Effects of drinking hydrogen-rich water in men at risk of peripheral arterial disease: a randomized placebo-controlled trial

Abstract

Aims Smoking, hypertension, hyperlipidemia, and diabetes are considered to increase the incidence of peripheral arterial disease (PAD). They can activate endogenous free radicals, cause inflammation and oxidative stress, and lead to endothelial cell dysfunction. Hydrogen (H2) has been proven to decrease oxidative stress, improve cell function, and reduce chronic inflammation. The purpose of this research was to validate the role of H2 in individuals who are at risk of PAD.
Methods Sixty subjects were randomly assigned to placebo (PBO) group or H2-rich water (HRW) group and drank either bottled pure water or H2-rich water (245 mL/time, 3 times/d) for ten weeks.
Results The pulse wave velocity was ameliorated in the HRW group with no significant change in the ankle-brachial index. The serum total cholesterol of the HRW group was significantly reduced compared to the placebo group. In addition, compared to baseline, the levels of lipoprotein(a) was decreased, the malondialdehyde content was reduced, the superoxide dismutase activity was increased, and the expression of intercellular cell adhesion molecule-1 was decreased significantly in the HRW group. The oxidized phospholipid of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphatidylcholine level in the HRW group were significantly reduced compared to the placebo group. Finally, H2 significantly improved the antioxidant, antiinflammatory, and antiapoptotic abilities of high-density lipoprotein (HDL).
Conclusions Drinking HRW can improve vascular sclerosis indicators, improve dyslipidemia, reduce vascular oxidative stress and inflammation, and improve HDL function. H2 may be used to prevent and relieve PAD caused by major risk factors such as smoking, hypertension, hyperlipidemia, and diabetes.

Full text

Page 1/20
Effects of drinking hydrogen-rich water in men at
risk of peripheral arterial disease: a randomized
placebo-controlled trial
Qianqian Gu
Shandong First Medical University & Shandong Academy of Medical Sciences
Boyan Liu
Shandong First Medical University & Shandong Academy of Medical Sciences
Junli Xue
Shandong First Medical University & Shandong Academy of Medical Sciences
Min Zhao
Shandong First Medical University & Shandong Academy of Medical Sciences
Xiaoyi Zhang
Shandong First Medical University & Shandong Academy of Medical Sciences
Mingyue Wang
Shandong First Medical University & Shandong Academy of Medical Sciences
Mengyu Zhang
Shandong First Medical University & Shandong Academy of Medical Sciences
Yazhuo Xue
Shandong First Medical University & Shandong Academy of Medical Sciences
Shucun Qin (  scqin@sdfmu.edu.cn )
Shandong First Medical University & Shandong Academy of Medical Sciences
Research Article
Keywords: peripheral arterial disease (PAD), atherosclerotic diseases, chronic inammation
DOI: https://doi.org/10.21203/rs.3.rs-136097/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License

Page 2/20
Abstract
Aims Smoking, hypertension, hyperlipidemia, and diabetes are considered to increase the incidence of
peripheral arterial disease (PAD). They can activate endogenous free radicals, cause inammation and
oxidative stress, and lead to endothelial cell dysfunction. Hydrogen (H2) has been proven to decrease
oxidative stress, improve cell function, and reduce chronic inammation. The purpose of this research
was to validate the role of H2 in individuals who are at risk of PAD.
Methods Sixty subjects were randomly assigned to placebo (PBO) group or H2-rich water (HRW) group
and drank either bottled pure water or H2-rich water (245mL/time, 3 times/d) for ten weeks.
Results The pulse wave velocity was ameliorated in the HRW group with no signicant change in the
ankle-brachial index. The serum total cholesterol of the HRW group was signicantly reduced compared
to the placebo group. In addition, compared to baseline, the levels of lipoprotein(a) was decreased, the
malondialdehyde content was reduced, the superoxide dismutase activity was increased, and the
expression of intercellular cell adhesion molecule-1 was decreased signicantly in the HRW group. The
oxidized phospholipid of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphatidylcholine level in the HRW group
were signicantly reduced compared to the placebo group. Finally, H2 signicantly improved the
antioxidant, antiinammatory, and antiapoptotic abilities of high-density lipoprotein (HDL).
Conclusions Drinking HRW can improve vascular sclerosis indicators, improve dyslipidemia, reduce
vascular oxidative stress and inammation, and improve HDL function. H2 may be used to prevent and
relieve PAD caused by major risk factors such as smoking, hypertension, hyperlipidemia, and diabetes.
Introduction
Atherosclerotic diseases, including peripheral arterial disease (PAD), coronary artery disease, and cerebral
artery disease are the leading causes of death worldwide. PAD is an abnormal narrowing of the arteries,
and mainly includes disease of the aortoiliac, femoropopliteal, and infrapopliteal arterial segments. The
symptomatic manifestations of PAD include leg pain, intermittent claudication, pain at rest, gangrene
when the limb is severely ischemic, and even amputation1. Currently, there are more than 202million
patients with PAD worldwide, and it is predicted that up to 45million patients with PAD will die from
coronary or cerebrovascular disease over a 10-year period2. Smoking, hypertension, hypercholesteremia,
and diabetes are the four major risk factors for PAD 3. Smoking is a particularly strong risk factor for PAD
with an obvious dose-response relationship, and heavy smokers are four times more likely to develop PAD
than nonsmokers4. Diabetes, hypertension, and hypercholesterolemia are also highly associated with
PAD, with an approximately two- to three-fold increased risk. The incidence of PAD is also increased with
age5,6.
Planned exercise and lifestyle improvements are practical ways to reduce the risk and delay the progress
of PAD. Currently, PAD is mainly treated with antiplatelet therapy, anticoagulant therapy, statins,

