The Effects of 24-Week, High-Concentration Hydrogen-Rich Water on Body Composition, Blood Lipid Profiles and Inflammation Biomarkers in Men and Women with Metabolic Syndrome: A Randomized Controlled Trial

Abstract

Purpose
Metabolic syndrome is associated with several medical risk factors including dyslipidemia, hyperglycemia, and obesity, which has become a worldwide pandemic. The sequelae of this condition increase the risk of cardiovascular and neurological disease and increased mortality. Its pathophysiology is associated with redox dysregulation, excessive inflammation, and perturbation of cellular homeostasis. Molecular hydrogen (H2) may attenuate oxidative stress, improve cellular function, and reduce chronic inflammation. Pre-clinical and clinical studies have shown promising effects of H2-rich water (HRW) on specific features of metabolic syndrome, yet the effects of long-term, high-concentration HRW in this prevalent condition remain poorly addressed.

Methods
We conducted a randomized, double-blinded, placebo-controlled trial in 60 subjects (30 men and 30 women) with metabolic syndrome. An initial observation period of one week was used to acquire baseline clinical data followed by randomization to either placebo or high-concentration HRW (> 5.5 millimoles of H2 per day) for 24 weeks.

Results
Supplementation with high-concentration HRW significantly reduced blood cholesterol and glucose levels, attenuated serum hemoglobin A1c, and improved biomarkers of inflammation and redox homeostasis as compared to placebo (P < 0.05). Furthermore, H2 tended to promote a mild reduction in body mass index and waist-to-hip ratio.

Conclusion
Our results give further credence that high-concentration HRW might have promising effects as a therapeutic modality for attenuating risk factors of metabolic syndrome.

Full text

ORIGINAL RESEARCH
The Effects of 24-Week, High-Concentration
Hydrogen-Rich Water on Body Composition,
Blood Lipid Profiles and Inflammation Biomarkers
in Men and Women with Metabolic Syndrome:
A Randomized Controlled Trial
This article was published in the following Dove Press journal:
Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy
Ty l e r W L e B a r o n ,
1,2
Ram B
Singh,
3
Ghizal Fatima,
4
Kumar
Kartikey,
3
Jagdish P Sharma,
3
Sergej M Ostojic,
5,6
Anna
Gvozdjakova,
7
Branislav Kura,
2
Mami Noda,
8
Viliam Mojto,
9
Mohammad Arif Niaz,
10
Jan
Slezak
1
1
Centre of Experimental Medicine,
Institute for Heart Research, Slovak
Academy of Sciences, Bratislava, Slovak
Republic;
2
Molecular Hydrogen Institute,
Enoch, UT, USA;
3
Hospital and Research
Institute, Moradabad, India;
4
Era Medical
College, Lucknow, India;
5
Applied
Bioenergetics Lab, Faculty of Sport and
PE, University of Novi Sad, Novi Sad,
Serbia;
6
Faculty of Health Sciences,
University of Pécs, Pécs, Hungary;
7
Medical Faculty, Pharmacobiochemical
Laboratory of 3rd Medical Department,
Comenius University Bratislava,
Bratislava, Slovakia;
8
Laboratory of
Pathophysiology, Graduate School of
Pharmaceutical Sciences, Kyushu
University, Fukuoka, Japan;
9
Third
Internal Clinic, Faculty of Medicine,
Comenius University, Bratislava, Slovakia;
10
Center of Nutrition Research,
International College of Nutrition,
Moradabad, India
Purpose: Metabolic syndrome is associated with several medical risk factors including
dyslipidemia, hyperglycemia, and obesity, which has become a worldwide pandemic. The
sequelae of this condition increase the risk of cardiovascular and neurological disease and
increased mortality. Its pathophysiology is associated with redox dysregulation, excessive
inflammation, and perturbation of cellular homeostasis. Molecular hydrogen (H
2
) may
attenuate oxidative stress, improve cellular function, and reduce chronic inflammation. Pre-
clinical and clinical studies have shown promising effects of H
2
-rich water (HRW) on
specific features of metabolic syndrome, yet the effects of long-term, high-concentration
HRW in this prevalent condition remain poorly addressed.
