l o s e
such as wheat bran, soy f l o u r , and vegetable f l o u r reduced a v a i l a -
b i l i t y of iron, while p u r i f i e d c e l l u l o s e had no e f f e c t . Further
studies with c e l l u l o s e i n both p u r i f i e d and natural form w i l l be
necessary to determine i f the reduction i n manganese uptake produced
by c e l l u l o s e could lead to adverse n u t r i t i o n a l consequences.

Pectin. Pectic substances are a complex group of i n d i g e s t i b l e

polysaccharides that are c l a s s i f i e d as f i b e r . Although the n u t r i -
t i o n a l and physiological e f f e c t s of pectins have been previously
noted (47,48), the d i v e r s i t y of t h e i r structure and chemistry has
complicated research concernin
a b i l i t y . I t i s known tha
magnesium, calcium, and iron (47). However, the physiological
significance of t h i s phenomenon may be determined by the type and
dose of pectin and the subjects under study. For example, Sandberg
et a l . (49) showed that 15 g of c i t r u s pectin reduced apparent
absorption of i r o n , but not of calcium, phosphorous, magnesium, and
zinc i n ileostomy patients. Likewise, Monnier et a l . (50) demon-
strated that pectin caused a decrease i n iron absorption i n patients
with idiopathic hemochromatosis.
Although l i t t l e i s known about the effects of pectins on the
absorption of manganese, administration of pectin has been shown to
increase the excretion of b i l e acids i n humans (48). In the only
previous study related to the influence of pectin on manganese
metabolism, f u c c i d a n — a polyuronic acid of seaweed o r i g i n — w a s found
to reduce the uptake of manganese i n t i e d - o f f segments of r a t
jejunum by up to 77% (51).
The e f f e c t of a high-methoxyl pectin (15 g) derived from apples
(Spreda/USA Co., Prospect, KY) was investigated i n our laboratory
using the 40 mg manganese tolerance test and the same subjects who
participated i n the tests with c e l l u l o s e . The 15 g doses of
c e l l u l o s e and pectin were chosen since these are amounts obtainable
from normal diets high i n f i b e r . As shown i n Figure 4, the addition
of pectin produced a pronounced depression of the plasma response to
o r a l manganese (MhCl^)• Manganese uptake was s i g n i f i c a n t l y (p<0.05)
lower at hours 2 and 3 postdose than during the baseline t e s t . In
addition, t o t a l area under the response curve f o r pectin (1.40 ug/L)
was s i g n i f i c a n t l y lower than that determined with manganese alone
(3.19 ug/L). Thus the reduction i n plasma appearance of manganese
was more pronounced with pectin than when alpha c e l l u l o s e was
administered to the same subjects. Since pectin may bind b i l e s a l t s
more e f f e c t i v e l y than c e l l u l o s e (42), t h i s could explain the d i f f e r -
e n t i a l effects of pectin and c e l l u l o s e on manganese uptake.

Phytate. Phytic acid i s an organic polyphosphate found widely i n

plants, p a r t i c u l a r l y cereals, nuts and legumes. It has been shown
to complex with various divalent cations i n the g a s t r o i n t e s t i n a l
tract and thus reduce mineral b i o a v a i l a b i l i t y (33,44,52). Davis et
a l . (53) reported that feeding a diet based on i s o l a t e d soybean

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
11. BALES ET AL. Plasma Uptake of Manganese 119

protein, which i s known to be high i n phytate concentration, i n t e r -

fered with u t i l i z a t i o n of manganese and thus increased the require-
ment i n chicks. Furthermore, Davies and Nightingale (40) found a
marked reduction i n whole-body retention of manganese i n young rats
fed a d i e t providing 10 g/kg phytate.
Experiments i n t h i s laboratory examined the e f f e c t s of small
(100 and 300 mg) doses of sodium phytate administered i n capsule
form on plasma uptake of 50 mg of MnCl . As shown i n Figure 5, the
300 mg dose of phytate produced a moderate reduction i n manganese
response, which was manifested c h i e f l y at hours three and four of
the t e s t . The r e l a t i v e l y modest e f f e c t of phytate on manganese
uptake i s somewhat unexpected, since i t has been shown that phytic
acid may be a more potent i n h i b i t o r of trace element absorption than
dietary fibers (35). However, the doses of sodium phytate ad-
ministered to our subjects were much smaller than the t y p i c a l d a i l y
intake of 600 to 800 mg (54). Thus we are now investigating the
e f f e c t s of larger dose

Conclusions

In a series of manganese tolerance t e s t s , three dietary components

— c e l l u l o s e , pectin, and phytate—were found to reduce plasma uptake
of manganese. Although the amount of manganese administered (40-50
mg) i n these tolerance tests was much larger than that t y p i c a l l y
consumed (0.9 to 7.0 mg per day) (55,56), the r e s u l t s were s i m i l a r
to those obtained i n b i o a v a i l a b i l i t y studies with other trace
elements. Thus i t appears that diets high i n f i b e r and phytates
also reduce the b i o a v a i l a b i l i t y of manganese.

-1-2 ! i i 1 i
0 1 2 3 4

Hour* ofter dot*

Figure 5: Effect of 100 and 300 mg of sodium phytate on plasma
uptake of 50 mg of manganese (n=3).

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
120 NUTRITIONAL BIOAVAILABILITY O F MANGANESE

Acknowledgments

This work was supported i n part by USDA grant #84-CRCR-l-1497 and

NIH Biomedical Research Support Grant RR07-091-21.

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RECEIVED December 1, 1986

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Chapter 12

Manganese and Lipid Metabolism as Affected

by Dietary Manganese and Fat
1
Jan M. Johnson and Constance Kies
Department of Human Nutrition and Food Service Management,
University of Nebraska, Lincoln, NE 68583

Two studies were conducted to investigate the

effects of dietary manganese and fat on manganese
and lipid metabolism. In Study I, 80 male, weanling
rats were fed tw level f dietar fat (5% d
25%). Serum, live
body weight chang
greater in rats fed the diet with 25% fat. Within
each level of fat, total liver lipids decreased
and liver cholesterol increased as level of dietary
manganese increased. Serum cholesterol levels were
highest when manganese was fed at 50 and 500 mg/kg
diet. Manganese intake, fecal manganese excretion
and whole blood manganese increased as level of
dietary manganese increased. Dietary treatments
had no effect on liver manganese concentrations.
In Study II, young adult human subjects were fed
two laboratory-controlled diets containing either
30% of total calories from fat (approximately 100
mg cholesterol; 10:10:10 PUFA to MUFA to SFA
ratio) or 40% of total calories from fat (approx-
imately 600 mg cholesterol; 4:14:14 fatty acid
ratio). Two levels of manganese were fed (5 and
45 mg Mn/day) within each level of fat. The higher
level of dietary fat generally increased fecal
excretion of manganese and increased serum lipids.
Dietary supplementation with 40 mg of manganese
increased both fecal excretion and whole blood
concentration of the mineral but had no effect
on serum lipids or fecal fat.

At least i n part because atherosclerosis and coronary heart

disease continue to be the number one cause of death among North
Americans, interest i n interactions between dietary constituents

7
Current address: Department of Home Economics, Illinois State University,
Normal, IL 61761

0097-6156/87/0354-0123$06.00/0
© 1987 American Chemical Society

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
124 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

remains high. Several reports of an association of manganese with

steroid biosynthesis and l i p i d metabolism have been published (_1).
Curran and Clute (_1) demonstrated an i n v i t r o increase i n chol-
esterol synthesis i n rat l i v e r c e l l clusters injected with manganese.
In a similar experiment, Curran (2) injected several t r a n s i t i o n
elements (V, T i , Cr, Mn, Fe, Co, Ni, Cu and Zn) i n t r a p e r i t o n e a l l y
into r a t s . After one hour the rats were s a c r i f i c e d and the l i v e r s
from each group were pooled and incubated with sodium a c e t a t e - l - C ^ .
Manganese and chromium were found to increase incorporation of
acetate into cholesterol by 100 percent, whereas vanadium depressed
cholesterol synthesis by 50 percent.
Manganese acts as a cofactor of mevalonate kinase and farnesyl
pyrophosphate synthetase. Mevalonate kinase and possibly one other
manganese-activated enzyme are necessary for the formation of
mevalonate from acetate (3). Farnesyl pyrophosphate synthetase acts
to add one 5-carbon unit to geranyl pyrophosphate to make farnesyl
pyrophosphate (4) (Figur
L i t t l e i s known of th
t e r o l metabolism. Doisy (6) observed a decrease i n serum choles
t e r o l (from 206 to 80 mg/dl) in a single manganese d e f i c i e n t human
subject. Reports of other human studies conducted to determine the
influence of dietary manganese on cholesterol metabolism were not
f ound.
Recently, Klimis-Tavantzis and coworkers (7,8) reported results
of a series of studies designed to investigate the effects of
dietary manganese deficiency on cholesterol and l i p i d metabolism in
two experimental animal models. Day-old chicks were fed a manganese-
d e f i c i e n t (4.8 ug/g) or a manganese-supplemented (104.8 Ug/g) diet
for 4 weeks a f t e r which an i n j e c t i o n of estrogen was given. Mangan-
ese deficiency did not s i g n i f i c a n t l y a l t e r plasma cholesterol or
l i v e r cholesterol. When older (36-week-old) laying hens were given
similar diets (.7), they demonstrated decreased hepatic manganese
and cholesterol concentrations. These hens also tended to have i n -
creased t o t a l l i v e r l i p i d concentrations.
Weanling, Wistar and RICO (genetically hypercholesterolemic)
rats were placed on manganese-deficient (0.12 ug Mn/g) or manganese-
s u f f i c i e n t (100.12 ug Mn/g) d i e t s . Plasma t o t a l , VLDL- and HDL-
cholesterol l e v e l s , and l i v e r cholesterol and l i p i d concentrations
were not affected by the treatment used. These r e s u l t s suggest that
dietary manganese deficiency does not r e s u l t i n s i g n i f i c a n t a l t e r a -
tions in cholesterol and l i p i d metabolism i n the rat (8).
Manganese has a further r o l e as a l i p o t r o p i c agent. Amdur and
associates (9) found that hepatic l i p i d concentration was increased
-
by manganese deficiency. Plumlee et a l . (10) conducted four experi-
ments to determine the e f f e c t of manganese deficiency i n swine and
found that t o t a l body fat and l i v e r l i p i d concentrations were i n -
creased by manganese deficiency.
It has been c h a r a c t e r i s t i c of n u t r i t i o n studies to use one
nutrient a l t e r a t i o n experimental design to investigate one possible
e f f e c t . However, i n order to elucidate dietary relationships with
pathological conditions, i t may be necessary to use interaction-type
studies. Therefore, the o v e r a l l objective of the research conducted
in our laboratories was to investigate the effect of dietary f a t -
manganese interactions on cholesterol synthesis.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
12. JOHNSON AND KIES Manganese and Lipid Metabolism 125

Experimental Plan - Study I

Two studies were used to investigate the e f f e c t s of dietary manganese

and fat on manganese and l i p i d metabolism. The purpose of the f i r s t
study was to determine the interactions among four levels of dietary
manganese and two levels of dietary f a t on manganese and l i p i d
status of male weanling r a t s .
Male, weanling, Sprague-Dawley inbred rats (Harlan/Sprague/
Dawley, Madison, WI) were used. After three days of adjustment, the
80 rats were randomly assigned to one of eight dietary treatment
groups. Two levels of dietary fat (5 percent and 25 percent of the
diets by weight) and four l e v e l s of dietary manganese (5, 50, 500
and 5000 mg/kg diet) were fed.
The composition of the basal 5 percent and 25 percent f a t diets
i s shown i n Table I. A l l ingredients were purchased from Teklad
(Madison, WI) except for the hydrogenated vegetable o i l and the corn
starch which were purchase
Corn starch was used to
d i e t s . The animals were allowed feed and d i s t i l l e d water ad libitum
for 56 days. Treatment variations are shown i n Table I I .

Table I. Composition of Experimental Rations

Amount/kg
Ingredient 5% Fat 25% Fat

Casein 20 g 20 g
DL-methionine 300 mg 300 mg
Crisco shortening 5 g 25 g
Corn starch 45 g 25 g
Sucrose 20 g 20 g
Cellulose ^ 5 g 5 g
AIN mineral mix 3.5 g 3.5 g
AIN vitamin mix 1 g 1 g
Choline b i t a r t r a t e 200 mg 200 mg
Mineral mix without manganese. Manganese to supply 5, 50, 500 and
5000 mg/kg diet as manganese carbonate (Teklad, Madison, WI) was
added at the expense of sucrose to create rations varied i n man-
ganese content.

Feed intakes and body weights were recorded on a weekly basis.

