nutrients

Article
Associations of Changes in Blood Lipid Concentrations with
Changes in Dietary Cholesterol Intake in the Context of a
Healthy Low-Carbohydrate Weight Loss Diet: A Secondary
Analysis of the DIETFITS Trial
Monica Vergara 1 , Michelle E. Hauser 2,3,4 , Lucia Aronica 5 , Joseph Rigdon 6 , Priya Fielding-Singh 7 ,
Cynthia W. Shih 6 and Christopher D. Gardner 5, *

1 Department of Health Research & Policy, Stanford University School of Medicine, Stanford, CA 94305, USA;
mvergara@alumni.stanford.edu
2 General Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305, USA;
mehauser@stanford.edu
3 Medical Service-Obesity Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304, USA
4 Internal Medicine-Primary Care, Fair Oaks Health Center, San Mateo County Health System,
Redwood City, CA 94063, USA
5 Stanford Prevention Research Center, Stanford University School of Medicine, Stanford, CA 94305, USA;
laronica@stanford.edu



6

 Quantitative Sciences Unit, Stanford University School of Medicine, Stanford, CA 94305, USA;
jrigdon@wakehealth.edu (J.R.); cshih@alumni.stanford.edu (C.W.S.)
Citation: Vergara, M.; Hauser, M.E.; 7 Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA;
Aronica, L.; Rigdon, J.; Fielding-Singh, priya.fielding-singh@hci.utah.edu
P.; Shih, C.W.; Gardner, C.D. * Correspondence: cgardner@stanford.edu; Tel.: +1-650-725-2751
Associations of Changes in Blood
Lipid Concentrations with Changes Abstract: In 2015, the Dietary Guidelines for Americans (DGA) eliminated the historical upper limit
in Dietary Cholesterol Intake in the
of 300 mg of dietary cholesterol/day and shifted to a more general recommendation that cholesterol
Context of a Healthy
intake should be limited. The primary aim of this secondary analysis of the Diet Intervention
Low-Carbohydrate Weight Loss Diet:
Examining the Factors Interacting With Treatment Success (DIETFITS) weight loss diet trial was to
A Secondary Analysis of the
evaluate the associations between 12-month changes in dietary cholesterol intake (mg/day) and
DIETFITS Trial. Nutrients 2021, 13,
1935. https://doi.org/10.3390/
changes in plasma lipids, particularly low-density lipoprotein (LDL) cholesterol for those following a
nu13061935 healthy low-carbohydrate (HLC) diet. Secondary aims included examining high-density lipoprotein
(HDL) cholesterol and triglycerides and changes in refined grains and added sugars. The DIETFITS
Academic Editor: Jogchum Plat trial randomized 609 healthy adults aged 18–50 years with body mass indices of 28–40 kg/m2 to
an HLC or healthy low-fat (HLF) diet for 12 months. Linear regressions examined the association
Received: 20 April 2021 between 12-month change in dietary cholesterol intake and plasma lipids in 208 HLC participants
Accepted: 29 May 2021 with complete diet and lipid data, adjusting for potential confounding variables. Baseline dietary
Published: 4 June 2021
cholesterol intake was 322 ± 173 (mean ± SD). At 12 months, participants consumed an average of
460 ± 227 mg/day of dietary cholesterol; 76% consumed over the previously recommended limit
Publisher’s Note: MDPI stays neutral
of 300 mg/day. Twelve-month changes in cholesterol intake were not significantly associated with
with regard to jurisdictional claims in
12-month changes in LDL-C, HDL-C, or triglycerides. Diet recall data suggested participants’ increase
published maps and institutional affil-
in dietary cholesterol was partly due to replacing refined grains and sugars with eggs. An increase in
iations.
daily dietary cholesterol intake to levels substantially above the previous 300 mg upper limit was
not associated with a negative impact on lipid profiles in the setting of a healthy, low-carbohydrate
weight loss diet.

Copyright: © 2021 by the authors.

Keywords: dietary cholesterol; low-carbohydrate; LDL cholesterol; HDL cholesterol; triglycerides;
Licensee MDPI, Basel, Switzerland.
human study; weight loss trial; healthy adults; diet quality; eggs
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Nutrients 2021, 13, 1935. https://doi.org/10.3390/nu13061935 https://www.mdpi.com/journal/nutrients