Methods: We conducted a randomized, double-blinded, placebo-controlled trial in 60 sub-
jects (30 men and 30 women) with metabolic syndrome. An initial observation period of one
week was used to acquire baseline clinical data followed by randomization to either placebo
or high-concentration HRW (> 5.5 millimoles of H
2
per day) for 24 weeks.
Results: Supplementation with high-concentration HRW significantly reduced blood cho-
lesterol and glucose levels, attenuated serum hemoglobin A1c, and improved biomarkers of
inflammation and redox homeostasis as compared to placebo (P< 0.05). Furthermore, H
2
tended to promote a mild reduction in body mass index and waist-to-hip ratio.
Conclusion: Our results give further credence that high-concentration HRW might have
promising effects as a therapeutic modality for attenuating risk factors of metabolic
syndrome.
Keywords: metabolism, fasting blood glucose, cholesterol, inflammation, oxidative stress,
hydrogen water
Introduction
The prevalence of metabolic syndrome is considered a growing epidemic in
countries worldwide, and is characterized by various medical conditions including
visceral obesity, hyperglycemia, insulin resistance, hypertension, and dyslipidemia.
1
The sequelae of this condition increase the risk of cardiovascular and neurological
disease and increased mortality. Its pathophysiology is associated with redox
dysregulation, excessive inflammation, and perturbation of cellular homeostasis.
2
Correspondence: Jan Slezak
Centre of Experimental Medicine,
Institute for Heart Research, Slovak
Academy of Sciences, Dúbravská Cesta 9,
Bratislava 841 04, Slovak Republic
Tel +421 903 620 181
Email jan.slezak@savba.sk
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There is no approved drug to prevent or treat metabolic
syndrome. Modifications to diet and lifestyle including
caloric restriction and exercise are currently recommended
and if implemented can be effective.
3
However, stresses of
daily life, lack of time, and sufficient motivation are often
cited as reasons that prevent people from making sufficient
modifications until after they develop symptoms.
Nevertheless, even after symptoms emerge, many still do
not make the needed changes, and, as a corollary, develop
the associated diseases that otherwise could have been
prevented.
4
Molecular hydrogen (H
2
gas) has been demonstrated to
attenuate oxidative stress, improve cellular function, and
reduce chronic inflammation,
5
many of which are asso-
ciated with the pathology and etiology of metabolic syn-
drome and its associated diseases.
1
Molecular hydrogen
modulates signal transduction, protein phosphorylation
cascades, gene expression, autophagy, miRNA expression,
as well as has important metabolic effects.
5,6
H
2
can
induce the Keap1/Nrf2 signaling pathway,
7
promote mito-
chondrial biogenesis,
8
and the cytoprotective mitochon-
drial unfolded protein response.
9
H
2
has been proposed
to act as an exercise mimetic and redox adaptogen via
activating hormetic pathways.
10
Inhalation of H
2
gas suppressed brain damage induced
by middle-cerebral artery occlusion in rats,
11
and
improved cognitive scores and reduced brain injury in
patients with acute cerebral infarction.
12
Additionally, H
2
gas dissolved in water to make H
2
-rich water (HRW) has
also been shown to have therapeutic and ergogenic effects
in pre-clinical and clinical studies
10,13
such as, mild cog-
nitive impairments,
14
metabolic syndrome,
15
and submax-
imal exercises.
10,16,17
Furthermore, as has been reviewed
recently,
5
molecular hydrogen may be a novel approach
for the treatment of cardiovascular diseases. For example,
as illustrated in the recent review,
5
H
2
attenuates radiation-
induced heart disease and myocardial ischemia-
reperfusion injury in rats by decreasing inflammation,
apoptosis, sarcoplasmic and oxidative stress, and by reg-
ulating microRNAs and autophagy.
5
In APOE knockout
mice, ingestion of HRW prevented the development of
atherosclerosis,
18
and H
2
also protected against drug-
induced cardiac hypertrophy and dysfunction.
