Context:

We have found that hydrogen (dihydrogen [H2]) decreases plasma low-density lipoprotein (LDL) cholesterol levels and improves high-density lipoprotein (HDL) function in patients with potential metabolic syndrome in a before-after self-controlled study.

Objective:

The purpose of this study was to further characterize the effects of H2-rich water (0.9 L/day) on the content, composition, and biological activities of plasma lipoproteins on patients with hypercholesterolemia and their underlying mechanisms in a double-blinded, randomized, and placebo-controlled trial.

Design:

This was a case-control study.

Setting:

The setting was the Zhoudian community, Tai'an, China.

Patients:

A total of 68 patients with untreated isolated hypercholesterolemia were randomly allocated to either drinking H2-rich water (n = 34) or placebo water (n = 34) for 10 weeks.

Results:

HDL isolated from the H2 group showed an increased ability to promote the ATP-binding cassette transporter A1–mediated cholesterol efflux ex vivo. Plasma pre-β-HDL levels were up-regulated although there were no changes in plasma HDL-cholesterol levels. Moreover, other HDL functions, assessed in protection against LDL oxidation, inhibition of oxidized-LDL–induced inflammation, and protection of endothelial cells from oxidized-LDL–induced apoptosis, were all significantly improved by H2 treatment. In addition, H2 treatment increased the effective rate in down-regulating plasma levels of total cholesterol (47.06% vs 17.65%) and LDL cholesterol (47.06% vs 23.53%). Western blot analysis revealed a marked decrease in apolipoprotein B100 and an increase in apolipoprotein M in plasma of the H2 group. Finally H2 treatment resulted in a significant reduction in the levels of several inflammatory and oxidative stress indicators in whole plasma and HDL particles.

Conclusions:

H2 activates ATP-binding cassette transporter A1–dependent efflux, enhances HDL antiatherosclerotic functions, and has beneficial lipid-lowering effects. The present findings highlight the potential role of H2 in the regression of hypercholesterolemia and atherosclerosis.

High-density lipoproteins (HDLs) possess key atheroprotective biological properties including cellular cholesterol efflux capacity and antioxidative and anti-inflammatory activities (1). Within the circulating HDL particle population, small discoidal pre-β-HDL particles display elevated cellular cholesterol efflux capacity, afford potent protection of atherogenic low-density lipoprotein (LDL) against oxidative stress, and attenuate inflammation (1). The antiatherogenic properties of HDL can, however, be compromised in metabolic diseases, such as hypercholesterolemia associated with accelerated atherosclerosis (2, 3). Functional HDL deficiency is intimately associated with alterations in intravascular HDL metabolism and structure. Deficient HDL function may act to accelerate atherosclerosis in hypercholesterolemia. New therapies for normalization of attenuated antiatherogenic HDL function and lowering lipids might be an effective way to reduce the significant burden of cardiovascular disease in patients (4, 5).

Hydrogen (dihydrogen [H2]), as the lightest and most abundant chemical element that can reduce oxidative stress, is considered a novel antioxidant (6) and has come to the forefront of therapeutic medical gas research. Accumulated evidence in a variety of biomedical fields using clinical and experimental models for many diseases proves that H2, administered either through gas inhalation or consumption of an aqueous H2-containing solution, can act as a feasible therapeutic strategy in different disease models. For example, supplementation with H2-rich water was demonstrated to have a beneficial role in prevention of type 1 and type 2 diabetes and insulin resistance (7, 8), chronic liver inflammation (9), acute oxidative stress, and focal brain ischemia/reperfusion injury (6). In addition, we have reported that consumption of H2-saturated saline for 8 weeks prevented atherosclerosis in apolipoprotein E-knockout (apoE−/−) mice (10) and that H2 not only decreases plasma total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels in high-fat diet-fed hamsters (11) but also improves HDL function and decreases plasma LDL-C levels in patients with potential metabolic syndrome in a before-after self-controlled study (12). However, whether H2 has similar effects in a placebo-controlled study and how H2 affects HDL function have not yet been reported. Given the fact that functional HDL deficiency is intimately associated with alterations in its composition and enzymatic activities (13), we hypothesized that H2 modulates HDL subfractions and then improves HDL functions. Therefore, the aim of the present study was to further characterize the effects of H2-rich water (0.9 L/d) on the content, composition, and biological activities of plasma lipoproteins and 10-year risk for atherosclerotic cardiovascular disease (ASCVD) in patients with hypercholesterolemia in a double-blinded, randomized, and placebo-controlled trial.

Materials and Methods

Subjects

The study protocol was approved by the Ethics Committee of TaiShan Medical University. All participants recruited from ZhouDian community residents provided written informed consent to participate before enrollment in the study. We recruited 68 subjects (35–60 years old) with hypercholesterolemia, defined as plasma TC of ≥5.18 mmol/L and/or LDL-C of ≥2.59 mmol/L (12, 14). All patients had been complying with lifestyle intervention but had not received any hypolipidemic agent for at least 3 months before the beginning of the study. We excluded patients with any acute and chronic inflammatory processes, stage 2 or 3 hypertension, unstable coronary artery disease, myocardial infarction or stroke within 6 months preceding the study, symptomatic congestive heart failure, diabetes, autoimmune disorders, thyroid diseases, chronic pancreatitis, impaired renal or hepatic function, nephrotic syndrome, and body mass index of >35 kg/m2. Throughout the entire study period, all participants continued to comply with lifestyle modifications.