Nutrients 2021, 13, 1935 2 of 14

1. Introduction
Poor diet is a key risk factor for cardiovascular disease (CVD), the leading cause of
death in the United States [1]. Until recently, a “heart-healthy diet” included a recommen-
dation to limit dietary cholesterol to no more than 300 mg/day [2–5]. This recommendation
was based on a body of evidence that included both interventional studies and obser-
vational epidemiology studies suggesting links between dietary cholesterol and blood
concentrations of LDL cholesterol (LDL-C), a major risk factor for CVD [6–8]. However,
evidence has failed to show a clear dose–response relationship between dietary cholesterol
intake and LDL-C levels [9–11]. A lack of clear evidence for selecting 300 mg/day of dietary
cholesterol as a specific upper limit led to the removal of the upper limit in the 2015–2020
Dietary Guidelines for Americans (DGA), although there is still a general recommendation
to limit cholesterol intake [12].
A 2019 American Heart Association (AHA) Science Advisory position statement
reviewed the existing evidence on the associations between dietary cholesterol, LDL-C, and
CVD with the aim of providing recommendations to clinicians [13]. Discrepancies between
different studies’ findings were examined and highlighted the importance of considering
two important features: (1) confounding with saturated fat, which is found in most foods
high in cholesterol, and (2) overall diet quality and food patterns, which may modify the
effect of cholesterol on LDL-C and CVD risk [14]. Eggs, in particular, have been singled out
in debates about the health impacts of dietary cholesterol as they are the most consumed
high-cholesterol food in the United States yet have relatively low levels of saturated fat. A
recent prospective study of a half-million people in nine European countries reported an
inverse relationship between egg intake and ischemic heart disease [15]. There is a paucity
of data on the effects of a high-cholesterol diet or a diet high in egg consumption where
this intake occurs in the context of an overall high-quality diet.
The Diet Intervention Examining the Factors Interacting with Treatment Success
(DIETFITS) randomized controlled weight loss trial provides an opportunity to examine
the effect of increased dietary cholesterol intake on blood concentrations of LDL-C in
the context of consuming a high-quality diet. In addition to focusing on weight loss, the
intervention aimed to help participants in one arm reduce carbohydrate intake via a healthy
low-carbohydrate (HLC) diet. Many of the low-carbohydrate foods regularly chosen by
participants in this arm were animal-derived, particularly eggs, and were therefore high
in cholesterol. Extensive data collection over a 12-month period (i.e., baseline, 3, 6, and
12 months) included plasma lipid measurements and multiple 24-hour diet recalls [16,17].
In this secondary analysis of the DIETFITS trial, the primary objective was to assess the
associations between 12-month change in dietary cholesterol intake and 12-month change in
plasma lipids, particularly LDL-C. Secondary objectives included exploration of the extent
to which changes in dietary cholesterol intake were due to changes in egg consumption
and whether changes in egg consumption were associated with changes in refined grain
and added sugar intake.

2. Materials and Methods

2.1. Study Design
Detailed methods of the DIETFITS trial were published previously [16]. In brief, it
was a randomized controlled weight loss trial of 609 participants assigned to follow either
HLC or healthy low-fat (HLF) diets. The original trial employed a parallel study design
with the primary aim of assessing the effects of both diets on weight change and whether
these effects were modified by genetic factors (i.e., multilocus genotype pattern) or insulin
secretion over a 12-month period. The original randomized controlled trial was powered to
detect this effect modification, with a sample size of around 300 per arm. Participants were
instructed to focus on eating high-quality diets based on whole foods while achieving the
lowest intake of either net carbohydrate (HLC) or fat (HLF) they could maintain long-term;
there was no specific caloric restriction target. Informed written consent was obtained from
all participants.
Nutrients 2021, 13, 1935 3 of 14

Participants were men and premenopausal women aged 18 to 50 years with body
mass indices (BMI, kg/m2 ) of 28 to 40, who were otherwise generally healthy. Exclusion
criteria included pregnancy, lactation, being within 6 months postpartum, or planning to
become pregnant during the study; diabetes, metabolic disorders, or cancer; cardiovascular,
renal, or liver disease; or use of any medications that altered lipids, blood pressure, weight,
or energy expenditure. The trial was conducted between January 2013 and May 2016. The
intervention was a 12-month protocol that consisted of 22 diet-specific group education
sessions with registered dietitian nutritionists and clinical health educators. The study
was single-blinded, as subjects could not be blinded to their diet assignment. All data
were collected at baseline, 3, 6, and 12 months [17]. Of the 609 participants, 304 were
randomized to the HLC arm. This secondary analysis included participants in the HLC
arm with complete plasma lipid and diet data at baseline and 12 months (n = 208).
Dietary intake data were collected via three, unannounced, 24-hour dietary recalls
within a two-week window at each data collection time point. Food composition and
energy data were collected using the Nutrition Data System for Research (NDSR) software
from the University of Minnesota Nutrition Coordinating Center. A standardized, multiple-
pass interview approach was used for dietary recalls [16]. Blood samples used for plasma
lipid analysis were collected from participants after a fast of at least 10 h. Plasma lipid
data from fasting blood samples were analyzed by a laboratory certified by the lipid
standardization program of the National Heart, Lung, and Blood Institute of the Centers
for Disease Control and Prevention (Krauss Lab, Children’s Hospital Oakland Research
Institute, Oakland, CA, USA). The LDL-C concentrations (normal range < 200 mg/dL)
were calculated using the Friedewald equation (note that only one participant at only one
time point had a triglyceride measurement over 400 mg/dL). HDL-C and triglyceride
levels were measured using standard methods [16].
Data collectors were blinded to diet assignment. Participant data were managed in
Research Electronic Data Capture (RedCap)—an electronic data-capture tool hosted at
Stanford University [18]. Participants provided informed consent as approved by the
Stanford University Human Subjects Committee.

