Hydrogen-rich water as a modulator of gut microbiota?

Abstract

Hydrogen-rich water (HRW) is an innovative functional drink with many professed benefits for human health, including good intestinal viability and gut microbiota upregulation. A source of molecular hydrogen, HRW might be a convenient medium to deliver this bioactive gas to the gastrointestinal tract, and perhaps modulate the activity of both hydrogen-producing and hydrogen-consuming bacteria, abundant members of the intestinal microbiota community. This paper summarizes the findings from previous studies evaluating a response of gut microbiota to HRW intake and discusses possible mechanisms and medical consequences of this interaction. It appears that only a handful of rodent studies and one human randomized-controlled trial investigated how drinking HRW affects gut microbiota, with all studies published from 2018 onwards. HRW-induced protection of the gut barrier integrity and upregulation of butyrate-producing bacteria were seen in most studies, with HRW ameliorated clinical features of gut microbiota disturbances, including diarrhea rate, weight, and fluid loss. However, no well-powered multicentric trial evaluated the effectiveness of HRW consumption so far in common gastrointestinal diseases with gut flora scenario, including inflammatory bowel disease, irritable bowel syndrome, gastroenteritis and colitis of infectious origin. HRW might be an up-and-coming compound that might tune endogenous H2 homeostasis and modulate gut microbiota but it should still be perceived as an experimental drink and not widely recommended to the general public.

Full text

Journal of Functional Foods 78 (2021) 104360
Available online 14 January 2021
1756-4646/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Hydrogen-rich water as a modulator of gut microbiota?
Sergej M. Ostojic *
FSPE Applied Bioenergetics Lab, University of Novi Sad, Novi Sad, Serbia
ARTICLE INFO
Keywords:
Hydrogen-rich water
Microbiota
Inammatory bowel disease
Methanogens
Butyric acid
ABSTRACT
Hydrogen-rich water (HRW) is an innovative functional drink with many professed benets for human health,
including good intestinal viability and gut microbiota upregulation. A source of molecular hydrogen, HRW might
be a convenient medium to deliver this bioactive gas to the gastrointestinal tract, and perhaps modulate the
activity of both hydrogen-producing and hydrogen-consuming bacteria, abundant members of the intestinal
microbiota community. This paper summarizes the ndings from previous studies evaluating a response of gut
microbiota to HRW intake and discusses possible mechanisms and medical consequences of this interaction. It
appears that only a handful of rodent studies and one human randomized-controlled trial investigated how
drinking HRW affects gut microbiota, with all studies published from 2018 onwards. HRW-induced protection of
the gut barrier integrity and upregulation of butyrate-producing bacteria were seen in most studies, with HRW
ameliorated clinical features of gut microbiota disturbances, including diarrhea rate, weight, and uid loss.
However, no well-powered multicentric trial evaluated the effectiveness of HRW consumption so far in common
gastrointestinal diseases with gut ora scenario, including inammatory bowel disease, irritable bowel syn-
drome, gastroenteritis and colitis of infectious origin. HRW might be an up-and-coming compound that might
tune endogenous H
2
homeostasis and modulate gut microbiota but it should still be perceived as an experimental
drink and not widely recommended to the general public.
1. Introduction
Hydrogen-rich water (HRW, or hydrogen-infused water) is an
emerging functional drink with purported benecial effects on human
health. Over 150 studies with HRW were published in the past decade or
so, with human trials reported in 2019–2020 alone have shown ad-
vantageous effects of consuming HRW in patients with non-alcoholic
fatty liver disease (Korovljev, Stajer, Ostojic, LeBaron, & Ostojic.
2019), metabolic syndrome (LeBaron et al., 2020), in elite athletes to
relieve psychometric fatigue (Mikami et al., 2019) and improve per-
formance (Botek, Krejˇ
cí, McKune, and Sl´
adeˇ
ckov´
a, 2020), and healthy
adults to reduce inammatory responses and prevent apoptosis (Sim
et al., 2020), to quote but a few recent reports. Although many
contentious issues surround its medicinal properties (Ostojic, 2019),
HRW is an apparent source of molecular hydrogen (H
2
), a bioactive gas
that is believed to act as a selective antioxidant, anti-inammatory,
antiapoptotic and signaling agent (for more details see Ohta, 2014). A
unique molecular target for H
2
remains unknown yet few studies imply
its possible role in the ne-tuning of homeostasis (Ishibashi, 2019;
LeBaron, Kura, Kalocayova, Tribulova, & Slezak, 2019), perhaps similar
to other naturally-occurring gases such as NO, H
2
S and CO. Besides other
plausible targets, exogenous H
2
delivered by HRW can have an effect on
gut microbiota, a complex community of over 100 trillion microbial cells
which inuence human physiology, metabolism, nutrition and immune
function (Guinane & Cotter, 2013). The fact that intestinal microbiota
produces and utilizes endogenous hydrogen gas by itself (approximately
12 L of gaseous hydrogen per day) makes HRW performance in the
human gut even more convoluted. To address this, I summarized nd-
ings from previous studies that evaluated a response of gut ora to HRW
intake and discussed possible mechanisms and medical consequences of
the interaction.