Page 3/20
antihypertensive therapy, and medications to improve circulatory ow7. These therapies are mainly aimed
at the cause of PAD, and applied to patients with symptomatic PAD; however, such drugs are usually
accompanied by side effects. At present, 17β-estradiol 8, ginsenoside Rb39, recombinant human Relaxin-
210 and active substances from red wine 11 have shown promise in improving vascular damage caused
by smoking. However, their curative effects have certain limitations. Patients with typical PAD symptoms
account for only 20%, and approximately 50% of patients are asymptomatic12. People generally lack
awareness of the diagnosis and treatment of PAD, and are unlikely to choose long-term treatment to slow
the progression of PAD. Therefore, for asymptomatic patients with PAD, it is of great signicance to
choose a safe treatment with no side effects in order to prevent and alleviate the progression of the
disease.
Hydrogen (H2) is a bioactive gas that has benecial effects in diseases such as metabolic syndrome 13,14,
type 2 diabetes 15, chronic liver inammation 16 ,and focal brain and ischemia/reperfusion injury 17. the
mechanisms of which are thought to be related to its antioxidative, antiinammatory, and antiapoptotic
properties. In addition, it has been previously demonstrated that H2 has a protective effect on endothelial
cells. Indeed, a previous report showed that H2-saturated water could promote the recovery of blood
perfusion in a mouse PAD model by increasing angiogenesis and decreasing the level of oxidative stress
18. However, the effect of H2 in individuals at risk of PAD has not yet been demonstrated. In this study, we
performed a randomized placebo-controlled trial to characterize the effect of H2-rich water drinking on
PAD in men at risk of PAD, and its effects on oxidative stress and inammatory factors. This is the rst
randomized controlled trial of H2 on risk of PAD.
Results
Subject's baseline characteristics
The selection process is shown in Fig.1. 59 subjects participate in the study, and one subject withdrew at
week 10 due to his work outside. The specic baseline clinical indicators are shown in Table1. The study
subjects were divided into the placebo (PBO) group or the H2-rich water (HRW) group at random. The
study subjects each drank three bottles of placebo water or H2-rich water (245mL/bottle) per day for 10
weeks. Blood samples were collected at the beginning and after the 10-week trial.

Page 4/20
Table 1
Baseline level of general information of the two groups.
variable PBO (n = 29) HRW (n = 30) P
value
Number of cases
(percentage) Number of cases
(percentage)
Age    0.31
 40–55 16 (53.3%) 12 (40%) 
 55–65 9 (30%) 11 (36.6%) 
≥ 65 5 (16.6%) 7 (23.3%) 
BMI    0.79
 27-29.9 10 (33.3%) 11 (36.6%) 
≥ 30 20 (66.6%) 19 (63.3%) 
Drink alcohol    0.61
 Often 16 (53.3%) 18 (60%) 
 occasionally 8 (26.6%) 7 (23.3%) 
 No 6 (20%) 5 (16.6%) 
Activity    0.66
 Often 15 (50%) 16 (53.3%) 
 occasionally 9 (30%) 10 (33.3%) 
 No 6 (20%) 4 (13.3%) 
Smoking index    0.12
 < 400 4 (13.3%) 9 (30%) 
≥ 400 26 (86.6%) 21 (70%) 
Disease condition    
 hypertension 10 9 
 Diabetes 14 12 
 Hyperlipidemia 8 8 
Systolic blood
pressure    0.19
HRW, H2-rich water group. PBO, placebo group. BMI, body mass index.

Page 5/20
variable PBO (n = 29) HRW (n = 30) P
value
Number of cases
(percentage) Number of cases
(percentage)
 < 140 16 (53.3%) 21 (70%) 
≥ 140 14 (46.6%) 9 (30%) 
Diastolic blood
pressure    0.10
 < 90 17 (56.6%) 23 (76.6%) 
≥ 90 13 (43.3%) 7 (23.3%) 
HRW, H2-rich water group. PBO, placebo group. BMI, body mass index.

Breath hydrogen measurement following administration of H2-rich water
As shown in Fig.2, after drinking H2-rich water (650–700µmol/L, 245mL), the exhaled H2 concentration
rose rapidly over the course of 5min and reached the peak in the 10th min, with a value of approximately
7ppm. Subsequently, the concentration gradually decreased and returned to baseline in the 60th min.
There was no signicant difference between the results of males and females (data not shown).
Effect of H2 on PWV and ABI
Table2 depicts the changes in ABI and PWV after 10 weeks of H2-rich water intervention. The low value
of ABI (the minimum value of the measured ABI of the left and right lower limbs) and the ABI of the left
and right lower limbs were not signicantly different between the HRW group and the PBO group). The
low value of PWV (the minimum value of the measured PWV of the left and right lower limbs) and the
PWV of the left lower limb decreased signicantly after drinking H2-rich water (P < 0.05). The PWV of the
right lower limb also showed a decreasing trend after drinking H2-rich water (P > 0.05).

Page 6/20
Table 2
Effect of H2 on PWV and ABI
measure HRW group P PBO group P P§
0-week 10-week 0-week 10-week
Left lower limb
ABI 1.17 ± 0.10 1.22 ± 0.14 0.08 1.17 ±
0.11 0.16 ±
0.14 0.90 0.21
Right lower
limb ABI 1.11 ± 0.11 1.16 ± 0.15 0.11 1.15 ±
0.15†
1.14 ±
0.17 0.64‡0.72
ABI low value 1.10 ± 0.12 1.12 ± 0.16 0.47 1.14 ±
0.10†
1.11 ±
0.17 0.98‡0.55
PWV left, cm/s 1583.5 ±
269.90 1430.16 ±
228.53 0.01* 1421 ±
412†
1520 ±
315 †0.19‡0.39‡
PWV right,
cm/s 1561.13 ±
272.93 1502.53 ±
258.41 0.17 1439 ±
421†
1454 ±
313.5†0.08‡0.79‡
PWV low value,
cm/s 1583.5 ±
269.90 1391.56 ±
201.88 0.0004* 1427 ±
326†
1386 ±
267.5†0.56‡0.50‡
P§represents the 10-week comparison between the hydrogen-rich water group and the placebo group.
† Non-normal data is represented by median and interquartile range and the remaining results are
shown as mean ± SD. ‡Statistical analysis was performed by nonparametric tests for nonparametric
data, and Student's t test for normally distributed data. *P < 0.05.