2.2. Statistical Analysis

Descriptive statistics were used to summarize baseline diet and health characteristics,
demographic information, and dietary cholesterol intake. Statistical tests (chi-square tests
for categorical variables, Fisher’s exact test with Monte Carlo approximation used for
cells with n < 5, one-way ANOVA for continuous variables) were also performed to assess
differences in demographic and baseline characteristics between HLC participants included
in this secondary analysis (n = 208) and those with missing data (n = 96) who were excluded.
Least squares regression was used to compare 12-month change in dietary cholesterol
intake (per 100 mg) with 12-month change in plasma LDL-C (mg/dL). Models were run
both unadjusted and adjusted for age, sex, baseline dietary cholesterol intake, 12-month
change in saturated fat intake, and 12-month weight change. While the regression analyses
were done using continuous data, for ease of visual comparison 12-month changes in lipid
variables (i.e., LDL-C, HDL-C, and triglycerides) are displayed in tables and figures by
tertiles of 12-month change in dietary cholesterol intake (i.e., low change, moderate change,
and high change). Correlations between 12-month change in dietary cholesterol intake and
12-month changes in LDL-C, HDL-C, and triglycerides were computed using Pearson’s
correlation coefficients, and 12-month changes in lipids are displayed in box plots by tertile
of dietary cholesterol intake.
For comparison to the HLC analyses described above, all descriptive and primary
analyses were repeated for the HLF group. Because the focus of this analysis is on the HLC
group, and due to limitations of the number of tables and figures in the main paper, some
of the HLF data and findings are only available in the supplemental materials.
Nutrients 2021, 13, 1935 4 of 14

Significance levels for all analyses were set at an alpha of 0.05. All statistical analyses
were performed using SAS University Edition (SAS Studio 3.6; SAS Institute Inc., Cary,
NC, USA).

3. Results
3.1. Baseline Characteristics of the Study Population
Of the 304 participants randomized to the HLC arm (Figure A1), 208 (68%) had
complete diet and lipid data at baseline and 12 months and were included in this complete
case analysis. There were no significant differences between the HLC subjects included in
the analysis (n = 208) and those excluded (n = 96) with regard to sex, race, education, or
baseline diet, but those with missing data were younger (38 ± 6.9 years vs. 40 ± 6.6 years,
mean ± standard deviation (SD), heavier (99 ± 15 kg vs. 95 ± 16 kg), and had lower mean
LDL-C level (109 ± 25 mg/dL vs. 116 ± 26 mg/dL) (Table A1).
Participants reported consuming a mean of 322 ± 173 (SD) mg of dietary cholesterol
per day at baseline, with 103 participants (~50%) reporting consuming >300 mg/day. The
following characteristics were not significantly different among the tertiles of 12-month
change in dietary cholesterol intake: age, sex, race, education, baseline weight, and plasma
lipids (Table 1). Baseline levels of cholesterol intake were not significantly different between
the tertiles. Baseline carbohydrate intake was similar among the tertiles (p = 0.18). There
were inverse relationships between 12-month change in dietary cholesterol intake and
baseline levels of cholesterol (mg), total fat (g), saturated fat (g), protein (g), and calories
(kcal; all p < 0.01). At baseline, the lowest, middle, and highest tertiles of 12-month dietary
cholesterol change consumed 414 ± 200, 307 ± 148, and 245 ± 120 mg/day (mean ± SD),
respectively (Table 1). Baseline diet and demographic data for the HLF arm are shown in
Table A2.
Table 1. Baseline demographics of participants by tertiles of 12-month change in dietary cholesterol intake (mg) 1 in the
healthy low-carbohydrate arm (HLC).

HLC-Tertile 2
Total Lowest Middle Highest
(n = 208) (n = 69) (n = 70) (n = 69) p-Value 3

Age 40 ± 6.6 40 ± 6.6 40 ± 6.7 41 ± 6.3 0.45

Gender, n (%)
Female 125 (60) 45 (65) 42 (60) 38 (55) 0.48
Male 83 (40) 24 (35) 28 (40) 31 (45)
Race, n (%) 4
White 151 (73) 52 (75) 48 (70) 51 (74) 0.66
Other 56 (27) 17 (25) 21 (30) 18 (26)
Education, n (%)
High School 7 (3) 4 (6) 1 (1) 2 (3)
College Graduate 118 (57) 39 (57) 39 (56) 40 (58) <0.001 5
Postgrad Degree 83 (40) 26 (37) 30 (43) 27 (39)
Body Weight (kg) 95 ± 16 95 ± 15 94 ± 16 94 ± 17 0.94
Baseline Diet
Calories (kcal) 2198 ± 638 2338 ± 613 a 2271 ± 702 a 1985 ± 537 b 0.002
Carbohydrates (g) 243 ± 73 252 ± 72 246 ± 78 231 ± 69 0.18
Fat (g) 91 ± 34 100 ± 32 a 96 ± 36 a 78 ± 28 b 0.0002
Saturated Fat (g) 30 ± 13 34 ± 14 a 32 ± 12 a 25 ± 10 b <0.0001
Protein (g) 92 ± 29 101 ± 30 a 92 ± 28 ab 85 ± 26 b 0.003
Cholesterol (mg) 322 ± 173 414 ± 200 a 307 ± 148 b 245 ± 120 c <0.0001
Lipids (mg/dL)
LDL-C 6 116 ± 26 119 ± 27 115 ± 24 114 ± 27 0.43
HDL-C 6 50 ± 9 51 ± 8 51 ± 9 49 ± 9 0.54
Triglycerides 125 ± 105 128 ± 62 119 ± 53 131 ± 65 0.48
1 Data are expressed as means ± standard deviation (SD) unless otherwise indicated; 2 HLC: healthy low-carbohydrate, tertiles are based on
12-month change in dietary cholesterol intake from baseline (mg/day); 3 p-values calculated by chi-squared tests for categorical variables
and one-way ANOVA for continuous variables for the three tertile columns; significance level set at α = 0.05; 4 207/208 participants
reported their race/ethnicity; 5 Fisher’s exact test with Monte Carlo approximation used to calculate p-value for cells with less than 5;
a,b,c for baseline characteristics with p-value < 0.05 across tertiles from one-way ANOVA, pairwise differences are indicated by superscripts;

pairs with a shared superscript are not different as determined by unpaired t-test; 6 LDL-C: low-density lipoprotein cholesterol, HDL-C:
high-density lipoprotein cholesterol.
Nutrients 2021, 13, 1935 5 of 14

