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Annu. Rev. Nutr. 1991. 11 :121-40
Copyright © 1991 by Annual Reviews Inc. All rights reserved

THE INTERACTIONS BETWEEN

COPPER, MOLYBDENUM, AND
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SULPHUR IN RUMINANT
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NUTRITION

N. F. Suttle
Department of Biochemistry, Moredun Research Institute, Edinburgh EH17 7JH,
Scotland

KEY WORDS: thiomolybdates, molybdenosis, hypocuprosis, copper deficiency, molybdenum

toxicity

CONTENTS

INTRODUCTION .. . . . . . . . . . . . . ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . 122

THIOMOLYBDATES: NUTRITIONAL DOUBTS AND CERTAINTIES .... . ........... 123
Qualitative Evidence for Thiomolybdate Formation in vivo . . .... . . . . . . . . . . . . . . . . . . . . . . . . 123
Qualitative Evidence for Thiomolybdate Inhibition of Copper Absorption ... . . . . . . . . . 123
Qualitative Evidence for Absorption of Thiomolybdates ......... . . . . . . . . . . . . . . . . . . . . . . . . . 124
Quantitative Evidence for Thiomolybdate Excretion in Feces................... . . . . . . . . . 124
Systemic Effects of Thiomolybdates .................................. . . . . . . . . . . . . . . . . . . . . . . . . . 125
Conclusions on Physiological Importance of Thiomolybdates....... . . . . . . . . . . . . . . . . . . . . . 129
NUTRITIONAL VARIABLES AFFECTING THE RUMEN INTERACTION . . . .. . . . . .. 130
Sulphur.................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 130
Molybdenum ....................................................................................... 131
Copper ........... . ............................................. .. ................................ .. . 131
Undigested Organic Matter .......................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Sulphide-Trapping Agents ..... . ................................................................. 131
Copper-Trapping Agents..................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Post-RuminaIInteractions....................................................................... 132
ASSESSING RISKS OF MOLYBDENUM-INDUCED DISORDERS...................... 133
Analysis of Soils .................................................................................. 133
Analysis of Herbage and Drinking Water....................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Analysis of Blood, Tissue, and Excreta ...................................................... 135

121
0199·9885/9110715-0121$02.00
 122 SUTTLE

TREATING MOLYBDENUM-INDUCED DISORDERS ......................... ........... . 136

TREATING DISORDERS OF COPPER EXCESS ............. ................... ...........
. . 136
CONCLUSIONS....................................................................................... 137

INTRODUCTION

The interaction between copper (Cu), molybdenum (Mo), and sulphur (S) in
ruminant nutrition is probably unique in its effects on health and production.
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No other interaction in ruminants is known to have the capacity to swing the

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nutritional status of its unsuspecting host from deficiency to toxicity while

wholly natural foodstuffs are consumed. Sheep consuming a complete diet,
low in S and Mo and with a modest 12-20 mg Cu/kg dry matter (DM), can
succumb to Cu toxicity (80) while others grazing pasture of similar Cu content
but high in Mo and S can give birth to lambs suffering from the Cu deficiency
disease swayback (2). Bovine Cu deficiency is endemic in regions throughout
the world,and a frequent feature is the presence of high Mo concentrations in
the pastures (47). Affected animals may show only mild symptoms such as
loss of coat condition and poor growth,but under more extreme conditions
stiffness of gait,infertility,diarrhoea,and emaciation are seen (15,50). The
biochemical explanations for the diverse clinical signs are by no means clear,
and no other nutrient interaction in ruminants needs to be understood and
managed with quite the same urgency as that between Cu, Mo, and S.
The complex history of the interaction in ruminants can be summarized by
referring to three earlier reviews. By the mid-1970s it was clear that events in
the rumen explained why the interaction had marked effects on ruminant
species (56). Cu, Mo, and S (from organic or inorganic sources) could
combine in the rumen to foml an unabsorbable triple complex,possibly Cu
tetrathiomolybdate (CuMoS4), thus depleting the tissues of Cu. By the end of
the decade (58),considerable support for the "copper thiomolybdate" hypoth­
esis had accrued and a systemic dimension had been added to it: Cu could be
rendered unavailable post- as well as pre-absorptively by the highly reactive
metabolites thiomolybdates (TMs), formed in the sulphide-rich environment
of the rumen. Recently an unexpected twist has been added by the suggestion
that Mo rather than Cu may be the dominant interactant,exerting toxic effects
on metabolism that Cu can negate (9, 24,48,49). That is,the problem may
be one of Mo excess rather than Cu deficiency: molybdenosis not hypocupro­
sis. The third and most recent review found that the case for the alternative
"TM toxicity hypothesis" was unproven (63), but the issue is so important that
it will provide the framework for this review. Nutritionally valid hypotheses
can and must be established before the outstanding practical problems and
possibilities presented by the Cu-Mo-S antagonism can be addressed.
 CU x MO x S INTERACTIONS IN RUMINANTS 123

