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A Moderate Interleukin‐6 Reduction, Not a Moderate Weight Reduction,

Improves the Serum Iron Status in Diet‐Induced Weight Loss with Fish Oil
Supplementation

Article  in  Molecular Nutrition & Food Research · July 2018

DOI: 10.1002/mnfr.201800243

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RESEARCH ARTICLE
Fish Oil www.mnf-journal.com

A Moderate Interleukin-6 Reduction, Not a Moderate Weight

Reduction, Improves the Serum Iron Status in Diet-Induced
Weight Loss with Fish Oil Supplementation
Shih-Yi Huang, Nindy Sabrina, Yi-Wen Chien, Yi-Chun Chen, Shyh-Hsiang Lin,
and Jung-Su Chang*

1. Introduction
Background & Aims: Whether moderate weight loss or a reduction in IL-6
improves the serum iron status in overweight (OW) and obese adults The prevalence of metabolic syndrome
(MetS) in Taiwan has risen over the
supplemented with or without fish oil is explored. past two decades, and these trends co-
Methods and Results: In total, 93 OW/obese Taiwanese adults with ࣙ2 incide with expansion of the obesity
metabolic components are randomized to a 12-week calorie-restricted diet epidemic.[1] Excess intake of energy and
with meal replacement alone (CRMR, n = 45) or supplemented with fish oil a decrease in physical activity are closely
(CRMRF, n = 48). Mean reductions in the %body weight and serum IL-6 are associated with the obesity epidemic.
For overweight (OW) and obese peo-
7.5% versus 5.9% and 21% versus 35% for the CRMR and CRMRF groups,
ple, a hypocaloric diet or the combina-
respectively. In the CRMRF group, a moderate loss of IL-6 (reduced ࣙ35%) tion of a hypocaloric diet and moder-
also significantly improves the serum iron and transferrin saturation ate exercise can induce weight loss, and
compared to those with loss of <35% in the mean serum IL-6 or those of the >5% weight loss can improve a num-
CRMR group who has a moderate loss of IL-6 (reduced ࣙ21%) (all p < 0.05). ber of cardiometabolic profiles.[2,3] Al-
In contrast, modest weight loss does not improve the serum iron status. though an energy-restricted diet, particu-
larly the standard energy-restricted diet,
Conclusions: Fish oil is ineffective (45–65% carbohydrate, 10–20% protein,
as an adjunct for weight or fat loss but has beneficial effects on preserving the and <35% fat), is the cornerstone of
lean body mass. A significant improvement in the iron status is only observed obesity treatment,[4] no consensus has
in those with moderate loss of serum IL-6 supplemented with fish oil. yet been reached on the most effective
hypocaloric dietary programs for treating
obesity or MetS.[5]
Prof. S.-Y. Huang, MSc. N. Sabrina, Prof. Y.-W. Chien, Prof. Y.-C. Chen, Obesity is characterized as a state of low-grade inflammation,[6]
Prof. S.-H. Lin, Prof. J.-S. Chang and increased systemic inflammation is associated with cen-
School of Nutrition and Health Sciences tral obesity,[7] dysregulated iron metabolism,[8] and a risk of
College of Nutrition MetS.[9] Increased IL-6 levels are known to upregulate hepcidin
Taipei Medical University
synthesis.[10,11] Hepcidin is the master regulator of iron home-
Taipei 110, Taiwan
E-mail: susanchang@tmu.edu.tw ostasis, and elevated hepcidin can lead to lower circulating iron
Prof. S.-Y. Huang, Prof. Y.-W. Chien, Prof. J.-S. Chang levels but higher iron retention within tissues due to hepcidin-
Graduate Institute of Metabolism and Obesity Sciences mediated ferroportin degradation.[12,13] In addition, abnormal
College of Nutrition levels of both IL-6 and iron biomarkers (e.g., serum ferritin) are
Taipei Medical University independent predictors of central obesity,[7] liver injury,[13] and
Taipei 110, Taiwan
MetS.[14–16]
MSc. N. Sabrina
Nutrition Program Fish oil (FO) or n-3 long chain PUFA supplementation was
Faculty of Food Technology and Health shown to reduce inflammation[6] and the risk of MetS.[17–21] How-
Sahid Jakarta University ever, the effects of FO on weight loss and the body composition
Jakarta 12870, Indonesia are still uncertain.[22–24] Although 5–10% weight loss is routinely
Prof. Y.-W. Chien, Prof. J.-S. Chang recommended for obese people,[3] the effects of moderate weight
Nutrition Research Center
loss and FO supplementation on the iron status are not known.
Taipei Medical University Hospital
Taipei 110, Taiwan The current study was designed to explore whether the addition
Prof. J.-S. Chang of supplemental omega-3 fatty acids in conjunction with a
Chinese Taipei Society for the Study of Obesity hypocaloric diet (1) would provide greater improvement in body
CTSSO compositions, and (2) whether a moderate weight loss or a
Taipei 110, Taiwan reduction in IL-6 could improve the serum iron status over a
DOI: 10.1002/mnfr.201800243