Effect of H2 on lipid proles
After 10 weeks of intervention, the TC level in the HRW group was signicantly lower than that in the PBO
group (P < 0.05, Table3). The serum Lp(a) levels were signicantly decreased after 10 weeks of H2
treatment in the HRW group (P < 0.05, Table3). In addition, the levels of TG, VLDL, and LDL-C showed a
slight decreasing trend in the HRW group after 10 weeks of intervention compared with the HRW group
before intervention or the PBO group (Table3). No obvious changes in Apo A, Apo B, and HDL-C levels
were observed (Table3).

Page 7/20
Table 3
Effect of H2 on lipid proles, oxidative and inammatory biomarkers
measure HRW group P PBO group P P§
0-week 10-week 0-week 10-week
TC, mmol/L 5.24 ±
1.21 5.34 ±
0.82†0.82‡5.39 ±
0.77 5.69 ± 0.84 0.004* 0.047*
TG, mmol/L 1.19 ±
0.64†
1.53 ±
1.18†0.22‡1.30 ±
0.65†
1.42 ±
0.64†0.82‡0.43‡
Lp(a), mg/L 2.06 ±
0.50 1.98 ± 0.54 0.01* 1.98 ±
0.36 1.99 ± 0.38 0.50 0.55
LDL-C, mmol/L 1.21 ±
0.27 1.23 ± 0.27 0.70 1.22 ±
0.20 1.24 ± 0.21 0.27 0.72
VLDL, mmol/L 0.85 ±
0.43†
0.86 ± 0.23 0.22 ‡0.83 ±
0.3†
0.86 ± 0.30 0.79‡0.98
HDL-C (mmol/L) 1.44 ±
0.32 1.41 ± 0.35 0.70 1.30 ±
0.28 1.32 ± 0.28 0.72 0.95
ApoB, g/L 1.24 ±
0.21 1.24 ± 0.21 1.00 1.22 ±
0.20†
1.23 ±
0.24†0.72‡0.90‡
ApoA, g/L 1.48 ±
0.23 1.43 ± 0.21 0.34 1.38 ±
0.33†
1.46 ± 0.27 0.85‡0.60
MDA, mmol/L 3.93 ±
1.68 3.12 ± 0.69 0.03* 3.38 ±
1.39 3.32 ± 0.85 0.86 0.98
SOD activity,
U/mL 12.47 ±
1.84 13.39 ±
0.88†
0.002*
‡
12.71 ±
1.84 †
12.98 ±
1.91†0.06‡0.44‡
PAZPC,relative
peak area —— 0.08 ± 0.02 —— —— 0.09 ± 0.03 —— 0.04
PGPC,relative
peak area —— 0.05 ± 0.02 —— —— 0.06 ± 0.02 —— 0.27
PONPC,relative
peak area —— 0.0056 ±
0.0004 —— —— 0.0059 ±
0.0008†
—— 0.09‡
POVPC,relative
peak area —— 0.0045 ±
0.002 —— —— 0.0046 ±
0.001 —— 0.85
P§represents the comparison between the hydrogen-rich water group and the placebo group at 10-
week.*P < 0.05. †Non-normal data is represented by median and interquartile range and the remaining
results are shown as mean ± SD. ‡Statistical analysis was performed by nonparametric tests for
nonparametric data, and Student's t test for normally distributed data.

Page 8/20
measure HRW group P PBO group P P§
0-week 10-week 0-week 10-week
ICAM-1, pg/mL 5.32 ±
0.39 5.30 ± 0.37 0.003* 5.11 ±
0.85†
5.10 ±
0.83†0.16‡0.42‡
MMP-1, pg/mL 2.67 ±
0.32 2.66 ± 0.30 0.82 2.69 ±
0.31 2.64 ± 0.27 0.35 0.85
CCL1, pg/mL 1.35 ±
0.35†
1.49 ±
0.25†0.19‡1.48 ±
0.20†
1.35 ±
0.35†0.33‡0.26‡
P§represents the comparison between the hydrogen-rich water group and the placebo group at 10-
week.*P < 0.05. †Non-normal data is represented by median and interquartile range and the remaining
results are shown as mean ± SD. ‡Statistical analysis was performed by nonparametric tests for
nonparametric data, and Student's t test for normally distributed data.

Effect of H2 on oxidative and inammatory biomarkers
The serum levels of MDA decreased and the activity of SOD increased in the HRW group after 10 weeks
of intervention (P < 0.05, Table3). The levels of oxidized phospholipids also decreased, especially PAzPC
(P < 0.05, Table3). The HRW group demonstrated signicant attenuation of the inammatory biomarker
of ICAM-1 (P < 0.05, Table3). The concentration of MMP-1 and CCL1 was not signicantly different
following intervention (Table3).
H2 improves the oxidation and the functional properties of HDL
H2 signicantly reduced thiobarbituric acid-reactive substances generated by LDL oxidation (Fig.3A),
indicating that H2 can improve HDL antioxidant function. We detected cell viability by CCK-8 to verify the
antiapoptotic function of HDL after H2 intervention. We found that H2 inhibited endothelial cell apoptosis
induced by ox-LDL (Fig.3B). Ox-LDL can induce HUVECs to increase the adhesion of monocytes to
endothelial cells. Following incubation with HDL, we found that the adhesion of monocytes in the HRW
group was signicantly reduced after H2 intervention (Fig.3C), showing that H2 can enhance the
antiinammatory effect of HDL.
Discussion
We performed a randomized placebo-controlled trial and found that H2 can relieve vascular sclerosis,
regulate lipid metabolism disorder, improve antioxidant capacity, and reduce inammation in people who
are at risk of PAD. Since ischemia/reperfusion injury that occurs during PAD is accompanied by an
increase in reactive oxygen species (ROS) formation, antioxidant therapy may be a viable
countermeasure 19. H2, as a novel antioxidant, has been shown to have roles in various diseases, and a