19
However, most studies with HRW have been con-
ducted using relatively low concentrations of H
2
.
20
For
example, an early study in a mouse model of Parkinson’s
disease
21
suggested that a low H
2
concentration (≈40 μM)
may be as effective as a higher H
2
concentration (≈800
μM). However, even this higher H
2
concentration was not
high enough to result in detectable increases in brain H
2
concentration.
22
It was subsequently determined that H
2
-
induced secretion of neuroprotective gastric ghrelin,
which, as a 2nd messenger, mediated the neuroprotective
effects of HRW.
22
However, the mechanism appears more
complicated since the protective effects of HRW were still
observed in a ghrelin-KO mice model of Parkinson’s
disease.
23
Nevertheless, it appears that a higher concentra-
tion of H
2
is at least as effective as, and often more
effective than, a lower H
2
concentration. For example, it
has been demonstrated that high-concentration hydrogen
produced via magnesium was more effective than low-
concentration H
2
contained in alkaline ionized water in
attenuating non-alcoholic fatty liver disease (NAFLD) in
mice fed a high-fat diet.
24
Similarly, in a randomized con-
trolled pilot study in patients with NAFLD, we found that
high-concentration HRW significantly decreased liver fat
as measured by dual-echo magnetic resonance imaging.
25
In addition, supplementation with high-concentration
HRW in middle-aged overweight women significantly
reduced body fat percentage and decreased fasting insulin
levels.
26
In addition to the H
2
concentration being impor-
tant, the duration of use is also an important consideration.
Although HRW has been studied in subjects with potential
metabolic syndrome for up to 10 weeks, no study has
determined the effect long-term (24-week), high-
concentration HRW in this population. Despite hydrogen’s
ability to ostensibly induce hormesis, and therefore poten-
tially elicit adverse effects, there are no studies either in
cells, animals, or humans, even at very high doses, where
clear adverse effects have been reported.
10
We therefore
evaluated the effects of 24-week intervention with high-
concentration HRW on body composition, blood lipid pro-
files and inflammation biomarkers in men and women with
metabolic syndrome.
Methods and Subjects
Sixty subjects of Indian ethnicity (30 men and 30 women;
age 43.2 ± 10.0 years) with metabolic syndrome were
recruited to participate in this double-blinded, placebo-
controlled interventional trial. Subjects participated in this
study if they met at least three of the five inclusion criteria
including prehypertension/hypertension (systolic blood pres-
sure [BP] > 130 mmHg and/or diastolic BP > 85 mmHg),
prediabetes/diabetes (fasting glucose > 110 mg/dL), central
obesity (waist circumference [WC] > 90 cm for men, and
WC > 80 cm for women), and dyslipidemia (high-density
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lipoprotein [HDL] < 40 mg/dL for men and < 50 mg/dL for
women; triglycerides [TG] > 200 mg/dL). Exclusion criteria
included cancer, chronic dysentery, human immunodefi-
ciency virus infection, stroke, myocardial infarction, preg-
nancy or use of contraceptives, and other chronic diseases.
The study was conducted in Moradabad India, and all parti-
cipants were recruited by pamphlet distribution, local news-
papers, and announcements on hospital notice boards.
Ethical clearance was obtained from the Hallberg Hospital
and Research Institute ethic committee (Moradabad), with
the trial registered within the Drug Controller of India
(Clinical Trial Registration #2018/03/012487). Written
informed consent was obtained from all participants, and
the trial was conducted in accordance with the Declaration
of Helsinki, and this statement was added to the methods.
An initial observation period of one week was used to
acquire baseline clinical metrics and biochemical data
(Table 1), with no differences found between the HRW
and the placebo group. Subjects were then randomized in
a double-blind fashion to either intervention (HRW) or
placebo group by computer-generated random numbers.