THIOMOLYBDATES: NUTRITIONAL DOUBTS AND

CERTAINTIES

Qualitative Evidence for ThiomoLybdate Formation in vivo

Thirteen years passed before the "copper thiomolybdate hypothesis," first
proposed in 1974 (56), gained the necessary experimental corroboration with
the demonstration that TMs could be formed in the rumen under nutritionally
relevant dietary conditions and that they remain stable during transit to the
ileum. Using displacement chemistry and characteristic elution profiles of the
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radio-labelled species, Price et al (52) showed that tri- (MoOSl- or TM3)

and tetrathiomolybdate (MoSi- or TM4) accounted for approximately 41 and
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34%, respectively, of the displaceable 99Mo bound to the solid phase of the
rumen contents of sheep, 16 h after placing 99Mooi- in the rumen; little
2 2
mono- (M003S - or TMJ) or dithiomolybdate (M002S2 - or TM2) was
found. The diet used was grass pellets containing Cu and Mo in a 1 : 1 ratio at
commonly encountered concentrations (6 mg/kg DM). Within individual
sheep the proportion of displaceable TM4 found on the solid phase in the
duodenal and ileal digesta was similar to that found in the rumen. This
observation confirmed the suggestion that, while TMs per se were unstable in
acid solution and unlikely to escape hydrolysis in the abomasum, association
with the solid phase in the rumen conferred stability upon them (3). Further­
more, association with the solid phase would draw the reaction

to the right (66) and give more time for it to occur by delaying outflow from
the rumen (39). What then are the consequences (in terms of the antagonism
of Cu metabolism) of this stable association between TMs and the solid phase
of rumen digesta?

Qualitative Evidence for ThiomoLybdate Inhibition

of Copper Absorption
Allen & Gawthorne (3) showed that addition of TM4 to the rumen increased
the binding of Cu to protozoa, bacteria, and particularly to undigested feed
particles; the addition of Mool- had the same effect. Using neutral detergent
to break down complexes, they found that the bound Cu was associated with
high molecular weight proteins. Others found that preformed TM4 reduced
the absorption of Cu when added to the diet of sheep (66). Furthermore, when
preformed CuTM3 and CuTM4 were placed in the rumen they were quantita-
 124 SUTTLE

tively recovered from the feces with their characteristic spectra (seen after
KCN extraction) intact (72). Digesta solids from sheep given dietary molyb­
denum have a poor capacity to replete cytochrome oxidase activity in the
intestinal mucosa of Cu-depleted rats (51). These findings together suggest
strongly that the "higher" TMs (TM3 and TM4) cause Cu to be irreversibly
bound to high molecular weight proteins and thus reduce Cu absorption. The
lesser substituted oxythioanions are probably not involved because TM2 did
not impair Cu absorption when added to a low S diet; it only impaired Cu
absorption with a high S diet, which would presumably convert TM2 to TM3
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and TM4 (66).

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Qualitative Evidence for Absorption of Thiomolybdates

Before TMs can either add a systemic dimension to their antagonism or exert
direct toxic effects of their own on tissue metabolism, some TMs must first be
absorbed. Price et al (52) showed that small amounts of TM3 and larger
amounts of what was probably TM I were detectable in the duodenal super­
natant 16 h after dosing with 99Mo. Release of TMs probably occurred in the
abomasum, since acidification of rumen solids in vitro (simulating passage
through the abomasum) released TM3 (but not TM4). TM3 alone was detect­
able in the plasma after 16 h, suggesting that TMI (i.e. most of the absorbable
TM) was probably broken down prior to or during absorption. Thus, TM3
alone remained both stable and absorbable, although the proportion of the
dose that finally entered the hepatic portal vein as TM3 and was recovered in
the peripheral blood was not calculated. Presumably, the absorbed TM3 had
been reversibly bound to the solid phase independently of Cu.
Mason and his associates (for reviews see 38,39) have shown that in sheep,
cattle, and deer, labelled TM3 and TM2 were detectable in plasma after large
single doses of 99Mo04 were placed in the sheep's rumen. The same group
detected T� in plasma after ruminal administration, but they concluded that
under normal conditions TM3 was the more likely mediator of any systemic
effects of TM on Cu metabolism (40). The appearance of labelled TM3 in the
bloodstream after its administration via the duodenum confirmed their view
(29). However, the extent of such systemic effects is probably much smaller
when Mo is presented at comparable concentrations to Cu via the diet. A
better perspective of the quantitative importance of TM absorption can be
gained from data for TM excretion in feces.

Quantitative Evidence for Thiomolybdate Excretion in Feces

Since Cu and Mo can be concomitantly and irreversibly bound in reactions
with protein, the inhibition of Mo absorption should be no less than that of Cu
absorption following CuTM complex formation. The complexed elements
 CU x MO x S INTERACTIONS IN RUMINANTS 125

would be equally unavailable to function or antagonize systemically. Raising

the Mo concentration of semi-purified diets or fresh grass to a I : I ratio with
Cu reduces Cu absorption by a factor of three (59); a stoichiometric reduction
in the absorption of Mo (i.e. fourfold in terms of atomic mass) would be
expected. Experiments indicate a smaller though still substantial inhibitory
effect of TM formation on Mo absorption. Addition of S to diets containing
Mool - or TM2 reduced the absorption of Mo from 60 to 30%, probably
through the formation of unabsorbable TM3 and TM4 (66). Price et al (52)
found that half of the 99Mo in rumen (strained), duodenal, and ileal digesta
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was nondisplaceable and presumably nonabsorbable. Thus evidence exists for

substantial though incomplete formation of nonabsorbable TMs under suitable
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rumen conditions. Where it is not present in excess of eu [i.e. the norm for
most Mo-enriched pastures (47)], most of the dietary Mo is probably excreted
in the feces and is not absorbed. If and when TM3 enters the bloodstream,
what are the likely consequences for Cu and Mo metabolism?