Page 9/20
previous report has demonstrated the therapeutic effect of H2 in PAD in mice 18. Therefore, it is important
to further verify the effect of H2 in patients with PAD.
We measured the H2 concentration in the exhaled gas of patients in order to study the
in vivo
kinetics of
H2 after drinking H2-rich water. Our results demonstrated that it took 10min for H2 to reach the peak, and
it gradually returned to baseline in 60min. These results were consistent with previous reports that
showed that the breath H2 concentration reaches the maximum 10–15 minutes after intake, and then
decreases gradually, returning to baseline levels 45–150min after drinking H2-rich water20–22. The peak
concentration in the current study was approximately 7ppm after drinking 245mL of H2-rich water; this is
lower than previous reports that found peak breath hydrogen levels of 30–60ppm after drinking 200–
300mL of H2-rich water20–22.
PWV and ABI are becoming increasingly important in the assessment of arterial stiffness. ABI refers to
the pressure ratio of the left and right upper arms to the left and right ankles, and has a normal range of
1.0–1.4. PWV is the most widely used measure of arterial stiffness, and an increase in the propagation
speed of the PWV in the artery is associated with an increase in arterial stiffness. The uctuating
conduction velocity between two heartbeats is used to judge the elasticity of arterial wall23. The normal
PWV is less than 1400cm/s, and our study showed that H2 had a signicant impact on PWV following
10-week intervention.
The lipid-lowering effect of H2-rich water has been veried in patients with potential metabolic syndrome
24. In our study, the TC level was lower in the HRW group than the PBO group, and the 10-week H2
intervention reduced the Lp(a) level in individuals at risk of PAD. Furthermore, our previous study also
showed the reducing effect of H2-rich water on TC in patients with potential metabolic syndrome or
hypercholesterolemia 13,25. In addition, studies have shown that the serum TC levels were signicantly
decreased in ethanol-induced fatty liver and nonalcoholic fatty liver mice after H2-rich water treatment
26,27. It is well known that a high cholesterol level is one of the most important risk factors for PAD.
Therefore, elucidation of the mechanism by which H2 decreases serum cholesterol will provide solid
evidence for the application of H2 in PAD therapy.
Lp(a) is a cholesterol-rich, LDL-like particle, with specic apolipoprotein (a) and apolipoprotein B-100. The
Lp(a) levels of plasma independently predict atherosclerotic cardiovascular disease (CVD) and PAD,
although its mechanism of action in atherosclerosis remains unclear 28. Therapy to lower the plasma
levels of Lp(a) has gained much attention in recent years, and our results reveal that H2-rich water can
reduce the level of plasma Lp(a). Lp(a) is regarded as a preferential carrier of oxidized phospholipids
(OxPLs) in human plasma 29, and we demonstrated that H2-rich water can reduce the levels of oxidized
phospholipids, especially PAzPC. Moreover, a previous study showed that H2 can suppress the
autoxidation of linoleic acid and PAPC in a pure chemical system30. The mechanism by which H2
decreased the formation of OxPLs
in vivo
is worthy of further study.

Page 10/20
PAD is an atherosclerotic disease of arterial vessels, in which the cycles of ischemia and reperfusion
induced by PAD leads to increased mitochondrial ROS19. Smoking is one of the main risk factors for PAD,
and Smokers with hypertension, hyperlipidemia, and diabetes will increase the incidence of PAD. Cigarette
products are known to activate the production of oxidative free radicals in the body 31. In our study, H2, as
a novel antioxidant, could signicantly reduce MDA and signicantly increase the activity of SOD. MDA is
the product of lipid peroxidation and a marker of oxidative stress, while SOD is an antioxidant enzyme
that can remove ROS from the body 32. Studies have shown a reduction in serum MDA and the increase
in SOD activity in patients with potential metabolic syndrome 33 or type 2 diabetes 20 after H2 treatment.
Previous reports have also shown that H2 can inhibit formation of MDA in ethanol/acetaminophen-
induced fatty liver 34 or liver ischemia reperfusion injury in mice 35, and reduce MDA and increase SOD
activity to relieve oxidative stress induced by chronic intermittent hypoxia in rats 36. Thus, H2 may reduce
the formation of lipid peroxides in people at risk PAD by improving antioxidant enzyme activity.
ICAM-1 plays key roles in immune-mediated and inammatory processes; it can be induced by interleukin-
1 and tumor necrosis factor, and expressed by the vascular endothelium, macrophages, and lymphocytes.
ICAM-1 is also involved in local plaque formation, and has been shown to be an independent predictor of
the development and progression of PAD 37. Previous studies have shown that H2 can inhibit the
expression of ICAM-1 and reduce the inammatory response in different animal models, including a
pressure ulcer mouse model 38, sepsis mouse model 39, ,and noise-induced hearing loss guinea pig model
40. In our study, the reduced expression of serum ICAM-1 may have led to the alleviation of PAD.
HDL is known to have vasoprotective actions and antiatherogenic effects41. The underlying mechanism
is mainly related to its function in promoting cholesterol eux from macrophage foam cells and
stimulating endothelial cell NO production to improve endothelial cell function41,42. HDL also plays roles
in the reduction of inammation and oxidative stress 43, and is benecial to endothelial cells by
protection of cytokine-induced monocyte adhesion 44. It has been shown that H2 can improve HDL
function by enhancing the cholesterol eux ability mediated by HDL, preventing LDL oxidation, and
reducing ox-LDL-induced endothelial cell apoptosis and monocyte adhesion to endothelial cells 25,33. In
this study, we isolated HDL from different groups before and after intervention and tested the functions
in
vivo
. Our results showed that H2 can improve the antioxidant, antiinammatory, and antiadhesion effects
of HDL, which may improve oxidation and inammation, and reduce vascular damage in individuals at
risk of PAD.
This study shows the effects of H2 on relieving vascular sclerosis by antioxidation and antiinammatory
mechanisms, as well as improving HDL function. It is likely that H2 exerts antioxidant and
antiinammatory effects by improving SOD activity and HDL function; this, in turn, decreases the levels
of MDA, OxPLs, ICAM-1, CCL-1, and even Lp(a), thereby alleviating artistic stiffness as measured by PWV.
At the same time, H2 exerts a lipid-lowering effect by reducing plasma TC, which also functions to lower
the risk of PAD .