All subjects were asked to maintain the same lifestyle
throughout the study. Moreover, data on food, tobacco,
and alcohol intake and physical activity were obtained by
dietary diaries and assessed by a dietitian. The data was
collected again after 24 weeks of the intervention. High-
concentration HRW was prepared via hydrogen-producing
tablets (HRW Natural Health Products Inc., New
Westminster BC, Canada) while the placebo was prepared
as described previously
16,25
with the final placebo drink
similar in taste, dissolution, and appearance to HRW. The
participants consumed 1 tablet 3 x daily in 250 mL of 12-
18°C water. They were advised to drink the product in one
gulp as soon as the tablet finished dissolving on an empty
stomach/morning. This method of H
2
administration
would provide >5.5 millimoles H
2
/day. The concentration
of molecular hydrogen produced via these tablets was
determined by H
2
Analytics (Las Vegas, USA) via gas
chromatography (SRI 8610C; California USA).
The laboratory data were obtained following an overnight
fast (10–12 hrs) at 08:00 to 09:00 am. Height was measured
usingameasuringstandafterremovingshoes.Bodyweight
was measured in underclothes after removing shoes. Waist
circumference was measured with anthropometric tape as the
largest horizontal circumference between iliac crest and
costal margin. The hip girth was measured at the greatest
circumference at the level of greater trochanters. Heart rate
was measured by auscultation for 5 mins at rest at the supine
position. Fasting blood glucose was measured after an over-
night fast. Thiobarbituric acid reactive substances (TBARS),
malondialdehyde (MDA), diene conjugate, vitamin E and
C, nitrate, and angiotensin-converting enzyme were
measured by colorimetric methods using a UV–VIS
Spectrophotometer (Electronics Corporation of India, Ltd).
Glycosylated hemoglobin (HbA1c) was assayed by HPLC
using DIO machine (Bio-Rad Laboratories, Inc, Hercules,
CA). Fasting blood sugar, lipid profiles, and C-reactive pro-
tein (CRP) were determined by Pictus 500 Diatron kits
(Medicon Hellas S.A., Gerakas, Greece). Tumor necrosis
factor-alpha (TNF-α) and interleukin 6 (IL-6) were analyzed
with an enzyme-linked fluorescent assay on Vidas machines
(Vidas Biomerieux, Marcy I’Étoile, France). The inter-and
intra assay coefficients of variation of these markers are
shown in Table 2.
The number of participants recruited was in accordance
with a minimal sample size (n = 48) calculated by power
analysis (G*Power 3.1, Heinrich Heine University,
Table 1 Baseline Characteristics of the Study Participants of
Indian Ethnicity. Values are Mean ± SD
HRW
(n = 30)
Placebo
(n = 30)
P
Female (%) 53.3 53.3
Age (years) 43.4 ± 9.2 42.9 ± 7.6 0.81
Weight (kg) 70.5 ± 12.2 72.8 ± 12.3 0.47
Height (cm) 155.9 ± 8.8 153.2 ± 7.7 0.22
Body mass index (kg/m
2
) 28.9 ± 4.8 31.1 ± 5.4 0.10
Waist-hip circumference 1.00 ± 0.08 0.96 ± 0.05 0.05
Total cholesterol (mg/dL) 187.7 ± 32.4 184.3 ± 37.4 0.71
Low-density cholesterol (mg/dL) 109.0 ± 34.4 105.5 ± 42.0 0.72