3.2. Changes in Cholesterol Intake

At 12 months, mean intake of dietary cholesterol was 460 ± 227 (SD) mg/day in the
HLC group—an increase of 42.9% from baseline—and most participants (76%) reported
consuming >300 mg/day. From baseline to 12 months, mean dietary cholesterol for the
total HLC group included in the analyses increased by 137 ± 206 mg/day. In comparison,
the total HLF group decreased cholesterol consumption by 82 ± 161 and had a mean intake
of 238 ± 128 at 12 months. In the HLC group, the lowest tertile had a 12-month mean
change of −102 ± 131 mg/day, and the middle and highest tertiles increased by 112 ± 56
and 401 ± 185 mg/day, respectively (Table 2a,b). The interquartile ranges for 12-month
change in dietary cholesterol intake (mg/day) were (−148.4, −20.0), (62.7, 153.5), and
(274.1, 476.2) for the lowest, middle, and highest tertiles of the HLC arm, respectively.

Table 2. (a) Mean changes from baseline in diet, weight, and lipids by tertile of 12-month change in dietary cholesterol
intake for (a) the healthy low-carbohydrate arm. (b) Mean changes from baseline in diet, weight, and lipids by tertile of
12-month change in dietary cholesterol intake for the healthy low-fat arm (HLF).

(a)
HLC-Tertile
Total Lowest Middle Highest
(n = 208) (n = 69) (n = 70) (n = 69)
Total dietary cholesterol intake at 12 months (mg) 460 ± 227 312 ± 159 420 ± 160 647 ± 214
12-month change in dietary cholesterol intake (mg) 137 ± 206 -102 ±131 112 ± 56 401 ± 185
Range (min, max) (−760, 1332) (−760, 26) (32, 230) (230, 1332)
Calories (kcal) −507 ± 617 −664 ± 584 −578 ± 643 −277 ± 559
Carbohydrates (g) −112 ± 76 −104 ± 68 −104 ± 83 −128 ± 75
Saturated Fat (g) −1.9 ± 14 −7.7 ± 14 −3.3 ± 13 5.4 ± 11
Body Weight (kg) −6.3 ± 6.8 −4.4 ± 6.4 −6.5 ± 6.5 −8.0 ± 6.9
LDL-C (mg/dL) 3.5 ± 20 2.8 ± 20 2.7 ± 18 5.1 ± 23
HDL-C (mg/dL) 2.8 ± 6.5 2.2 ± 6.2 2.4 ± 5.8 3.9 ± 7.4
Triglycerides (mg/dL) −25 ± 48 −17 ± 44 −24 ± 46 −35 ± 52
(b)
HLF-Tertile
Total Lowest Middle Highest
(n = 208) (n = 69) (n = 70) (n = 69)
Total dietary cholesterol intake at 12 months (mg) 238 ± 128 181 ± 108 216 ± 107 316 ± 127
12-month change in dietary cholesterol intake (mg) −82 ± 161 −265 ±103 −61 ± 39 79 ± 74
Range (min, max) (−655, 365) (−655, −144) (−144, −4.4) (0, 365)
Calories (kcal) −485 ± 627 −723 ± 700 −422 ± 593 −310 ± 509
Carbohydrates (g) −36 ± 81 −38 ± 90 −37 ± 84 −33 ± 71
Saturated Fat (g) −11 ± 12 −19 ± 12 −8.8 ± 11 −6.6 ± 10.2
Body Weight (kg) −5.6 ± 7.3 −7.0 ± 7.6 −5.5 ± 8.1 −4.1 ± 5.7
LDL-C (mg/dL) −2.1 ± 20 −3.9 ± 18 −0.8 ± 24 −1.6 ± 18
HDL-C (mg/dL) 0.2 ± 6.0 −0.5 ± 6.2 0 ± 6.0 1.0 ± 5.6
Triglycerides (mg/dL) −11 ± 55 −14 ± 51 −13 ± 55 −6.1 ± 59
Data expressed as means ± standard deviation; positive change indicates an increase from baseline. HLC: healthy low-carbohydrate, HLF:
healthy low-fat, LDL-C: low-density lipoprotein cholesterol, HDL-C: high-density lipoprotein cholesterol.