Feces were collected d a i l y and composited into 7-day l o t s . At the
end of the study, a 12-hour fasting blood sample was collected from
each r a t . The brain and l i v e r of each animal was excised and frozen.
Liver, whole blood and feed manganese was measured using a
Varian Techtron Atomic Absorption Spectrophotometer Model 1275.
Total l i v e r l i p i d was extracted from l y p h i l i z e d tissue and determined
by the method described by Folch et a l . (11). Serum t o t a l chol-
esterol and HDL-cholesterol were also enzymatically assayed (12).
Fecal fat analyses were performed using the Goldfisch method (13).

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
126 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

Table I I . Dietary Treatment Variations

Treatment Fat Manganese1

Number (R/kR) (mg/kg)

1 5 5
2 25 5
3 5 50
4 25 50
5 5 500
6 25 500
7 5 5000
8 25 5000
Manganese carbonate (Teklad, Madison, WI).

Results and Discussion - Study I

Mean serum t o t a l cholestero

consuming the high f a t d i e t s , serum t o t a l cholesterol levels were
highest when 50 mg Mn/kg diet was fed, although the value was not
s i g n i f i c a n t l y higher than values attained on other high f a t d i e t s .
For rats consuming the low f a t d i e t s , mean serum cholesterol levels
were s i g n i f i c a n t l y higher f o r the 50 and 500 mg Mn/kg diet t r e a t -
ments (P<0.05). Perhaps the lower f a t intake allowed the effect of
manganese intake on l i p i d metabolism to become more pronounced.
Figure 3 shows the mean serum HDL-cholesterol l e v e l s of rats
fed low and high f a t dietary treatments as affected by l e v e l of
dietary manganese. HDL-cholesterol l e v e l s were higher f o r rats fed
the high f a t diets than f o r rats fed the low f a t diets at each l e v e l
of dietary manganese except at the 500 mg Mn/kg diet l e v e l . When
the data were grouped according to dietary manganese alone, serum
HDL-cholesterol l e v e l s of rats fed the lowest l e v e l of manganese
were s i g n i f i c a n t l y higher than were those of rats consuming the
highest l e v e l of manganese.
As can be seen i n Figure 4, l i v e r l i p i d concentrations were
higher f o r rats fed high f a t diets (within each l e v e l of dietary
manganese) than f o r rats fed low f a t d i e t s . These differences were
s i g n i f i c a n t f o r each l e v e l of dietary manganese fed (P<0.05).
Within each l e v e l of f a t fed, l i v e r t o t a l l i p i d concentrations de-
creased as l e v e l of dietary manganese increased.
Figure 5 i l l u s t r a t e s the increase i n l i v e r cholesterol concen-
t r a t i o n which occurs with an increase i n dietary manganese. Within
each l e v e l of manganese fed, l i v e r cholesterol concentrations were
higher f o r rats consuming the high f a t d i e t s , although these
differences were only s i g n i f i c a n t at the lowest and highest l e v e l s
of dietary manganese (P<0.05).
Mean l i v e r manganese concentrations are shown i n Table I I I .
Means did not d i f f e r s i g n i f i c a n t l y . Liver manganese concentrations
did not seem to r e f l e c t the l e v e l of dietary manganese consumed.
Whole blood l e v e l s of manganese r e f l e c t e d differences i n l e v e l s
of dietary manganese. As shown i n Figure 6, whole blood manganese
concentrations of rats fed both low f a t and high f a t diets tended to
increase as l e v e l of dietary manganese increased.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
12. JOHNSON AND KIES Manganese and Lipid Metabolism 127

3 a c e t y l CoA
i
mevalonate
i
j *mevalonate kinase
J

3-phospho-5-pyrophopho-mevalonate
t
isopentenyl pyrophosphate
i
geranyl pyrophosphate
i
1 * f a r n e s y l pyrophosphate
j synthase
farnesyl pyrophosphate

squalene

lanosterol

Figure 1. Pathway of cholesterol biosynthesis.

80"

5 50 500 5000

mg M n / k g diet

Figure 2. Serum t o t a l cholesterol l e v e l s (mg/dl) as affected by

dietary manganese and f a t .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
128 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

90H

Serum
HDL
85 4
Cholesterol
(mg/dl)
80

75

70

50 500 5000

mg M n / k g diet

Figure 3. Serum HDL-cholesterol l e v e l s (mg/dl) as affected by

dietary manganese and f a t .

65

Liver
Lipid 60
Concentration
(mg/g w e t
tissue)
25% fat
55

50

5% f a t
45

50 500 5000

mg M n / k g diet

Figure 4. Liver l i p i d concentrations (mg/g wet tissue) as

affected by dietary manganese and f a t .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 JOHNSON AND KIES Manganese and Lipid Metabolism 129

550

•25% fat

500

450
Liver
57. f a t
Cholesterol
(mg/100 g
wet t i s s u e )
400

350

300

t 500 5000
50

mg M n / k g diet

Figure 5. Liver cholesterol concentrations (mg/100 g wet tissue)

as affected by dietary manganese and f a t .

7.0

6.5

Whole
Blood
Mn 6.0
(ug/dl)

5.5

5.0

5 50 500 5000
mg M n / k g diet
Figure 6. Whole blood manganese l e v e l s (ug/dl) as affected by
dietary manganese and f a t .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
130 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

Table I I I . Mean Liver Manganese Concentrations (yg/g wet tissue)

in Rats Fed Varying Levels of Manganese and Fat

Dietary Dietary Fat (%)

Manganese 5 25
mg/kg

5 4.0 + 1.0 5.0 ± 0.9

50 3.7 + 1.7 3.5 ± 1.4
500 4.7 + 2.4 4.0 ± 1.7
5000 4.5 + 2.7 3.8 ± 2.1

As shown i n Figure 7, rats excreted approximately f i v e to six

times more fat when receiving the high fat diets compared to the
low f a t d i e t s . Since the high fat diets contained f i v e times as
much fat as did the low fat diets i t appeared that f e c a l fat excre-
tion of the rats was proportional t th f a t content f th d i e t s
This i s perhaps due to th
u t i l i z e f a t . For the hig
rats fed the lowest and highest l e v e l s of dietary manganese were
s i g n i f i c a n t l y d i f f e r e n t from each other and from excretions of rats
fed the other two levels of dietary manganese (P<0.05). With the
lwo fat r a t i o n , however, there were no s i g n i f i c a n t differences i n
f e c a l fat excretion attributable to l e v e l of dietary manganese.
Body weight changes (g/8 week period) as affected by dietary
manganese and fat are shown i n Figure 8. Rats consuming 50 mg Mn/
kg diet gained the most weight on each l e v e l of dietary f a t , while
those consuming the diets containing the lowest and highest l e v e l s
of manganese gained the l e a s t . No s i g n i f i c a n t main e f f e c t s of
dietary manganese occurred. Thus, s i g n i f i c a n t differences i n body
weight change may be attributed to differences i n dietary fat alone.

Experimental Plan - Study II

The purpose of the second study was to determine the effect of

changes i n kind and amount of dietary f a t , with or without manganese
supplementation, on blood serum cholesterol and t r i g l y c e r i d e levels
and on manganese status of human adults. The project was comprised
of a 5-day pre-period and four, 14-day experimental periods.
During the experimental periods, two constant, laboratory-
controlled diets were fed. The "usual" U.S. diet (U.F.) was
formulated to contain 40 percent of t o t a l calories from f a t , 600 mg
cholesterol, and polyunsaturated to monounsaturated to saturated
f a t t y acids i n a r a t i o of 4:14:14. The modified fat diet (M.F.)
contained 30 percent of t o t a l c a l o r i e s from f a t , approximately 100
mg cholesterol, and a polyunsaturated to monounsaturated to saturated
fatty acid r a t i o of approximately 10:10:10.
The four experimental periods were divided into parts A and B,
with each part composed of two experimental periods. Within each
part the following variations were used: basal diet alone (either
U.F. or M.F.) or the basal diet plus a 40 mg manganese supplement
(as manganese gluconate amino acid chelate) (Table IV).

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
JOHNSON AND KIES Manganese and Lipid Metabolism

0.18

0.16

0.14

0.12
25% f a t
Fecal
Fat 0.10
ig/day)

0.08

0.06

0.04

5% f a t
0.02

50 500 5000

mg M n / k g diet
Figure 7. Mean fecal fat excretion (g/day) as affected by
dietary manganese and f a t .

310

300

290

Body
Weight
Change 280
(g)
25% fat
270

260

250 5% f a t

T.
5 50 500 5000

mg M n / k g diet

Figure 8. Body weight change (g/8 week period) as affected by

dietary manganese and f a t .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
132 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

Table IV. Experimental Plan f o r Study II

No. of
Period* Days Diet Type and Modification

Pre-period 5 Self-selected, self-recorded

Part A
1 14 Controlled, usual U.S. diet
2 14 Controlled, usual U.S. diet
+ 40 mg Mn/day

Part B
1 14 Controlled, f a t modified diet
2 14 Controlled, f a t modified diet
+ 40 mg Mn/day
^"Fourteen subjects; orde
cross-over design.

Subjects were 14 healthy men and women who l i v e d i n the human

metabolic unit of the Department of Human N u t r i t i o n and Food Service
Management at the University of Nebraska-Lincoln. A l l meals were
prepared and consumed i n the metabolic diet kitchen. Subjects made
complete urine and f e c a l c o l l e c t i o n s throughout the study and
donated blood samples biweekly.
The manganese content of the basal diets and of urine, feces
and whole blood was measured using a Varian Techtron Atomic
Absorption Spectrophotometer Model 1275. Urine samples were read
d i r e c t l y using a graphite furnace attachment (Model GTA-95). Fecal
composites were analyzed f o r f a t content (13). Serum t o t a l chol-
esterol and HDL-cholesterol were enzymatically assayed (12,14).
Serum t r i g l y c e r i d e s were assayed spectrophotometrically based on the
method of Fletcher (15).

Results and Discussion - Study II

The mean manganese intakes are shown i n Table V. Mean manganese

intakes d i f f e r e d s i g n i f i c a n t l y among dietary treatments. Whole
wheat bread, a good source of manganese, supplied extra c a l o r i e s
to those subjects who began losing weight during the experimental
portion of the study. Since weight loss was more prevalent during
the M.F. periods than when the U.F. diet was fed, the mean manganese
intake was higher when the M.F. diet was fed. Manganese intakes
during the supplemented periods were at least s i x times greater than
during the non-supplemented periods. Therefore, effects of manganese
intakes on manganese and l i p i d metabolism may be attributed to
supplementation versus non-suppiementation.
Fecal manganese losses (Table V) were s i g n i f i c a n t l y higher
during periods of manganese supplementation than during non-
supplemented periods. S i g n i f i c a n t l y more manganese was excreted
during the U.F. + Mn period than during the M.F. + Mn period suggest-
ing that manganese u t i l i z a t i o n from t h i s supplement was affected by
differences i n f a t content of the two diets (either t o t a l amount or
type of dietary f a t ) .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
12. JOHNSON AND KIES Manganese and Lipid Metabolism 133

T a b l e V. Mean Manganese I n t a k e s (mg/day) and F e c a l Manganese

E x c r e t i o n s (mg/day) o f Humans Fed V a r y i n g L e v e l s o f Manganese
and F a t
1
Diet
Pre- MF MF+Mn UF UF+Mn

b
Manganese i n t a k e 5.68 d
7.18 e
47.20 a
5.85 d
45.61
(mg/day) ±2.70 ±0.85 ±0.80 ±0.88 ±0.54

F e c a l manganese 3.68 e
5.55 e
32.29 b
5.73 e
46.89 a

(mg/day) ±1.55 ±2.43 ±12.14 ±2.84 ±11.69

* D i e t Code: P r e - = Self-selected diet
UF = U s u a l U.S. d i e t
MF = Modified f a t diet
Μη = Manganese supplement (40 mg/day)

As shown i n T a b l e V I
d i f f e r s i g n i f i c a n t l y among t h e f o u r d i e t a r y treatment p e r i o d s . No
e x p l a n a t i o n can be o f f e r e d f o r t h e h i g h u r i n a r y manganese l o s s e s
that occurred during the pre-period.
Mean manganese b a l a n c e s a r e a l s o shown i n T a b l e V I . Manganese
b a l a n c e s were s i g n i f i c a n t l y h i g h e r d u r i n g t h e MF+Mh p e r i o d than
d u r i n g any o t h e r p e r i o d . T h i s agrees w i t h t h e d a t a on f e c a l mangan­
ese e x c r e t i o n and i n d i c a t e s t h a t s u p p l e m e n t a l manganese may be
absorbed and r e t a i n e d by t h e body more e f f i c i e n t l y d u r i n g consump­
t i o n o f a l o w f a t d i e t than d u r i n g consumption o f a h i g h f a t d i e t .