High-density cholesterol (mg/dL) 41.7 ± 4.2 41.8 ± 2.3 0.96
Very low-density cholesterol
(mg/dL)
37.3 ± 17.9 36.8 ± 20.6 0.92
Triglycerides (mg/dL) 189.8 ± 93.3 184.4 ± 102.8 0.83
C-reactive protein (mg/dL) 0.5 ± 0.2 0.6 ± 0.5 0.33
Glucose (mg/dL) 121.5 ± 61.0 123.9 ± 43.4 0.86
Hemoglobin A1c (%) 5.8 ± 0.9 6.2 ± 1.2 0.17
Tumor necrosis factor alpha (μM) 4.8 ± 1.2 4.8 ± 1.3 0.97
Interleukin 6 (μM) 1.9 ± 0.7 1.6 ± 0.6 0.10
Thiobarbituric acid reactive
substances (μM)
2.5 ± 0.3 2.5 ± 0.3 0.31
Malondialdehyde (μM) 3.4 ± 0.2 3.4 ± 0.2 0.66
Diene conjugates (μM) 27.8 ± 1.0 28.3 ± 0.8 0.03
Vitamin E (μM) 23.0 ± 2.3 23.0 ± 1.5 0.95
Vitamin C (μM) 20.7 ± 2.5 20.7 ± 2.5 0.99
Nitrite (μM) 0.63 ± 0.06 0.66 ± 0.04 0.04
Angiotensin-converting enzyme
(μM)
85.2 ± 7.8 84.5 ± 8.8 0.72
Heart rate (beat/min) 86 ± 7 86 ± 7 0.76
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Düsseldorf, Germany), with effects size set at 0.30, alpha
error probability 0.05, power 0.80 for two groups and two
measurements of study outcomes. Subject baseline data were
analyzed using a two-tailed two-sample t-test. Two-way
mixed model ANOVA with repeated measures (treatment
vs time interaction) adjusted for age and gender was used
to establish if any significant differences existed between
patients’responses over time of intervention. The statistical
significance was set at P≤0.05. All values are reported as
mean ± SD. Data were analyzed using the SPSS program
(version 21.0) (SPSS Inc., Chicago, IL, USA).
Results
All subjects completed the study and both interventions
were well tolerated with no ill-reported effects. HRW
favorably affected all outcomes at 24-week follow-up as
compared to placebo (P< 0.05), except for TBARS,
a marker of lipid peroxidation (P= 0.309) (Table 3).
Other markers of oxidation (MDA, D-conjugate)
decreased while vitamins E and C increased in the HRW
group. This was accompanied by a significant reduction in
HR, BMI and WHR after HRW intervention (P< 0.05).
HRW induced a significant reduction in total cholesterol
by approximately 18.5 mg/dL (P< 0.05), and in triglycer-
ides levels by ~ 47 mg/dL (P< 0.05). Fasting blood
glucose also decreased after 24-week HRW intervention
from 121.5 ± 61.0 mg/dL to 103.1 ± 33.0 mg/dL, with an
accompanying 12% reduction in HbA1C (P< 0.05).
Table 2 Inter- and Intra-Assay Coefficients of Variation for the
Measured Biomarkers (CV)
Biomarker Inter CV Intra CV
TC 1.50 1.70
LDL-C 0.91 1.80
HDL-C 1.11 1.25
VLDL-C 1.02 1.36
TG 0.95 1.46
CRP 1.91 1.48
FBS 0.97 0.98
HbA1c 1.16 1.79
TNF 1.25 1.29
IL-6 1.82 1.88
TBARS 1.91 1.95
MDA 0.87 0.96
D conjugate 2.10 2.06
Vit E 1.18 1.27
Vit C 1.57 1.98
Nitrite 2.11 2.20
ACE 1.18 1.62
Table 3 Changes in Body Composition and Biochemical Variables from Baseline to 24 Weeks. Values are Mean ± SD
HRW Placebo P*
Baseline Follow Up Baseline Follow Up
Body mass index (kg/m
2
) 28.9 ± 4.8 28.2 ± 4.9
†
31.1 ± 5.4 31.3 ± 5.3 < 0.001
Waist-hip circumference 1.00 ± 0.08 0.99 ± 0.07
†
0.96 ± 0.05 0.96 ± 0.05 0.03