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3-month treatment period in OW and obese Taiwanese adults
with ࣙ2 metabolic components.
2. Experimental Section
2.1. Ethical Statement
This single-center open-label parallel-arm controlled trial was
conducted at the School of Nutrition and Health Sciences, Taipei
Medical University and its affiliated hospitals, Taipei Medical
University Hospital (TMUH) and Wan Fang Hospital (WFH) in
Taipei, Taiwan. Subjects were recruited through local commu-
nity advertisements, and the intervention program was carried
out over the course of 12 weeks, with an initial and final visit
to either TMUH or WFH between May 2012 and March 2013.
This study was approved by the Joint Institutional Review Board
of Taipei Medical University (no. 201110026), was conducted in
accordance with the Declaration of Helsinki, and was registered
at ClinicalTrials.gov (NCT01768169). Written informed consent
was obtained from all participants.
2.2. Study Design and Data Collection
The concept and procedure of the random allocation were accord-
ing to the publication of Kim and Shin.[25] Briefly, the simple ran-
domization was used to obtain random numbers and assigning
random numbers to each dietary treatment.[26] Randomization of
study subjects was generated by a computer program to prevent
the selection bias by choosing subjects for different treatment
groups entirely by chance. Except for the dieticians, investigators
and staff were kept blind to dietary treatment of the participants.
The anticipated mean difference in the change of body weight by
fish oil supplementation was obtained from Hill et al.[27] and Ke-
shavarz et al.[28] Based on their data, we and Keshavarz et al.[28]
estimated a corresponding SD of weight change to be 4.8[27] or
3.4.[28] At 80% power and 5% significance level, the calculated
sample size was 23 or 50 per intervention group, respectively. We
thus initially recruited 50 participants per arm.
2.3. Inclusion and Exclusion
Subjects were included if they were i) ࣙ20 years, ii) overweight
(OW) or obese, and iii) had ࣙ2 components of MetS. The body–
mass index (BMI) was calculated as the mass (kg) height (m)–2 .
Being OW and obese were defined based on criteria of the World
Health Organization for Asia, which defines being OW as a BMI
of ࣙ24 to <27 kg m–2 and being obese as a BMI of ࣙ27 kg m–2 .[29]
Central obesity was defined as a waist circumference (WC) of
ࣙ90 cm in men and ࣙ80 cm in women. MetS was defined as
patients with at least two of the following criteria based on the
modified National Cholesterol Education Program Adult Treat-
ment Panel III for the Asia-Pacific: i) a systolic blood pressure
of ࣙ130 mm Hg or diastolic blood pressure of ࣙ85 mm Hg,
ii) fasting plasma glucose (FPG) of ࣙ100 mg dL–1 , iii) high-
density lipoprotein cholesterol (HDL-C) of <40 mg dL–1 in men
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or <50 mg dL–1 in women, and iv) fasting triglycerides (TGs) of
ࣙ150 mg dL–1 .[29] Participants were excluded if they had i) a his-
tory of diabetes, coronary heart disease, cardiovascular disease,
of renal, liver, endocrine, or psychiatric diseases, ii) alcohol, sub-
stance abuse, or a smoker, iii) cancer or immunodeficiency, iv)
pregnant, planning a pregnancy, or breastfeeding, v) sensitive or
allergic to fish products, and vi) having used n-3 PUFA supple-
ments or medicines (lipid-lowering, antihypertensive, or hypo-
glycemic medication).
2.4. Calorie-Restricted Meal-Replacement Dietary Intervention
A subject’s total daily energy requirement was determined by reg-
istered dieticians prior to the intervention. The basal metabolic
rate was calculated using the Harris–Benedict equation and an
energy deficiency of 500–800 calories d–1 was applied to the to-
tal energy requirements of each participant. The weight reduc-
tion target was 0.5–1 kg per week. Subjects were randomly as-
signed to a calorie-restricted meal-replacement (CRMR) diet or
a CRMR with fish oil supplementation (CRMRF) group. The
CRMRF group received ten capsules of fish oil (Herbalifeline;
Herbalife, Los Angeles, CA, USA) daily, which contained a to-
tal of 2.13 g of n-3 PUFAs (including 1.28 g of eicosapentaenoic
acid [EPA] and 0.85 g of docosahexaenoic acid [DHA]). Partic-
ipants underwent a weekly group dietary counseling program
by registered dieticians for 10 weeks. Participants were asked to
prepare their breakfast according to a sample menu or informa-
tion given by dietitians. A calorie-restricted lunch box (500 kcal
per lunch box containing 53% carbohydrates, 17% protein, and
30% fat) was prepared and handed to participants during the 12-
week intervention period. Dinner consisted of 8 g of low-calorie,
high-protein nutrition drink mix powder (Herbalife) dissolved in
240 mL of skimmed milk. The low-calorie, high-protein nutri-
tional drink contained a total of 275 kcal (60% carbohydrates,
35% protein, and 5% fat). Final macronutrients of the total calo-
ries of the CRMR (55.3% carbohydrates, 17% proteins, and 27.7%
fat) and CRMRF diets (53.8% carbohydrates, 16.8% proteins, and
29.4% fat) were similar. Compliance was monitored by checking
the remaining Herbalife nutrition drink mix powder and fish oil
capsules that had been given to participants during the group di-
etary counseling sessions.
2.5. Evaluation of the Body Composition
The body composition was recorded at the baseline and at week
12 by dual-energy X-ray absorptiometry (DEXA; GE Healthcare,
Madison, WI, USA) by an experienced technologist.[30] DEXA was
calibrated according to standard procedures recommended by
the manufacturer, and DEXA data were analyzed using Lunar en-
CORE 2006 software vers. 10.50.086 (GE Healthcare). Body com-
positions were obtained as the total body-fat mass of the trunk,
and android and gynoid regions using Lunar Prodigy DEXA, and
results are expressed as the total fat mass (kg), lean body mass
(kg), total body fat mass (%), android fat mass (%), and gynoid
fat mass (%).[30] The body composition, BMI, and WC were cal-
culated during each of two visits (at the baseline and week 12).