3.3. Correlations between Changes in Cholesterol Intake and Lipid Profile

The 12-month changes in cholesterol intake (Figure 1A) were not significantly corre-
lated with changes in LDL-C (r = 0.04, p = 0.53), HDL-C (r = 0.1, p = 0.16), or triglycerides
(r = −0.1, p = 0.06) for the HLC arm. Levels of LDL-C were relatively stable across all time
points for all tertiles of the HLC arm (Figure 1B). Similar lack of significant correlations
(LDL-C (r = 0.09, p = 0.18), HDL-C (r = 0.1, p = 0.15), triglycerides (r = 0.03, p = 0.69)) and
Nutrients 2021, 13, 1935 6 of 14

Nutrients 2021, 13, x FOR PEER REVIEW 7 of 16

LDL-C relative stability across time points for all tertiles were observed for the HLF arm
(Figure 2).

Figure 1. (A) Box plots of 12-month changes in dietary cholesterol intake by tertile of 12-month
Figure 1. (A) Box plots of 12-month changes in dietary cholesterol intake by tertile of 12-month
change in dietary cholesterol for healthy low-carb (HLC). Boxes represent interquartile range (IQR);
change in dietary cholesterol for healthy low-carb (HLC). Boxes represent interquartile range
center line in box is median, “X” within the box is the mean; upper and lower tails are 1.5X IQR.
(IQR); center line in box is median, “X” within the box is the mean; upper and lower tails are 1.5X
(B)
IQR.Box(B)plots of 12-month
Box plots changes
of 12-month in LDL
changes cholesterol
in LDL intake
cholesterol by tertile
intake of 12-month
by tertile change
of 12-month in low-
change in
density lipoprotein (LDL) cholesterol for healthy low-carb (HLC). Boxes represent
low-density lipoprotein (LDL) cholesterol for healthy low-carb (HLC). Boxes represent interquar-interquartile
range (IQR);
tile range center
(IQR); line line
center in box is median,
in box “X” “X”
is median, within the box
within is the
the box is mean; upper
the mean; and and
upper lower tails are
lower
1.5X
tails IQR.
are 1.5X IQR.

The 12-month change in LDL-C for each 100 mg increase in daily dietary cholesterol
intake in the HLC arm was not statistically significant in either the unadjusted model
(0.37 mg/dL; 95% confidence interval (CI): −0.14 to 6.2) or the model adjusted for age,
gender, baseline dietary cholesterol intake, and 12-month changes in saturated fat intake
and weight (0.01 mg/dL; 95% CI: −1.4 to 1.5) (Table 3a,b). The 12-month changes in
HDL-C and triglycerides per 100 mg increase in daily dietary cholesterol intake were also
not statistically significant for either the HLC or the HLF arm.
 Figure 1. (A) Box plots of 12-month changes in dietary cholesterol intake by tertile of 12-month
change in dietary cholesterol for healthy low-carb (HLC). Boxes represent interquartile range
(IQR); center line in box is median, “X” within the box is the mean; upper and lower tails are 1.5X
Nutrients 2021, 13, 1935 IQR. (B) Box plots of 12-month changes in LDL cholesterol (LDL-C) intake by tertile of 12-month
7 of 14
change in LDL-C for healthy low-carb (HLC). Boxes represent interquartile range (IQR); center
line in box is median, “X” within the box is the mean; upper and lower tails are 1.5X IQR.

(n = 208) (n = 208)

(n = 208) (n = 208)

Figure 2. Levels of both dietary cholesterol intake and low-density lipoprotein cholesterol (LDL-C) (unadjusted for saturated
fat or other factors) in the healthy low-carbohydrate (HLC) and healthy low-fat (HLF) arm at baseline, 3, 6, and 12 months:
(A) HLC cholesterol; (B) HLC LDL-C; (C) HLF cholesterol; (D) HLF LDL-C.

Table 3. (a) Linear regression model estimates for 12-month changes (mg/dL) in LDL-C, HDL-C, and triglycerides associated
with each 100 mg increase in daily dietary cholesterol intake among those assigned to follow a healthy low-carbohydrate
diet (HLC). (b) Linear regression model estimates for 12-month changes (mg/dL) in LDL-C, HDL-C, and triglycerides
associated with each 100 mg increase in daily dietary cholesterol intake among those assigned to follow a healthy low-fat
diet (HLF).

(a)
Model #2 1 Model #3 2
Model #1
Outcome Adjusted Estimate Adjusted Estimate
Unadjusted Estimate (95% CI)
(95% CI) (95% CI)
LDL-C (mg/dL) 0.37 (−0.7 to 1.5) 0.30 (−1.0 to 1.6) 0.01 (−1.4 to 1.4)
HDL-C (mg/dL) 0.26 (−0.1 to 0.6) 0.12 (−0.3 to 0.6) 0.05 (−0.4 to 0.5)
Triglycerides (mg/dL) −2.52 (−5.2 to 0.1) −0.92 (−4.0 to 2.1) 0.06 (−3.1 to 3.3)
(b)
Model #3 2
Model #1 Model #2 1
Outcome Adjusted Estimate
Unadjusted Estimate (95% CI) Adjusted Estimate(95% CI)
(95% CI)
LDL-C (mg/dL) 1.17 (−0.5 to 2.7) 1.40 (−1.0 to 3.8) 0.57 (−1.9 to 3.0)
HDL-C (mg/dL) 0.37 (−0.13 to 0.88) 0.24 (−0.5 to 0.9) 0.10 (−0.6 to 0.8)
Triglycerides (mg/dL) 0.95 (−3.7 to 5.65) 0.71 (−5.4 to 6.9) 0.61 (−5.8 to 7.0)
LDL-C: low-density lipoprotein cholesterol, HDL-C: high-density lipoprotein cholesterol, CI: confidence interval. 1 Adjusted for age,
gender, baseline dietary cholesterol intake (mg/day), and 12-month change in weight (kg); 2 adjusted for age, gender, baseline dietary
cholesterol intake (mg/day), and 12-month changes in both saturated fat intake (g) and weight (kg).
Nutrients 2021, 13, 1935 8 of 14