T a b l e V I . Mean U r i n a r y Manganese E x c r e t i o n ( y g / d a y ) ,
Manganese B a l a n c e (mg/day) and Whole B l o o d Manganese ( y g / d l ) i n
Humans Fed V a r y i n g L e v e l s o f Manganese and F a t

Dietl
Pre- MF MF+Mn UF UF+Mn

ab
U r i n a r y manganese 9.86 a
6.99 b
7.80 ab
7.93 ab
7.70
(yg/day) ±4.11 ±3.04 ±3.07 ±3.19 ±2.40

b
Manganese balance 1.70 b
1.63 b
14.91 a
0.11 b
-1.37
(mg/day) ±3.21 ±2.45 ±12.47 ±2.77 ±11.72

b e a
Whole b l o o d manganese 2.43° 2.70 b
2.90 a
2.55 2.91
(yg/dl) ±0.36 ±0.28 ±0.15 ±0.29 ±0.17
D i e t Code: Pre- = Self-selected diet
UF = U s u a l U.S. d i e t
MF = Modified f a t diet
Mn = Manganese supplement (40 mg/day)

Mean whole b l o o d manganese l e v e l s (Table V I ) were s i g n i f i c a n t l y

h i g h e r d u r i n g p e r i o d s o f manganese s u p p l e m e n t a t i o n . T h i s would n o t
have been e x p e c t e d d u r i n g t h e UF+Mn p e r i o d s i n c e manganese b a l a n c e
f o r t h i s p e r i o d was -1.37 mg/day. T h i s would suggest t h a t t h e h i g h
f e c a l manganese e x c r e t i o n d u r i n g t h i s p e r i o d was a c t u a l l y due t o an

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
134 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

i n c r e a s e i n endogenous s e c r e t i o n o f manganese i n t o t h e g a s t r o i n t e s -
t i n a l t r a c t r a t h e r than t o a decreased a b s o r p t i o n o f t h e m i n e r a l .
Serum l i p i d v a l u e s a r e shown i n T a b l e V I I . There were no s i g n i -
f i c a n t e f f e c t s o f e i t h e r d i e t a r y f a t o r manganese on serum c h o l -
e s t e r o l l e v e l s during the experimental periods. However, t o t a l
c h o l e s t e r o l l e v e l s tended t o be lower when t h e MF d i e t was f e d than
when t h e UF d i e t was used (P<0.10).
Serum H D L - c h o l e s t e r o l v a l u e s were s i g n i f i c a n t l y h i g h e r f o r t h e
UF d i e t t r e a t m e n t s than f o r t h e MF d i e t t r e a t m e n t s (P<0.05). There
were no d i f f e r e n c e s , however, i n manganese-supplemented v e r s u s non-
supplemented p e r i o d s w i t h i n t h e same l e v e l of d i e t a r y f a t .
W h i l e mean t r i g l y c e r i d e l e v e l s were h i g h e r f o r t h e UF and UF+Mn
p e r i o d s t h a n f o r t h e lower f a t p e r i o d s , t h e d i f f e r e n c e was o n l y
s i g n i f i c a n t f o r t h e manganese-supplemented (MF+Mn v e r s u s UF+Mn,
P<0.005) p e r i o d . No e f f e c t o f d i e t a r y manganese on serum t r i g l y c e r -
i d e l e v e l s was seen.

T a b l e V I I . Mean
T r i g l y c e r i d e L e v e l s (mg/dl) i n Humans Fed V a r y i n g L e v e l s o f
Manganese and F a t

Dietl
Pre- MF MF+Mn UF UF+Mn

Serum t o t a l 207.4 a
169.l b
177.0 b
192.8 ab
191.l a b

cholesterol ±34.2 ±30.8 ±30.8 ±33.7 ±48.7

(mg/dl)

Serum HDL- 46.7 b

47.6 b
48.0 b
63.6 a
62.6 a

cholesterol ±9.0 ±5.74 ±6.46 ±6.58 ±9.65

(mg/dl)
a
Serum t r i g l y c e r i d e s 55.6 C
80.2 a b
73.3 b
88. l 88.3 a

(mg/dl) ±14.6 ±11.5 ±8.1 ±13.9 ±11.5

D i e t Code: Pre-= Self-selected diet
UF = U s u a l U.S. d i e t
MF = M o d i f i e d f a t d i e t
Mn = Manganese supplement (40 mg/day)

No s i g n i f i c a n t d i f f e r e n c e s were found i n mean f e c a l f a t e x c r e -

t i o n s ( T a b l e V I I I ) . F a i l u r e t o d e t e c t d i f f e r e n c e s i n f e c a l f a t may
i n d i c a t e that the increase i n t o t a l f a t content of the usual versus
the m o d i f i e d f a t d i e t i s compensated f o r by an i n c r e a s e i n a b s o r p -
t i o n o f f a t . I n c r e a s e d a b s o r p t i o n o f f a t , t h e n , c o u l d account f o r
the h i g h e r serum H D L - c h o l e s t e r o l and t r i g l y c e r i d e s l e v e l s caused by
the two h i g h e r f a t t r e a t m e n t s .
In c o n c l u s i o n , serum, l i v e r and b r a i n l i p i d concentrations,
body weight change and f e c a l f a t e x c r e t i o n s were g r e a t e r i n r a t s f e d
a d i e t w i t h 25% f a t t h a n i n r a t s f e d a lower f a t d i e t . W i t h i n each
l e v e l of f a t , t o t a l l i v e r l i p i d s d e c r e a s e d and l i v e r c h o l e s t e r o l
c o n c e n t r a t i o n i n c r e a s e d as l e v e l o f d i e t a r y manganese i n c r e a s e d .
However, i n 14 a d u l t , human s u b j e c t s f e d two l e v e l s o f d i e t a r y f a t ,
d i e t a r y manganese had no e f f e c t on serum l i p i d p a r a m e t e r s o r f e c a l
fat excretion.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
12. JOHNSON AND KIES Manganese and Lipid Metabolism 135

Table V I I I . Mean F e c a l F a t E x c r e t i o n s (g/day) o f Humans Fed

V a r y i n g L e v e l s o f Manganese and F a t
1
Diet
Pre- MF MF+Mn UF UF+Mn

a
F e c a l f a t (g/day) 2.39 a
2.21 a
2.36 a
1.57 a
2.21
±1.40 ±1.10 ±1.33 ±0.68 ±1.09
^iet Code: Pre= - Self-selected diet
UF = U s u a l U.S. d i e t
MF = M o d i f i e d f a t d i e t
Μη = Manganese supplement (40 mg/day)

Acknowledgment s

P u b l i s h e d as Paper Number 8243 J o u r n a l S e r i e s Agricultural

Research D i v i s i o n , U n i v e r s i t
Supported by U.S.D.A.,
and W-143 and U n i v e r s i t y o f Nebraska A g r i c u l t u r a l Research D i v i s i o n
P r o j e c t 91-031.

Literature Cited
1. Curran, G.L.; Clute, O.L. J. Biol. Chem. 1953, 204, 215-219.
2. Curran, G. J. Biol. Chem. 1954, 210, 765-770.
3. Amdur, B.; Rilling, H.; Bloch, K. J . Chem. Soc. 1957, 79,
2646-2647.
4. Benedict, C.; Kett, J.; Porter, J. Arch. Biochem. Biophys.
1965, 110, 611-621.
5. Bloch, K.S. Science 1965, 150, 19-28.
6. Doisy, E.A., Jr. In Trace Element Metabolism in Animals;
Hoekstra, W.G.; Suttie, J.W.; Ganther, H.E.; Mertz, W., Ed.;
University Park Press: Baltimore, 1974; p. 668-670.
7. Klimis-Tavantzis, D.J.; Kris-Etherton, P.M.; Leach, R.M., Jr.
J. Nutr. 1983, 113, 320-327.
8. Klimis-Tavantzis, D.J.; Leach, R.M., Jr.; Kris-Etherton, R.M.
J. Nutr. 1983, 113, 328-336.
9. Amdur, M.O.; Norris, L.C.; Heuser, G.F. J . Biol. Chem. 1946,
164, 783-784.
10. Plumbee, M.P.; Thrasher, D.M.; Beeson, W.M.; Andrews, F.N.;
Parker, H.E. J. Ani. Sci. 1956, 15, 352-367.
11. Folch, J.; Lees, M.; Stanley, G.H. J . Biol. Chem. 1957, 226,
497-507.
12. Allain, C.A.; Poon, L.S.; Chan, C.S.G.; Richmond, W.; Fu, P.C.
Clin. Chem. 1974, 20, 470-475.
13. . In AOAC Official Method of Analysis; Williams, S.,
Ed.; Byrd Press: Richmond, 1984; 14th ed., p. 159.
14. Lopes-Virella, M.F.; Stone, P.; Eliss, S.; Colwell, J.A. Clin.
Chem. 1977, 23, 882-884.
15. Fletcher, M.J. Clin. Chem. Acta. 1968, 22, 393-397.
RECEIVED July 17, 1987

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Chapter 13

Manganese Availability for Humans

Effect of Selected Dietary Factors

Constance Kies, K. D. Aldrich, Jan M. Johnson, C. Creps, C. Kowalski,

and R. H. Wang
Department of Human Nutrition and Food Service Management,
University of Nebraska, Lincoln, NE 68583

The objective of the project was to determine the

effect of several dietary constituents on manganese
bioavailability of young adult humans In a series
of studies, huma
laboratory-controlle
background against which several different dietary
variations could be tested during treatment periods
ranging from seven to 28 days. Manganese contents
of food, feces, urine and whole blood were analyzed
using a carbon rod attachment on an atomic absorption
spectrophotometer. At low manganese intake levels,
manganese bioavailability was apparently enhanced by
ascorbic acid and by meat-containing diets but was
possibly inhibited by iron, ascorbic acid (at high
manganese intake levels) and by some dietary fiber
sources.
Recommended and/or projected changes i n the American diet i n d i r e c t l y
may change intakes of s p e c i f i c nutrients or of a v a i l a b i l i t y of
s p e c i f i c nutrients i n unpredicted or unpredictable ways. Dietary
f i b e r , ascorbic acid and dietary f a t have a l l been shown to affect
the a v a i l a b i l i t y of some nutrient minerals. Hence, i t i s not
unreasonable to assume that a l t e r a t i o n s i n intake of these dietary
constituents might have a direct effect on the b i o a v a i l a b i l i t y of
manganese or might i n d i r e c t l y affect manganese b i o a v a i l a b i l i t y by,
for example, a f f e c t i n g the a v a i l a b i l i t y of other minerals with which
manganese competes for absorption s i t e s . Stress on reduced c a l o r i c
intake to avoid obesity has received a p a r t i c u l a r l y attentive
audience among American women. Ideally, dietary reduction i n c a l o r -
ies i s achieved v i a decreased consumption of empty-calorie foods
which leaves consumption of vitamins and minerals unchanged. In
"real world" s i t u a t i o n s , a decrease i n c a l o r i c consumption almost
invariably leads to a decreased intake of a l l nutrients.
Impact of dietary factors on manganese b i o a v a i l a b i l i t y and on
manganese n u t r i t i o n a l status have not been extensively investigated.
The objective of the current project was to determine e f f e c t s of

0097-6156/87/0354-0136506.00/0
© 1987 American Chemical Society

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
13. KIES ET AL. Manganese Availability for Humans 137

a l t e r a t i o n s i n i r o n , a s c o r b i c a c i d , f i b e r and meat i n t a k e on mangan-

ese u t i l i z a t i o n by young human a d u l t s .