Systemic Effects of Thiomolybdates

A vast amount of information on the systemic effects of preformed TMs in
ruminants has been generated, mostly by Mason and his co-workers (for
reviews see 38, 39). These effects mainly involve the inhibition of Cu
metabolism and support the concept, though not necessarily the reality, of a
systemic component to the "copper thiomolybdate hypothesis."

PLASMA COPPER When free TM3 (or TM4 ) is mixed with plasma, it reacts
with albumin and causes Cu to be bound at a site other than the normal
N-terminal locus; at the same time, eu increases the affinity of the site for
TMs (79). The complex with albumin is so strong that it precipitates with the
protein upon acidification, which explains earlier findings of an abnormal
trichloracetic acid (TeA) insoluble fraction in the plasma of sheep exposed to
Mo and S (cf 58). The Cu-TM-albumin complex prepared in vitro has a longer
biological half-life than does copper-albumin when administered to cattle
(25). The delayed clearance of intravenously administered 64CU from plasma
following administration of excess Mo (25 mg/kg diet DM; 55) and of stable
Cu following the intravenous administration of TM3 (44) suggests strongly
that the Cu:TM:albumin complex can form in vivo; however, it has only been
detected following an excessive intake of Mo or following parenteral adminis­
tration of TM3 or TM4.
The important physiological effects of the formation of the Cu-TM-albumin
complex in the bloodstream could be twofold: first, to restrict the availability
of Cu for ceruloplasmin synthesis from absorbed Cu delivered to the liver via
the hepatic portal vein; secondly, to restrict the availability of the absorbed
TM in an effective detoxification mechanism. While the TMs (TM2' TM3,
 126 SUTTLE

and TM4) can reversibly inhibit the diamine oxidase activity of ceruloplasmin
in vitro (30, 35), even TM4 does not do so in vivo (19).

BILIARY COPPER EXCRETION TMs can deplete body reserves of eu by

interfering with the enterohepatic circulation of eu via biliary secretion.
Studies with cannulated sheep showed that the intravenous administration of
TM3 or TM4 enhanced biliary eu secretion (18, 28) and fecal Cu excretion
(41). The removal of Cu from liver can be so swift and substantial after
parenteral administration of TM4 that sheep can be saved from Cu poisoning
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when on the brink of a hemolytic crisis (19, 23). The extent to which
endogenous Cu losses are enhanced following the addition of Mo to the diet
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is, however, debatable; Smith et al (55) found no such enhancement, whereas

it has been implied by others (66). It should be noted that increased
endogenous losses of eu could arise through complex formation with biliary
eu within the intestines as well as systemically.

URINARY COPPER EXCRETION The urinary route of excretion for Cu has

always been regarded as of minor importance when compared with the biliary
route, but this perspective has been formed in the context of excess rather than
deficient supplies of Cu. In terms of unavoidable losses, urinary Cu excretion
may constitute 25% of the total loss and is higher in young than in old animals
(62). Addition of Mo and S to diets with adequate Cu increased urinary eu
excretion (37) and did so independently of the margin of Mo excess (77).
Diuretic effects of the sulphate supplement may contribute to the enhanced
urinary Cu excretion, and the modest increase « I mg Cu per day) may
disappear if dietary Cu status is reduced (37). Injection of TM3 did not
increase urinary Cu excretion in short-term studies (28, 41), but this finding
would be expected if eu was initially bound in nonexchangeable forms to
albumin. Mason et al (42) have suggested, by analogy with the metabolism of
thiotungstates, that Cu:TM:albumin complexes will eventually be hydrolyzed
and their constituent Mo excreted in urine; previously complexed Cu may be
simultaneously released. Long-term feeding of Mo increased the loosely
bound fraction of plasma eu (65) and may thus have increased urinary eu
losses.

COPPER AND MOLYBDENUM IN TISSUES Gawthome (17) suggested that

TMs profoundly altered the equilibria between free Cu and cupro-proteins,
including those with enzyme activity, throughout the body. Under in vitro
conditions, TMs removed eu from metallothionein, which has a very high
3
binding constant (lOiS) for Cu (4). The intravenous administration of 5S_
labelled TM3 to cattle enabled these effects to be demonstrated in vivo. The
oxythioanion was traced to the liver where it arrived intact and shifted eu
 CU x MO x S INTERACTIONS IN RUMINANTS 127

from metallothionein to a high-molecular weight 3sS-labelled protein (75).

Histochemical studies showed that intravenous TM4 removed Cu from both
the lysosomes and cytosol of hepatocytes (33); these effects probably explain
the increased biliary Cu secretion seen in TM4-treated sheep. Wang et al have
suggested that the initial increase in plasma TCA-insoluble Cu following the
parenteral administration of TM3 represented Cu removed from liver stores,
since less was found as liver Cu stores were progressively depleted (76).
However, in ruminants receiving Mo in pasture or forage the most vulnerable
tissue would be the intestinal mucosa, exposed to TMs liberated during
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digestion, rather than the liver. No studies of placental permeability to TMs

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have been carried out. Mo crosses the placenta, but it seems highly unlikely
that TM-protein complexes will do so, although they may accumulate there
with harmful effects on placental metabolism.
The turnover of CuTM- or TM-tissue protein complexes has not been
studied but it is probably slow, contributing to a retention of body Mo.
Dietary molybdate increased Cu and Mo concentrations in kidney in sheep
(58) while addition of S to the diet increased whole body retention of Mo
despite a reduction in Mo absorption (21). The presence of abnormal, un­
reactive Cu-TM-protein complexes in tissues would complicate the assess­
ment of tissue as well as plasma Cu status when vast excesses of Mo are
ingested, giving falsely high estimates of available tissue Cu.