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Figure 1. Flowchart diagram of participant selection (n = 93). Subjects were randomly assigned to a CRMR (n = 45) or a CRMRF (n = 48) group for 12
weeks intervention.
2.6. Clinical and Blood Biochemical Assesments
Fasting blood samples were collected from overnight-fasted par-
ticipants at the baseline and week 12, and serum and plasma
were stored at −80 °C until being analyzed. Heparinized whole
blood was collected to measure hemoglobin (Hb). Serum iron
and the total iron-binding capacity (TIBC) were measured using
a ferrozine-based colorimetric method. The percentage transfer-
rin saturation (%TS) was determined as (serum iron/TIBC) ×
100. Serum ferritin (SF) was measured by an electrochemilumi-
nescent immunoassay and was quantitated with a Roche Modu-
lar P800 analyzer (Mannheim, Germany). Serum hepcidin (DRG
International; Marburg, ST, USA) and IL-6 (R&D Systems, Min-
neapolis, MN, USA) were analyzed by an ELISA, according to the
manufacturer’s instructions. Serum C-reactive protein (CRP) lev-
els were evaluated using an automated analyzer (Toshiba TBA-
c16000, Toshiba, Tokyo, Japan). Serum aspartate aminotrans-
ferase (AST) and alanine aminotransferase (ALT) were measured
using a colorimetric method.
2.7. Definition of Iron Deficiency and Iron-Deficiency Anemia
Anemia was defined as Hb of <13 g dL–1 for men and post-
menopausal women, and Hb of <12 g dL–1 for reproductive-aged
women. Iron depletion was considered if any of the two iron indi-
cators showed abnormal values: SF <20 ng mL–1 and %TS <30%.
An iron deficiency was noted if both iron indicators showed ab-
normal values: SF ˂12 ng mL–1 and %TS <15%. Iron-deficiency
anemia (IDA) was defined as SF ˂12 ng mL–1 , %TS <15%, and
Hb <12 g dL–1 .
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2.8. Statistical Considerations
Statistical analyses were conducted using SPSS 19 (IBM, Ar-
monk, NY, USA). Categorical and continuous variables are pre-
sented as the number (percentage, %) and mean ± SD, respec-
tively. Percent (%) changes were calculated as ([end point value
– baseline value]/baseline value) × 100%. The change was cal-
culated as the end point value – baseline value. The Mann–
Whitney U test was used to compare data between groups. The
Wilcoxon signed-ranks test for paired samples was used to com-
pare changes within groups over the time course of the study. The
time × diet interaction is presented as a p-value derived from a
repeated-measures analysis of variance (ANOVA) after adjusted
for baseline serum IL-6 and AST levels. p < 0.05 was considered
statistically significant.
3. Results
3.1. Participants’ Characteristics Prior to the Intervention
Figure 1 shows flowchart diagram of participant selection. In
total, 25 men and 68 women were included in the study, and
participants were randomly allocated to the FO supplementation
(CRMRF = 48 people) or placebo group (CRMR = 45 people).
The mean age was 49.0 ± 13.6 years (CRMR: 48.3 ± 13.48 years
and CRMRF: 49.7 ± 13.9 years), and the mean BMI was 29.4
± 4.21 kg m–2 (CRMR: 29.5 ± 4.0 kg m–2 and CRMRF: 29.3 ±
4.4 kg m–2 ). Table 1 shows no differences in age, gender, BMI,
body composition, or iron profiles. However, the CRMRF group

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Table 1. Baseline characteristics of the participants stratified by diet mass compared to –0.54 ± 1.4 kg of lean mass in the CRMRF
(n = 93). group (p = 0.001, Table 2). No statistically significant differences
in changes in the WC, android fat mass, or iron biomarkers
a) b)
Variables Diet p-Value were observed in either group.

c)
CRMR (n = 45) d)
CRMRF (n = 48)