Study design

The study was a 10-week, randomized, placebo-controlled, double-blinded design. Patients were randomized by computer in a 1:1 allocation to either H2 or placebo water therapy. Participants and researchers were blinded after assignment to interventions. The patients consumed 0.9 L/d (0.3 L/time, 3 times/d) of H2-rich pure water or 0.9 L/d (0.3 L/time, 3 times/d) of placebo water in the ZhouDian community hospital for 10 weeks. Both waters were provided in 300-mL unlabeled aluminum pouches obtained from Beijing Hydrovita Biotechnology Company, and the H2 concentration was maintained between 0.5 and 0.6 mM as measured by an H2 sensor (Unisense) for 5 minutes continuously after the sealed pouches were opened. The water was administered to the patients by 2 resident physicians every day, and the water was drunk by patients within 5 minutes after the sealed pouches were opened. The blood samples were collected at baseline (0 week) and after 10 weeks of drinking H2 water.

Plasma analysis

Plasma levels of lipids, oxidative stress and oxidizability, and inflammatory factors were measured as detailed in the Supplemental Methods.

Lipoprotein preparation and lipid analysis in HDL3

The plasma of every 5 to 6 patients was pooled and fasted plasma lipoproteins were fractionated by ultracentrifugation at 40 000 rpm in a Beckman Optima LE-80K into very low-density lipoprotein (density = <1.006 g/mL), LDL (density = 1.006–1.063 g/mL), and HDL3 (density = 1.125–1.21 g/mL) as described previously (15). The sphingosine-1-phosphate (S1P) and ceramide contents in HDL3 were measured as described previously via liquid chromatography-tandem mass spectrometry (16, 17) The phospholipid concentration in HDL3 was measured by using a kit (catalog no. 433–36201; Wako).

HDL-induced ex vivo cholesterol efflux from bone marrow macrophages and RAW264.7 macrophages

We measured plasma high-density lipoprotein cholesterol (HDL-C)–mediated efflux in bone marrow macrophages with or without glyburide, a commonly used ATP-binding cassette transporter A1 (ABCA1) chemical inhibitor (18, 19). Next, we measured ABCA1-stimulated efflux in RAW264.7 macrophages with or without cAMP, which can induce ABCA1 activity. Detail are found in the Supplemental Methods.

Measuring antioxidant properties of HDL

LDL (100 μg of protein/mL) was incubated with freshly prepared CuSO4 (10 μM) in the presence or absence of isolated HDL3 (200 μg of protein/mL). After incubation at 37°C for 2 hours, the extent of LDL oxidation was assessed by measurement of thiobarbituric acid-reactive substance formation (20) via a spectrophotometric method according to the manufacturer's instructions (Nanjing Jiancheng Biochemistry).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis

Human umbilical vein endothelial cells (HUVECs) were grown to confluence in 96-well plates and pretreated with or without HDL (100 μg of protein/mL) for 2 hours and then stimulated with oxidized low-density lipoprotein (ox-LDL) (100 μg of protein/mL) for 12 hours. Cells treated with medium only were used as a negative control. Detection of cell viability and apoptosis by MTT and flow cytometry analysis were performed as detailed in the Supplemental Methods.

Endothelial cell-monocyte adhesion assay

Monocyte adhesion assays were performed under static conditions as described previously (21) with minor modifications as detailed in the Supplemental Methods.

Estimation of 10-year risk for ASCVD

Based on the 2013 ACC/AHA Guideline on the Assessment of Cardiovascular Risk (22), 10-year risk was defined as the risk of developing a first ASCVD event and calculated by using a web-based calculator (available at http://my.americanheart.org/cvriskcalculator).

Statistical analysis

Statistical analysis was performed by the Student t test and one-way and two-way ANOVAs with GraphPad Prism version 4.0. Multiple comparisons between the groups were performed using the Turkey method. Results are expressed as means ± SD. P values of <.05 were considered significant.

Results

Subject characteristics

The baseline demographics of subjects are presented in Supplemental Table 1. The 68 subjects enrolled in the study included subjects who had TC of ≥5.18 mmol/L (n = 57), LDL-C of ≥2.59 mmol/L (n = 68), and body mass index of 19.8 to 32.9 kg/m2 (n = 68). All subjects showed mean normal clinical levels of baseline biometric parameters and normal clinical chemistry and hematology results. No smokers or drinkers were recruited in the study.