Procedures (General)

I n a s e r i e s o f s t u d i e s r a n g i n g from 14 t o 56 days e a c h , a d u l t human

s u b j e c t s were f e d c o n s t a n t , measured, l a b o r a t o r y c o n t r o l l e d d i e t s .
D i e t s were based on o r d i n a r y foods and i n c l u d e d t h e f o l l o w i n g i t e m s :
m i l k , u n e n r i c h e d 70% f l o u r bread ( p l u s wheat bran i n some s t u d i e s ) ,
r e a d y - t o - e a t o a t o r c o r n based b r e a k f a s t c e r e a l s , green beans,
tomato and orange j u i c e ( o r a p p l e j u i c e i n some s t u d i e s ) , peanut
b u t t e r , ground beef and tuna ( o r soy i s o l a t e p r o d u c t s ) , peaches,
p e a r s , p o t a t o e s and r i c e . Water was a l l o w e d ad l i b i t u m . Jelly,
b u t t e r and s o f t d r i n k s were used t o a d j u s t c a l o r i c i n t a k e f o r each
s u b j e c t t o t h a t needed f o r weight maintenance. Thus, some d i f f e r -
ences i n t h e b a s a l d i e t s o c c u r r e d among s u b j e c t s b u t d i e t s were
maintained constant f o
were superimposed on thes
o f each study as d e s c r i b e greate
food was p r e p a r e d and eaten under s u p e r v i s i o n o f p e r s o n n e l i n t h e
human n u t r i t i o n m e t a b o l i c u n i t s p e c i a l d i e t k i t c h e n , Department o f
Human N u t r i t i o n and Food S e r v i c e Management, U n i v e r s i t y o f Nebraska.
Each s t u d y was d i v i d e d i n t o e x p e r i m e n t a l p e r i o d s o f 7 t o 28 days
each. W i t h i n each s t u d y , a l l s u b j e c t s r e c e i v e d a l l e x p e r i m e n t a l
v a r i a b l e s a c c o r d i n g t o a randomized, c r o s s - o v e r d e s i g n .
S u b j e c t s were a l l s t u d e n t s o r employees o f t h e U n i v e r s i t y o f
Nebraska who m a i n t a i n e d t h e i r u s u a l work, study and s o c i a l a c t i v i -
t i e s except f o r t h e e a t i n g o f t h e e x p e r i m e n t a l d i e t s and n o t h i n g
e l s e , making c o l l e c t i o n s o f e x c r e t a , g i v i n g b l o o d samples, and f i l l -
i n g o u t v a r i o u s q u e s t i o n n a i r e s . A l l were assumed t o be i n good
h e a l t h as e v a l u a t e d from h e a l t h h i s t o r i e s by m e d i c a l p e r s o n n e l o f
the U n i v e r s i t y o f Nebraska H e a l t h C e n t e r . S i g n i n g o f s u b j e c t consent
forms was r e q u i r e d o f a l l p a r t i c i p a n t s p r i o r t o p a r t i c i p a t i o n . This
p r o j e c t was approved f o r human s u b j e c t p a r t i c i p a t i o n by t h e U n i v e r -
s i t y o f Nebraska I n s t i t u t i o n a l Review Board f o r t h e P r o t e c t i o n o f
Human Research S u b j e c t s .
S u b j e c t s made complete c o l l e c t i o n s o f u r i n e and s t o o l s t h r o u g h -
out each s t u d y . Feces f o r each s u b j e c t were d i v i d e d i n t o p e r i o d
l o t s r e p r e s e n t i n g food eaten d u r i n g each p e r i o d by use o f f e c a l dyes
( b r i l l i a n t b l u e and carmine r e d ) and c o l o r e d g l a s s beads, composited,
mixed and sampled f o r l a t e r a n a l y s e s . U r i n e f o r each s u b j e c t f o r
each day was composited on t h e b a s i s o f t i m e , d i l u t e d t o a c o n s t a n t
volume w i t h d i s t i l l e d w a t e r , mixed, sampled and f r o z e n f o r l a t e r
a n a l y s e s . F a s t i n g b l o o d samples were drawn a t t h e b e g i n n i n g o f each
study and a t t h e end o f each e x p e r i m e n t a l p e r i o d .
Food, u r i n e , f e c e s and whole b l o o d were a n a l y z e d f o r manganese
c o n t e n t s u s i n g a V a r i a n Model 1150 o r 1275 Atomic A b s o r p t i o n Spec-
trophotometer w i t h carbon r o d attachment a c c o r d i n g t o manual d i r e c -
t i o n s . A l t h o u g h manganese c o n t e n t s o f u r i n e were measured, r e s u l t s
a r e n o t r e p o r t e d i n t h i s paper because o f t h e m i n u t e , unchangeable
amounts w h i c h were found. Whole b l o o d manganese v a l u e s a l s o w i l l n o t
be r e p o r t e d s i n c e t h i s a n a l y s i s was n o t performed on samples from a l l
s u b j e c t s f o r a l l s t u d i e s . Those r e s u l t s which were o b t a i n e d i n d i -
c a t e d t h a t whole b l o o d manganese l e v e l s were r e s i s t a n t t o change i n
the s h o r t term s t u d i e s w h i c h comprised t h i s p r o j e c t .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
138 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

I n e v a l u a t i o n o f d a t a o b t a i n e d , i t was assumed t h a t an i n c r e a s e
i n f e c a l manganese l o s s a t s i m i l a r l e v e l s o f manganese i n t a k e denoted
a d e c r e a s e i n manganese a b s o r p t i o n and u t i l i z a t i o n . Manganese i n t h e
f e c e s may r e p r e s e n t exogenous, unabsorbed food manganese o r may be
endogenous manganese w h i c h has been s e c r e t e d i n t o t h e g a s t r o i n t e s -
tinal tract. S i n c e t h i s endogenous manganese can be r e c y c l e d , an
i n c r e a s e i n f e c a l manganese l o s s due t o an i n c r e a s e i n endogenous
f e c a l manganese l o s s may even-so be due t o d i e t a r y c o n s t i t u e n t s .
Apparent manganese r e t e n t i o n was c a l c u l a t e d by s u b t r a c t i n g t h e
f e c a l manganese l o s s from t h e d i e t a r y i n t a k e . The p e r c e n t manganese
r e t e n t i o n was c a l c u l a t e d by d i v i d i n g t h e manganese r e t e n t i o n by t h e
manganese i n t a k e and m u l t i p l y i n g t h e r e s u l t i n g f i g u r e by 100. An
i n c r e a s e i n apparent manganese r e t e n t i o n o r p e r c e n t manganese r e t e n -
t i o n was assumed t o i n d i c a t e an i n c r e a s e i n manganese b i o a v a i l -
ability. T h i s would be c o n s i d e r e d good i n s i t u a t i o n s i n v o l v i n g low
manganese i n t a k e s but t h e r e v e r s e would be t r u e i f manganese t o x i -
c i t i e s are the issue.
Data f o r each study
These t e s t s i n c l u d e d a n a l y s i , desig
1
s t u d y w a r r a n t e d , Duncan s M u l t i p l e Range Test and/or o r t h o g o n a l
contrast.

Manganese U t i l i z a t i o n from Omnivore and V e g e t a r i a n Diets

Americans a r e b e i n g encouraged t o i n c r e a s e t h e i r i n t a k e o f p l a n t -
based foods w h i l e d e c r e a s i n g t h e i r i n t a k e s o f a n i m a l o r i g i n
products. S i n c e meat, m i l k , p o u l t r y and f i s h a r e known t o c o n t a i n
o n l y s m a l l amounts o f manganese, t h i s recommendation would be
e x p e c t e d t o have l i t t l e e f f e c t on t h e manganese i n t a k e l e v e l s o f
humans. P l a n t p r o d u c t s such as soy w h i c h a r e o f t e n used i n p l a c e o f
meat i n v e g e t a r i a n d i e t s may c o n t a i n h i g h e r amounts o f manganese t h a n
does meat but c o n c u r r e n t l y t h e s e foods c o n t a i n g r e a t e r amounts o f
p h y t a t e s w h i c h may i n h i b i t manganese u t i l i z a t i o n .
The e f f e c t o f s u b s t i t u t i n g 100 g / s u b j e c t / d a y o f soy i s o l a t e -
based p r o d u c t s f o r t h e 50 g o f ground beef and 50 g o f t u n a / s u b j e c t /
day i n t h e u s u a l b a s a l d i e t on manganese u t i l i z a t i o n was i n v e s t i -
gated i n one 56-day study a t t h e U n i v e r s i t y o f Nebraska. The p r o j e c t
i n v o l v e d two 28-day p e r i o d s a r r a n g e d a c c o r d i n g t o a randomized c r o s s -
o v e r d e s i g n f o r each s u b j e c t . The s u b j e c t s were 10 a d u l t women who
l i v e d and a t e a l l meals i n t h e d e p a r t m e n t a l m e t a b o l i c u n i t . D u r i n g
one p e r i o d t h e s u b j e c t s r e c e i v e d t h e meat d i e t w h i l e , d u r i n g t h e
o t h e r p e r i o d , t h e m e a t - f r e e ( s o y - c o n t a i n i n g ) d i e t was g i v e n .
As shown i n T a b l e I , s u b s t i t u t i o n o f 50 g o f ground beef and
50 g o f t u n a f i s h / s u b j e c t / d a y w i t h 100 g / s u b j e c t / d a y o f soy i s o l a t e
based p r o d u c t s r e s u l t e d i n a s i g n i f i c a n t decrease i n apparent man-
ganese r e t e n t i o n o f t h e s u b j e c t s (p<0.05). Because o f t h e s l i g h t l y
h i g h e r manganese content o f t h e beef and tuna f i s h t h a n o f t h e soy
i s o l a t e replacement p r o d u c t s , t h e m e a t - c o n t a i n i n g d i e t p r o v i d e d
s l i g h t l y more manganese than d i d t h e m e a t - f r e e d i e t . Even s o , f e c a l
manganese l o s s e s were g r e a t e r than when t h e m e a t - f r e e d i e t was f e d
t h a n when t h e m e a t - c o n t a i n i n g d i e t s were used. T h i s s u g g e s t s t h a t
the manganese i n meat i s v e r y a v a i l a b l e and, t h a t meat enhances t h e
b i o a v a i l a b i l i t y o f manganese from t h e whole d i e t .
Because o f t h e change i n l e v e l o f d i e t a r y p r o t e i n , i t i s n o t
p o s s i b l e t o p o s i t i v e l y determine whether q u a l i t y o f p r o t e i n o r l e v e l

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
13. KIES ET AL. Manganese Availability for Humans 139

of p r o t e i n was t h e c a u s a t i v e f a c t o r i n change i n manganese r e t e n t i o n .

However, i n f l u e n c e o f l e v e l o f d i e t a r y p r o t e i n on manganese b i o -
a v a i l a b i l i t y has been i n v e s t i g a t e d i n s e v e r a l o t h e r s t u d i e s w i t h no
s i g n i f i c a n t e f f e c t b e i n g demonstrated ( 1 - 3 ) . N u m e r i c a l l y , however,
manganese b a l a n c e s o f human s u b j e c t s i n t h e Greger and Snedeker
s t u d y (1) were more p o s i t i v e when r e c e i v i n g t h e h i g h p r o t e i n d i e t
than when r e c e i v i n g t h e low p r o t e i n d i e t . V a r i a t i o n i n p r o t e i n
i n t a k e between t h e h i g h and low p r o t e i n d i e t s was c o n s i d e r a b l y
g r e a t e r i n t h a t study than was t r u e i n t h e c u r r e n t s t u d y . I n two
o t h e r s t u d i e s conducted a t t h e U n i v e r s i t y o f N e b r a s k a , s o u r c e o f
d i e t a r y p r o t e i n was demonstrated t o i n f l u e n c e manganese n u t r i t i o n a l
s t a t u s i n human s u b j e c t s ( 4 - 6 ) . Thus, i t i s p r o v a b l e t h a t p r o t e i n
q u a l i t y r a t h e r t h a n p r o t e i n q u a n t i t y was t h e f a c t o r i n f l u e n c i n g man-
ganese u t i l i z a t i o n . A l t h o u g h n o t measured, soy i s o l a t e p r o d u c t s
a l s o may c o n t a i n p h y t a t e s w h i c h may have had an a d v e r s e e f f e c t on
manganese u t i l i z a t i o n ( 7 ) .

T a b l e I . Manganese (Mn
Meat-Free D i e t s

V a l u e when receiving:1»2
Parameter + meat d i e t - meat d i e t
Number o f s u b j e c t s 10 10
a
Mn i n t a k e , mg/day 4.94 a
4.53
a
Mn f e c a l l o s s , mg/day a
4.66 ±0.22 4.71 ±0.35
Apparent Mn r e t e n t i o n , mg/day a
+0.28 ±0.90 -0.18b±0.07
^Values w i t h d i f f e r e n t l e t t e r s u p e r s c r i p t s a r e s i g n i f i c a n t l y
d i f f e r e n t from one another (p<0.05).
2
Based on r e c a l c u l a t e d d a t a s u p p l i e d by A l d r i c h ( 5 ) .
Wheat Bran as a Source o f Manganese f o r Humans

Wheat b r a n and whole wheat c e r e a l s q u a n t i t a t i v e l y c o n t a i n h i g h

amounts o f manganese and a r e o f t e n l i s t e d as p a r t i c u l a r l y v a l u a b l e
s o u r c e s o f manganese. However, z i n c a l s o i s c o n t a i n e d i n a p p r e c i a b l e
amounts i n wheat b r a n and whole wheat p r o d u c t s b u t i s p o o r l y absorbed
by t h e human from t h e s e s o u r c e s . T h i s has been a t t r i b u t e d t o e i t h e r
the p h y t a t e o r t h e f i b e r c o n t e n t s o f t h e s e p r o d u c t s o r a c o m b i n a t i o n
of t h e s e two d i e t a r y f a c t o r s . These same f a c t o r s may a l s o a f f e c t
the a b s o r p t i o n o f manganese.
I n a s e r i e s o f f o u r s t u d i e s o f 24 days each, t h e e f f e c t s o f
f e e d i n g two c e r t i f i e d wheat brans on manganese u t i l i z a t i o n o f human
a d u l t s was i n v e s t i g a t e d . The two wheat b r a n s were o b t a i n e d from The
American A s s o c i a t i o n o f C e r e a l Chemists. T h i s o r g a n i z a t i o n has
o b t a i n e d and s t o c k p i l e d t h e s e brans f o r i n v e s t i g a t i o n a l purposes so
as t o reduce v a r i a t i o n s i n f i n d i n g s among l a b o r a t o r y i n v e s t i g a t i o n s
of wheat b r a n as a s o u r c e o f f i b e r . Each 24-day s t u d y was d i v i d e d
i n t o t h r e e e x p e r i m e n t a l p e r i o d s o f seven days each d u r i n g w h i c h t h e
b a s a l d i e t a l o n e , t h e b a s a l d i e t p l u s h a r d r e d s p r i n g wheat b r a n
(21 g / s u b j e c t / d a y ) o r t h e b a s a l d i e t p l u s s o f t w h i t e s p r i n g wheat
b r a n (21 g / s u b j e c t / d a y ) was f e d . P e r i o d s were randomly a r r a n g e d .
Brans were i n c o r p o r a t e d i n t o an u n e n r i c h e d , 70% e x t r a c t e d wheat f l o u r
b r e a d . A t o t a l o f 38 s u b j e c t s p a r t i c i p a t e d i n t h e s e s t u d i e s .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
140 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