Total cholesterol (mg/dL) 187.7 ± 32.4 169.2 ± 26.1
†
184.3 ± 37.4 184.4 ± 38.6 < 0.001
Low-density cholesterol (mg/dL) 109.0 ± 34.4 102.5 ± 28.0 105.5 ± 42.0 106.0 ± 43.3
†
0.06
High-density cholesterol (mg/dL) 41.7 ± 4.2 40.4 ± 1.8
†
41.8 ± 2.3 42.3 ± 2.4
†
0.01
Very low-density cholesterol (mg/dL) 37.3 ± 17.9 28.0 ± 11.3
†
36.8 ± 20.6 37.3 ± 20.5
†
< 0.01
Triglycerides (mg/dL) 189.8 ± 93.3 142.4 ± 65.0
†
184.4 ± 102.8 185.6 ± 101.3 < 0.01
C-reactive protein (mg/dL) 0.5 ± 0.2 0.5 ± 0.1
†
0.6 ± 0.5 0.6 ± 0.5 0.04
Glucose (mg/dL) 121.5 ± 61.0 103.1 ± 33.0
†
123.9 ± 43.4 126.4 ± 42.3
†
< 0.01
Hemoglobin A1c (%) 5.8 ± 0.9 5.1 ± 0.2
†
6.2 ± 1.2 6.1 ± 1.2 < 0.001
Tumor necrosis factor alpha (μM) 4.8 ± 1.2 3.9 ± 0.6
†
4.8 ± 1.3 4.8 ± 1.3 < 0.001
Interleukin 6 (μM) 1.9 ± 0.7 1.6 ± 0.2
†
1.6 ± 0.6 1.7 ± 0.6 < 0.01
Thiobarbituric acid reactive substances (μM) 2.5 ± 0.3 1.6 ± 0.3
†
2.5 ± 0.3 2.5 ± 0.3 0.31
Malondialdehyde (μM) 3.4 ± 0.2 2.7 ± 0.2
†
3.4 ± 0.2 3.5 ± 0.2 < 0.001
Diene conjugates (μM) 27.8 ± 1.0 26.7 ± 0.5
†
28.3 ± 0.8 28.3 ± 0.8 < 0.001
Vitamin E (μM) 23.0 ± 2.3 26.8 ± 1.9
†
23.0 ± 1.5 23.1 ± 1.1 < 0.001
Vitamin C (μM) 20.7 ± 2.5 24.2 ± 1.8
†
20.7 ± 2.5 20.8 ± 2.4 < 0.001
Nitrite (μM) 0.63 ± 0.06 0.68 ± 0.06
†
0.66 ± 0.04 0.65 ± 0.03 < 0.001
Angiotensin-converting enzyme (μM) 85.2 ± 7.8 80.7 ± 5.8
†
84.5 ± 8.8 83.8 ± 8.7
†
< 0.001
Heart rate (beat/min) 86 ± 7 83 ± 5
†
86 ± 7 85 ± 5 0.02
Notes: *P-value from two-way mixed ANOVA (treatment vs time interaction).
†
Indicates significant difference baseline vs follow-up at P≤0.05 for each intervention.
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Furthermore, HRW significantly attenuated the inflamma-
tory markers, such as TNF-α, IL-6, and CRP (P<0.05).
Discussion
Uncontrolled metabolic syndrome increases the risk of car-
diovascular disease. For example, the risk factors that are
associated with metabolic syndrome play causative roles in
the development of atherosclerosis, which further leads to
coronary artery disease, stroke, and myocardial infarction.
27
Atherosclerosis develops when LDL cholesterol infiltrates
the subendothelial space and gets oxidized, which promotes
inflammation and subsequent migration and transformation
of vascular smooth muscle cells.
28
This process is further
exacerbated in the presence of hyperglycemia due to the
increased formation of advanced-glycated end products
(AGEs), which is when the reducing end of glucose mole-
cules reacts and combines with proteins and creates protein
cross-linking. AGEs further promote inflammation, oxida-
tion, and cellular damage contributing to cardiovascular
disease.
28
Accordingly, in our study we determined if high
concentration HRW would improve the various biomarkers
of metabolic syndrome that are casually involved in the
development of cardiovascular disease namely dyslipidemia
(HDL, LDL, VLDL, TG), inflammation (TNF-α,IL-6,
CRP), oxidative stress, (MDA, TBARS, diene conjugates,
vitamins E and C,) and hyperglycemia (glucose, HbA1c).