COPPER-DEPENDENT FUNCTIONS When molybdenum is presented via the

diet, the effects of TM4 and insoluble Cu:TM4 complex formation should be
confined to the gut and to the reduction of Cu absorption; and they should not
differ in any way from the effects of straightforward Cu deficiency lucidly
described by Fell (13). Systemic effects of TM3 are possible and gain interest
because of their uncertainty. In the course of absorption across the intestinal
epithelium, this powerful sequestrating agent may remove Cu from superox­
ide dismutase and from cytochrome oxidase (67), which may lead to impaired
local mitochondrial integrity and cell function that manifests itself as di­
arrhoea. Addition of only 3 mg Mo/kg DM to the diet of rats as TM4 produced
gross changes in gut morphology with mitochondrial lesions (14). Elsewhere,
Wang et al (75) estimated that up to 10-15% of an intravenous dose of TM3
was found in bovine liver: Distribution of Cu within subcellular fractions was
altered, but functional consequences were not studied.
The distribution of TM effects on Cu-dependent functions within the body
may not be the same as that caused by straightforward dietary Cu depletion
because of the abnormal binding of eu to what some believe is its principal
carrier protein, albumin. When excess Mo was added to silage and fed to
cattle, coat changes and diarrhoea were seen even though circulating Cu and
ceruloplasmin concentrations remained high (76). Repeated injections ofTM4
 128 SUTTLE

into sheep reduced wool crimp and strength, similar effects to those seen in
Cu deficiency, even though liver and blood Cu concentrations were normal
(20). One possible explanation is that TMs accumulate in the skin and
produce local Cu-depletion in developing hair or fleece. Connective tissue
may be another vulnerable site, given the prominence of periosteal and
cartilagenous tissue changes in lambs exposed to Mo (22). Physiological
barriers may partially protect the fetus and organs such as the brain and spinal
cord from the harmful effects of any circulating TMs.
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MOLYBDENUM METABOLISM AND FUNCTION Effects of TM formation on

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Mo metabolism and function have been largely ignored, but three areas are of
particular interest. The first concerns xanthine oxidoreductase, which can act
as an oxidase (type 0) or dehydrogenase (type D). Is Mo that is absorbed as
TMs available for oxidoreductase synthesis? Does it form complexes that
affect specific activity of the enzyme or its conversion from one mode of
action to another? The conversion from type D to type 0 is stimulated by
Or deprivation and is believed to occur in the joints of rheumatoid arthritis
sufferers (5). Stiffness of gait has long been recognized as a feature of cattle
introduced to Mo-rich pasture (15), but sheep are not affected. Far higher
levels of type 0 activity are found in bovine than in ovine tissues (10), and it
is possible that arthritic changes occur in the joints of grazing cattle but not in
the joints of sheep. Inhibition of the cupro-enzyme cytochrome oxidase could
initiate or increase conversion to type 0 by impairing cell respiration, thus
giving the Cu x Mo antagonism yet another intriguing dimension. The
greater susceptibility of cattle than sheep to Mo-rich pasture, first noted in the
1940s, has yet to be explained, and the answer may be found in Mo rather
than Cu metabolism.
The second area of interest concerns the affinity of Mo for bi-hydroxy
groups (78) such as those on catechol estrogens. Impairment of fertility in
heifers exposed to Mo is believed to involve interference with estrogen
metabolism (48). Interactions of Mo, whether as Mo04 or TM3, with bi­
hydroxy groups on other molecules such as catecholamines and dihydroxy
vitamin D seem possible. Increases in catecholamine concentrations in the
intestinal mucosa have been reported in cattle given Mo. However, a Cu­
containing enzyme, dopamine B hydroxylase, is also involved in catechol­
amine metabolism, and such effects could not be attributed to TM toxicity
rather than Cu deficiency without further study.
The third area concerns proteinases, which are widely distributed in
mammalian tissues and have a variety of functions including immune defense
and blood clotting. Observations that dietary Mo enhanced the pathogenicity
of nematode infections of the gut in lambs while reducing worm burdens (67,
68) led to the study of proteinase activity in the parasite. Proteinases are
 CU x MO x S INTERACTIONS IN RUMINANTS 129

important for parasite entry and migration through the mucosa and for the
digestion of nutrient protein. Proteinase activity was reduced in Trichostron­
gyLus vitrinus recovered from lambs fed molybdate, and the effect was
reproduced by exposing cultured worms to Mool - (with no sulphide source)
in vitro (31). Since other indicators of metabolic activity were not impaired,
specific effects of Mo on proteinases may exist that are independent of TM
formation. Proteinase activities in the host's intestinal mucosa were also
affected by exposure to dietary Mo (D. Knox and N. Suttle, unpublished
data).
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Thus, many more pathways whereby Cu x Mo X S interactions mediate

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their effects on ruminants may possibly exist than have so far been consid­
ered. The involvement of a Mo-sensitive pathway in the Cu x Mo interaction
could influence the biochemical methods used in the detection and prevention
of ill health in stock grazing molybdeniferous pastures, as is discussed in the
latter part of this review.