As shown i n T a b l e I I , a l t h o u g h wheat brans i n t h i s s t u d y con-

t a i n e d c o n s i d e r a b l e amounts of manganese, a p p a r e n t l y l i t t l e o r none
of t h i s was a v a i l a b l e t o the human s u b j e c t s . W i t h t h e f e e d i n g o f
h a r d r e d o r s o f t w h i t e wheat b r a n supplemented b r e a d , f e c a l l o s s e s
of manganese were s i g n i f i c a n t l y i n c r e a s e d (p<0.05). Apparent mangan-
ese r e t e n t i o n was n o t s i g n i f i c a n t l y d i f f e r e n t when t h e r e d b r a n bread
was f e d t h a n when t h e no b r a n b r e a d was used; however, s i g n i f i c a n t l y
l e s s manganese was r e t a i n e d by the s u b j e c t s when t h e w h i t e b r a n
bread was f e d than when e i t h e r o f t h e o t h e r t e s t breads were used.
T h i s s u g g e s t s t h e p o s s i b i l i t y t h a t n o t o n l y was t h e manganese from
the w h i t e b r a n u n a v a i l a b l e but t h a t manganese a v a i l a b i l i t y from t h e
r e s t of t h e d i e t was a d v e r s e l y a f f e c t e d .
E x p e r i m e n t a l p e r i o d s i n t h i s study were o n l y seven days i n
l e n g t h ; hence, w i t h t i m e i t i s p o s s i b l e t h a t some a d a p t a t i o n might
have o c c u r r e d . I n an e a r l i e r p r o j e c t , Johnson e t a l . (8) f e d s i m i l a r
d i e t s w i t h t h e same h a r d r e d and s o f t w h i t e wheat b r a n supplements t o
a d u l t human s u b j e c t s f o
determined f o r each o f t h
R e c e n t l y , Schwartz e t a l . (9) r e p o r t e d t h a t over a 48-day f e e d i n g
p e r i o d apparent manganese a b s o r p t i o n s i g n i f i c a n t l y improved w i t h
t i m e . However, from the d a t a p r e s e n t e d f o r weeks 2-4 as compared t o
weeks 5-7, f e c a l l o s s e s o f manganese were v i r t u a l l y unchanged but t h e
apparent i n c r e a s e i n a b s o r p t i o n was a f u n c t i o n o f t h e i n c r e a s e i n
manganese i n t a k e (mean 1.1 mg/subject/day) i n weeks 5-7 as compared
t o weeks 2-4.

T a b l e I I . E f f e c t o f Wheat Bran on Manganese (Mn) U t i l i z a t i o n

i n Humans

Basal Diet
1
Parameter Alone +Red B r a n +White B r a n l
Number o f s u b j e c t s 59 59 59
c a b
Mn i n t a k e , mg/day 5.34 8.46 7.90
c a b
F e c a l Mn, mg/day 4.17 ±0.32 7.12 ±0.69 6.46 ±0.05
a a
Apparent Mn r e t e n t i o n , mg/day 1.17 ±0.18 1.34 ±0.21 0.44^10.27
a b C
Apparent Mn r e t e n t i o n , % 21.9 15.8 6.4
*21 g / s u b j e c t / d a y , AACC c e r t i f i e d r e d and w h i t e wheat b r a n s .
2
Values w i t h d i f f e r e n t l e t t e r s u p e r s c r i p t s are s i g n i f i c a n t l y
d i f f e r e n t from one another (p<0.05).
Based on r e c a l c u l a t e d d a t a s u p p l i e d by A l d r i c h (5) and Johnson ( 2 6 ) .

E f f e c t o f H e m i c e l l u l o s e ( P s y l l i u m F i b e r ) on Manganese U t i l i z a t i o n

Wheat b r a n c o n t a i n s s e v e r a l d i f f e r e n t forms o f d i e t a r y f i b e r
i n c l u d i n g h e m i c e l l u l o s e . Some but n o t a l l p u r i f i e d d i e t a r y f i b e r
s o u r c e s have been found t o have an adverse e f f e c t on manganese b i o -
a v a i l a b i l i t y (10).
I n a s e r i e s o f 28-day s t u d i e s , t h e e f f e c t s o f h e m i c e l l u l o s e on
manganese u t i l i z a t i o n were i n v e s t i g a t e d . As i n t h e p r e v i o u s s t u d i e s
on wheat b r a n , t h e h e m i c e l l u l o s e (20 g / s u b j e c t / d a y ) was i n c o r p o r a t e d
i n t o a bread p r o d u c t . D u r i n g t h e two e x p e r i m e n t a l p e r i o d s , s u b j e c t s
r e c e i v e d e i t h e r t h e h e m i c e l l u l o s e e n r i c h e d b r e a d o r t h e bread w i t h o u t
h e m i c e l l u l o s e . P u r i f i e d p s y l l i u m f i b e r was used as a s o u r c e o f mixed

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
13. KIES ET AL. Manganese Availability for Humans 141

h e m i c e l l u l o s e . Because o f i t s l o n g h i s t o r y o f u s e as a b u l k l a x a t i v e
and because o f i t s f r e q u e n t use as a f i b e r source i n p u r i f i e d o r
s e m i - p u r i f i e d d i e t s i n r e s e a r c h s t u d i e s i n v o l v i n g human s u b j e c t s ,
the use o f p s y l l i u m f i b e r as a source o f h e m i c e l l u l o s e i s p a r t i c u -
l a r l y i n t e r e s t i n g t o r e s e a r c h s c i e n t i s t s i n human n u t r i t i o n . A t o t a l
o f 60 s u b j e c t s p a r t i c i p a t e d i n t h e s t u d i e s .
As shown on T a b l e I I I , t h e s u b j e c t s f e d 20 g o f h e m i c e l l u l o s e
from p u r i f i e d p s y l l i u m f i b e r e x c r e t e d s i g n i f i c a n t l y more manganese
i n t h e f e c e s than when they were f e d bread w i t h o u t t h e h e m i c e l l u l o s e
supplement. U n l i k e wheat b r a n , p u r i f i e d p s y l l i u m f i b e r ( s o l d
c o m m e r c i a l l y as a b u l k l a x a t i v e ) c o n t a i n s no manganese o r p h y t a t e s ;
hence, any change i n f e c a l manganese e x c r e t i o n when p s y l l i u m f i b e r i s
added t o human d i e t s c a n p r o b a b l y be c r e d i t e d t o t h e m i x t u r e o f
hemicellulose comprising t h i s product.

T a b l e I I I . Manganese U t i l i z a t i o n as A f f e c t e d by P s y l l i u m

Basal D i e t >
Parameter Alone + Hemicellulose
Number o f s u b j e c t s 30 30
Mn i n t a k e , mg/day 5.34 a
5.43a
a
F e c a l Mn, mg/day 3.01 ±0.56 3.98 ±0.62
b

b
Apparent Mn r e t e n t i o n , mg/day 2.33 ±0.41 1.45 ±0.53
a

b
Apparent Mn r e t e n t i o n , % 43.63 a
26.70
20 g/day as p s y l l i u m
Values w i t h d i f f e r e n t l e t t e r s u p e r s c r i p t s a r e s i g n i f i c a n t l y
d i f f e r e n t from one another (p<0.05).

Manganese U t i l i z a t i o n from Spinach

Spinach i s a l s o an e x c e l l e n t source o f manganese. However, s p i n a c h

c o n t a i n s h i g h amounts o f s o l u b l e f i b e r and o x a l i c a c i d . Both o f
these f a c t o r s have been found t o i n h i b i t t h e u t i l i z a t i o n o f i r o n .
U s i n g two 5-day p e r i o d s i n a c r o s s - o v e r d e s i g n , manganese u t i l i z a -
t i o n from s p i n a c h was determined. S u b j e c t s (12) consumed t h e i r
n o r m a l , s e l f - s e l e c t e d , s e l f - r e c o r d e d d i e t s . D u r i n g one o f t h e two
randomly arranged p e r i o d s , s u b j e c t s were asked t o e a t one 8 o z . can
of s p i n a c h .
The s p i n a c h p r o v i d e d an a d d i t i o n a l 1.10 mg manganese/subject/
day. However, f e c a l l o s s e s o f manganese were i n c r e a s e d by an almost
i d e n t i c a l amount and no s i g n i f i c a n t e f f e c t on t h e apparent n e g a t i v e
manganese b a l a n c e s e x h i b i t e d by these s u b j e c t s was found (Table I V ) .
Thus, a l t h o u g h s p i n a c h c o n t a i n s h i g h amounts o f manganese, i t s
c l a s s i f i c a t i o n as a good source of manganese must be q u e s t i o n e d s i n c e
t h i s i s e v i d e n t l y n o t manganese which can be absorbed.

Tea as a Source o f Manganese

Tea i s a p l a n t o r i g i n food c o n t a i n i n g s u r p r i s i n g l y h i g h amounts o f

manganese. However, t e a a l s o c o n t a i n s h i g h amounts o f t h e p o l y -
p h e n o l i c substance t a n n i n which h a s been found t o have a p r o f o u n d l y
adverse e f f e c t on the u t i l i z a t i o n o f such d i v e r s e n u t r i e n t s as p r o -

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
142 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

T a b l e I V . Manganese U t i l i z a t i o n as A f f e c t e d by Spinach Consumption

Mn Mn e x c r e t i o n Mn
Diet intake Urine Feces Balance
mg/day mg/day mg/day mg/day
a
Self-selected 0.89 b
0.031 a
1.21 b
-0.35
Self-selected +
a
spinach 1.76 a
0.029 a
1.96 a
-0.23

V a l u e s w i t h d i f f e r e n t l e t t e r s u p e r s c r i p t s i n each column a r e
s i g n i f i c a n t l y d i f f e r e n t from one another (p<0.05).

t e i n , t h i a m i n and i r o n . U s i n g d a t a from l a b o r a t o r y c o n t r o l l e d
s t u d i e s employing use o f c o n s t a n t d i e t s , responses o f 10 t e a d r i n k i n g
s u b j e c t s (4 g o f d r y i n s t a n t t e a p e r day) were compared t o 10 non-tea
or n o n - c o f f e e d r i n k i n g , age, sex matched c o n t r o l s from t h e same
studies.
Tea d r i n k i n g r e s u l t e d i n an i n c r e a s e i n f e c a l manganese e x c r e -
t i o n a p p r o x i m a t e l y e q u a l t o t h a t added t o t h e d i e t s i n t h e form o f
t e a . Hence, no s i g n i f i c a n t improvement i n manganese r e t e n t i o n
o c c u r r e d as t h e r e s u l t o f t e a d r i n k i n g . T h i s suggests t h a t e i t h e r
t h e p o l y p h e n o l s o r o t h e r c o n s t i t u e n t s o f t e a rendered t h e manganese
i n t e a e s s e n t i a l l y unusable t o t h e human (Table V ) .

T a b l e V. Manganese U t i l i z a t i o n as A f f e c t e d by Tea Consumption

Mn Mn e x c r e t i o n Mn
Diet intake Urine Feces Balance
mg/day mg/day mg/day mg/day
a
Basal 2.15 b
0.014 a
2.20 b
-0.06
o.oua
a
Basal + t e a 3.41 a
3.66 a
-0.26
10 age, s e x , weight matched subjects

V a l u e s w i t h d i f f e r e n t l e t t e r s u p e r s c r i p t s i n each column a r e
s i g n i f i c a n t l y d i f f e r e n t from one another (p<0.05).

A s c o r b i c A c i d Supplement Use and Manganese U t i l i z a t i o n

Manganese e x i s t s i n s e v e r a l d i f f e r e n t v a l e n c e s t a t e s but i s thought

to be absorbed i n t h e reduced s t a t e (+2) as i s i r o n . Hence, a d d i t i o n
of a s c o r b i c a c i d might be expected t o enhance t h e apparent a b s o r p t i o n
of manganese.
I n a s e r i e s o f f i v e s t u d i e s o f 14 days each, t h e e f f e c t s o f
a s c o r b i c a c i d on manganese u t i l i z a t i o n were i n v e s t i g a t e d . The u s u a l
b a s a l d i e t was m o d i f i e d t o lower t h e a s c o r b i c a c i d c o n t e n t by sub-
s t i t u t i n g a p p l e j u i c e f o r t h e u s u a l l y - f e d orange and tomato j u i c e s .