Page 11/20
This study has several limitations. First, the number of participants is small, and an expanded sample is
needed to verify the results. Second, the dose-effect of H2-rich water was not studied, and a further study
is needed to determine the best dose. Third, the intervention only lasted for 10 weeks, and the effect of
long-term intervention needs further verication. Finally, besides the antioxidative and antiinammatory
properties, the molecular mechanisms of H2 need further study, especially its regulatory effects on lipid
metabolism disorder and its effect on Lp(a).
In conclusion, our data show that H2−rich water can improve vascular sclerosis indicators and lipid
disorders, and reduce oxidative stress and inammatory factor inltration. H2 may be used in adjunctive
therapy for alleviating PAD.
Methods
Subjects and study design
The study was a 10-week, randomized, placebo-controlled trial. The study protocol was authorized by the
Ethics Committee of Shandong First Medical University (NO.2019121, Date 08/10/2019). This study was
registered in the Chinese Clinical Trial Registry (www.chictr.org.cn, Registration number Chi CTR
2000035232, Date: 04/08/2020). All participants who were eligible and agreed to participate in the
randomized assignment were required to sign a written informed consent before participating in the
study. This study followed CONSORT guidelines. All methods were performed in accordance with the
relevant guidelines and regulations for research involving humans. Sixty-three subjects over 40years old
were enrolled from Zhoudian community (Tai’an, China). The enrollment conditions were as follows:
Current smoker or quit smoking within the past 10 years, ankle-brachial index (ABI) < 1.0 or smoking index
(smoking intensity × duration of smoking) ≥ 200, with or without risk factors for diabetes, high blood
pressure, and hyperlipidemia, and able to complete the questionnaire independently or with the help of
the researcher.
H2-rich water and placebo water
Bottled H2-rich water, in which the nano-scale hydrogen bubbles are physically mixed with pure water, was
purchased from Beijing Huoli Qingyuan Co., Ltd. (China). The placebo was pure water, which was
consistent with H2-rich water in terms of appearance, dosage, and packaging. The water was drunk by
subjects within 15min of opening the sealed cap. The H2 concentration of the H2-rich water was between
650 to 700µmol/L when the bottle was opened, as measured by the H2 sensor (Unisense, Aarhus,
Denmark) in our laboratory.
Breath hydrogen measurement after H2-rich water administration
We recruited 10 participants (5 males and 5 females) met the following criteria: 20–30years old, without
intestinal disease, hypoglycemia, or major diseases, and not taking intestinal ora drugs. After fasting for
12h, the participant drank a bottle of H2-rich water over the course of 1min. The exhaled breath was

Page 12/20
collected every 5min thereafter, and the H2 concentration was measured using a sensor gas
chromatograph (SGHA-P1; FIS Co. Ltd., Hyogo, Japan).
Analysis of vascular stiffness and elasticity index
Peripheral arterial stiffness and elasticity were assessed by automatic ABI and pulse wave velocity (PWV)
measurement using a noninvasive vascular screening device (BP-203RPEIII, Omron, Japan) with
participants in the supine position after resting for 10min. The ABI refers to the pressure ratio of the left
and right upper arms to the left and right ankles, and PWV refers to the speed at which the blood from the
heartbeat travels to the periphery in the form of waves, forming a pulse wave and propagating in the
arteries.
Serum lipid analysis
Serum lipoprotein(a) (Lp[a]) was measured by the latex immunoturbidimetric method, and total
cholesterol (TC), triacylglycerols (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein
cholesterol (LDL-C), very low-density lipoprotein (VLDL-C), apolipoprotein A (Apo A), and Apo B were
measured by enzymatic methods on a chemical autoanalyzer (Hitachi Co, Tokyo, Japan).
Serum oxidative stress and inammatory factors
Serum levels of malondialdehyde (MDA) were determined following spectrophotometric measurement of
thiobarbituric acid-reactive substances (TBARS) using a commercial kit (Nanjing Jiancheng Biochemistry,
China). The activity of superoxide dismutase (SOD) was tested according to the manufacturer’s
instructions (Nanjing Jiancheng Biochemistry, China). Serum concentrations of intercellular cell adhesion
molecule-1 (ICAM-1), matrix metalloproteinase-1 (MMP-1) and Chemokine (C-C Motif) Ligand 1 (CCL1)
were measured by Luminex kits (Univ-bio, Shanghai, China).
Oxidized phospholipids in serum
Lipids were extracted from plasma using a methyl-tert-butyl ether method with
dimyristoylphosphatidylcholine (14:0/14:0 PC) as an internal standard. Lipid extracts were analyzed
using a Shimadzu LC-20 AD binary pump system interfaced with an ABI 4000QTrap mass spectrometer
(Sciex, Framingham, MA, USA). Chromatographic separation was performed using a Waters Symmetry
C18 column (3.5µm, 2.1mm i.d. × 100mm) at 40°C with a ow rate of 0.3mL/min. The injection
volume was 10µL; the mobile phase comprised solvent A (acetonitrile/water, 60:40, v/v) and solvent B (2-
propanol/acetonitrile, 90:10, v/v), with both solvents containing 10mM ammonium acetate and 0.1%
acetic acid. Isocratic elution was performed for 16min with 95% B. Detection of oxidized phospholipids
was accomplished during the multiple reaction monitoring mode with positive-ion detection using m/z
184 as the product ion. The parent ions of 1-palmitoyl-2-(5-oxo-valeroyl)-sn-glycero-3-phosphatidylcholine
(POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphatidylcholine (PGPC), 1-palmitoyl-2-azelaoyl-sn-
glycero-3-phosphatidylcholine (PAzPC), and 1-palmitoyl-2-(9-oxo-nonanoyl)-sn-glycero-3-
phosphatidylcholine (PONPC) were 594.4, 610.4, 666.5, and 650.5, respectively.
Antioxidant properties of HDL