Conclusions on Physiological Importance of Thiomolybdates

Formation of TMs, particularly TM3 and TM4, in the rumen reduces Cu
absorption in ruminants. Furthermore, as the Mo:Cu ratio in the diet
approaches and exceeds unity, TMs, notably TM3, can be absorbed in
sufficient quantities to change the binding of Cu to albumin. Whether other
changes seen in vitro (e.g. the removal of Cu from metallothionein) or
following the intravenous administration of TM3 occur even under extreme
dietary conditions remains questionable. Responses to Cu supplementation
have usually been obtained on pastures containing only a slight excess of Cu
over Mo and rarely> 8 mg Mo/kg DM (47). Under these conditions, Mo
intakes for a cow might not exceed 80-160 mg per day of which only a small
fraction « 1O%?) would be slowly absorbed as TM3 over 24 h. It is against
such figures that the "physiological" relevance of apparently modest in­
travenous doses of TM3 [e.g. 26 mg Mo for a 400-kg steer (75)] must be
judged. Estimates of the amounts of TM3 arriving at the liver under different
dietary conditions would help to place experiments employing parenteral TMs
in physiological and nutritional perspective.
The new hypothesis that harmful effects of Mo may arise chiefly from Mo
or TM toxicity is founded on the poor correlation between conventional
measurements of low blood and tissue Cu status and the occurrence of clinical
symptoms of Cu deficiency in cattle (9,24,48,49). It is clear,however,that
TMs associate extensively with Cu in unreactive forms, wherever they
appear. Under such circumstances,measurements of total blood, plasma, or
tissue eu will not reflect the ability of Cu enzymes to perform their essential
functions. Most of the clinical signs attributed to "thiomolybdate toxicity" are
the same as those produced by simple Cu deficiency and therefore are likely to
 130 SUTTLE

have arisen frorn irnpaired Cu rnetabolisrn (63). It is possible that fertility is

uniquely vulnerable to the effects of TMs on estrogen rnetabolisrn and alone
responds indirectly to Cu acting as an antidote. Much rnore work needs to be
done, however, to differentiate putative effects of TM toxicity frorn the
well-established, predorninant effects of induced Cu deficiency.

NUTRITIONAL VARIABLES AFFECTING THE RUMEN

INTERACTION
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The vast differences in Cu availability that have been recorded for various
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foodstuffs of low Mo content probably owe rnuch to the effects of diet type on
S rnetabolisrn (61); these effects in tum are arnplified by Mo and fiber, which
affect, respectively, the formation and stability of TMs. Whether or not the
effects of TMs are rnediated through Cu, an understanding of the dietary
factors likely to influence TM formation and absorption is critical to un­
derstanding and rnanipulating the Cu x Mo x S interaction.

Sulphur
Of the three principal cornponents in the interaction, S provides the rnost
opportunities for variation in outcorne because of alternative rnetabolic path­
ways frorn the rurnen. S leaves the rurnen extensively by absorption as
sulphide (S2- ) but also by outflow as undegraded protein S or in rnicrobial
protein. Only degraded protein S and inorganic S frorn the diet or saliva are
available for interaction with Mo and Cu in the rurnen. Partition of degradable
S depends on such factors as supply of degradable nitr:ogen, rate of eating,
rate of S degradation by rurnen rnicrobes, and the rate of arrival of readily
2
fermentable carbohydrate, which influences rurnen pH and hence S - absorp­
tion. Those factors that rnaxirnize the area under the rurnen sulphide con­
centration-versus-tirne curve are likely to increase the formation of higher
TMs if the diet is rich in Mo. For exarnple, continuous feeding of a readily
fermented, serni-purified diet, high in S but low in Mo, increased rurnen
sulphide concentrations and decreased Cu availability in sheep rnore than did
a once daily feeding (71). In addition to levelling the rate at which S entered
the rumen, continuous feeding may have increased the numbers of protozoa in
the rurnen, thus lowering Cu availability by increasing rurnen sulphide forma­
tion (26). Weak expression of the Cu x Mo x S antagonism in sheep without
rurnen protozoa (53) rnay be attributable to reduced "sulphide profiles" and
the decreased opportunities for TM3 and TM4 formation. Differences in the
outcorne of the Cu x Mo x S interaction in terms of Cu availability for sheep
continuously grazing grass or discontinuously fed conserved roughages and
serni-purified diets (59) rnay also involve the kinetics of S rnetabolisrn in the
rurnen.
 CU x MO x S INTERACTIONS IN RUMINANTS 131

Molybdenum
Fewer alternative pathways to leave the rumen are known for Mo than for S.
Mo is not absorbed from the rumen and little is known about its incorporation
into microbial protein. Mo generally occurs in readily soluble and releasable
forms in feeds and is unlikely to occur in undegradable forms that bypass the
Cu x Mo x S interaction in the rumen. The predominant sources of variation
in Mo metabolism are therefore likely to be Cu and S that bind Mo to the solid
phase.
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Copper
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Cu is extensively bound to particulate matter in the rumen and is not absorbed

from the rumen; the bonds may not be completely broken down by digestion
further down the gastrointestinal tract. Some Cu may thus be protected from
interaction with Mo in the rumen, but may remain unavailable for absorption.
Cu protected via association with protein that escapes rumen degradation
may, however, have enhanced availability when it is released in the intestine.
The vulnerability of the ruminant to Cu deficiency is chiefly determined by
these equilibria and by the readiness with which the small fraction of Cu
ingested in feeds and potentially available is then complexed via the interac­
tion with Mo x S or S per se.