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
13. KIES ET AL. Manganese Availability for Humans 143

D u r i n g t h e two randomly arranged e x p e r i m e n t a l p e r i o d s o f seven days

each, t h e s u b j e c t s r e c e i v e d t h e e x p e r i m e n t a l d i e t s a l o n e o r w i t h a
200 mg/subject/day amino a c i d supplement. A t o t a l o f 57 s u b j e c t s
p a r t i c i p a t e d i n t h e s t u d i e s i n which t h e o n l y manganese s u p p l i e d was
by t h e b a s a l d i e t (4.91-4.95 m g / s u b j e c t / d a y ) .
A s c o r b i c a c i d s u p p l e m e n t a t i o n o f d i e t s s i g n i f i c a n t l y enhanced
the apparent r e t e n t i o n o f manganese (p<0.05) a t l o w l e v e l s o f man-
ganese i n t a k e . When a s c o r b i c a c i d was added t o t h e d i e t s , f e c a l
manganese l o s s e s were s i g n i f i c a n t l y i n c r e a s e d i n comparison t o v a l u e s
when t h e d i e t was f e d a l o n e (p<0.05) as shown on T a b l e V I . At l e a s t
at two l e v e l s o f manganese i n t a k e , a s c o r b i c a c i d a p p a r e n t l y enhances
manganese u t i l i z a t i o n .

Table V I . E f f e c t o f A s c o r b i c A c i d on Manganese U t i l i z a t i o n i n
Humans

Parameter
Number o f s u b j e c t s 57 57
b
Mn i n t a k e , mg/day 4.91 a
4.95
b
F e c a l Mn, mg/day a
4.19 ±0.15 3.95 ±0.05
a
Apparent Mn r e t e n t i o n , mg/day b
+0.72 ±0.05 +1.00 ±0.05
a
Apparent Mn r e t e n t i o n , % 14.66 b
20.20
Values w i t h d i f f e r e n t l e t t e r s u p e r s c r i p t s are s i g n i f i c a n t l y
d i f f e r e n t from one another (p<0.05).
^Based on r e c a l c u l a t e d d a t a s u p p l i e d by K o w a l s k i (17) and Wang ( 1 6 ) .

I r o n Supplement Use and Manganese U t i l i z a t i o n

A s c o r b i c a c i d i s known t o enhance t h e b i o a v a i l a b i l i t y o f i r o n by
r e d u c i n g i r o n from the +3 t o t h e +2 v a l e n c e s t a t e , t h e a b s o r b a b l e
v a l e n c e s t a t e (11-14). I n one s t u d y , t h e e f f e c t o f f e r r o u s fumarate
s u p p l e m e n t a t i o n on manganese u t i l i z a t i o n from h i g h and low manganese
c o n t a i n i n g d i e t s was i n v e s t i g a t e d . The 28-day study was d i v i d e d
i n t o f o u r , 7-day, randomly-arranged p e r i o d s . D u r i n g these p e r i o d s
the seven s u b j e c t s r e c e i v e d t h e b a s a l d i e t a l o n e , t h e b a s a l d i e t
p l u s f e r r o u s fumarate ( t o s u p p l y 20 mg i r o n / s u b j e c t / d a y ) , t h e b a s a l
d i e t p l u s manganese g l u c o n a t e ( t o s u p p l y 40 mg manganese/subject/day)
o r t h e b a s a l d i e t p l u s a c o m b i n a t i o n o f t h e f e r r o u s fumarate and
manganese g l u c o n a t e supplements. Supplements were g i v e n i n t a b l e t
form a t t h e b r e a k f a s t meal.
S t u d i e s , i n c l u d i n g t h e p r e s e n t one, designed t o i n v e s t i g a t e
e f f e c t s o f i n c r e a s i n g d i e t a r y l e v e l s o f i r o n on manganese u t i l i z a -
t i o n have g e n e r a l l y i n d i c a t e d ad a d v e r s e e f f e c t o f d i e t a r y i r o n on
manganese a b s o r p t i o n from t h e i n t e s t i n a l t r a c t (15-22). As shown i n
T a b l e V I I , f e r r o u s fumarate s u p p l e m e n t a t i o n o f e i t h e r l o w - l e v e l o r
h i g h - l e v e l manganese d i e t s r e s u l t e d i n i n c r e a s e d f e c a l manganese
l o s s e s and decreased manganese r e t e n t i o n i n comparison t o v a l u e s when
the a p p r o p r i a t e c o n t r o l l e d d i e t s were f e d (p<0.05).

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
144 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

Table V I I . Manganese U t i l i z a t i o n as A f f e c t e d by I r o n Intake

a t Two L e v e l s o f Manganese I n t a k e
1 2
Basal Diet »
Parameter Alone + Mn J
+ Fe^ + Fe + Μη
Mn i n t a k e , mg/day 44.56^ 4.56 b
44.56a
F e c a l Mn, mg/day 4.78d 37.20b 7.22 e
41.44a
±0.77 ±3.10 ±0.93 ±2.54
Apparent Mn r e t e n t i o n , -0.24 d
+7.36a -2.58 e
+3.12 b

mg/day ±0.09 ±0.97 ±0.88 ±0.08

V a l u e s w i t h d i f f e r e n t l e t t e r s u p e r s c r i p t s were s i g n i f i c a n t l y
d i f f e r e n t from one another (p<0.05).
Based on r e c a l c u l a t e d d a t a by Creps ( 1 5 ) .
3
40 mg/day as manganese g l u c o n a t e amino a c i d c h e l a t e .
4

20 mg/day as f e r r o u s f u m a r a t e

Conclusion
Other d i e t a r y v a r i a b l e s b e s i d e s those d i s c u s s e d i n t h e p r e s e n t paper
may a l s o a f f e c t manganese b i o a v a i l a b i l i t y and r e t e n t i o n by i n f l u ­
e n c i n g e i t h e r a b s o r p t i o n o r e x c r e t o r y mechanisms. F o r example,
i n c r e a s e d i n t a k e o f d i e t a r y c a l c i u m has been found t o i n h i b i t appar­
ent a b s o r p t i o n o f manganese (3,17). R e s u l t s of s t u d i e s designed t o
i n v e s t i g a t e e f f e c t o f d i e t a r y phosphorus on manganese b i o a v a i l a b i l i t y
have g i v e n mixed r e s u l t s (23-25). I n o t h e r s t u d i e s conducted a t t h e
U n i v e r s i t y o f N e b r a s k a , apparent manganese a b s o r p t i o n from v a r i o u s
p l a n t s o u r c e s by humans was found t o be v e r y poor i n comparison t o
t h a t e x h i b i t e d by manganese s a l t supplements o r manganese s u p p l i e d
by a mixed food d i e t (5,26).
On t h e b a s i s o f d a t a p r e s e n t e d i n t h i s p a p e r , a p p a r e n t l y some
of t h e b e s t s o u r c e s o f manganese i n terms o f amount o f manganese
c o n t a i n e d p e r r e a s o n a b l e s e r v i n g p o r t i o n a r e , i n f a c t , poor s o u r c e s
on t h e b a s i s o f u t i l i z a b l e manganese. T h e r e f o r e , i t may be t h a t
manganese c o n t e n t o f food s h o u l d be e x p r e s s e d i n u n i t s o f u t i l i z a b l e
manganese r a t h e r than i n g r a v i m e t r i c u n i t s p e r s e .

Acknowledgments

P u b l i s h e d as U n i v e r s i t y o f Nebraska A g r i c u l t u r a l Research D i v i s i o n
J o u r n a l A r t i c l e S e r i e s No. 8062. Supported by Nebraska A g r i c u l t u r a l
Research D i v i s i o n P r o j e c t 91-031 and USDA, CSRS P r o j e c t W-143.

Literature Cited
1. Greger, J . L . and Snedeker, S.N. J . Nutr. 1980, 110, 2243.
2. Price, N.O.; Bunce, G.E. and Engel, R.W. Am. J . Clin. Nutr.
1970, 23, 258.
3. Price, N.O. and Bunce, G.E. Nutr. Reports Int. 1972, 5, 275.
4. Rojhani, A. 1984. M.S. Thesis, University of Nebraska-Lincoln.
5. Aldrich, K.D. 1984. M.S. Thesis, University of Nebraska­
-Lincoln.
6. Koszewski, W.M. 1984. M.S. Thesis, University of Nebraska­
-Lincoln.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
13. KIES ET AL. Manganese Availability for Humans 145

7. Davis, N.T. and Nightingale, R. Br. J. Nutr. 1975, 34, 243.

8. Johnson, J.; Kies, C. and Fox, H. XIII International Congress
of Nutrition, 1985 (Abstract).
9. Schwartz, R.; Apgar, J. and Wien, E.M. Am. J . Clin. Nutr. 1986,
43, 444.
10. Corrington, J. 1982. M.S. Thesis, University of Nebraska­
-Lincoln.
11. Kies, C. In Nutritional Bioavailability of Iron; Kies, C., Ed.;
American Chemical Society: Washington, DC, 1982, p. 183.
12. Bowering, J.; Sanchez, A.M. and Irwin, M.I. J . Nutr. 1976, 106,
985.
13. Hallberg, L. In Nutrition Review's Present Knowledge in
Nutrition, 5th Ed. Nutrition Foundation, Inc.: Washington, DC,
1984, p. 459.
14. Cook, J.D.; March, I.Α.; and Lynch, S. Am. J. Clin. Nutr.
1977, 30, 235.
15. Creps, C. 1984. M.S
16. Wang, R.H. 1984. M.S
17. Kowalski, C. 1983. M.S. Thesis, University of Nebraska
-Lincoln.
18. Gruden, N. Reprod. Nutr. Develop. 1980, 20(SA), 1539.
19. Gruden, N. Nutr. Reports Int. 1984, 30, 553.
20. Gruden, N. Nutr. Reports Int. 1977, 15, 577.
21. Gruden, N. and Buben, M. Nutr. Reports Int. 1981, 24, 943.
22. Gruden, N. and Buben, M. Nutr. Reports Int. 1981, 25, 849.
23. Spencer, H.; Asmussen, C.R.; Holtzman, R.B. and Kramer, L.
Am. J. Clin. Nutr. 1979, 12, 1867.
24. Schaible, P.J. and Bandemer, S.L. Poult. Sci. 1942, 21, 8.
25. Pound, W.G.; Walter, E.F., Jr.; and Kirtland, D. J . Ani. Sci.
1978, 46, 686.
26. Johnson, J. 1983. M.S. Thesis, University of Nebraska-Lincoln.
RECEIVED May 15, 1987

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Chapter 14

Manganese Usage in Humans as Affected

by Use of Calcium Supplements
Susan D. McDermott and Constance Kies
Department of Human Nutrition and Food Service Management,
University of Nebraska, Lincoln, NE 68583

Calcium has long been implicated as a dietary factor

which inhibits the absorption of manganese. Since
manganese is better absorbed in the oxidized (+2)
than in the reduce
which increases gastrointestina
alkalinity) would be expected to inhibit manganese
utilization. Calcium carbonate, a commonly used
antacid by humans as well as a calcium nutrient
supplement, has been found to have a greater adverse
effect on apparent manganese absorption in humans
than does milk.