H2 treatment increases pre-β1-HDL without decreasing lecithin:cholesterol acyltransferase (LCAT) activity

It is well known that HDLs can be divided into major subfractions: those with α mobility and the pre-β-migrating HDL (23). In HDL subfractions, apolipoprotein (apo) A-I–-containing lipoproteins with pre-β electrophoretic mobility are called pre-β1-HDLs (24), have attracted attention as the first acceptors of cellular free cholesterol, and are regulated by LCAT (25). As shown in Figure 1A, the H2 group showed a significant increase in the plasma pre-β-HDL level. Because plasma LCAT is critical for HDL maturation, we analyzed whether the changes were influenced by LCAT activity (Figure 1B). There were no differences in the activity of LCAT between the groups, indicating that H2 increased pre-β-HDL without decreasing LCAT activity. Furthermore, we evaluated the lipid levels in HDL3 and, as shown in Figure 1C, the phospholipid was significantly decreased by H2 treatment, supporting the finding that the small lipid-poor apoA-I–containing lipoprotein particle was increased by H2. In addition, S1P and ceramide contents in HDL3 were not altered by H2 (Figure 1D).

Figure 1.

A, Levels of plasma pre-β-HDL. B, Activity of LCAT in plasma. C, S1P and ceramide measured by liquid chromatography-tandem mass spectrometry in HDL3. D, Phospholipid concentration in HDL3 after drinking of placebo (control) or H2 water at week 10. *, P < .05; **, P < .01. n = 34 each group. Con, control.

H2 treatment increases cholesterol efflux via ABCA1 ex vivo

We examined whether cholesterol efflux via the ABCA1 pathway was enhanced by H2, because H2 increased pre-β1-HDL after treatment. HDL3 from patients who had been treated with H2 or placebo for 10 weeks was used for ex vivo cholesterol efflux studies with bone marrow macrophages from mice and RAW264.7 macrophages. As shown in Figure 2, A and B, we measured plasma HDL3-mediated efflux in bone marrow macrophages with or without glyburide, a commonly used ABCA1 chemical inhibitor. HDL3 from the H2 group had significantly greater cellular cholesterol efflux capacity than HDL3 from the control group (Figure 2A). However, no significant difference was seen with the use of glyburide (Figure 2B). Similar results were observed regarding cholesterol efflux using RAW264.7 macrophages. The H2 group had significantly greater total efflux than the control group (Figure 2C). In addition, with RAW264.7 macrophages in which ABCA1 was stimulated by 3′,5′-cAMP, total efflux in the H2 group was significantly greater than that in the control group (Figure 2D).

Figure 2.

Effects of H2 on cholesterol efflux ex vivo.

Plasma of every 5 to 6 patients was pooled, and HDL3 was isolated by ultracentrifugation from the plasma. Cultured bone marrow macrophage (BMM) or RAW264.7 cells were incubated for 24 hours with or without glyburide or cAMP. The cellular cholesterol efflux was determined in the presence of HDL3 taken from patients after 10 weeks of H2 or placebo water administration. A and B, Total (without glyburide) (A) and ABCA1-independent (with glyburide) (B) cholesterol efflux in bone marrow macrophage cells with acetylated LDL. C and D, Total (without cAMP) (C) and ABCA1-stimulated (with cAMP) (D) efflux in RAW264.7 cells with acetylated LDL. n = 3 to 4 pooled plasma samples. *, P < .05. Con, control.

H2 improves the other functional properties of HDL3 particle besides cholesterol efflux

First, the biological effect of H2 on the antioxidative functionality of HDL3 was tested, namely, the protection of LDL particles from oxidation. As shown in Figure 3A, H2 treatment significantly inhibited the formation of thiobarbituric acid–reactive substances, which was supported by the decreased plasma ox-LDL levels in patients treated with H2 water (Figure 3B). Second, the biological effect of H2 on the antiapoptotic functionality of HDL3 in HUVECs was determined. As shown in Figure 3, C and D, treatment of cells with ox-LDL led to a reduction in cell viability and a dramatic elevation in the numbers of apoptotic cells, which were prevented by HDL pretreatment. Furthermore, the inhibition effects of HDL isolated from the H2 group on cell apoptosis were significantly higher than those of HDL isolated from the placebo control group.

Figure 3.

H2 improves functional antioxidative and antiapoptotic properties of the HDL particle.

Plasma of every 5 to 6 patients was pooled, and HDL3 was isolated by ultracentrifugation from the plasma. HDL function was determined as protection of LDL against oxidation (n = 3–4 pooled plasma samples) (A), plasma levels of ox-LDL (n = 34) (B), protection of endothelial cells from ox-LDL–induced apoptosis by MTT assay (C) and flow cytometry analysis (D) (n = 3–4 pooled plasma samples). Assays were performed as detailed in Materials and Methods. *, P < .05; ***, P < .001 vs control group; ###, P < .001 vs ox-LDL group; &, P < .05 vs HDL-Con group. HDL-Con, HDL3 extracted from patients drinking placebo water for 10 weeks; HDL-H2, HDL3 extracted from patients drinking H2 water for 10 weeks. Con, control; TBARS, thiobarbituric acid-reactive substances; UL, upper left; UR, upper right; LL, lower left; LR, lower right.