Undigested Organic Matter

Diets that allow significant amounts of organic matter to pass undigested from
the rumen should theoretically provide plentiful binding sites for products of
the Cu x Mo x S interaction; readily digested feeds such as cereals and
brassicas should not, and therefore they yield their Cu more readily for
absorption (11). Furthermore, the high fermentable carbohydrate content of
digestible feeds may lower rumen pH and enhance Cu availability by increas­
ing absorption of S2- and breakdown of TMs; this would explain why Mo
(2.5-5. 0 mg/kg DM) did not accelerate the rate of depletion of liver Cu in
sheep given a whole grain diet (N. F. Suttle, unpublished data). Rumen pH is
also relatively low in animals on silage diets, and the high availability of Cu in
Mo-rich silage (60) may be attributable to a combination of rapid digestibility
and low rumen pH. Further evidence of reduced potency of the Cu X Mo X S
interaction on silage diets comes from the study of Wang et al (76) in which it
took 13-14 weeks for diarrhoea to be induced in steers given silage containing
35 mg Mo/kg DM. Similar Mo concentrations in pasture cause immediate
diarrhoea or "teartness" (15).

Sulphide-Trapping Agents
Any agent that competes with molybdate for sulphide in the rumen is likely to
influence the course of the Cu X Mo x S inter action. Iron (Fe) is potentially
 132 SUTTLE

the most important example for grazing animals because large quantities of Fe
are ingested in soil though only a small fraction is likely to be available for
sulphide trapping. Inhibition of Cu metabolism by soluble Fe supplements in
sheep has been partly attributed to trapping of Sl- as FeS in the rumen,
2
followed by release of S - in the acid abomasum to form CuS (64). Brebner
(8) found that the inhibitory effects of soil ingestion on Cu availability in
sheep were correlated with the pyrophosphate-extractable Fe content of the
soil. In the presence of Mo, rumen-soluble Fe may thus reduce TM formation
while continuing to deplete the ruminant of Cu by a different mechanism (CuS
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formation). The two antagonists should therefore have less than an additive
Annu. Rev. Nutr. 1991.11:121-140. Downloaded from www.annualreviews.org

effect when combined, as has indeed been found in calves (9, 24). The
formation of polymeric complexes involving Fe, Mo, and S has been sug­
gested (54), but this would only lead to less than additive Cu-depleting effects
if the polymeric complexes had an affinity for Cu that was less than the
combined affinities of sulphide (released from FeS) and TMs. Other metals
that form acid-labile sulphides could have similar effects to Fe (e.g. Mn), but
those that form acid-insoluble sulphides (such as lead and cadmium) could be
protective by restricting both TM and CuS formation.

Copper-Trapping Agents
In theory, agents other than S2-, which prevent Cu from reacting with TMs in
the rumen, could alter the course of the Cu x Mo x S antagonism; the net
outcome in terms of Cu status would thus depend on the reversibility of any
bonds formed. Fe as Fe203 inhibits Cu absorption independently of any
involvement of sulphide (71). This Fe X Cu antagonism may be explained by
the fact that Fe203 adsorbs Cu even under mildly acid conditions (N. Suttle,
unpublished data); other oxides, for example, of Mn, may do the same. Since
the metal oxides do not compete for sulphide, they would not reduce TM
formation. The effects of Fe203 and Mo as inhibitors of Cu absorption may
still not be fully additive because they compete for a small pool of absorbable
Cu in the rumen. By binding Cu, however, Fe203 may lessen the strength of
binding between TM3 or TM4 and the solid phase and may enhance the
prospects for systemic effects of TMs.

Post-Ruminal Interactions
To confine consideration of the important interactions between Cu, Mo, and S
to the rumen would be a mistake. The cecum, for example, has been shown to
play an important role in S metabolism in ruminants, and it would be
surprising if there was not some reformation of thiomolybdates in the alka­
line, S2- -rich environment provided in this organ. Reactions with the more
concentrated but much changed cecal digesta are hard to predict, but con­
tinuation of Cu x Mo x S interactions in the cecum is clearly possible and
 CU x MO x S INTERACTIONS IN RUMINANTS 133

merits study. Indeed these interactions may be responsible for the most
debilitating consequence of the antagonism, i.e. the onset of diarrhoea.

ASSESSING RISKS OF MOLYBDENUM-INDUCED

DISORDERS

A variety of approaches attempt to confirm or predict the extent to which

excess Mo affects the health and productivity of grazing livestock. Some have
focused on the soil, some on herbage, others on the animal; some restrict
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analysis to Mo while others include the principal interactants, Cu and S. New

knowledge about the pivotal role of TMs in the Cu x Mo x S should now
Annu. Rev. Nutr. 1991.11:121-140. Downloaded from www.annualreviews.org

influence the approaches taken.