3.4. Qualitative Analysis of Sources of Dietary Cholesterol Increase

The study health educators that advised DIETFITS study participants throughout the
trial indicated that many of those assigned to the HLC arm frequently consumed eggs for
breakfast, substituting for previous habitual breakfast fare. Figure A2 shows the mean
intake of eggs and refined grains for each tertile of 12-month change in dietary cholesterol
intake at each time point in the study in the HLC arm. Mean intake was 0.86 ± 0.9 eggs/day
at baseline and 1.45 ± 1.1 eggs/day at 12 months for all participants (n = 200, p < 0.001;
egg data missing for 8 participants). The average egg contains approximately 207 mg of
cholesterol, meaning participants’ mean dietary cholesterol intake from eggs was 178 mg
and 315 mg at baseline and 12 months, respectively [19]. A secondary objective in this
study was to explore the extent to which changes in dietary cholesterol intake were due to
changes in egg consumption and whether changes in egg consumption were associated
with changes in refined grain and added sugar intake. Participants in the highest tertile ate
an average of one egg per day at baseline and two eggs per day at 12 months, while those
in the lowest tertile ate approximately one egg per day at both time points. All tertiles
reduced both sugar and refined grain consumption.

4. Discussion
In this secondary analysis of the DIETFITS weight loss trial, substantial 12-month
changes in dietary cholesterol were not significantly associated with 12-month changes in
LDL-C, HDL-C, or triglycerides among those assigned to follow a healthy, low-carbohydrate
diet. The findings were similar before and after adjustment for age, gender, baseline dietary
cholesterol intake, and 12-month changes in saturated fat intake and weight.
The one-third of the study population with the greatest 12-month change in mean
dietary cholesterol intake increased from 245 mg/day to nearly 650 mg/day—more than
double the 300 mg/day upper limit recommended in the 2010 and prior Dietary Guidelines
for Americans. Notably, this portion of the study population also substantially increased
average egg consumption from approximately one egg/day to two eggs/day. The calorie
reduction in each tertile at each time point after baseline appears to be largely due to
decreases in refined grains and added sugars, with some but not all of those calories being
replaced by an increase in egg consumption for the middle and high tertiles.
These results are supportive of the change in recent guidelines that eliminate recom-
mending a specific upper cut point for cholesterol intake. From 1968 to 2015, both the
American Heart Association (AHA) and the Dietary Guidelines for Americans (DGA) rec-
ommended no more than 300 mg/day of dietary cholesterol, stating that dietary cholesterol
intake should be restricted due to its positive association with total and LDL cholesterol
concentrations [2,3,5–7,20–23]. Consumption of eggs, especially egg yolks, was advised
against for those aiming for heart health [2]. However, recent updates to the AHA and DGA
guidelines have eliminated specific dietary cholesterol target recommendations, reflecting
a shift away from emphasizing specific nutrients (e.g., cholesterol) and foods (e.g., eggs) to-
ward a greater focus on heart-healthy dietary patterns such as healthy Mediterranean-style,
DASH-style, and vegetarian-style diets [13].
This study’s findings align with the 2020 AHA Scientific Advisory position statement’s
general recommendation to focus primarily on establishing healthy dietary patterns, rather
than on trying to limit cholesterol intake. However, our findings are not consistent with the
AHA’s specific recommendation regarding amount. The AHA recommendation is to limit
egg intake to current levels, with healthy individuals being able to include a whole egg
or equivalent daily. Our findings suggest a potentially modified recommendation on the
basis of impact on LDL-C concentrations, i.e., up to two eggs/day. While the HLC group
in the DIETFITS trial reported a wide range of 12-month changes in dietary cholesterol,
no significant association with 12-month changes in LDL-C was observed. Mean LDL-C
levels remained relatively stable for participants regardless of change in dietary cholesterol
intake over 12 months. More specifically, egg intake also did not seem to be associated
with LDL-C levels. The third of the study population that had the least change in dietary
Nutrients 2021, 13, 1935 9 of 14