Third, the effect of H2 on the anti-inflammatory properties of HDL3 was tested, including protection of ox-LDL–induced monocyte adhesion to endothelial cells and secretion of adhesion molecules and inflammatory factor, including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and IL-6. As shown in Figure 4, A and B, after incubation of HUVECs for 6 hours with ox-LDL, adhesion of monocytes to HUVECs was significantly increased, which was prevented by HDL preincubation. The prevention effect of HDL3 isolated from the H2 group on adhesion was markedly higher than that of HDL3 isolated from control group. Furthermore, ox-LDL–induced expression of ICAM-1 and VCAM-1 and secretion of IL-6 were significantly decreased by preincubation with HDL3 isolated from the H2 group compared with preincubation with HDL3 isolated from the control group (Figure 4, C, D, and E). These data suggested that the anti-inflammatory function of HDL3 was improved by H2.

Figure 4.

H2 improves the anti-inflammatory property of the HDL particle.

Plasma of every 5 to 6 patients was pooled, and HDL3 was isolated by ultracentrifugation from the plasma. HDL function was determined as inhibition of ox-LDL–induced THP-1 monocytes adhesion to endothelial cells (A and B), inhibition of ox-LDL–induced secretion of adhesion molecules (C and D), including ICAM-1 and VCAM-1 in endothelial cells by Western blots, and protection of endothelial cells from ox-LDL–induced secretion of cytokine by ELISA (E). Assays were performed as detailed in Materials and Methods. n = 3 to 4 pooled plasma samples. ***, P < .001 vs control group; #, P < .05; ##, P < .01; ###, P < .001 vs the ox-LDL group; &, P < .05; &&, P < .01 vs the HDL-Con group. HDL-Con, HDL3 extracted from patients drinking placebo water for 10 weeks; HDL-H2, HDL3 extracted from patients drinking H2 water for 10 weeks. Con, control.

Effects of H2 on plasma lipid and lipoprotein profiles

The plasma lipid levels of each individual are presented in Table 1. Plasma biochemical analysis showed that the effective rate of H2 to lower plasma TC and LDL-C levels were increased compared with those of the placebo group (TC, 47.06% vs 17.65%; LDL-C, 47.06% vs 23.53%). However, the plasma levels of HDL-C (Table 1), triacylglycerols, and glucose (Supplemental Table 2) were not altered by consumption of H2.

Table 1.