Analysis of Soils
Analysis of soils for Mo represents one of the most successful applications of
geochemistry to the improvement of animal health. Thornton (73) used stream
sediment reconaissance in the UK to predict soil Mo and delineate large areas
underlain by molybdeniferous shales where grazing animals were likely to
become hypocupremic. Soil scientists in Canada (12) and Scotland (11) have
begun to map areas of risk using extractable Mo in the soil to predict herbage
Mo, with levels above 5 mg Mo/kg OM in herbage as the threshold. The
well-known effects of soil pH, herbage species, and season on Mo uptake
(e.g. 11, 36, 69) impose limits on the predictability of herbage Mo from soil
Mo. Furthermore, the use of extractable soil Mo cannot resolve these prob­
lems when the procedure standardizes pH (e. g. neutral ammonium acetate)
and ignores plant factors. Herbage Mo did not correlate with extractable soil
Mo in the acid soils of western Kenya (pH range 4.8-5.6; ,.z =11.3%);
although the range of herbage Mo was small (0. 1 to 4.2 mg Mo/kg DM), the
higher levels could induce eu deficiency (27). As a first line of attack,
analysis of the soil for Mo remains a useful approach; it predicts the approx­
imate capacity of the overlying herbage to produce TMs, however they may
act in the body. Whether extraction of soil Mo can and does improve
prediction of ill health remains to be seen.

Analysis of Herbage and Drinking Water

An early attempt to use herbage analysis to predict health problems was made
by Kubota (32), who used legume Mo concentrations to map the soils of the
USA in terms of their Mo status. The ratio of agonist to antagonist should be
important and Cu:Mo ratios in herbage have been used for predictive purposes
(e.g. 6, 45). However, a recent survey indicates that the risk threshold may
not be a fixed ratio (e.g. 3: 1) and that the tolerable ratio declines from 5: 1 to
 134 SUTTLE

2: 1 as pasture Mo concentrations increase from 2 to 10 mg Mo/kg DM (69).

The Agricultural Research Council (1) went one step further by using the
equations of Suttle & McLauchlan (70) for semi-purified diets to predict the
outcome of the full Cu x Mo x S interaction in terms of available Cu.
However, when the approach was tested under grazing conditions in Canada
for prediction of hypocupremia, it was unsuccessful (7).
Since then, studies have shown that different equations are needed not only
for pasture but for various types of conserved pasture that differed by several
orders of magnitude in Cu availability at the same Mo and S concentrations
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(60, 61). The new "grass equation" has been applied to field problems in the
Annu. Rev. Nutr. 1991.11:121-140. Downloaded from www.annualreviews.org

UK, although it could not accommodate particularly high Mo or S pastures

(36). Further difficulties of applying the latest equations in the field were
illustrated by Jumba (27), who sought to predict available Cu concentrations
for different grass species in western Kenya. Most species were sampled at
the mature hay stage while a few, such as Kikuyu grass, remained lush and·
green. The fresh grass equation predicted 2-3 times lower available eu
concentrations than the hay equation for Kikuyu but was considered in­
appropriate for other grass species. The small effects of Mo and S on liver eu
in grazing Merinos in Armidale, NSW (34), compared with those expected
for a temperate grazed sward (59), would probably be more appropriately
predicted by the equation for hay. In two studies, one in lambs (69) and the
other in cattle (7), inclusion of herbage S in prediction equations did not
improve relationships with growth or plasma Cu, but the failure may have
been due to the absence of pastures low in S. Boila et al (7) also pointed out
that in some regions the drinking water was a significant source of S.
Predicting the outcome of Cu x Mo (± S) interactions in the above manner
makes two important assumptions: First, that factors such as Fe can be
ignored, and secondly that the impairment of health is due solely to induced
eu deficiency. In the case of Fe the assumption is necessary because only
limited quantitative information on the effects of Fe on eu absorption has
been published (64, 71) and none relates to Mo-supplemented diets in which
the effect of Fe may be diminished (9,24). This assumption may only lead to
error during periods of limited pasture supply and high soil ingestion when Fe
intakes are maximal. The second assumption may be more serious. If the TM
toxicity hypothesis is valid in some circumstances, prediction of health risks
will have to be based on estimates of Mo excess or capacity for TM forma­
tion. The need for alternative models for prediction will be best demonstrated
by any failure of the appropriate available Cu model (i.e. forage equation) to
predict risks of ill health.
The greater sensitivity of cattle than sheep to the Cu x Mo antagonism has
long been recognized, but recent work has revealed further differences be­
tween species of grazing ruminants that imply a need for yet more specific
 CU x MO x S INTERACTIONS IN RUMINANTS 135

equations for predicting risks of ill health. Mason et al (43) have shown that
the conversion of Mo to TM4 in the rumen is less marked in deer than in sheep
or cattle. Others have suggested that addition of Mo to silage depleted liver
Cu more in sheep than in deer (16), but interpretation was complicated by
species differences in initial Cu stores. Zervas et al (81) have shown that goats
accumulate far less Cu in their livers than do sheep on the same low Mo diet.
It would be surprising if the Cu x Mo x S antagonism did not also vary
between the two species. Species differences may reflect differences in the
extent of enterohepatic recycling of Cu as well as differences in rumen S
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metabolism.
Annu. Rev. Nutr. 1991.11:121-140. Downloaded from www.annualreviews.org