2. Research studies on HRW and gut microbiota
A handful of rodent studies and one human trial investigated how
drinking HRW affects gut microbiota, with all studies appeared from
2018 onwards (Table 1). Arguably the rst study, published in January
2018 by a Chinese research group, evaluated whether HRW adminis-
tration affects radiation-induced small intestine toxicity in an animal
model (Xiao et al., 2018). The authors reported that force-fed mice
E-mail address: sergej.ostojic@chess.edu.rs.
*
Address: FSPE Applied Bioenergetics Lab, University of Novi Sad, Lovcenska 16, Novi Sad 21000, Serbia.
Contents lists available at ScienceDirect
Journal of Functional Foods
journal homepage: www.elsevier.com/locate/jff
https://doi.org/10.1016/j.jff.2021.104360
Received 31 October 2020; Received in revised form 15 December 2020; Accepted 22 December 2020

Journal of Functional Foods 78 (2021) 104360
2
gavaged with HRW (H
2
0.80 mM) for 5 days experienced an ameliora-
tion of radiation-mediated gastrointestinal toxicity, illustrated by
improved tract functions and epithelial integrity, stabilized small in-
testine MyD88 (myeloid differentiation primary response gene 88, an
essential modulator of the innate immune response to gut pathogens),
and counterbalanced a radiation-induced lower abundance of Bacter-
oidia, Betaproteobacteria and Coriobacteria, and a higher relative abun-
dance of Phycisphaerae, Planctomycetia and Sphingobacteria spp. A few
months later, Japanese authors have explored the effects of HRW on the
intestinal environment, including microbial composition and short-
chain fatty acid (SCFA) contents (Higashimura et al., 2018). Six-week-
old mice were administered HRW (H
2
0.32 mM) or normal water (H
2
0.00 mM) for 4 weeks in ad libitum drinking protocol. At follow-up,
HRW-treated animals experienced a signicantly increased weight of
cecal contents (a marker of intestinal fermentation) as compared to the
control group. The animals treated with HRW also produced signi-
cantly more certain SCFAs, including propionic acid, isobutyric acid,
and isovaleric acid, and exhibited a distinct microbiota composition that
clustered separately from that of the control mice. For instance, drinking
HRW favored a lower relative abundance of Bidobacterium, Clos-
tridiaceae, Coprococcus, Ruminococcus, and Sutterella, and a higher
abundance of Parabacteroides, Rikenellaceae, Butyricimonas, Prevotella,
and Candidatus arthromitus, as evaluated by fecal samples 16S rRNA
sequencing. Ikeda and co-workers (2018) investigated the impact of
HRW therapy as a countermeasure against bacterial translocation in a
murine model of sepsis. Either 15 mL/kg of normal saline or super-
saturated hydrogen-rich saline (3.5 mM) were gavaged daily for 7 days
following cecal ligation and puncture, and hydrogen intervention pre-
vented the expansion of Enterobacteriaceae and Lachnospiraceae, and
ameliorated intestinal hyper-permeability after a ligation. Another trial
(Zheng et al., 2018) explored the intestinal microbiota response to 25-
day oral administrations of HRW (10 mL/kg body weight; H
2
0.6 mM)
and lactulose (a synthetic non-absorbable sugar) in female piglets fed a
Fusarium mycotoxin-contaminated maize. HRW treatment provoked
increased H
2
concentrations in the mucosa of the stomach and duo-
denum, and decreased the diarrhea rate in Fusarium mycotoxin-fed
piglets. This was accompanied by higher levels of colon butyrate, and
higher levels of acetate, butyrate, and total SCFAs in the caecum of
animals treated with HRW. The populations of selected bacteria in
different intestinal segments were also affected by HRW treatment, with
the abundance of Escherichia coli was lower and Bidobacterium
abundance higher in HRW group in the ileum, as compared to the group
that received Fusarium mycotoxin-contaminated diet. In the colon, the
abundance of methanogenic Archaea and sulfate-reducing bacteria was
higher in HRW versus a contaminated diet. A succeeding study by the
same group (Ji, Zhang, Zheng, Yao, 2019) essentially conrmed above
ndings, with 25-day oral administration of HRW (10 mL/kg body