In this study, we found that a 24-week intervention with
high-concentration HRW improved several biomarkers of car-
diometabolic health in mid-age men and women with meta-
bolic syndrome, including BMI, WHR, resting HR, blood
lipids and glucose, inflammation and redox homeostasis. The
favorable changes in blood cholesterol need to be cautiously
interpreted since the absolute change was relatively low, and
HDL decreased by ~ 1.3 mg/dL. HDL cholesterol is considered
to be beneficial due to its role inreverse cholesterol transport.
29
However, the ratios of total cholesterol or triglycerides to HDL
are better predictors of cardiovascular disease than total cho-
lesterol, with lower ratios correlating with a lower risk for heart
disease.
30
Since we found that HRW significantly lowered total
cholesterol (by ~ 18.5 mg/dL), the ratio of total cholesterol to
HDL favorably decreased by ~ 7.2%, whereas it stayed the
same in the placebo group. Similarly, the risk ratio of triglycer-
ides to HDL auspiciously decreased by 22.9% in the HRW
group, yet stayed about the same in the placebo group. Our data
also show that HRW essentially lowered the mean glucose
level from the upper range to the lower range of the prediabetic
criteria, which was also accompanied by a 12% reduction of
HbA1C.
These favorable changes in cholesterol and glucose are
corroborated with a few discrepancies in several previous
clinical trials. For example, Song et al reported that HRW,
supplying 0.5 millimoles H
2
/day, for 10 weeks in patients
with potential metabolic syndrome decreased total serum
cholesterol and LDL-C levels, improved HDL function and
redox status (eg increased serum superoxide dismutase
[SOD] and decreased MDA), and reduced inflammation (eg
serum TNF-α).
31
However, whereas our study showed sig-
nificant improvements in BMI, WHR, and fasting glucose,
their study only reported a potential, albeit non-significant,
downward trend in these parameters. Similarly, an earlier
randomized, placebo-controlled, crossover study in patients
with type 2 diabetes or impaired glucose tolerance demon-
strated that ingestion of HRW (~ 0.6 millimoles/day) slightly
improved cholesterol, significantly decreased markers of
oxidative stress (eg urinary 8-isoprostanes) and increased
serum SOD.
32
However, in contrast to our study, there were
no statistically significant changes in either BMI, CRP,
HbA1c, or fasting blood glucose. Perhaps the higher dose of
H
2
and longer duration of our study compared to those
studies could account for the differences. Additionally, the
subjects in our study had significantly higher baseline glu-
cose levels (~ 122 mg/dL vs 108 mg/dL). Lastly, although not
tested in our study, the previous study
32
reportedthatin4of6
subjects with impaired glucose tolerance, HRW normalized
the oral glucose tolerance test, and that 1 hr plasma insulin
levels were significantly increased compared to baseline.
32
An open-label 8-week study on 20 subjects with poten-
tial metabolic syndrome demonstrated that HRW (~ 1 milli-
mole H
2
/day) increased SOD level by 39% and decreased
TBARs by 43%.
15
Although a decrease in TBARS was not
detected in our study, we found a decrease in the more
specific marker of lipid peroxidation MDA, as well as
increased levels of vitamins C and E, which collectively
suggest that HRW favorably modulates oxidative processes.
Similar to our study, the open-label trial revealed that HRW
decreased the total cholesterol to HDL ratio by 13%.
However, in our study, the primary change was a decrease
in total cholesterol, whereas in the open-label study, it was
an increase in HDL cholesterol. Additionally, in contrast to
our findings, HRW did not decrease BMI, triglycerides or
fasting blood glucose. However, the triglyceride and fasting
glucose level was significantly higher in the subjects in our
study compared to those in the open-label study (~ 143 mg/
dL vs 190 mg/dL; 88 mg/dL vs 122 mg/dL, respectively).
Again, it may be due to our study condition with a higher
dose of H
2
and the longer time duration.
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The underlying molecular mechanisms that mediate these
effects induced by HRW need further study. However, H
2
appears to influence metabolism and bioenergetics.