Manganese can exist i n several d i f f e r e n t oxidation states including

I, I I , I I I , IV, VI and VII although the most stable s a l t s are
those i n the oxidation states I I , IV, VI and VII (_1). Although
research on manganese needs of humans i s very l i m i t e d , manganese as
a nutrient requirement of plants and factors a f f e c t i n g manganese
u t i l i z a t i o n of s o i l manganese by plans has received extensive inves-
t i g a t i o n (1). Total manganese i s generally accepted as being a poor
predictor of manganese e x t r a c t a b i l i t y (or a v a i l a b i l i t y ) from s o i l to
the plant. One of the most important agronomic c h a r a c t e r i s t i c s
a f f e c t i n g manganese e x t r a c t a b i l i t y i s that of s o i l pH. As pH of
s o i l s r i s e (as may occur with addition of lime-calcium oxide to
s o i l s ) , o x i d a t i o n of manganese II to manganese III and IV i s favored
which reduces the s o l u b i l i t y of manganese and, consequently, i t s
a v a i l a b i l i t y to plants. Conversely, manganese t o x i c i t y i n plants i s
usually found i n acid s o i l s with pH lower than 5.5.
Manganese i n the human i s also thought to be absorbed maximally
in the duodenum i n the II valence state. Therefore, as with man-
ganese uptake by plants, the pH of the upper g a s t r o - i n t e s t i n a l tract
might be expected to be of importance i n the absorption of manganese
by the human.
Calcium carbonate preparations for many years have been used
by humans i n large amounts on a s e l f - p r e s c r i p t i o n basis or as rec-
ommended by physicians i n control or treatment of upper gastro-
i n t e s t i n a l d i s t r e s s conditions which are thought to be related to
gastric acid production. These include dyspepsia, peptic u l c e r s ,

0097-6156/87/0354-0146S06.00/0
© 1987 American Chemical Society

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
14. McDERMOTT AND KIES Manganese Use and Calcium Supplements 147

g a s t r i t i s , e s o p h a g a t i t u s and h i a t a l h e r n i a . A n t a c i d c a l c i u m carbon-
a t e p r o d u c t s a r e a v a i l a b l e i n s e v e r a l forms i n c l u d i n g t a b l e t s ,
g e l a t i n c a p s u l e s , chewable w a f e r s , gum, and c a r a m e l s ; hence, c a l c i u m
r e l e a s e from t h e s e p r o d u c t s designed t o i n c r e a s e pH (lower a c i d i t y )
i n the g a s t r o - i n t e s t i n a l t r a c t would be expected t o v a r y w i t h t i m e .
Recent concern r e l a t i v e t o the h i g h i n c i d e n c e o f o s t e o p o r o s i s
i n e l d e r l y , female, Americans has r e s u l t e d i n i n c r e a s e d usage o f
c a l c i u m supplements. C a l c i u m carbonate c o n t a i n s more c a l c i u m per
weight u n i t o f the s a l t than does c a l c i u m phosphate, c a l c i u m l a c t a t e
o r c a l c i u m g l u c o n a t e ; hence, e i t h e r c a l c i u m carbonate o r one o f t h e
c a l c i u m carbonate forms such as o y s t e r s h e l l s o r d o l o m i t e i s most
o f t e n the c a l c i u m s a l t c h o i c e f o r u s e i n a supplement s i n c e fewer
c a p s u l e s need t o be taken ( 2 ) . S i n c e c a l c i u m carbonate p r e p a r a t i o n s
marketed as a n t a c i d s are o f t e n s o l d a t a p r i c e lower than a r e t h o s e
c a l c i u m carbonate p r e p a r a t i o n s marketed as c a l c i u m supplements,
c a l c i u m a n t a c i d p r e p a r a t i o n s a r e c u r r e n t l y b e i n g used as c a l c i u m
supplements.
C a l c i u m has l o n g bee
b i o a v a i l a b i l i t y o f manganese. E x c e s s i v e i n t a k e s o f c a l c i u m o r
phosphorus have been shown t o i n c r e a s e the d a i l y r e q u i r e m e n t s f o r
manganese i n swine (3-5) p r o b a b l y due t o decreased a b s o r p t i o n o f
t h i s m i n e r a l . However, c o m p a r a t i v e l y l i t t l e i n f o r m a t i o n i s a v a i l a b l e
on the comparative e f f e c t s o f d i f f e r e n t sources o f c a l c i u m on man-
ganese u t i l i z a t i o n i n humans.
E f f e c t s o f c a l c i u m phosphate and c a l c i u m l a c t a t e w i t h and w i t h -
out a s c o r b i c a c i d s u p p l e m e n t a t i o n were examined i n s t u d i e s r e p o r t e d
by K o w a l s k i ( 6 ) . C a l c i u m phosphate was found t o have a g r e a t e r neg-
a t i v e e f f e c t on apparent manganese a b s o r p t i o n i n a d u l t humans as
judged on p e r c e n t a g e o f d i e t a r y manganese r e c o v e r e d i n f e c e s i n com-
p a r i s o n t o v a l u e s when c a l c i u m l a c t a t e o r no c a l c i u m supplements
were g i v e n (Table I ) . A s c o r b i c a c i d supplements tended t o negate
the n e g a t i v e e f f e c t s o f c a l c i u m s u p p l e m e n t a t i o n .

Table I . C a l c i u m (Ca) Supplementation and Manganese (Mn)

B i o a v a i l a b i l i t y i n Human A d u l t s

Parameter Alone
_____ +Ca

Study 1: ( w i t h 200 mg a s c o r b i c a c i d ) (Ca phosphate)

# Subjects 8 8
Mn i n t a k e , mg/day 2.82 2.82
b
F e c a l Mn, mg/day 1.55 2.03*
a
Apparent Mn r e t e n t i o n , mg/day +1.27 +0.79,
a
% Mn r e t e n t i o n 45.04 28.01

Study 2: ( w i t h no added a s c o r b i c a c i d ) (Ca l a c t a t e )

# Subjects 10 10
Mn i n t a k e , mg/day 4.03 4.03
a 3 , 5 7
F e c a l Mn, mg/day 3.81 a
Apparent Mn r e t e n t i o n , mg/day +0.12 +0.46^
a
% Mn r e t e n t i o n 2.98 11.41
Means w i t h d i f f e r e n t l e t t e r s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t
at p<0.05.

American Chemical Society

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In Nutritional Bioavailability 20036
Manganese; Kies, C.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
148 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

Comparative e f f e c t s of c a l c i u m l a c t a t e and m i l k on apparent

manganese u t i l i z a t i o n by humans a r e shown on T a b l e I I ( 7 ) . I n t h i s
s t u d y , 10 a d u l t human s u b j e c t s were f e d 900 mg c a l c i u m from m i l k o r
916 mg of c a l c i u m from c a l c i u m l a c t a t e / s u b j e c t / d a y . C a l c i u m p r o v i d e d
by the b a s a l d i e t was m a i n t a i n e d c o n s t a n t . The i n c r e a s e i n f e c a l
manganese e x c r e t i o n w i t h the c a l c i u m l a c t a t e supplemented d i e t i n
comparison to v a l u e s when m i l k was the supplemental manganese s o u r c e
suggests t h a t c a l c i u m s u p p l i e d by m i l k had a l e s s e r adverse e f f e c t
on manganese a b s o r p t i o n than d i d t h a t from c a l c i u m l a c t a t e (Table
II).

Table I I . Manganese S t a t u s as A f f e c t e d by M i l k and C a l c i u m L a c t a t e

Mn E x c r e t i o n Mn
Diet Mn Intake Urine Feces Balance
mg/day mg/day mg/day mg/day

Basal diet + 2.40

milk

Basal diet + 2.34 a

0.0137 b
3.00 a
-0.67 b

Ca l a c t a t e
Means w i t h d i f f e r e n t l e t t e r s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t
at p<0.05.

E f f e c t s of s e v e r a l d i f f e r e n t , c o m m e r c i a l l y - a v a i l a b l e , calcium
supplements on apparent manganese u t i l i z a t i o n by humans i n compari-
son t o m i l k were i n v e s t i g a t e d by McDermott ( 8 ) . D u r i n g e i g h t r a n -
domly a r r a n g e d e x p e r i m e n t a l p e r i o d s , 20 a d u l t women were f e d a con-
s t a n t , measured, l a b o r a t o r y c o n t r o l l e d d i e t p r o v i d i n g 400 mg o f
c a l c i u m / s u b j e c t / d a y and 6.29 mg of manganese/subject/day. The h i g h
manganese c o n t e n t of t h i s d i e t was p r i m a r i l y p r o v i d e d by whole g r a i n
ground wheat p r o d u c t s . To t h i s was added a p p r o x i m a t e l y 600 mg o f
c a l c i u m / s u b j e c t / d a y from m i l k o r from the t e s t c a l c i u m supplement
p r e p a r a t i o n s . The p r i m a r y o b j e c t i v e of t h i s p r o j e c t was t o determine
comparative e f f e c t i v e n e s s o f these p r o d u c t s r e l a t i v e t o c a l c i u m
s t a t u s ( 2 ) ; however, measurements of parameters r e l a t i v e t o mangan-
ese u t i l i z a t i o n were i n c l u d e d to meet the secondary o b j e c t i v e .
S u b j e c t s made complete c o l l e c t i o n s o f u r i n e and s t o o l s t h r o u g h -
out t h i s s t u d y . As i s t r u e w i t h o t h e r human s t u d i e s conducted i n
t h i s l a b o r a t o r y , f e c e s were d i v i d e d i n t o l o t s r e p r e s e n t i n g food
eaten d u r i n g each e x p e r i m e n t a l p e r i o d by use of o r a l l y g i v e n f e c a l
dyes and beads g i v e n at the b e g i n n i n g and end of each e x p e r i m e n t a l
p e r i o d . U r i n e was p r o c e s s e d i n t o p e r i o d l o t s on the b a s i s of t i m e .
F e c a l manganese l o s s e s w h i l e s u b j e c t s r e c e i v e d the d i f f e r e n t
c a l c i u m supplements are g i v e n i n Table I I I . Because two o f the
s u b j e c t s (both A m e r i c a n / B l a c k by n a t i o n a l i t y / r a c e c l a s s i f i c a t i o n )
were found to be l a c t o s e i n t o l e r a n t , d a t a f o r t h e s e two i n d i v i d u a l s
were o m i t t e d i n c a l c u l a t i o n s o f mean f i g u r e s . F e c a l manganese
l o s s e s were l o w e s t s u g g e s t i n g b e s t manganese a b s o r p t i o n when m i l k
was used as the c a l c i u m supplement. G r e a t e s t f e c a l l o s s e s of mangan-
ese o c c u r r e d when the v a r i o u s forms of c a l c i u m carbonate were g i v e n
( c a l c i u m c a r b o n a t e , d o l o m i t e and o y s t e r s h e l l c a l c i u m ) .
I t i s r e a s o n a b l e t o suppose t h a t m i l k had a l e s s e r e f f e c t i n
r a i s i n g upper g a s t r o i n t e s t i n a l t r a c t pH than d i d the c a l c i u m

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
14. McDERMOTT AND KIES Manganese Use and Calcium Supplements 149

Table I I I . E f f e c t o f D i f f e r e n t Calcium (Ca) Supplements on Manganese (Mn)

U t i l i z a t i o n of Humans*

Sources of supplemental calcium;

Oyster Ca Ca Ca M u l t i Ca
Parameter Milk S h e l l 2 Dolomite^ Carbonate^ G l u c o n a t e 5
L a c t a t e ^ Supplement

Basal d i e t Ca,
mg/day 637 637 637 637 637 637 637

Supplement Ca,
mg/day 592 500 520 500 500 505 500

Basal d i e t Mn, 6.2 6.

mg/day ±1.4 ±1.

F e c a l Mn l o s s , 5.33 bc
6.27 ab
6.60 a
6.97 a
5.60 b
6.01 ab
5.99 ab

mg/day ±2.35 ±2.20 ±2.70 ±3.84 ±5.21 ±4.28 ±3.67

a
Urinary Mn l o s s ., 0.116 a
0.119 a
0.126 a
0.210 3
0.082 a
0.104 a
0.112
mg/day ±0.046 ±0.032 ±0.031 ±0.024 ±0.046 ±0.050 ±0.060

b
Mn balance, +0.75 ab
-0.09 bc
-0.33 bc
-0.78 c
+0.32 b
+0.29 b
+0.20
mg/day ±2.7 ±2.5 ±1.5 ±1.8 ±3.0 ±2.8 ±4.7
R e c a l c u l a t e d from McDermott ( 8 ) .
O y s t e r s h e l l c a l c i u m with v i t a m i n D, 2 t a b l e t s s u p p l i e d 500 mg calcium. Oyster shell
powder and v i t a m i n D, Walgreens L a b o r a t o r i e s , Inc., Chicago, IL 60632.

Dolomite calcium, 4 t a b l e t s s u p p l i e d 520 mg calcium carbonate and 320 mg magnesium

as magnesium carbonate. N u t r i - P l u s N u t r i t i o n a l Products, Los Angeles, CA 91331.
*Bio C a l Calcium, 1 t a b l e t s u p p l i e d 500 mg calcium as calcium carbonate. Miles
L a b o r a t o r i e s , Inc., E l k h a r t , IN 46515.
Mega C a l calcium with v i t a m i n D, 1 t a b l e t s u p p l i e d 500 mg c a l c i u m and 100 mg
v i t a m i n D, d e r i v e d from egg s h e l l s , o y s t e r s h e l l s , calcium l a c t a t e , calcium
gluconate, calcium carbonate, non-fat d r i e d m i l k , malted m i l k and d a i r y sweet
whey. H o l i s t i c Products Corp., East Rutherford, NJ 07073.
C a l c i u m gluconate, 8 t a b l e t s s u p p l i e d 500 mg calcium, Pioneer S p e c i a l t y Foods,
Fargo, ND 58102.
Calcium l a c t a t e , 6 t a b l e t s s u p p l i e d 505 mg calcium, General N u t r i t i o n Corp.,
P i t t s b u r g h , PA 15222.