Page 13/20
HDL was isolated from the pooled serum by ultracentrifugation (n = 3 samples for each group, each
comprising the serum of 4–5 subjects) as described 45. LDL (100µg/mL) from healthy people and HDL
(200µg/mL) isolated from each group were incubated with freshly prepared CuSO4 (10µmol/L) at 37°C
for 2h. The extent of LDL oxidation was assessed by measuring the level of MDA via a
spectrophotometric method according to the manufacturer’s instructions (Nanjing Jiancheng
Biochemistry, China).
Endothelial cell - monocyte adhesion assay
The monocyte adhesion assay was slightly modied as described previously46. Human umbilical vein
endothelial cells (HUVECs) were cultured at 37°C in a humidied 95% air-5% CO2 atmosphere, grown to
70–80% conuence in 96-well plates, and stimulated with ox-LDL (100µg/mL) in the presence or
absence of HDL (100µg/mL) for 24h. THP-1 monocytes at a density of 2 × 105 were labeled with
10µmol/L 2',7'-bis(2-carboxyethyl)-5(6)-carboxyuorescein, acetoxymethyl ester (BCECF-AM) at 37°C for
1h in RPMI-1640 medium and rinsed with serum-free RPMI-1640 medium. HUVECs in 96-well plates were
washed three times and incubated with 100µL THP-1 cells for 1h. Then, each well was rinsed three times
with PBS to remove unbound THP-1 cells. THP-1 cells bound to HUVECs were visualized with a
uorescent microscope (Nikon, Japan) at 4 elds per × 100 high-power-eld well. Experiments were
performed at least three times and the selection of high-power elds to count separate wells was
performed at random.
Cell viability determined by CCK-8 assay
HUVECs were seeded in 96-well plates and pretreated with or without HDL (100µg /mL) for 6h and
stimulated with ox-LDL (100µg/mL) for 18h. The viability of the HUVECs was measured by CCK-8 assay
(Med Chem Express, USA), and the absorbance was measured at 450nm using a microplate
spectrophotometer system (Tecan, Sweden). The percentage viability was calculated using the following
formula: HUVECs viability % = (OD sample– OD blank)/ (OD control– OD blank) × 100%33.
Statistical analysis
Descriptive statistics were used to compare the baseline characteristics of the subjects in the two groups
(means ± SD). Statistical analysis was performed by Student's t test for normally distributed data and by
nonparametric tests for nonparametric data. The SPSS program (version 22.0) was used for all statistical
analyses, and all data were plotted with GraphPad Prism 8. P-values < 0.05 were considered signicant.
Declarations
Conict of Interest:
The authors have no conicts of interest to report.
Funding:

Page 14/20
This research was funded by the National Natural Science Foundation of China (grant number
81770855), Taishan Scholars Program of Shandong Province (grant number ts201511057) and
Academic promotion programme of Shandong First Medical University (grant numbers 2019QL010,
2019PT009).
Author contributions
S.Q., Y.X, B.L., and Q.G., participated in study conception and design. Q.G., B.L., J.X. M.Z., X.Z., M.W., and
M.Z. performed the acquisition of data. Q.G., B.L., and J.X. participated in analysis and interpretation of
data. B.L. and Q.G. drafted the manuscript and S.Q. helped in critical review of the manuscript. S.Q.
obtained the funding. S.Q. and Y.X. supervised the study. All the authors have read and approved the
submitted manuscript.
Acknowledgments
This research was funded by the National Natural Science Foundation of China (grant number
81770855), Taishan Scholars Program of Shandong Province (grant number ts201511057) and
Academic promotion programme of Shandong First Medical University (grant numbers 2019QL010,
2019PT009).The authors thank Xiao Liu for her assistance with subject recruitment, Meiyuan Liu, Yujuan
Sun and Mei Li from Zhoudian community for their help, as well as all the volunteers who participated in
this study.
References
1 Writing Committee Members, G.-H. M., Gornik HL, et al. . 2016 AHA/ACC Guideline on the
Management of Patients With Lower Extremity Peripheral Artery Disease: Executive Summary.
Vascular
Medicine
22(3) NP1 –NP43, doi:DOI: 10.1177/1358863X17701592 (2017).
2 Lozano, R.
et al.
Global and regional mortality from 235 causes of death for 20 age groups in
1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010.
The Lancet
380,
2095-2128, doi:10.1016/s0140-6736(12)61728-0 (2012).
3 Fowkes, F. G. R.
et al.
Comparison of global estimates of prevalence and risk factors for
peripheral artery disease in 2000 and 2010: a systematic review and analysis.
The Lancet
382, 1329-
1340, doi:10.1016/s0140-6736(13)61249-0 (2013).
4 Hisamatsu, T.
et al.
Smoking, Smoking Cessation, and Measures of Subclinical Atherosclerosis
in Multiple Vascular Beds in Japanese Men.
Journal of the American Heart Association
5(9):e003738,
doi:10.1161/jaha.116.003738 (2016).
5 Mohammedi K, Woodward M, Hirakawa Y & al., e. Microvascular and Macrovascular Disease
and Risk for Major Peripheral Arterial Disease in Patients With Type 2 Diabetes.
Diabetes Care
2016;39(10):1796-1803, doi:10.2337/dc16-0588/-/DC1 (2016).