33
For
example, we previously demonstrated that HRW treatment
increased mitochondrial coenzyme Q9 concentration, which
enhanced mitochondrial respiratory chain function (ie com-
plex I and complex II) and subsequent increase in ATP
production rat myocardium.
34,35
In another study in mice
lacking the leptin receptor, and in normal mice fed a high-
fat diet, HRW reduced oxidative stress, reduced fatty liver
deposits, and decreased plasma glucose, insulin, and trigly-
ceride levels. This effect was comparable to a 20% caloric
restriction.
36
HRW increased energy expenditure as mea-
sured by oxygen consumption and induced the hepatic hor-
mone, fibroblast growth factor 21 (FGF-21), which
stimulates fatty acid and glucose expenditure.
36
In streptozo-
tocin-induced type 1 diabetic mice, H
2
induced translocation
of glucose transporter-4 via activation of phosphatidylinosi-
tol-3-OH kinase (PI3K), protein kinase C (PKC), and AMP-
activated protein kinase (AMPK).
37
This study demonstrated that HRW induced significant
improvements in clinically relevant metrics of blood biomar-
kers and biometric data in subjects with metabolic syndrome.
Compared to previous studies, it may also indicate that high
doses of H
2
are more effective than lower doses at least in
metabolic syndrome. However, more dose-dependent studies
in this area are needed. Moreover, several limitations should
be considered when interpreting our study. We only per-
formed analysis during the final 24 weeks instead of at
4-week follow-ups, which prevented us from finding impor-
tant temporal changes in the various parameters. We also did
not investigate gender- or age-dependent effects, which may
be important since metabolic parameters are influenced by
both sex and age.
38
Additionally, although subjects were
instructed to consume HRW on an empty stomach, we
could not ensure that this occurred. There may be differences
in the biological effects of H
2
if HRW is ingested with or
without food intake since following ingestion of normal
fibers from the diet, bacterial production of H
2
gas signifi-
cantly increases.
39
Lastly, we did not measure the temporal
changes or pharmacokinetics of H
2
in the blood and breath of
the subjects. Therefore, the suggested molecular mechanisms
as demonstrated in vitro or in animal studies may be different
than those in our study since the cellular H
2
concentration
may be significantly different. Future research should inves-
tigate if there are sexually dimorphic responses to H
2
therapy,
the molecular mechanisms of H
2
at physiologically relevant
H
2
concentrations, and also the comparison of the effects of
different doses, durations, and methods of administration (eg
drinking vs inhaling).
Conclusion
In conclusion, the results from our study suggest that
supplementation with high-concentration HRW produced
via H
2
-producing tablets improves body composition,
favorably modulates fatty acid and glucose metabolism,
and improves inflammation and redox homeostasis in sub-
jects with metabolic syndrome. Therefore, long-term treat-
ment with high-concentration hydrogen-rich water may be
used as an adjuvant therapy to decrease the features of
metabolic syndrome. However, a larger prospective clin-
ical trial is warranted to further determine the biological
effects of HRW in this subject population.
Data Sharing Statement
The data presented in this article constitutes all data that
the authors plan on making publicly available.
Acknowledgments
We thank Mr. Alex Tarnava, CEO of HRW Natural Health
Products Inc. for kindly donating DrinkHRW tablets for
this study.
Author Contributions
All authors made substantial contributions to conception and
design, acquisition of data, or analysis and interpretation of
data; took part in drafting the article or revising it critically
for important intellectual content; gave final approval of the
version to be published; and agree to be accountable for all
aspects of the work.
Funding
This study was partially supported by Slovak Research and
Development Agency (APVV)-0241-11, APVV-15-0376;
ITMS 26230120009; Scientific grant agency of the
Ministry of Education of the Slovak Republic (VEGA) 2/
0063/18, and by HRW Natural Health Products Inc.
Disclosure
TWL reports personal fees from medical/academic confer-
encesincluding travel reimbursement, honoraria, and speak-
ing and consultancy fees from various academic and
commercial entities regarding molecular hydrogen. All
other authors report no conflict of interest.
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