Effects of H2 on the Levels of Plasma Lipids in Each Individual Patient

Patient No. TC LDL-C HDL-C
Placebo (Control) H2 Placebo (Control) H2 Placebo (Control) H2
Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM)
1 10.25 10.1 10.87a 8.28a 8.36 8.52 8.98a 6.74a 1.46 1.23 1.32 1.32
2 6.93 7.45 7.53 7.67 5.76a 5.06a 6.33 6.1 1.36 1.42 1.18 1.23
3 7.14 6.87 7.02 7.85 5.6 5.13 5.96 6.34 1.84 1.54 1.24 1.42
4 6.68 7.09 7.23 8.15 4.96 5.16 5.58 6.11 1.14 1.15 1.62 1.6
5 6.49 6.77 6.71a 5.29a 4.92 4.87 5.4a 4.15a 1.74 1.8 1.36 1.07
6 5.98 6.32 6.73a 6.01a 4.84 4.51 5.07a 4.35a 1.59 1.78 1.77 1.59
7 5.99a 5.23a 5.96a 4.06a 4.63a 3.3a 4.98a 2.28a 1.31 1.75 0.82 0.8
8 6.26a 5.47a 6.19a 5.5a 4.58a 3.96a 4.93a 4.15a 1.55 1.24 1.59 1.32
9 6.2 6.34 5.9a 5.3a 4.57 4.27 4.9a 4.18a 1.29 1.39 1.4 1.53
10 6.02 6.14 6.31 7.61 4.57 4.35 4.68 5.55 1.71 1.4 1.67 1.58
11 6.25 5.67 6.52a 5.84a 4.34a 3.42a 4.54a 3.82a 1.33 1.33 1.36 1.15
12 6.49 6.58 6a 4.28a 4.33 4.23 4.52a 2.21a 2.33 2.32 1.37 1.46
13 5.86 5.86 7.19 7.21 4.3 4.95 4.47 5.09 1.34 1.63 1.14 1.1
14 7.32a 4.77a 6.1a 5.41a 4.25a 3.23a 4.37a 3.84a 1.09 1.19 1.81 1.51
15 7.73a 6.08a 6.98a 4.05a 4.2 4.05 4.35a 2.77a 0.93 1.66 1.26 1.23
16 7.38 7.7 5.85 6.26 4.2 6.02 4.28 4.51 0.93 1.15 1.29 1.36
17 5.04 5.59 6.36 6.31 4.18 4.03 4.22 4.52 1.43 1.11 1.87 1.23
18 6.66 7.19 6.61a 4.57a 4.14 4.79 4.03a 2.49a 0.98 1.24 1.57 2.05
19 5.34 5.79 5.92 5.94 4.13 4.04 3.97 3.81 1.42 1.66 1.34 1.51
20 7.16 7.96 5.34 5.34 3.97 3.63 3.95 3.95 1.5 1.64 1.3 1.3
21 6.05 6.06 6.01 5.94 3.96 3.92 3.94 3.94 1.7 1.57 1.76 1.76
22 5.18 5.18 5.17 5.65 3.9 3.9 3.85 4.11 1.21 1.22 1.51 1.35
23 6.12 6.12 4.64 5.37 3.86 3.86 3.83 3.78 1.21 1.21 1.01 1.09
24 5.77 5.73 5.61 6.22 3.8 3.59 3.76 3.59 1.9 2.09 1.73 1.89
25 5.06 5.03 5.84 6.52 3.79a 3.33a 3.69 3.54 1.41 1.37 2.08 2.59
26 6.06 6.09 5.7 5.73 3.76 3.78 3.57 3.27 1.9 2.1 2.3 2.19
27 5.61a 4.26a 5.24 6.02 3.69a 2.21a 3.55 3.71 1.52 1.48 1.5 1.19
28 5.77 6.88 4.76a 4.01a 3.66 4.04 3.3a 2.68a 1.09 1.24 1.71 1.58
29 4.6 5.21 4.75 4.95 3.62 4.17 3.28 3.82 1.11 1.12 1.27 1.15
30 5.08 5.64 5.13a 4.57a 3.58 4.09 3.21a 2.81a 1.3 1.25 1.7 1.8
31 4.64a 4.09a 4.46a 4.7a 3.16a 2.23a 3.13a 2.76a 1.8 1.94 0.7 0.96
32 5.22 5.02 5.01a 4.5a 3.06 2.87 2.68 2.44 2.29 2.03 1.5 1.48
33 4.13 6.61 4.26 4.34 2.71 2.7 2.59a 2.21a 1.28 1.57 1.64 1.89
34 3.96 4.33 5.96a 2.8a 2.59 2.74 2.59a 1.41a 1.66 1.56 1.19 1.34
Mean 6.071176 6.094706 6.054706 5.654412 4.234412 4.086765 4.308235 3.853824 1.460294 1.511176 1.467059 1.459412
SD 1.177096 1.188817 1.192591 1.300073 1.001956 1.154532 1.203839 1.218013 0.34524 0.320858 0.329504 0.366796
Effective rate, % 17.65 47.06 23.53 47.06
Patient No. TC LDL-C HDL-C
Placebo (Control) H2 Placebo (Control) H2 Placebo (Control) H2
Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM)
1 10.25 10.1 10.87a 8.28a 8.36 8.52 8.98a 6.74a 1.46 1.23 1.32 1.32
2 6.93 7.45 7.53 7.67 5.76a 5.06a 6.33 6.1 1.36 1.42 1.18 1.23
3 7.14 6.87 7.02 7.85 5.6 5.13 5.96 6.34 1.84 1.54 1.24 1.42
4 6.68 7.09 7.23 8.15 4.96 5.16 5.58 6.11 1.14 1.15 1.62 1.6
5 6.49 6.77 6.71a 5.29a 4.92 4.87 5.4a 4.15a 1.74 1.8 1.36 1.07
6 5.98 6.32 6.73a 6.01a 4.84 4.51 5.07a 4.35a 1.59 1.78 1.77 1.59
7 5.99a 5.23a 5.96a 4.06a 4.63a 3.3a 4.98a 2.28a 1.31 1.75 0.82 0.8
8 6.26a 5.47a 6.19a 5.5a 4.58a 3.96a 4.93a 4.15a 1.55 1.24 1.59 1.32
9 6.2 6.34 5.9a 5.3a 4.57 4.27 4.9a 4.18a 1.29 1.39 1.4 1.53
10 6.02 6.14 6.31 7.61 4.57 4.35 4.68 5.55 1.71 1.4 1.67 1.58
11 6.25 5.67 6.52a 5.84a 4.34a 3.42a 4.54a 3.82a 1.33 1.33 1.36 1.15
12 6.49 6.58 6a 4.28a 4.33 4.23 4.52a 2.21a 2.33 2.32 1.37 1.46
13 5.86 5.86 7.19 7.21 4.3 4.95 4.47 5.09 1.34 1.63 1.14 1.1
14 7.32a 4.77a 6.1a 5.41a 4.25a 3.23a 4.37a 3.84a 1.09 1.19 1.81 1.51
15 7.73a 6.08a 6.98a 4.05a 4.2 4.05 4.35a 2.77a 0.93 1.66 1.26 1.23
16 7.38 7.7 5.85 6.26 4.2 6.02 4.28 4.51 0.93 1.15 1.29 1.36
17 5.04 5.59 6.36 6.31 4.18 4.03 4.22 4.52 1.43 1.11 1.87 1.23
18 6.66 7.19 6.61a 4.57a 4.14 4.79 4.03a 2.49a 0.98 1.24 1.57 2.05
19 5.34 5.79 5.92 5.94 4.13 4.04 3.97 3.81 1.42 1.66 1.34 1.51
20 7.16 7.96 5.34 5.34 3.97 3.63 3.95 3.95 1.5 1.64 1.3 1.3
21 6.05 6.06 6.01 5.94 3.96 3.92 3.94 3.94 1.7 1.57 1.76 1.76
22 5.18 5.18 5.17 5.65 3.9 3.9 3.85 4.11 1.21 1.22 1.51 1.35
23 6.12 6.12 4.64 5.37 3.86 3.86 3.83 3.78 1.21 1.21 1.01 1.09
24 5.77 5.73 5.61 6.22 3.8 3.59 3.76 3.59 1.9 2.09 1.73 1.89
25 5.06 5.03 5.84 6.52 3.79a 3.33a 3.69 3.54 1.41 1.37 2.08 2.59
26 6.06 6.09 5.7 5.73 3.76 3.78 3.57 3.27 1.9 2.1 2.3 2.19
27 5.61a 4.26a 5.24 6.02 3.69a 2.21a 3.55 3.71 1.52 1.48 1.5 1.19
28 5.77 6.88 4.76a 4.01a 3.66 4.04 3.3a 2.68a 1.09 1.24 1.71 1.58
29 4.6 5.21 4.75 4.95 3.62 4.17 3.28 3.82 1.11 1.12 1.27 1.15
30 5.08 5.64 5.13a 4.57a 3.58 4.09 3.21a 2.81a 1.3 1.25 1.7 1.8
31 4.64a 4.09a 4.46a 4.7a 3.16a 2.23a 3.13a 2.76a 1.8 1.94 0.7 0.96
32 5.22 5.02 5.01a 4.5a 3.06 2.87 2.68 2.44 2.29 2.03 1.5 1.48
33 4.13 6.61 4.26 4.34 2.71 2.7 2.59a 2.21a 1.28 1.57 1.64 1.89
34 3.96 4.33 5.96a 2.8a 2.59 2.74 2.59a 1.41a 1.66 1.56 1.19 1.34
Mean 6.071176 6.094706 6.054706 5.654412 4.234412 4.086765 4.308235 3.853824 1.460294 1.511176 1.467059 1.459412
SD 1.177096 1.188817 1.192591 1.300073 1.001956 1.154532 1.203839 1.218013 0.34524 0.320858 0.329504 0.366796
Effective rate, % 17.65 47.06 23.53 47.06
a