Age (years) 48.29 ± 13.40 49.73 ± 13.88 0.513 3.3. Moderate Weight Loss did not Improve the Serum
Male (n, %) 14 (31.1%) 11 (22.9%) 0.373 Iron Status
Body weight (kg) 79.40 ± 14.79 75.72 ± 17.30 0.117
BMI (kg m–2 ) 29.45 ± 3.97 29.29 ± 4.35 0.779 Mean percent body weight losses in the CRMR and CRMRF
Waist circumference (cm) 93.92 ± 9.95 93.67 ± 12.40 0.460 groups were –7.51 ± 5.25% and –5.91 ± 2.78%, respectively
Central obesity (n, %) 44 (97.8%) 47 (97.9%) 0.963
(Table 2, p < 0.05). We next stratified participants by the mean
reduction in body weight: 7% for the CRMR and 6% for the
Body composition
CRMRF group (Table 3). Moderate weight losses (ࣙ7% for
Total fat mass (kg) 30.72 ± 8.21 28.99 ± 9.28 0.258
the CRMR and ࣙ6% for the CRMRF group) were associated
Lean body mass (kg) 45.78 ± 10.09 43.19 ± 9.76 0.230
with greater changes in the WC and body compositions (fat,
Total body fat (%) 40.11 ± 7.24 39.73 ± 6.45 0.456 android, and gynoid fat masses) compared to those who failed
Android fat (%) 48.26 ± 6.37 48.90 ± 5.08 0.979 to achieve moderate weight loss (Table 3, all p < 0.05). When
Gynoid fat (%) 43.08 ± 8.44 42.03 ± 7.60 0.278 comparing both groups who achieved moderate weight loss, FO
A/G ratio 1.15 ± 0.17 1.19 ± 0.17 0.103 supplementation was associated with smaller reductions in body
Iron status weight (–10.8 ± 3.10 and –8.23 ± 2.22 kg, p = 0.002), total fat
Serum iron (μg dL–1 ) 78.69 ± 34.16 79.33 ± 27.72 0.564 mass (–21.57 ± 7.87 and –16.31 ± 6.48 kg, p = 0.009), and CRP
Serum hepcidin (ng mL–1 ) 144.2 ± 3.7 143.5 ± 3.9 0.765 (–39.51 ± 38.15 and –9.20 ± 44.78 ng mL–1 , p = 0.019) but higher
Serum Ferritin (ng mL–1 ) 118.62 ± 94.95 142.17 ± 126.91 0.634
preservation of the lean body mass (–4.66 ± 3.44 and –1.56 ± 3.38
kg, p = 0.003) (all p < 0.05) (Table 3). However, moderate weight
Hemoglobin (μg dL–1 ) 14.08 ± 3.08 14.19 ± 1.57 0.675
loss did not improve the serum iron status in either group.
Transferrin Saturation (%) 26.12 ± 11.76 27.18 ± 9.35 0.368
Anemia (n, %) 5 (11.1%) 2 (4.2%) 0.205
IDA (n, %) 1 (2.2%) 1 (2.1%) 0.963
Iron deficiency (n, %) 3 (6.7%) 2 (4.2%) 0.593
3.4. A Moderate Serum IL-6 Reduction Improved the Serum Iron
Status in the FO-Supplemented Group
Iron depletion (n, %) 6 (13.3%) 3 (6.3%) 0.248
Inflammatory status
Because serum IL-6 levels are closely associated with the serum
CRP (ng mL–1 ) 0.44 ± 0.43 0.38 ± 0.38 0.567
iron status, we stratified participants by the mean reduction in
IL-6 (pg mL–1 ) 3.14 ± 1.83 4.31 ± 2.49 0.010 serum IL-6 levels: 21% for the CRMR and 35% for the CRMRF
AST (U L–1 ) 25.04 ± 13.57 29.29 ± 10.64 0.007 group (Tables 2 and 4). Table 4 shows that in the CRMRF group,
ALT (U L–1 ) −20.47 ± 25.68 −7.70 ± 38.70 0.358 a moderate loss of IL-6 (a ࣙ35% drop in the mean serum IL-6
level) significantly improved serum iron and transferrin satura-
a)
Continuous data are presented as mean ± SD, categorical data are presented
as numbers (percentages); b) p-Value was analyzed using the Mann–Whitney test tion (%TS) compared to those who did not achieve a moderate
for continuous variables and Chi-square for categorical variables; c) CRMR, calorie- IL-6 reduction (a <35% reduction in the mean serum IL-6 level
restriction meal-replacement diet; d) CRMRF, calorie-restriction meal-replacement in the CRMRF group) or to the CRMR group who had achieved a
diet with fish oil supplementation. moderate IL-6 reduction (a ࣙ21% reduction in the mean serum
IL-6 level in the CRMR group) (all p < 0.05). Notably, the relation-
ship between the IL-6 reduction and serum iron levels was less
had higher baseline levels of serum IL-6 and AST compared to evident in the CRMR group.
the CRMR group (both p < 0.05, Table 1).

3.2. Effects of the Interventions
Changes in anthropometric measurements, iron, and inflam-
matory biomarkers are shown in Table 2. Both groups lost on
average >5% of their initial body weight and exhibited signifi-
cant decreases in the BMI, WC, body composition, and serum
inflammatory biomarkers (CRP, IL-6, and ALT) (Table 2, all
p < 0.05). In general, FO supplementation induced slighter
losses of body weight, fat mass, and gynoid fat mass but had
greater preservation of the lean body mass (p < 0.05). Notably,
the CRMR group lost on average –1.58 ± 2.2 kg of lean body
4. Discussion
The present study was designed to investigate potential weight-
loss benefits of FO supplementation in OW/obese adults with ࣙ2
metabolic components. Our study found that both groups lost on
average >5% of their body weight, but a moderate weight reduc-
tion did not affect the systemic iron status or IL-6 levels (Table 3).
A significant improvement in the serum iron status was only ob-
served in those with a moderate IL-6 reduction supplemented
with FO. Overall, our study suggests that FO supplementation
did not work as an adjunct for body weight or fat loss, but the
addition of FO had beneficial effects of preserving the lean body

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Table 2. Intervention outcome at baseline and 12 weeks.