cholesterol intake consumed approximately one egg/day at both baseline and 12 months,
while the third of the study population that saw the greatest increase in cholesterol intake
also had the greatest increase in egg consumption, doubling mean intake from one egg/day
at baseline to two eggs/day at 12 months.
Notably, these findings occurred in the context of a weight loss study that involved
shifts to an overall more healthful dietary pattern including substantial reductions in added
sugars, refined grains, and overall energy intake. The AHA and DGA guidelines refer to
dietary intake broadly and not specifically to intake in the setting of a weight loss diet.
Regardless, it is clear that dietary cholesterol intake is not universally detrimental with
regard to blood lipid levels and warrants further investigation in the settings of weight
loss and weight neutral diets as well as among various dietary patterns.
In the landmark feeding studies of Hegsted and Keys from the 1950s and 1960s, satu-
rated fat and dietary cholesterol were both determined to adversely affect plasma lipids,
with saturated fat having the greatest impact [6,7,11]. However, Keys later published a
review of the literature concluding that dietary cholesterol did not, in fact, play a major
role in increasing serum cholesterol levels [24]. In line with Keys and the recent 2019 AHA
Scientific Advisory on cholesterol, Carson et al. conducted a meta-analysis of 11 controlled
studies and reported no significant association between dietary cholesterol and LDL-C [13].
Notably, the 11 studies selected from >50 randomized controlled trials [25] were all feeding
studies, and all had similar polyunsaturated/saturated fatty acid ratios between compar-
ison diets; the added rigor of these inclusion criteria provides a plausible explanation
for differences between the Advisory conclusions and previous studies [6,7,11,25]. Our
findings are aligned with the AHA Advisory report.
This study had several strengths. First, the study was conducted with a relatively
large sample size over 12 months. Second, the study population included a weight range
that was generalizable to much of the US population and had near equal representation
of the genders. Additionally, there was extensive dietary data assessment using the gold
standard for dietary data collection with 78% of participants completing 100% of the staff-
administered, unannounced, multipass, 24-hour dietary recalls. Finally, study participants’
wide variability in 12-month change in dietary cholesterol intake increased the likelihood
of identifying an association with LDL-C should an association exist.
The study also had notable limitations. First, this was a secondary analysis from a
randomized weight loss diet trial that was not designed to specifically test for causal effects
of changes in dietary cholesterol intake on plasma lipids. Second, this analysis focused on
only one of two intervention groups, thereby shifting to an observational design. Third, the
use of tertiles of 12-month change in cholesterol for presentation purposes led to several
important and potentially confounding differences at baseline between the tertiles (e.g.,
differential baseline cholesterol intake). However, this was addressed in the regression
analysis which was done using continuous variables and adjusted for potential confounders.
Complete data were available for only 68% of potential participants; fortunately, most
baseline characteristics were similar between participants and those excluded due to
missing data. Exceptions were age, baseline weight, and LDL-C. Despite using the most
rigorous type of dietary data assessment tool, by nature of self-report, the data are subject
to inaccuracies. In addition, energy (kcal) intake data were based on self-report, and
therefore energy intake was likely underreported; however, this is a common, unfortunate
but accepted aspect of nutritional trials. As we were looking at change in intake, we would
expect underreporting to be consistent across time for participants and, therefore, not to
affect our analyses to a large degree. Finally, as HLC-group participants’ egg consumption
increased, both their refined grain and sugar consumption decreased. While this suggests
that participants replaced some of the energy intake from carbohydrate-rich food sources
with eggs, future studies should rigorously analyze the dynamics and impacts of such
dietary shifts on LDL-C.
In conclusion, the results of this secondary analysis suggest that physicians concerned
about their patients’ blood lipid levels should take into consideration contextual factors in
Nutrients 2021, 13, 1935 10 of 14

determining how to counsel them about dietary cholesterol intake, particularly the source
of the cholesterol and the overall dietary quality pattern. Confusion or concern about
dietary cholesterol might be avoided if the important inter-relationships of nutrients, foods,
and food patterns are considered when advising patients on what to avoid and limit.

Author Contributions: Conceptualization, M.V., C.W.S. and C.D.G.; methodology, M.V., J.R., C.W.S.
and C.D.G.; validation, J.R.; formal analysis, M.V. and J.R.; investigation, M.V. and C.D.G.; resources,
C.D.G.; data curation, J.R.; writing—original draft preparation, M.V.; writing—review and editing,
M.V., M.E.H., L.A., J.R., P.F.-S., C.W.S., C.D.G., M.V. and C.W.S.; funding acquisition, C.D.G. All
authors have read and agreed to the published version of the manuscript.
Funding: M.E.H. and P.F.-S. were supported by the National Heart, Lung, and Blood Institute NIH
T32HL007034. L.A. was supported by the European Union’s Horizon 2020 Research and Innovation
Programme under grant agreement No. 701944. C.D.G. was supported by grant 1R01DK091831 from
the National Institute of Diabetes and Digestive and Kidney Diseases, and the Nutrition Science
Initiative (NuSI). Other support came from the Stanford Clinical and Translational Science Award
(CTSA) to Spectrum NIH UL1 TR001085.
Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki and approved by the Institutional Review Board of Stanford University
(protocol #22305, last approved 6-30-20).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the
study.
Data Availability Statement: Data described in the manuscript, code book, and analytic code will
be made available upon request pending application and approval by the corresponding author.
Acknowledgments: This study would not have been possible without the work of many research
team members. Jen-nifer Robinson and Antonella Dewell served as study coordinators. The team of
health educators included Rise Cherin, Susan Kirkpatrick, Jae Berman, Dalia Perelman, and Mandy
Murphy Car-roll. The diet assessment team included Sarah Farzinkhou, Valerie Alaimo, Margaret
Shimer Lawton, and Diane Demis. Various other important study roles in recruitment, screening,
blood sample management, innovation, and other tasks were played by Josephine Hau, Erin Avery,
Al-exandra Rossi, and Katherine Dotter. Excellent administrative support was provided by Alana
Koehler. All were affiliated with Stanford University at the time of the study, received compensa-tion
for their work, and have agreed to be acknowledged here. Finally, we would like to acknowledge the
participants of the DIETFITS study, without whom this research would not have been possible.
Conflicts of Interest: The authors declare no conflict of interest.
Nutrients 2021, 13, 1935 11 of 14

Appendix A

Figure A1. Participant flow of DIETFITS trial.