weight; H
2
0.6–0.8 mM) found to remarkably provide benecial effects
against Fusarium mycotoxin-induced apoptosis and intestinal leaking of
the small intestine in piglets, yet no detailed gut microbiota proles
were outlined. Bordoni and co-workers (2019) have recently explored
the effects of HRW on gut permeability and fecal microbiota in a rat
model of Parkinson’s disease induced by permethrin pesticide. In short,
a 15-day treatment with HRW (10 mL/kg body weight; H
2
0.4–0.9 mM)
improved intestinal barrier integrity corrupted by permethrin, pre-
served levels of occludin (a biomarker of tight junction integrity) in the
ileum, increased the levels of butyric acid in the feces, and preserved the
abundance of Lachnospira and Deuviitaleaceae while inducing a
higher abundance of butyrate-producing bacteria (e.g., Blautia, Lach-
nospiraceae, Ruminococcaceae, Papillibacter). The benecial effects of
HRW consumption on gut microbiota were corroborated in a recent rst-
in-human trial (Sha et al., 2019). Thirty-eight juvenile female football
players were subjected to a 2-month HRW drinking protocol (1.5–2.0 L/
day) using a randomized-controlled design. On top of other ndings, the
Table 1
The summary of studies evaluating the link between hydrogen-rich water (HRW) and gut microbiota.
Ref. Species n Model HRW Control Study
length
Outcomes in HRW
Xiao et al. 2018 Mouse 12 Radiation-induced intestinal
toxicity
0.80 mM Normal water 5 days ↑ epithelial integrity
↓ miR-1968-5p level
∅ abundance of enteric bacteria
Higashimura et al.
2018
Mouse 16 Intestinal environment 0.32 mM Normal water 4 weeks ↓ serum LDL-C and ALT
↑ propionic, isobutyric, and isovaleric acids
↑ relative abundance of 20 taxa
Ikeda et al. 2018 Mouse 36 Sepsis 3.5 mM Normal saline 7 days ↑ survival rates
∅ bacterial translocation
∅ intestinal hyperpermeability
↓ intestinal morphologic damage
↓ MDA, TNF-
α
, IL-1β, IL-6
Zheng et al. 2018 Piglet 24 Mycotoxin-contaminated diet 0.6 mM Lactulose 25 days ↓ diarrhea rate
↑ acetate, butyrate, total SCFAs
↑ relative abundance of specic taxa
Ji et al. 2019 Piglet 24 Mycotoxin-contaminated diet 0.6–0.8 mM Hydrogen-free
water
25 days ↓ apoptosis and intestinal leaking
∅ abnormal intestinal morphological
changes
↑ distribution and expression of CLDN3
Bordoni et al., 2019 Rat 58 Parkinson’s disease 0.4–0.9 mM Permethrin Vehicle 15 days ↑ intestinal barrier integrity
↑ butyric acid
↑ higher abundance of butyrate-producing
bacteria
∅ tight junction integrity
∅ abundance of Lachnospira and
Deuviitaleaceae
Sha et al. 2019 Human 38 Exercise training Unknown Normal water 2 months ↑ blood hemoglobin, MDA, SOD, TAC
↑ diversity and abundance of specic taxa
Guo et al. 2020 Mouse 30 Intestinal environment Unknown Deionized water
N2 nanobubble
water
5 weeks ↑ species diversity of fecal microbiota
↓ abundance of Mucispirillum and
Helicobacter
Abbreviations: ↑ ↓ ∅ denotes an increase, decrease or no change in a specic variable, respectively. LDL-C – low-density liporotein cholesterol; ALT – alanine tranferase;
MDA – malondialdehyde; TNF-
α
– tumor necrosis factor alpha; IL-1β – interleukin 1 beta; Ili-6 – interleukin 6; SCFA – short-chain fatty acids; CLDN3 – claudin 3; SOD –
superoxide dysmutase; TAC- total antioxidant capacity.
S.M. Ostojic

Journal of Functional Foods 78 (2021) 104360
3
authors reported that HRW led to higher abundance and diversity of gut
ora, an indicator of favorable microbial balance (Valdes, Walter, Segal,
& Spector, 2018).
In summary, it appears that cited research studies typically employed
a short- to medium-term duration of the intervention (e.g., ve days to
eight weeks), and dispensed mostly a moderately saturated HRW
(H
2
<1.0 mM) yet in rather heterogenous drinking protocols and across
various experimental conditions, which makes the comparison/inter-
pretation of ndings complicated. Nevertheless, HRW-driven protection
of the gut barrier integrity and an upgrade of butyrate-producing bac-
teria were seen as pertinent in most studies, with both effects being
segment-specic and occurring predominantly in the large intestine.