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
150 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

carbonate supplements. As previously discussed, an increase i n

g a s t r o i n t e s t i n a l tract pH would be expected to i n h i b i t the absorption
of manganese by oxidizing i t from i t s absorbable +2 valence state to
the +3 or +4 valence state. Milk diets were once customarily used
in the treatment of peptic u l c e r s . This practice was r a t i o n a l i z e d
on the basis that calcium i n the milk would neutralize the excess
gastric acid (9-12). However, current research suggests that the
protein i n milk a c t u a l l y stimulates gastric acid secretion. Hence,
unlike the other calcium supplements, milk might enhance rather than
i n h i b i t manganese absorption.
The possible adverse effect of calcium on manganese u t i l i z a t i o n
may be viewed i n either the p o s i t i v e or the negative sense since
manganese i s a toxic agent when consumed i n excessive amounts. Hard
water i s characterized by i t s high content of calcium and magnesium
in comparison to soft water. Absorption of the manganese isotope
5^Mn was found to be reduced s i g n i f i c a n t l y from a solution containing
calcium chloride i n compariso
water (13). In a second
cantly enhanced i n mice by chronic deprivation of a combination of
calcium and magnesium or by calcium deprivation alone but not by a
deprivation of magnesium alone i n comparison to ^Mn absorption i n
control mice. The authors concluded that water hardness may protect
against absorption of p o t e n t i a l l y toxic amounts of manganese. Con-
versely, i t could be concluded that the apparent adverse effect of
water hardness on manganese absorption could e l i c i t manganese d e f i -
ciency under conditions of low manganese intakes.
In conclusion, research r e s u l t s indicate that calcium apparent-
l y i n h i b i t s the absorption of manganese from the i n t e s t i n a l t r a c t .
Different sources of calcium apparently affect manganese to varying
degrees. Whether or not t h i s i s due to changes i n i n t e s t i n a l
a c i d i t y / a l k a l i n i t y , to possible competition between manganese and
calcium for absorption s i t e s , or to a combination of factors i s
unknown.

Acknowledgment s

Published as Nebraska A g r i c u l t u r a l Research D i v i s i o n Paper No. 8425.

Supported by Nebraska A g r i c u l t u r a l D i v i s i o n Project 91-031 and
USDA, CSRS Project W-143.

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7. Vigneau, L.E. M.S. Thesis, University of Nebraska-Lincoln,

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RECEIVED May 12, 1987

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Author Index

Aldrich, K . D., 136 Keen, Carl L., 9,21,56,105

Baker, David H., 35 Kies, Constance, 1,123,136,146
Bales, Connie W., 90,112 Kowalski, C , 136
Behmardi, Fares, 90 Lin, Pao-Hwa, 112
Bell, J. G., 9 Lonnerdal, Bo, 9,21
Carl, G. F., 105 McDermott, Susan D., 146
Chan, Wai-Yee, 80 Raghib, M Hassan, 80
Creps, C , 136 Rennert, Owen M., 80
Dougherty, Virginia, 112 Saltman, Paul, 46
Freeland-Graves, Jeann H. 90,112 Sandstrôm B. 9
Gallagher, Β. B., 105
Gruden, N., 67
Halpin, Kevin M , 35 Strause, Linda, 46
Hurley, L . S., 105 Wang, R. H., 136
Johnson, Jan M , 123,136 Zidenberg-Cherr, Sheri, 21,56

Affiliation Index

Louisiana State University Agricultural University of Illinois, 35

Center, 35 University of Nebraska, 1,123,136,146
Medical College of Georgia, 105 University of Oklahoma Health Sciences
University of California- Center, 80
Davis, 9,21,56,105 University of Texas at Austin, 90,112
University of California—San Diego, 46 Veterans Administration Medical
University of Gothenburg, 9 Center, 105

Subject Index

A Ataxia, 28, 105

Atherosclerosis, 123
Adolescent period Mn balance, 14
Adult period
balance studies, 92-96 Β
Mn deficiency, symptoms, 22
Age dependence, 72-74, 83-88, 91-104 Balance studies
Alcohol, 62 in adolescents, 14
Alkaline phosphatase, 91 in adults, 92-96
Anemia, 97 in children, 91-94
Antacids, 146-147 Bioavailability
Ascorbic acid dietary factors, 14-18, 112-121, 136-145
and Mn utilization, 142-143 in poultry feedstufts, 36-38
effect on Mn absorption, 18 inorganic Mn supplements, 38
152

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
 Author Index

Aldrich, K . D., 136 Keen, Carl L., 9,21,56,105

Baker, David H., 35 Kies, Constance, 1,123,136,146
Bales, Connie W., 90,112 Kowalski, C , 136
Behmardi, Fares, 90 Lin, Pao-Hwa, 112
Bell, J. G., 9 Lonnerdal, Bo, 9,21
Carl, G. F., 105 McDermott, Susan D., 146
Chan, Wai-Yee, 80 Raghib, M Hassan, 80
Creps, C , 136 Rennert, Owen M., 80
Dougherty, Virginia, 112 Saltman, Paul, 46
Freeland-Graves, Jeann H. 90,112 Sandstrôm B. 9
Gallagher, Β. B., 105
Gruden, N., 67
Halpin, Kevin M , 35 Strause, Linda, 46
Hurley, L . S., 105 Wang, R. H., 136
Johnson, Jan M , 123,136 Zidenberg-Cherr, Sheri, 21,56

Affiliation Index

Louisiana State University Agricultural University of Illinois, 35

Center, 35 University of Nebraska, 1,123,136,146
Medical College of Georgia, 105 University of Oklahoma Health Sciences
University of California- Center, 80
Davis, 9,21,56,105 University of Texas at Austin, 90,112
University of California—San Diego, 46 Veterans Administration Medical
University of Gothenburg, 9 Center, 105

Subject Index

A Ataxia, 28, 105

Atherosclerosis, 123
Adolescent period Mn balance, 14
Adult period
balance studies, 92-96 Β
Mn deficiency, symptoms, 22
Age dependence, 72-74, 83-88, 91-104 Balance studies
Alcohol, 62 in adolescents, 14
Alkaline phosphatase, 91 in adults, 92-96
Anemia, 97 in children, 91-94
Antacids, 146-147 Bioavailability
Ascorbic acid dietary factors, 14-18, 112-121, 136-145
and Mn utilization, 142-143 in poultry feedstufts, 36-38
effect on Mn absorption, 18 inorganic Mn supplements, 38
152

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
INDEX 153

Bioavailability—Continued Experimental studies

mechanism, 37 chick, 35-45
M n i n diet, 1-8 human
Mn in meat, 138 diet and Mn availability, 137-144
Mn salts, 114 effect of Ca supplements, 147
Mn stored in body tissue, 39-40 effect of dietary fat, 130-135
various types of diets, 14 Mn balance study, 91-102
Blood manganese, 105-111 plasma uptake, 113-119
Blood serum cholesterol, 130-135 mouse
Bone formation and resorption, 50-53 response to free radicals, 61
Bone metabolism, 46 uptake and distribution, 47,106
Bone particle implants, 50-53 rat
Brush border membrane vesicle, 12 age and Mn absorption, 10
Fe-Mn interaction, 69
low MnSOD activity, 59
C milk diet, Mn absorption, 81
Mn and lipid metabolism, 125
Cadmium-Mn interaction 74 Mn toxicity 24
Calcium
blood serum levels, 91
high-fiber diet, 116
supplements, 146-150 F
Calcium-Mn interaction, 42,98,144
Cellulose in diet, 116 Fiber
Childhood balance studies, 91-94 cellulose, 116-118
Cholesterol, 124,126 effect of plasma uptake, 113
Citrus pectin in diet, 118 effect on Zn absorption, 139
Cobalt, 41 hemicellulose, 140-141
Coccidial infections and Mn absorption, 43 high-fiber/high-phytate diet, 98
Congenital malformations, 23 pectin, 118
Copper phytate, 118-119
deficiency, 49 Free radicals, 61
plasma concentrations, 107,110
Copper-Mn relationship, 46
Coronary heart disease, 123 G

Galactosemia, 91
Glucocorticoids, 107,110

D
H
Dermatitis, 91
Dietary allowance of Mn, 90-104 Hemicellulose, 140-141
Dietary factors Hip abnormalities, 23
affecting Mn absorption, 14-18,36 Hydralazine syndrome, 91
balance in humans, 91-104 Hyperglycemia, 25
interactions with minerals, 41-42 Hypocholesterolemia, 91
milk diet, 68
Dose dependence, 69-70

Integumental losses, 96
Iron
bioavailability, 80
Ε cause of negative Mn balance, 97
effect on Mn absorption, 18,143,144
Epilepsy in human milk, 80-89
Mn blood concentrations, 2,23,91 inhibitory effect, 69-76
Mn-seizure relationship, 105-111 liver concentration, 63
Ethanol, 62 transport, 71-72

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
154 NUTRITIONAL BIOAVAILABILITY OF MANGANESE

Iron-citrus pectin, 118 Manganese—Continued

Iron-Mn interaction dietary factors, plasma uptake, 116-119
competitive, 97-98 dietary needs and intake, 3
milk diet, 67-79 dietary sources, 99
supplemental Mn, 42 enzyme systems, 2
excretion, 3,96,97
homeostasis, 35-45
L human intake levels, 6
in blood
Lactoferrin content of Mn, 18 concentrations and epilepsy, 2,23,91
Ligands, 18 relationship to epileptic
Lipid metabolism, 123-135 seizures, 105-111
Lipid peroxidation, 59 in foods, 4,5/
Lipid radicals, damage to mitochondrial in milk and infant formulas, 4
membranes, 59 interaction with Ca, 42,98,144
Lipids in diet, 136 interaction with Cd, 74
interaction with Cu, 46
interaction with Fe 69
M

metalloenzymes, 57
Magnesium plasma uptake, 112-121
effect of cellulose in diet, 116 requirements of humans, 90-104
effect of citrus pectin, 118 retention, 10,96,97
in water, 150 role in diseases, 22
toxicity symptoms, 24,100 toxicity, 23-27,68,100
Manganese transport, 69
absorption uptake and retention, 9-20
coccidial infections, 43 utilization, 138-144
different ages, 10 Manganese balance, adolescent period, 14
difficulty in determining, 96,97 Manganese superoxide dismutase
effect of ascorbic acid, 18 function, 57,61
from milk, 80 influence of dietary manganese, 58
overview, 3 physiological significance, 58
various organs, 112-113 Manganese tolerance test, 113-116
with Mn deficiency, 29 Metabolic responses to Mn-deficient diet, 28
balance study, human, 14,91-102 Metabolism
bioavailability bone, 46-55
Ca supplement, 146-150 dietary factors, 123-135
dietary factors, 136-145 effect of iron, 67-79
in diet, 1-8 manganese, 67,105-111
in meat, 138 manganese and lipid, 123
in milk, 80 Metalloenzymes, 57
in Mn salts, 114 Methylmalonic acidemia, 91
in poultry feedstuffs, 36-38 Milk
inorganic Mn supplements, 38 comparison of types, 82-83
mechanism, 37 effect on Mn absorption, 68-76,148
pH, 146 Mn uptake, 15,80,89
stored in body tissue, 39-40 transport of, 87
various types of diets, 14 Mitochondrial membranes, 59
with dietary fat, 130-135
deficiency
bone disorders, 46-47 Ν
dietary, 21-34
pancreatic function, 28-33 Neonatal period
rare in humans, 14 bioavailability of Mn in milk, 80-89
structural and physiological defects, 68 effect of age on Mn absorption, 83-88
symptoms, 2,22,90-91 excess Mn retention, 10
tissue and organ study, 28-33 Fe-Mn competition, 72

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
INDEX 155

Neonatal period—Continued Τ
low MnSOD activity, 58
Mn deficiency, 10
Mn uptake, 15
Nuts, 99 Tea, 99
Time dependence, 70
Tissue lipid peroxidation, 56-66
Ο Toxicity
in chicks, 43
Organ distribution in humans, 23-24
dietary Mn intake in chicks, 39 in rats, 24-27
lipids and dietary Mn, 126 margin of safety, 68
Mn absorption from milk, 82,85-88 Mn mine workers, 24
Mn and seizure frequency, 106 Mn supplements, 24
Mn uptake and distribution, 48 symptoms, 24,100
Mn-deficient diet, 28-33 water contaminated with Mn, 24
Osteopenia, 49 Triglyceride, 130-135
Osteoporosis
correlation with serum Mn
Mn concentration in bones
Mn deficiency, 23
U
Ozone
and dietary Mn deficiency, 61
Uptake process, 14,47
effect on body and lung weight, 61

Pancreatic Mn concentration and insulin

production, 25 V
Perosis, 36,47
pH, 150 Vegetarian diet, 95-96,138-139
Phenylketonuria (PKU), 22,91 Vitamin E , 63
Phosphorus
affected by dietary cellulose, 116
affected by dietary citrus pectin, 118
competition with Mn, 98 W
effect on Mn bioavailability, 144
serum levels, 91 Weanling period
Plasma uptake of Mn, 112-121 absorption of Mn in milk, 83-88
Protein, 94 F e - M n competition, 73-74
Wheat bran, 139-140
Whole grains, 99
R
Whole wheat, 94,139

Retention of Mn
from varying diets, 15
in various organs, 16
on surface of teeth, 49 Ζ
Rheumatoid arthritis, 91
Zinc
affected by dietary cellulose, 116
S affected by dietary citrus pectin, 118
effect on Mn in diet, 94
Safe intake levels, 6 in infants, 12
Seeds, 99 plasma concentration, 107,110
Soy products, 138 poorly absorbed from wheat, 139
Spinach, 141-142 serum concentration, 46

In Nutritional Bioavailability of Manganese; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1987.