Patients whose lipid level was decreased above 10% after drinking water compared with the lipid level before drinking water. The effective rate was calculated by the indicated patient numbers to total patient numbers.

Table 1.

Effects of H2 on the Levels of Plasma Lipids in Each Individual Patient

Patient No. TC LDL-C HDL-C
Placebo (Control) H2 Placebo (Control) H2 Placebo (Control) H2
Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM)
1 10.25 10.1 10.87a 8.28a 8.36 8.52 8.98a 6.74a 1.46 1.23 1.32 1.32
2 6.93 7.45 7.53 7.67 5.76a 5.06a 6.33 6.1 1.36 1.42 1.18 1.23
3 7.14 6.87 7.02 7.85 5.6 5.13 5.96 6.34 1.84 1.54 1.24 1.42
4 6.68 7.09 7.23 8.15 4.96 5.16 5.58 6.11 1.14 1.15 1.62 1.6
5 6.49 6.77 6.71a 5.29a 4.92 4.87 5.4a 4.15a 1.74 1.8 1.36 1.07
6 5.98 6.32 6.73a 6.01a 4.84 4.51 5.07a 4.35a 1.59 1.78 1.77 1.59
7 5.99a 5.23a 5.96a 4.06a 4.63a 3.3a 4.98a 2.28a 1.31 1.75 0.82 0.8
8 6.26a 5.47a 6.19a 5.5a 4.58a 3.96a 4.93a 4.15a 1.55 1.24 1.59 1.32
9 6.2 6.34 5.9a 5.3a 4.57 4.27 4.9a 4.18a 1.29 1.39 1.4 1.53
10 6.02 6.14 6.31 7.61 4.57 4.35 4.68 5.55 1.71 1.4 1.67 1.58
11 6.25 5.67 6.52a 5.84a 4.34a 3.42a 4.54a 3.82a 1.33 1.33 1.36 1.15
12 6.49 6.58 6a 4.28a 4.33 4.23 4.52a 2.21a 2.33 2.32 1.37 1.46
13 5.86 5.86 7.19 7.21 4.3 4.95 4.47 5.09 1.34 1.63 1.14 1.1
14 7.32a 4.77a 6.1a 5.41a 4.25a 3.23a 4.37a 3.84a 1.09 1.19 1.81 1.51
15 7.73a 6.08a 6.98a 4.05a 4.2 4.05 4.35a 2.77a 0.93 1.66 1.26 1.23
16 7.38 7.7 5.85 6.26 4.2 6.02 4.28 4.51 0.93 1.15 1.29 1.36
17 5.04 5.59 6.36 6.31 4.18 4.03 4.22 4.52 1.43 1.11 1.87 1.23
18 6.66 7.19 6.61a 4.57a 4.14 4.79 4.03a 2.49a 0.98 1.24 1.57 2.05
19 5.34 5.79 5.92 5.94 4.13 4.04 3.97 3.81 1.42 1.66 1.34 1.51
20 7.16 7.96 5.34 5.34 3.97 3.63 3.95 3.95 1.5 1.64 1.3 1.3
21 6.05 6.06 6.01 5.94 3.96 3.92 3.94 3.94 1.7 1.57 1.76 1.76
22 5.18 5.18 5.17 5.65 3.9 3.9 3.85 4.11 1.21 1.22 1.51 1.35
23 6.12 6.12 4.64 5.37 3.86 3.86 3.83 3.78 1.21 1.21 1.01 1.09
24 5.77 5.73 5.61 6.22 3.8 3.59 3.76 3.59 1.9 2.09 1.73 1.89
25 5.06 5.03 5.84 6.52 3.79a 3.33a 3.69 3.54 1.41 1.37 2.08 2.59
26 6.06 6.09 5.7 5.73 3.76 3.78 3.57 3.27 1.9 2.1 2.3 2.19
27 5.61a 4.26a 5.24 6.02 3.69a 2.21a 3.55 3.71 1.52 1.48 1.5 1.19
28 5.77 6.88 4.76a 4.01a 3.66 4.04 3.3a 2.68a 1.09 1.24 1.71 1.58
29 4.6 5.21 4.75 4.95 3.62 4.17 3.28 3.82 1.11 1.12 1.27 1.15
30 5.08 5.64 5.13a 4.57a 3.58 4.09 3.21a 2.81a 1.3 1.25 1.7 1.8
31 4.64a 4.09a 4.46a 4.7a 3.16a 2.23a 3.13a 2.76a 1.8 1.94 0.7 0.96
32 5.22 5.02 5.01a 4.5a 3.06 2.87 2.68 2.44 2.29 2.03 1.5 1.48
33 4.13 6.61 4.26 4.34 2.71 2.7 2.59a 2.21a 1.28 1.57 1.64 1.89
34 3.96 4.33 5.96a 2.8a 2.59 2.74 2.59a 1.41a 1.66 1.56 1.19 1.34
Mean 6.071176 6.094706 6.054706 5.654412 4.234412 4.086765 4.308235 3.853824 1.460294 1.511176 1.467059 1.459412