HRW also ameliorated clinical features of gut microbiota disturbances,
including diarrhea rate, weight and uid loss, and kept the intestinal
contents close to the normal state-containing stool. Still, no studies
evaluated the effectiveness of HRW consumption to modulate intestinal
microbiota in common gastrointestinal diseases with a gut ora sce-
nario, including inammatory bowel disease, irritable bowel syndrome,
gastroenteritis and colitis of infectious origin. In addition, no longitu-
dinal large-scale multicentric trials are available thus far, with only one
human RCT in healthy girls. Even so, a fact that all studies are published
in the past two years indicates that the HRW-gut microbiota case be-
comes a hot research topic in biomedicine.
3. Possible mechanisms of HRW action
HRW could modulate gut ora by assorted means (Fig. 1). The pri-
mary effect of drinking HRW is likely driven by delivering extra H
2
to
the metabolite milieu of the gut, a dynamic environment already rich in
this simple gas. Normally, intestinal H
2
is produced continuously by
several classes of hydrogen-releasing bacteria (e.g., Firmicutes and Bac-
teroidetes phyla), with a daily yield of approximately 13 L of hydrogen
gas (Hylemon, Harris, & Ridlon, 2018). On the other side, cross-feeding
microbes or hydrogenotrophs (e.g., methanogens, acetogens, sulfate-
reducing bacteria) sustain to utilize intestinal H
2
for its growth and
metabolism (Smith, Shorten, Altermann, Roy, & McNabb, 2019), with
H
2
-consuming microbes usually located in the distal intestine (Rey et al.,
2013). HRW may thereby provide a supplemental substrate for hydro-
genotrophs, leading to a higher abundance of these members of gut
ora, and possible increase of their metabolites in the gut (including
CH
4
, acetate, and H
2
S). Zheng and co-workers (2018) conrm this
speculation by nding an abundance of colonic methanogens and
sulfate-reducing bacteria (e.g., Methanobrevibacter smithii, Desulfovibrio
spp.) after 25-day HRW intervention, accompanied by higher levels of
acetate in the caecum of animals treated with HRW. This effect might be
a duration-dependent since short-term HRW gavage (e.g., 5 days) had no
signicant effect on enteric microbiota abundance, at least in total
abdominal irradiation model (Xiao et al., 2018). Besides reinforcing
hydrogenotrophs, a rise in hydrogen partial pressure after HRW con-
sumption could dampen the redox potential in the intestinal lumen
(Million & Raoult, 2018), favoring the growth of anaerobes and
butyrate-producing bacteria. This has been proven in previous studies
where HRW facilitated intestinal fermentation by increasing the relative
abundance of various anaerobic phyla (e.g., Deferribacteres, Bacter-
oidetes, Firmicutes) (Higashimura et al., 2018; Bordoni et al., 2019). The
above effects likely happen at once but could push cascade reactions of
HRW-triggered microbiota to mass-produce various biologically active
compounds (e.g., propionic acid, butyric acid, acetate, H
2
S) that can
further modulate gut microbiota metabolism per se, perhaps as a sec-
ondary upshot of HRW. For instance, propionic acid and H
2
S are known
to have many physiological functions that might be relevant for both
intestinal and systemic immunomodulation, gene expression and cell
signaling (Al-Lahham, Peppelenbosch, Roelofsen, Vonk, & Venema
2010; Blachier et al., 2010). This indirect effect might be accompanied
by another possible impact of HRW that occurs after a proportion of
exogenous H
2
passes through the gut mucosa wall into the circulation
and being transported to various organs; this by itself may produce ef-
fects relevant to the gut microbiota that are mediated by gastrin mod-
ulation (McCarty, 2015). Besides, hydrogen from HRW may also act as a
signaling agent and alter gene expression of several gut-specic meta-
bolic genes including proliferator-activated receptor-gamma
coactivator-1alpha and broblast growth factor 21 (Kamimura, Ichi-
miya, Iuchi, & Ohta, 2016), and reactome pathways related to collagen
biosynthesis and heat shock response (Nishiwaki et al., 2018). However,
Fig. 1. Possible mechanisms (red arrows) and open questions (blue arrows) of hydrogen-rich water action on gut microbiota. PK – pharmacokinetics.