Page 15/20
6 Selvin E, E. T. Prevalence of and Risk Factors for Peripheral Arterial Disease in the United States
Results From the National Health and Nutrition Examination Survey,1999–2000.
Circulation
110(6):738-
743, doi:10.1161/01.CIR.0000137913.26087.F0 ( 2004).
7 Firnhaber JM, P. C. Lower Extremity Peripheral Artery Disease: Diagnosis and Treatment.
Am
Fam Physician
99(6):362-369 (2019).
8 Resanovic, I.
et al.
Anti-atherogenic effects of 17beta-estradiol.
Horm Metab Res
45, 701-708,
doi:10.1055/s-0033-1343478 (2013).
9 Wang, M.
et al.
Ginsenoside Rb3 exerts protective properties against cigarette smoke extract-
induced cell injury by inhibiting the p38 MAPK/NF-kappaB and TGF-beta1/VEGF pathways in broblasts
and epithelial cells.
Biomed Pharmacother
108, 1751-1758, doi:10.1016/j.biopha.2018.10.018 (2018).
10 Pini, A.
et al.
Protection from cigarette smoke-induced vascular injury by recombinant human
relaxin-2 (serelaxin).
J Cell Mol Med
20, 891-902, doi:10.1111/jcmm.12802 (2016).
11 Schwarz, V.
et al.
Red Wine Prevents the Acute Negative Vascular Effects of Smoking.
Am J Med
130, 95-100, doi:10.1016/j.amjmed.2016.08.025 (2017).
12 Hiatt WR, G. J., Smith SC Jr, McDermott M, Moneta G, Oka R, Newman AB, Pearce WH. American
Heart Association Writing Group 1. Atherosclerotic Peripheral Vascular Disease Symposium II:
nomenclature for vascular diseases.
Circulation
118(25):2826-2829.,
doi:10.1161/CIRCULATIONAHA.108.191171 (2008).
13 Song, G.
et al.
Hydrogen-rich water decreases serum LDL-cholesterol levels and improves HDL
function in patients with potential metabolic syndrome.
Journal of lipid research
54(7), 1884–1893.
(2000).
14 Liu B, X. J., Zhang M, Wang M, Ma T, Zhao M, Gu Q, Qin S. Hydrogen inhalation alleviates
nonalcoholic fatty liver disease in metabolic syndrome rats.
Mol Med Rep
22(4):2860-2868,
doi:10.3892/mmr.2020.11364 (2020).
15 Amitani, H.
et al.
Hydrogen improves glycemic control in type1 diabetic animal model by
promoting glucose uptake into skeletal muscle.
PLoS One
8, e53913, doi:10.1371/journal.pone.0053913
(2013).
16 Gharib B
et al.
Anti-inammatory properties of molecular hydrogen: investigation on parasite-
induced liver inammation.
C R Acad Sci III
324(8):719-724, doi:doi:10.1016/s0764-4469(01)01350-6
(2001).
17 Ohsawa, I.
et al.
Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic
oxygen radicals.
Nature Medicine
13, 688-694, doi:10.1038/nm1577 (2007).

Page 16/20
18 Fu, J.
et al.
Hydrogen molecules (H2) improve perfusion recovery via antioxidant effects in
experimental peripheral arterial disease.
Molecular Medicine Reports
18(6):5009-5015,
doi:10.3892/mmr.2018.9546 (2018).
19 Koutakis, P.
et al.
Oxidative stress and antioxidant treatment in patients with peripheral artery
disease.
Physiol Rep
6, e13650, doi:10.14814/phy2.13650 (2018).
20 Kajiyama, S.
et al.
Supplementation of hydrogen-rich water improves lipid and glucose
metabolism in patients with type 2 diabetes or impaired glucose tolerance.
Nutr Res
28, 137-143,
doi:10.1016/j.nutres.2008.01.008 (2008).
21 Ito, M.
et al.
Drinking hydrogen water and intermittent hydrogen gas exposure, but not lactulose
or continuous hydrogen gas exposure, prevent 6-hydorxydopamine-induced Parkinson's disease in rats.
Med Gas Res
2, 15, doi:10.1186/2045-9912-2-15 (2012).
22 Shimouchi A, Nose K, Yamaguchi M, Ishiguro H & T., K. Breath Hydrogen Produced by Ingestion
of Commercial Hydrogen Water and Milk.
Biomark Insights
4:27-32, doi:doi:10.4137/bmi.s2209 ( 2009).
23 Kim HL, K. S. Pulse Wave Velocity in Atherosclerosis.
Front Cardiovasc Med
6, 41,
doi:10.3389/fcvm.2019.00041 (2019).
24 Nakao A, Toyoda Y, Sharma P, Evans M & N., G. Effectiveness of hydrogen rich water on
antioxidant status of subjects with potential metabolic syndrome-an open label pilot study.
J Clin
Biochem Nutr
46(2):140-149, 140-149, doi:doi:10.3164/jcbn.09-100 (2010).
25 Song, G.
et al.
Hydrogen Activates ATP-Binding Cassette Transporter A1-Dependent Eux Ex
Vivo and Improves High-Density Lipoprotein Function in Patients With Hypercholesterolemia: A Double-
Blinded, Randomized, and Placebo-Controlled Trial.
J Clin Endocrinol Metab
100, 2724-2733,
doi:10.1210/jc.2015-1321 (2015).
26 Lin, C. P., Chuang, W. C., Lu, F. J. & Chen, C. Y. Anti-oxidant and anti-inammatory effects of
hydrogen-rich water alleviate ethanol-induced fatty liver in mice.
World J Gastroenterol
23, 4920-4934,
doi:10.3748/wjg.v23.i27.4920 (2017).
27 Wang, X. & Wang, J. High-content hydrogen water-induced downregulation of miR-136 alleviates
non-alcoholic fatty liver disease by regulating Nrf2 via targeting MEG3.
Biol Chem
399, 397-406,
doi:10.1515/hsz-2017-0303 (2018).
28 Orsó E, S. G. Lipoprotein(a) and its role in inammation, atherosclerosis and malignancies.
Clin
Res Cardiol Suppl
12, 31-37, doi:10.1007/s11789-017-0084-1 (2017).
29 Bergmark, C.
et al.
A novel function of lipoprotein [a] as a preferential carrier of oxidized
phospholipids in human plasma.
J Lipid Res
49, 2230-2239, doi:10.1194/jlr.M800174-JLR200 (2008).