b) c)
Variables Diet p-Value Adjusted repeated ANOVA

CRMR (n = 45) CRMRF (n = 48) Diet × Time

Body weight (kg) 0.046

Baseline 79.4 ± 14.8 75.7 ± 17.3
Week 12 73.5 ± 14.4 71.3 ± 16.7
a)
p-Value <0.001 <0.001
Change −5.91 ± 4.44 −4.42 ± 2.42 0.036
Change (%) −7.51 ± 5.25 −5.91 ± 2.78 0.044
BMI (kg m–2 ) 0.077
Baseline 29.45 ± 3.97 29.29 ± 4.35
Week 12 27.27 ± 4.14 27.57 ± 4.35
a)
p-Value <0.001 <0.001
Change −2.19 ± 1.57 −1.70 ± 0.84 0.035
Change (%) −7.52 ± 5.25 −5.90 ± 2.78 0.039
Body composition
WC (cm)
Baseline 93.92 ± 9.95 93.67 ± 12.40 0.900
Week 12 87.84 ± 11.45 87.72 ± 12.27
a)
p-Value <0.001 <0.001
Change −6.09 ± 6.85 −5.95 ± 3.30 0.835
Change (%) −6.48 ± 7.45 −6.39 ± 3.57 0.803
Fat mass (kg) 0.003
Baseline 30.72 ± 8.21 28.99 ± 9.28
Week 12 26.23 ± 8.86 26.16 ± 9.33
a)
p-Value <0.001 <0.001
Change −4.49 ± 2.84 −2.83 ± 2.29 0.002
Change (%) −15.79 ± 10.22 −10.50 ± 8.02 0.009
Lean mass (kg) 0.008
Baseline 45.78 ± 10.09 43.19 ± 9.76
Week 12 44.20 ± 9.77 42.66 ± 9.39
a)
p-Value <0.001 0.010
Change −1.58 ± 2.21 −0.54 ± 1.41 0.001
Change (%) −3.37 ± 4.87 −1.07 ± 3.02 0.001
Android fat mass (%) 0.594
Baseline 48.26 ± 6.37 48.90 ± 5.08
Week 12 44.99 ± 7.81 46.27 ± 5.99
a)
p-Value <0.001 <0.001
Change −3.27 ± 3.56 −2.63 ± 2.37 0.782
Change (%) −7.06 ± 8.02 −5.54 ± 5.12 0.647
Gynoid fat mass (%) 0.010
Baseline 43.08 ± 8.44 42.03 ± 7.60
Week 12 39.80 ± 9.40 40.33 ± 8.01
a)
p-Value <0.001 <0.001
Change −3.28 ± 2.92 −1.71 ± 2.85 0.001
Change (%) −8.30 ± 7.73 −4.17 ± 6.91 0.002
Total body fat (%) 0.123
Baseline 40.11 ± 7.24 39.73 ± 6.45
Week 12 36.96 ± 8.56 37.38 ± 7.08
*
p-Value <0.001 <0.001

(Continued)

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Table 2. Continued.

b) c)
Variables Diet p-Value Adjusted repeated ANOVA

CRMR (n = 45) CRMRF (n = 48) Diet × Time

Change −3.15 ± 2.76 −2.35 ± 2.17 0.068

Change (%) −8.63 ± 7.97 −6.20 ± 5.85 0.108
Iron status
Serum iron (μg dL–1 ) 0.218
Baseline 78.69 ± 34.16 79.33 ± 27.72
Week 12 73.71 ± 24.21 82.85 ± 34.69
a)
P-value 0.299 0.576
Change −4.98 ± 35.61 3.52 ± 30.36 0.374
Change (%) 8.54 ± 55.03 8.69 ± 39.61 0.484
% TS 0.521
Baseline 26.12 ± 11.76 27.18 ± 9.35
Week 12 25.53 ± 9.03 28.09 ± 12.42
a)
p-Value 0.986 0.975
Change −0.59 ± 12.05 0.91 ± 10.37 0.988
Change (%) 13.45 ± 53.92 7.42 ± 43.23 0.442
Serum Ferritin (ng mL–1 ) 0.357
Baseline 118.6 ± 94.9 142.1 ± 126.9
Week 12 110.2 ± 104.6 142.6 ± 136.5
a)
p-Value 0.481 0.593
Change −0.59 ± 12.05 0.91 ± 10.37 0.358
Change (%) −0.34 ± 45.34 1.45 ± 29.22 0.701
Serum hepcidin (ng mL–1 ) 0.469
Baseline 144.2 ± 3.7 143.5 ± 3.9
Week 12 140.5 ± 20.3 132.2 ± 16.9
a)
p-Value 0.308 0.964
Change −2.75 ± 18.5 −11.3 ± 16.2 0.445
Change (%) −2.56 ± 12.4 −7.87 ± 22.2 0.442
Hemoglobin (μg dL–1 ) 0.744
Baseline 14.08 ± 3.08 14.19 ± 1.57
Week 12 14.15 ± 1.29 14.12 ± 1.53
a)
p-Value 0.767 0.337
Change 0.07 ± 2.96 −0.08 ± 0.69 0.641
Change (%) 0.25 ± 21.42 −0.60 ± 5.15 0.593
Inflammatory status
CRP (ng mL–1 ) 0.103
Baseline 0.44 ± 0.43 0.38 ± 0.38
Week 12 0.29 ± 0.33 0.32 ± 0.34
a)
p-Value 0.001 0.010
Change –0.15 ± 0.29 –0.06 ± 0.26 0.312
Change (%) -17.01 ± 68.78 -1.68 ± 52.81 0.072
IL-6 (pg mL–1 ) 0.045
Baseline 3.14 ± 1.83 4.31 ± 2.49
Week 12 2.00 ± 1.11 2.27 ± 1.32
a)
p-Value <0.001 <0.001
Change −1.13 ± 1.66 −2.04 ± 2.53 0.141
Change (%) −21.11 ± 52.74 −35.83 ± 40.08 0.140
AST (U L–1 ) 0.239
Baseline 25.04 ± 13.57 29.29 ± 10.64

(Continued)

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Table 2. Continued.