S.M. Ostojic

Journal of Functional Foods 78 (2021) 104360
4
this is a fairly simplied overview of how HRW may alter intestinal ora
while many questions remained unanswered, including a relative
contribution of each possible mechanism to the net-effect of HRW. It
remains particularly puzzling exactly how a relatively small quantity of
exogenous H
2
from HRW drives notable changes in gut microbiota since
only up to 0.04 L of hydrogen gas is supplied daily by drinking 2 L of
HRW while at least 300 times more H
2
is produced endogenously. This
could be due to a rather steep rise in gut H
2
levels that happens almost
immediately after HRW consumption (Zheng et al., 2018), a pattern that
might trigger acute mechanism(s) and/or cascade reactions described
above (or another unknown pathway), while endogenous H
2
is released
more gradually and perhaps being unable to elicit this response. A
clinical biotransformation study to describe the disposition of H
2
after
drinking isotopically labeled HRW is highly warranted to better un-
derstand the possible mechanism(s) and behavior of exogenous
hydrogen within the human body.
4. Can HRW be used as a prebiotic?
Prebiotics are food substances that could instigate the growth and
activity of healthy gut bacteria. A typical prebiotic is a non-digestible
specialized plant ber that purportedly enhances the fermentation in
the colon by being a substrate for Bidobacteria and Lactobacillus, pro-
tective endogenous enteric bacteria that may have favorable effects on
the host digestion and immunity (for a review see Holscher, 2017).
Although HRW has been found to modulate intestinal microbiota in the
above pilot studies, it should not be categorized either as a prebiotic or
probiotic (e.g., live microorganisms claimed to improve or restore the
gut ora) owing to the more complex and different behavior of H
2
in the
gut. HRW could be rather named as ‘hydrobiotic’, a unique compound
that aims to compensate and stabilize H
2
levels in the gut. A disbalance
in the intestinal cycling of hydrogen gas is recognized as a risk factor for
several diseases, including irritable bowel syndrome, inammatory
bowel disease (IBD), obesity, and Parkinson’s disease (Ostojic, 2018;
Smith et al., 2019). For instance, a low abundance of hydrogen-
producing bacteria has been demonstrated in patients with irritable
bowel syndrome (Pozuelo et al., 2015), with an abundance of several
bacterial taxa (e.g., Bacteroides, Ruminoccocus, Prevotella) correlates
negatively with the sensations of atulence and abdominal pain. An
apparent H
2
deciency may thus advance HRW as an experimental
therapeutics in those disorders. As a matter of fact, HRW was found to be
protective against IBD in an animal model (Shen et al., 2017). Although
gut microbiota proles were not evaluated to conrm ora-specic
modulation, 7-day HRW effectively alleviated the symptoms of food
toxin-induced IBD (e.g., change in weight, blood in the stool, and stool
consistency), ameliorated diarrhea, macroscopic and microscopic dam-
age of the colon, and protected colonic cells from oxidative stress and
inammation. Along with this, drinking ionized water (presumably rich
in hydrogen) for eight weeks improved quality of life in patients with
irritable bowel syndrome (Shin et al., 2018); no information has been
provided does ionized water modulates gut microbiota although the
authors suggested acceleration the growth of anaerobic bacteria (Lac-
tobacilli and Bidobacteria) after the intervention. Besides assumed
benecial effects on gut microbiota, drinking HRW could induce less
favorable consequences. For instance, HRW encourages sulfate-reducing
bacteria to produce hydrogen sulde. Hydrogen sulde (H
2
S) is a bio-
logically active gas that is normally produced in small amounts and has a
number of signaling functions; if present in large amounts it may show
pro-inammatory properties on the colonic mucosa and negatively
affect epithelial barrier in the colon (Blachier et al., 2010). Does HRW
induce over-production of H
2
S in the gut and how HRW-driven H
2
S
output alter intestinal ambiance remain currently unknown.
5. Conclusion
Preliminary ndings suggest that HRW may positively affect the gut
microbiota. However, this claim is still of very limited scope due to a
small body of work done in this area, and many unresolved biomedical
attributes of HRW consumption. The promising results from the pilot
studies, however, justify further scientic endeavors in this direction.
Further trials are highly warranted to detail mechanism(s) of HRW ac-
tion, its pharmacokinetics and pharmacodynamics, and HRW medium-
and long-term safety, while accounting for diverse microbial proles
among different individuals (both healthy populations and clinical pa-
tients), and HRW treatment dosages/protocols. HRW might be an up-
and-coming functional drink that could nely tune endogenous H
2
ho-
meostasis and adjust gut microbiota but it should still be perceived as an
experimental drink and not widely recommended to the general public.
Ethical statement
This is a review paper, which doesn’t include animal or human
experiments.
Author contributions
SMO solely contributed to all aspects of this paper. The corre-
sponding author had nal responsibility for the decision to submit for
publication.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Acknowledgments
None.
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