Page 17/20
30 Iuchi, K.
et al.
Molecular hydrogen regulates gene expression by modifying the free radical chain
reaction-dependent generation of oxidized phospholipid mediators.
Sci Rep
6, 18971,
doi:10.1038/srep18971 (2016).
31 Varela-Carver A, Parker H, Kleinert C & O., R. Adverse effects of cigarette smoke and induction of
oxidative stress in cardiomyocytes and vascular endothelium.
Curr Pharm Des
16(23):2551-2558.,
doi:doi:10.2174/138161210792062830 ( 2010).
32 Niizuma, K.
et al.
Mitochondrial and apoptotic neuronal death signaling pathways in cerebral
ischemia.
Biochim Biophys Acta
1802, 92-99, doi:10.1016/j.bbadis.2009.09.002 (2010).
33 Song, G.
et al.
Hydrogen-rich water decreases serum LDL-cholesterol levels and improves HDL
function in patients with potential metabolic syndrome.
Journal of lipid research
54(7), 1884–1893,
doi:https://doi.org/10.1194/jlr.M036640 (2013).
34 Lin, C.-P., Chuang, W.-C., Lu, F.-J. & Chen, C.-Y. Anti-oxidant and anti-inammatory effects of
hydrogen-rich water alleviate ethanol-induced fatty liver in mice.
World Journal of Gastroenterology
23,
4920 (2017).
35 Liu, Y.
et al.
Protective effects of hydrogen enriched saline on liver ischemia reperfusion injury by
reducing oxidative stress and HMGB1 release.
BMC gastroenterology
14, 12 (2014).
36 Li, W.
et al.
Hydrogen ameliorates chronic intermittent hypoxia-induced neurocognitive
impairment via inhibiting oxidative stress.
Brain Res Bull
143, 225-233,
doi:10.1016/j.brainresbull.2018.09.012 (2018).
37 Tzoulaki, I.
et al.
C-reactive protein, interleukin-6, and soluble adhesion molecules as predictors
of progressive peripheral atherosclerosis in the general population: Edinburgh Artery Study.
Circulation
112, 976-983, doi:10.1161/CIRCULATIONAHA.104.513085 (2005).
38 Fang, W.
et al.
Hydrogen gas inhalation protects against cutaneous ischaemia/reperfusion injury
in a mouse model of pressure ulcer.
J Cell Mol Med
22, 4243-4252, doi:10.1111/jcmm.13704 (2018).
39 Chen, H.
et al.
Molecular hydrogen protects mice against polymicrobial sepsis by ameliorating
endothelial dysfunction via an Nrf2/HO-1 signaling pathway.
Int Immunopharmacol
28, 643-654,
doi:10.1016/j.intimp.2015.07.034 (2015).
40 Chen, L.
et al.
Molecular mechanisms underlying the protective effects of hydrogen-saturated
saline on noise-induced hearing loss.
Acta Otolaryngol
137, 1063-1068,
doi:10.1080/00016489.2017.1328743 (2017).
41 Besler, C.
et al.
Mechanisms underlying adverse effects of HDL on eNOS-activating pathways in
patients with coronary artery disease.
J Clin Invest
121, 2693-2708, doi:10.1172/JCI42946 (2011).

Page 18/20
42 Tall, A. R., Yvan-Charvet, L., Terasaka, N., Pagler, T. & Wang, N. HDL, ABC transporters, and
cholesterol eux: implications for the treatment of atherosclerosis.
Cell Metab
7, 365-375,
doi:10.1016/j.cmet.2008.03.001 (2008).
43 Soran, H., Hama, S., Yadav, R. & Durrington, P. N. HDL functionality.
Curr Opin Lipidol
23, 353-366,
doi:10.1097/MOL.0b013e328355ca25 (2012).
44 Park SH, Park JH, Kang JS & YH., K. Involvement of transcription factors in plasma HDL
protection against TNF-alpha-induced vascular cell adhesion molecule-1 expression.
Int J Biochem Cell
Biol
35(2):168-182., 168-182., doi:doi:10.1016/s1357-2725(02)00173-5 (2003).
45 HAVEL RJ, E. H., BRAGDON JH. . The distribution and chemical composition of ultracentrifugally
separated lipoproteins in human serum.
J Clin Invest
34(9):1345-1353, doi: doi:10.1172/JCI103182
(1955).
46 Yang X, Wan M, Cheng Z, Wang Z & Q., W. Tofacitinib inhibits ox-LDL-induced adhesion of THP-1
monocytes to endothelial cells.
Artif Cells Nanomed Biotechnol
47(1):2775-2782,
doi:doi:10.1080/21691401.2019.1573740 (2019).
Figures
Figure 1

Page 19/20
Flow diagram of the study subjects
Figure 2
Exhaled H2 concentration after drinking hydrogen-rich water.

Page 20/20
Figure 3
Effect of H2 on the functional of HDL particle. The results are shown as mean ±SEM. (A)TBARS
(nmol/mL 30 μg LDL protein), (B) Cell viability, and (C) Monocyte Adhesion. *, P <0.05.