b) c)
Variables Diet p-Value Adjusted repeated ANOVA

CRMR (n = 45) CRMRF (n = 48) Diet × Time

Week 12 22.82 ± 7.23 24.35 ± 8.28

a)
p-Value 0.061 <0.001
Change −2.22 ± 12.14 −4.94 ± 9.90 0.088
Change (%) 1.02 ± 43.30 −12.76 ± 23.42 0.088
ALT (U L–1 ) 0.966
Baseline 20.47 ± 25.68 7.70 ± 38.70
Week 12 23.07 ± 11.64 27.25 ± 17.60
a)
p-Value <0.001 0.001
Change −6.69 ± 19.78 −6.85 ± 17.43 0.997
Change (%) −10.21 ± 42.49 −11.69 ± 35.39 0.842

Continuous data are presented as mean ± SD, categorical data are presented as numbers (percentages); a) Difference between baseline and 12 weeks was analyzed by Wilcoxon
signed rank test; b) Difference between CRMR and CRMRF was analyzed by Mann–Whitney test; c) Diet × time effect was assessed by repeated ANOVA after adjusted for
baseline serum IL-6 and AST levels.

Table 3. Changes of body composition and iron biomarkers stratified by mean reduction of percentage of body weight (n = 93).

Variables CRMR (n = 45) CRMRF (n = 48) b)

p-Value

a) a)
Body weight reduction p-Value Body weight reduction p-Value

< 7% (n = 17) ࣙ7% (n = 28) < 6% (n = 26) ࣙ6% (n = 22)

Age (years) 45.35 ± 13.94 50.07 ± 12.99 0.205 48.96 ± 13.47 50.64 ± 14.61 0.521 0.688
Male (n, %) 3 (17.6%) 11 (39.3%) 0.128
% Change of body weight −2.09 ± 3.04 −10.80 ± 3.10 0.0001 −3.95 ± 1.26 −8.23 ± 2.22 0.0001 0.002
% Change of WC 0.60 ± 2.89 −10.78 ± 5.90 0.0001 −4.65 ± 2.73 −8.44 ± 3.40 0.0001 0.218
Body composition
% Change of total fat mass −6.26 ± 5.25 −21.57 ± 7.87 0.0001 −5.57 ± 5.53 −16.31 ± 6.48 0.0001 0.009
% Change of lean body mass −1.25 ± 6.12 −4.66 ± 3.44 0.055 −0.66 ± 2.67 −1.56 ± 3.38 0.102 0.003
% Change of total body fat −2.86 ± 4.74 −12.14 ± 7.51 0.0001 −3.10 ± 4.33 −9.88 ± 5.30 0.0001 0.184
% Change of android fat −2.28 ± 4.07 −9.96 ± 8.48 0.0001 −2.97 ± 3.37 −8.59 ± 5.23 0.0001 0.830
% Change of gynoid fat −3.49 ± 6.52 −11.23 ± 6.97 0.0001 −0.60 ± 5.59 −8.38 ± 5.94 0.0001 0.125
Iron status
% Change of serum iron 17.05 ± 63.00 3.37 ± 50.10 0.691 3.13 ± 41.97 15.27 ± 36.49 0.196 0.127
% Change of serum Hepcidin −0.69 ± 2.12 −3.09 ± 8.66 0.606 0.56 ± 2.38 −5.47 ± 18.09 0.129 0.741
% Change of serum Ferritin −7.54 ± 44.67 4.02 ± 45.98 0.292 −3.11 ± 29.24 6.83 ± 28.93 0.153 0.860
% Change of Hb 77.16 ± 333.40 1.51 ± 7.09 0.615 0.39 ± 5.33 −1.22 ± 4.57 0.495 0.363
% Change of TS 15.90 ± 57.62 11.96 ± 52.58 0.925 1.11 ± 43.13 14.88 ± 43.14 0.192 0.681
Inflammatory biomarkers
% Change of CRP 20.06 ± 90.59 −39.51 ± 38.15 0.003 4.68 ± 58.87 −9.20 ± 44.78 0.788 0.019
% Change of IL-6 −15.50 ± 59.75 −24.52 ± 48.84 0.623 −33.90 ± 41.71 −38.10 ± 38.92 0.694 0.257
% Change of AST 2.73 ± 48.36 −0.02 ± 40.83 0.991 −13.01 ± 26.09 −12.45 ± 20.41 0.796 0.214
% Change of ALT −2.36 ± 53.91 −14.97 ± 34.01 0.150 −8.54 ± 41.01 −15.41 ± 27.82 0.642 0.769

Continuous data are presented as mean ± SD, categorical data are presented as numbers (percentages); p-Value was analyzed by Mann–Whitney test for continuous
a)

variables and Chi-square for categorical variables; b) p-Value was analyzed by Mann–Whitney test for mean reduction of percentage body weight ࣙ7% in CRMR compared
with mean reduction of percentage body weight ࣙ6% in CRMRF.

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Table 4. Changes of body compositions and iron biomarkers according to the mean percentage reduction of serum IL-6 (n = 93).