Analysis of Blood, Tissue, and Excreta

The limitations of blood and/or liver Cu concentration as the sole arbiter of Cu
responsiveness in grazing ruminants have long been recognized. While in­
terpretation can be improved and additional criteria of Cu status used (60),
further more drastic changes may be necessary. During the assessment of Cu
responsiveness in lambs on improved Scottish hill pastures, high in Mo, it
was noticed that some growth responses occurred in normocupremic lambs.
Furthermore, the responses waned on the pastures highest in Mo following the
oral administration of CuO needles even though normocupremia was main­
tained (69). Whether or not these effects were related to the direct or Cu­
mediated effects of TM, their existence suggests that conventional standards
(i. e. normal plasma Cu is > 9 /Lmol per liter) do not always predict the
outcome of the Cu X Mo x S interaction.
A novel approach, stemming from the study of Tangdilintin (72), might be
to extract and measure the CuTM complexes from feces to give an integrated
measure of TM formation on a particular diet. Measures of TMs in the
circulation may only be needed at extremely high Mo intakes, and then it may
prove necessary to distinguish "reactive" from "nonreactive" Cu in the blood­
stream. Ceruloplasmin concentration, or rather its ferroxidase activity, has
long been advocated as an alternative to plasma Cu concentration for assess­
ing Cu status in grazing animals; however, ceruloplasmin is an acute phase
protein and is greatly influenced by infection and even vaccination (Irene
Wadsworth, personal communication). If analytical difficulties can be over­
come, assay of SOD activity may be more stable and representative of
functional Cu status (69).
The need for alternative measures of blood Cu status may vary between
species. For example, TM formation is less likely to proceed beyond the TM3
stage in deer (43), and systemic effects may be more important in this species
than in those where the reaction proceeds readily to the TM4 (i.e. nonabsorb­
able) stage. When dietary Mo intakes were increased, TCA-insoluble Cu
increased more markedly in deer than in sheep on a high S diet (46).
 136 SUTTLE

TREATING MOLYBDENUM-INDUCED DISORDERS

The approach to and successful treatment of Mo-induced disorders in grazing

ruminants will be influenced by the mechanisms for induction of the disorder
and the sites affected. Chronically Cu-depleted animals with characteristic
hair, fleece, and bone abnormalities and impaired growth should respond
equally well to dietary Cu supplements, oral Cu boluses, and Cu injections. If
TMs have localized effects on the gut mucosa, these may be countered more
effectively by a continuous supply of Cu in the digesta than by a pulse of Cu
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from an injection that is then slowly dispensed from the liver. If TMs have
widespread sytemic (and toxic) effects on reproduction, it is questionable
Annu. Rev. Nutr. 1991.11:121-140. Downloaded from www.annualreviews.org

whether either oral or parenteral Cu would be capable of fully and in­

stantaneously overcoming them.
The whole strategy of treating and preventing Mo-induced disorders by eu
supplementation is therefore called into question by the TM toxicity hypoth­
esis, and-if it is proven-radical alternatives such as lowering Mo intakes
may have to be considered. Besides the obvious tactic of keeping soil pH as
low as possible to restrict Mo uptake by pasture, more use might be made of
sulphate as fertilizer and antagonist of Mo uptake by the plant. Cereals take up
Mo less avidly than grasses, and analyses of maize silage grown on the
infamous "teart" pastures of Somerset show remarkably low Mo con­
centrations (Alan Adamson, personal communication). Thus by matching
cropping policy to soil conditions and making more use of grazed or con­
served cereals, the harmful effects of Mo-rich soil types might be lessened.

TREATING DISORDERS OF COPPER EXCESS

The Cu x Mo x S interaction has been successfully exploited in the treatment

and prevention of Cu poisoning in sheep. In addition to the remarkable rates
of Cu depletion achieved by parenteral injections of TM4 (19, 23) already
referred to, the dietary antagonism has been used to prevent excessive Cu
accumulation in the liver. Effective concentrations of Mo are far lower than
those investigated in Scandinavia (74) because the protective effect soon
reaches a plateau with increasing concentration (57). This is fortunate because
Mo is still widely regarded as foe rather than friend and, in Europe, additions
to the diet are restricted by law to 2. 5 mg Mo/kg DM. Even at this low level,
risk of toxicity is reduced (57) and many feed compounders employ Mo
routinely as a Cu antagonist. The more that becomes known about the
systemic and toxic effects of TMs, the wiser that policy might prove. It is
important that minimal use is made of parenteral TMs or dietary Mo in
combatting Cu toxicity, because overuse of Mo by either route may have
prolonged adverse effects on fertility.
 CU x MO x S INTERACTIONS IN RUMINANTS 137

CONCLUSIONS

Considerable progress has been made in the last decade in unravelling the
complexities of the Cu x Mo x S interaction in ruminants. The principal
mechanism by which the interaction depletes the grazing animal of Cu is the
formation of unabsorbable complexes with TM3 and TM4 in the rumen and
their irreversible binding to the solid phase of the digesta. When Mo is present
in excess of Cu in S-rich diets, sufficient TM3 may be absorbed to inhibit
cupro-enzyme activity in the gut and peripherally. TM3 may also exert toxic
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effects on estrogen metabolism, resulting in impaired fertility, and on activity

for xanthine oxidoreductase, causing lameness; such effects might respond to
Annu. Rev. Nutr. 1991.11:121-140. Downloaded from www.annualreviews.org

Cu as an antidote. Resolution of the relative importance of gut and systemic

effects of the Cu x Mo x S interaction and of Cu- and Mo-mediated effects
will determine the success of monitoring, treating, and preventing the wide­
spread adverse effects that the Cu x Mo x S interaction currently has on the
health of grazing livestock throughout the world.

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