SD 1.177096 1.188817 1.192591 1.300073 1.001956 1.154532 1.203839 1.218013 0.34524 0.320858 0.329504 0.366796
Effective rate, % 17.65 47.06 23.53 47.06
Patient No. TC LDL-C HDL-C
Placebo (Control) H2 Placebo (Control) H2 Placebo (Control) H2
Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM) Before Drinking (mM) After Drinking (mM)
1 10.25 10.1 10.87a 8.28a 8.36 8.52 8.98a 6.74a 1.46 1.23 1.32 1.32
2 6.93 7.45 7.53 7.67 5.76a 5.06a 6.33 6.1 1.36 1.42 1.18 1.23
3 7.14 6.87 7.02 7.85 5.6 5.13 5.96 6.34 1.84 1.54 1.24 1.42
4 6.68 7.09 7.23 8.15 4.96 5.16 5.58 6.11 1.14 1.15 1.62 1.6
5 6.49 6.77 6.71a 5.29a 4.92 4.87 5.4a 4.15a 1.74 1.8 1.36 1.07
6 5.98 6.32 6.73a 6.01a 4.84 4.51 5.07a 4.35a 1.59 1.78 1.77 1.59
7 5.99a 5.23a 5.96a 4.06a 4.63a 3.3a 4.98a 2.28a 1.31 1.75 0.82 0.8
8 6.26a 5.47a 6.19a 5.5a 4.58a 3.96a 4.93a 4.15a 1.55 1.24 1.59 1.32
9 6.2 6.34 5.9a 5.3a 4.57 4.27 4.9a 4.18a 1.29 1.39 1.4 1.53
10 6.02 6.14 6.31 7.61 4.57 4.35 4.68 5.55 1.71 1.4 1.67 1.58
11 6.25 5.67 6.52a 5.84a 4.34a 3.42a 4.54a 3.82a 1.33 1.33 1.36 1.15
12 6.49 6.58 6a 4.28a 4.33 4.23 4.52a 2.21a 2.33 2.32 1.37 1.46
13 5.86 5.86 7.19 7.21 4.3 4.95 4.47 5.09 1.34 1.63 1.14 1.1
14 7.32a 4.77a 6.1a 5.41a 4.25a 3.23a 4.37a 3.84a 1.09 1.19 1.81 1.51
15 7.73a 6.08a 6.98a 4.05a 4.2 4.05 4.35a 2.77a 0.93 1.66 1.26 1.23
16 7.38 7.7 5.85 6.26 4.2 6.02 4.28 4.51 0.93 1.15 1.29 1.36
17 5.04 5.59 6.36 6.31 4.18 4.03 4.22 4.52 1.43 1.11 1.87 1.23
18 6.66 7.19 6.61a 4.57a 4.14 4.79 4.03a 2.49a 0.98 1.24 1.57 2.05
19 5.34 5.79 5.92 5.94 4.13 4.04 3.97 3.81 1.42 1.66 1.34 1.51
20 7.16 7.96 5.34 5.34 3.97 3.63 3.95 3.95 1.5 1.64 1.3 1.3
21 6.05 6.06 6.01 5.94 3.96 3.92 3.94 3.94 1.7 1.57 1.76 1.76
22 5.18 5.18 5.17 5.65 3.9 3.9 3.85 4.11 1.21 1.22 1.51 1.35
23 6.12 6.12 4.64 5.37 3.86 3.86 3.83 3.78 1.21 1.21 1.01 1.09
24 5.77 5.73 5.61 6.22 3.8 3.59 3.76 3.59 1.9 2.09 1.73 1.89
25 5.06 5.03 5.84 6.52 3.79a 3.33a 3.69 3.54 1.41 1.37 2.08 2.59
26 6.06 6.09 5.7 5.73 3.76 3.78 3.57 3.27 1.9 2.1 2.3 2.19
27 5.61a 4.26a 5.24 6.02 3.69a 2.21a 3.55 3.71 1.52 1.48 1.5 1.19
28 5.77 6.88 4.76a 4.01a 3.66 4.04 3.3a 2.68a 1.09 1.24 1.71 1.58
29 4.6 5.21 4.75 4.95 3.62 4.17 3.28 3.82 1.11 1.12 1.27 1.15
30 5.08 5.64 5.13a 4.57a 3.58 4.09 3.21a 2.81a 1.3 1.25 1.7 1.8
31 4.64a 4.09a 4.46a 4.7a 3.16a 2.23a 3.13a 2.76a 1.8 1.94 0.7 0.96
32 5.22 5.02 5.01a 4.5a 3.06 2.87 2.68 2.44 2.29 2.03 1.5 1.48
33 4.13 6.61 4.26 4.34 2.71 2.7 2.59a 2.21a 1.28 1.57 1.64 1.89
34 3.96 4.33 5.96a 2.8a 2.59 2.74 2.59a 1.41a 1.66 1.56 1.19 1.34
Mean 6.071176 6.094706 6.054706 5.654412 4.234412 4.086765 4.308235 3.853824 1.460294 1.511176 1.467059 1.459412
SD 1.177096 1.188817 1.192591 1.300073 1.001956 1.154532 1.203839 1.218013 0.34524 0.320858 0.329504 0.366796
Effective rate, % 17.65 47.06 23.53 47.06
a