Nutrients 2021, 13, 1935 12 of 14

Figure A2. (A) Egg, (B) refined grain, and (C) sugar consumption at each time point by tertile of 12-month change in dietary
cholesterol in the healthy low-carb arm (HLC).

Table A1. Missing data analysis.

Complete Missing
p-Value 1
(n = 208) (n = 96)
Age 40 ± 6.6 38 ± 6.9 0.02
Sex, n (%)
Female 125 (60) 54 (56) 0.5
Male 83 (40) 42 (44)
Race, n (%) 2
White 151 (73) 62 (68) 0.4
Other 56 (27) 29 (32)
Education, n (%)
High School 7 (3) 8 (8)
College Graduate 118 (57) 55 (58) 0.1
Postgrad Degree 83 (40) 32 (34)
Body Weight (kg) 95 ± 16 99 ± 15 0.04
Nutrients 2021, 13, 1935 13 of 14

Table A1. Cont.

Complete Missing
p-Value 1
(n = 208) (n = 96)
Baseline Diet
Calories (kcal) 2198 ± 638 2275 ± 686 0.3
Carbohydrates (g) 243 ± 73 253 ± 8 0.3
Fat (g) 91 ± 34 95 ± 34 0.3
Saturated Fat (g) 30 ± 13 32 ± 13 0.2
Protein (g) 92 ± 29 95 ± 34 0.4
Lipids (mg/dL)
LDL-C 116 ± 26 109 ± 25 0.03
HDL-C 50 ± 9 49 ± 9 0.4
Triglycerides 125 ± 105 133 ± 137 0.6
LDL-C: low-density lipoprotein cholesterol, HDL-C: high-density lipoprotein cholesterol. 1 p-value calculated by chi-square test for
categorical variables and t-test for continuous variables; significance level set at α = 0.05; 2 207/208 complete participants reported race;
91/96 missing participants reported race; 95/96 missing participants reported education level.

Table A2. Baseline demographics of participants by tertiles of 12-month change in dietary cholesterol intake (mg) 1 in the
healthy low-fat arm (HLF).

HLF-Tertile 2
Total Lowest Middle Highest
p-Value 3
(n = 208) (n = 69) (n = 70) (n = 69)
Age 39 ± 6.6 39 ± 7.0 39 ± 6.6 39 ± 6.1 0.86
Gender, n (%)
Female 115 (55) 32 (54) 45 (64) 38 (55) 0.10
Male 93 (45) 37 (46) 25 (36) 31 (45)
Race, n (%) 4
White 152 (74) 58 (85) 46 (66) 48 (71) 0.20
Other 54 (26) 10 (25) 24 (30) 21 (29)
Education, n (%) 5
High School 4 (2) 2 (6) 1 (1) 1 (1)
College Graduate 104 (50) 40 (57) 32 (46) 32 (47) <0.001 6
Postgrad Degree 99 (48) 26 (37) 37 (53) 36 (52)
Body Weight (kg) 97 ± 14 99 ± 14 95 ± 15 96 ± 14 0.17
Baseline Diet
Calories (kcal) 2209 ± 665 2435 ± 683 a 2140 ± 590 a 2051 ± 667 a 0.002
Carbohydrates (g) 251 ± 87 264 ± 100 252 ± 77 237 ± 80 0.19
Fat (g) 89 ± 34 103 ± 33 a 84 ± 32 b 81 ± 34 b 0.0001
Saturated Fat (g) 30 ± 13 34 ± 11 a 28 ± 12 b 27 ± 13 b 0.0004
Protein (g) 94 ± 27 106 ± 26 a 89 ± 22 b 87 ± 28 b <0.0001
Cholesterol (mg) 320 ±151 446 ± 149 a 278 ± 109 b 237 ± 110 c <0.0001
Lipids (mg/dL)
LDL-C 111 ± 31 107 ± 24 110 ± 34 115 ± 34 0.29
HDL-C 49 ± 9 48 ± 11 50 ± 8 50 ± 9 0.52
Triglycerides 130 ± 75 133 ± 68 131 ± 88 127 ± 67 0.89
HLF: healthy low-fat, LDL-C: low-density lipoprotein cholesterol, HDL-C: high-density lipoprotein cholesterol. 1 Data are expressed as
means ± standard deviation unless otherwise indicated; 2 tertiles are based on 12-month change in dietary cholesterol intake from baseline
(mg/day); 3 p-values calculated by chi-squared tests for categorical variables and one-way ANOVA for continuous variables for the three
tertile columns; significance level set at α = 0.05; 4 206/208 participants reported their race/ethnicity; 5 207/208 participants reported their
education level; 6 Fisher’s exact test with Monte Carlo approximation used to calculate p-value for cells with less than 5; a,b,c for baseline
characteristics with p-value < 0.05 across tertiles from one-way ANOVA, pairwise differences are indicated by superscripts; pairs with a
shared superscript are not different as determined by unpaired t-test.
Nutrients 2021, 13, 1935 14 of 14

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