Variables CRMR (n = 45) CRMRF (48) b)

p-Value

a) a)
serum IL-6 reduction p-Value Serum IL-6 reduction p-Value

< 21% (n = 19) ࣙ21% (n = 26) < 35% (n = 16) ࣙ35% (n = 32)

Age (years) 52.84 ± 10.48 44.96 ± 14.48 0.076 46.56 ± 15.10 51.63 ± 12.99 0.212 0.091
Male (n, %) 7 (36.8%) 7 (26.9%) 0.478 5 (27.8%) 6 (20.0%) 0.535 0.884
% Change of body weight −8.90 ± 5.12 −6.49 ± 5.20 0.161 −6.21 ± 2.76 −5.74 ± 2.82 0.537 0.114
% Change of WC −7.09 ± 7.76 −6.04 ± 7.34 0.491 −5.68 ± 2.68 −6.81 ± 4.00 0.365 0.356
Body compositions
% Change of total fat mass −18.84 ± 9.51 −13.56 ± 10.31 0.113 −10.13 ± 7.44 −10.71 ± 8.46 0.865 0.142
% Change of lean body mass −3.50 ± 3.55 −3.28 ± 5.71 0.613 −1.25 ± 3.76 −0.96 ± 2.54 0.469 0.008
% Change of total body fat −10.92 ± 7.58 −6.96 ± 7.97 0.135 −5.76 ± 5.52 −6.47 ± 6.11 0.701 0.650
% Change of android fat 46.74 ± 6.59 49.37 ± 6.09 0.251 49.17 ± 5.87 48.74 ± 4.65 0.670 0.882
% Change of gynoid fat 40.43 ± 8.68 45.01 ± 7.86 0.290 42.82 ± 7.50 41.56 ± 7.75 0.338 0.032
Iron status
% Change of serum iron 11.63 ± 46.47 6.28 ± 61.34 0.334 −7.41 ± 41.71 18.35 ± 35.58 0.006 0.049
% Change of serum Hepcidin −0.59 ± 2.90 –4.22 ± 12.12 0.372 –0.41 ± 2.25 −6.31 ± 22.29 0.340 0.290
% Change of serum Ferritin 7.69 ± 49.73 –6.22 ± 41.85 0.280 −6.21 ± 27.54 6.04 ± 29.68 0.110 0.254
% Change of hemoglobin −0.56 ± 4.76 2.49 ± 12.00 0.498 1.20 ± 3.77 −1.28 ± 5.48 0.108 0.150
% Change of TS 13.95 ± 45.36 13.08 ± 60.30 0.748 −8.63 ± 42.10 17.05 ± 41.64 0.007 0.617
Inflammatory biomarkers
% Change of CRP −9.78 ± 39.22 −22.29 ± 84.56 0.046 2.09 ± 49.88 –3.94 ± 55.20 0.443 0.029
% Change of IL-6 24.26 ± 51.73 −54.27 ± 16.12 0.0001 8.44 ± 27.45 –62.39 ± 14.69 0.0001 0.164
% Change of AST −2.67 ± 26.61 3.71 ± 52.65 0.401 −16.52 ± 17.31 −10.50 ± 26.44 0.544 0.324
% Change of ALT −22.22 ± 19.87 −1.43 ± 51.99 0.132 −22.72 ± 25.04 −5.07 ± 39.25 0.083 0.963

Continuous data are presented as mean ± SD, categorical data are presented as numbers (percentages); a) p-Value was analyzed by Mann–Whitney test for continuous
variables and Chi-square for categorical variables; b) p-Value: CRMR serum IL6 reduction ࣙ21% compared with CRMRF serum IL6 reduction ࣙ35% by Mann–Whitney test.

mass and improving the serum iron status, possibly through in-
terfering with IL-6 production.
To the best of our knowledge, this is the first weight-reduction
trial to report the beneficial effects of FO supplementation on im-
proving the serum iron status in OW and obese subjects with
MetS. A number of studies explored the effects of weight loss on
the iron status in obese individuals; however, results are incon-
sistent across studies.[8,26,31–34] This may partially be due to varia-
tions in the age (e.g., children versus adults), the degree of obesity
(e.g., healthy obese or with obesity comorbidities), the interven-
tion period and methods (e.g., dieting alone or the combination
of dieting, exercise, and surgery), and the type of dietary program
(e.g., high protein versus low protein). Our results agree with
those previous studies which showed that in some patients, diet-
induced weight loss is associated with a decline in serum iron
levels.[8,31,34,35] Cheng et al.[35] showed that an improved serum
iron status was only seen in those with a moderate weight re-
duction (>10%) who received a low-protein diet at 12 months
post-intervention, while no change in serum iron levels was seen
in those who received a high-protein diet with moderate weight
loss (>10%). Our study showed no significant effects of moderate
weight loss (ࣙ7% in the CRMR and ࣙ6% in the CRMRF group)
on serum iron levels in either group.
In the current study, a moderate loss in serum IL-6 improved
the serum iron status, and this effect was specific to the group
with FO supplementation. Our results raise the possibility that
the improvement in the serum iron status was due, at least in
part, to suppression of IL-6 synthesis by the addition of n-3 PU-
FAs. Although a moderate IL-6 reduction improved serum iron
status in FO-supplemented group, no statistical difference was
observed in the changes of serum hepcidin. This result is sim-
ilar to the finding of Cheng et al.[35] Cheng et al. conducted a
1 year diet-induced weight loss program in 36 obese women
and reported no changes of serum hepcidin levels despite the
fact that serum iron and %TS were improved in those who had
ࣙ10% weight loss at 1 year post-intervention.[35] The regulation
of hepcidin is complex and involves signals derived from multi-
organs, such as bone marrow (erythroid factors), liver and spleen
(storage iron), and adipose tissue (inflammation). In the case of
obesity, obesity-related inflammation triggers abnormal serum
hepcidin levels and high serum hepcidin, in turn, downregu-
late serum iron levels through the degradation of the iron ex-
porter ferroportin.[36] The relationship between IL-6 and hepcidin
is well established. The literature showed that IL-6 triggers hep-
cidin synthesis via signal transducer and activator of transcrip-
tion 3-dependent pathways,[10] and downregulation of hepcidin

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by treatment with an anti-IL-6 receptor antibody improved ane-
mia of inflammation.[17,37] FO or the n-3/n-6 fatty acid ratio has
potent anti-inflammation effects,[6] and a human study showed
that the addition of FO suppressed IL-6 synthesis by white blood
cells.[25] Our results reconfirmed the important role of IL-6 in
regulating systemic iron in obese individuals and suggest that
the addition of FO favorably influenced serum iron levels dur-
ing the weight-loss period. Currently, we do not know whether
FO directly regulates hepcidin or ferroportin expression or sim-
ply downregulates inflammation to influence iron status. Future
study is welcome to investigate the molecular pathway under-
lying the crosstalk between FO and iron metabolism in obese
model.
This study investigated whether the addition of supplemen-
tal FO influences body composition changes in a diet-induced
weight-loss trial. Optimal weight-loss dietary programs should
not only take into account the amount of weight lost but also
changes in the body composition (e.g., loss of body fat or vis-
ceral fat mass but preservation of the lean body mass), metabolic
profiles, dietary adherence, as well as weight maintenance af-
ter weight loss.[2–5] Although our study used a standard energy-
restricted diet, we replaced dinner with a high-protein milk drink,
which contained 35% protein. Meal replacement with a high-
protein milkshake is safe and has been intensively studied.[38,39]
The beneficial effects of a high-protein diet are linked to in-
creased satiety in the short-term, preservation of the lean mus-
cle mass, good dietary adherence, and maintenance of weight
loss,[39,40] but the long-term efficacy of a high-protein diet has still
not been clarified.[41] It was estimated that approximately 80% of
the weight lost was adipose tissues and ࣈ20% (ranging 14–26%)
was lean body mass (ࣈ0.2 kg of lean mass was lost for every 1
kg of weight loss).[42,43] In our study, the percentages weight loss
from the lean body mass in the CRMR and CRMRF groups were
25% and 11%, respectively. This suggests that meal replacement
with a high-protein milk drink had no beneficial effect on pre-
serving the lean body mass; however, the addition of 2.13 g of
FO (containing 1.2 g of EPA and 0.85 g of DHA) significantly
prevented the loss of lean body mass during the diet-induced
weight-loss period. Previous studies that investigated the role of
n-3 PUFAs in weight reduction showed variable results.[22–24] The
most common finding is that the addition of FO reduced the body
weight or body fat mass[23] ; however, our study failed to confirm
this. This may have been due in part to variations in study de-
signs, characteristics of subject at the baseline (e.g., age, gender,
severity of obesity, and MetS), and n-3 PUFA dosages. Nonethe-
less, our study supported the beneficial effects of n-3 PUFAs on
muscle anabolism or anti-catabolism.[44] Preventing loss of the
lean body mass during weight loss is important for middle-aged
and elderly subjects, because the skeletal muscle mass declines
by 0.2–0.5% per year after 50 years of age,[44] and weight rebound
after weight loss is associated with fat mass regain and not mus-
cle mass.[43]
Limitations of this study were similar to those often seen in
other weight-reduction studies, such as variations in age and the
severity of obesity, a gender imbalance (31% males in the CRMR
and 23% males in the CRMRF group), and a short duration
(12 weeks). In addition, subjects who enrolled in this trial were
recruited through advertisements in local communities in Taipei
and New Taipei City, which are the most affluent cities of Taiwan.
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Hence, our participants might have been better educated and
more motivated to lose weight compared to the general obese
Taiwanese population. The efficacy of weight loss was also influ-
enced by poor compliance during the Lunar New Year Festival as
well as adherence to take a total of ten tablets of FO (2.13 g n-3 PU-
FAs per day) and replace dinner with a high-protein milk drink.
The sample size of the study depends on the expected effect size.
In this study, the CRMR group lost on average –1.58 ± 2.2 kg of
lean body mass compared to –0.54 ± 1.4 kg of lean mass in the
CRMRF group (p = 0.001, Table 2). The mean difference between
two groups was 1.04 kg. Based on this result, the calculated
sample size is 39 participants per intervention group, with a
total of 78 participants required. The power to detect a difference
in changes of lean body mass between groups is 90%. Future
studies are required to validate our findings in a larger cohort
for a longer duration. Moreover, it is important to delineate the
cutoff point of serum IL-6 in predicting improvements in the
serum iron status as well as the underlying mechanisms of FO’s
action in improving iron metabolism in obese subjects.
5. Conclusions
FO supplementation showed no added benefits on body fat or
body weight loss, but the addition of n-3 PUFAs had beneficial
effects on preserving the lean body mass and improving the iron
status, particularly in those with a moderate IL-6 reduction.
Acknowledgements
S.Y.H. designed the study, supervised the clinical trial, and critically
reviewed the manuscript. N.S. carried out the initial data analyses
and data preparation. Y.W.C. ,Y.C.C., and S.H.L. supervised data collec-
tion and analysis. J.S.C. conceptualized the analysis, drafted the initial
manuscript, and approved the final manuscript as submitted. Dr. Jung-Su
Chang was supported by grants from Taipei Medical University Hospital
(107TMU-TMUH-11) and the Ministry of Science and Technology, Taiwan
(MOST106-2320-B-038-034).
Conflict of Interest
The authors declare no conflict of interest.
Keywords
calorie-restricted diet with meal replacement, interleukin-6, lean body
mass, omega-3 fatty acids, serum iron, weight loss
Received: March 9, 2018
Revised: July 23, 2018
Published online:
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