Patients whose lipid level was decreased above 10% after drinking water compared with the lipid level before drinking water. The effective rate was calculated by the indicated patient numbers to total patient numbers.

In addition, plasma LDL and HDL are particles composed of a variety of lipids and protein components; it is thus necessary to clarify which component of the lipoprotein is affected by H2 treatment. Consistent with the difference observed for LDL-C, the major proteins on LDL, apoB100, were significantly decreased by consumption of H2 water (Figure 5). However, the major protein on HDL, apoA-I, was not altered after intake of H2 water compared with that for the placebo control group (Figure 5), which is consistent with the changes observed for HDL-C. Moreover, based on the findings that apoM, which mainly exists in HDL particles, plays a role in HDL remodeling in humans and that pre-β-HDL and pre-β-HDL formation are positively associated with apoM, we tested the apoM contents in plasma because H2 increased pre-β1-HDL after treatment. As shown in Figure 5, plasma apoM levels were significantly increased in the H2 group compared with those in the control group.

Figure 5.

Effect of H2 on plasma levels of lipoproteins and an estimation of the 10-year risk for ASCVD.

A, Effect of H2 on plasma apoB, apoE, and apoA-I protein levels by Western blots. B, Densitometric quantitation of Western blot data (n = 4–8) by Quantity One software. n = 3 to 4 pooled plasma samples. C, Estimation of the 10-year risk for ASCVD (percentage) calculated by using a web-based Omnibus_Risk_Estimator. *, P < .05; **, P < .01. Con, control.

Risk assessment of H2 on ASCVD development in patients with hypercholesterolemia

Given that H2 treatment improves the biological activities of HDL in vitro, to support the potential role H2 of in the regression of atherosclerosis, we performed a risk assessment of H2 treatment on atherosclerosis development in patients with hypercholesterolemia. As shown in Figure 5C, the trend was for the 10-year ASCVD risk (percentage) was to be decreased by H2 treatment (P = .09).

Effect of H2 on plasma levels of antioxidative and inflammatory biomarkers

Changes in the biomarkers of oxidative and inflammatory status after 10 weeks of consumption of H2 water and placebo water are shown in Supplemental Figure 1. The plasma level of malondialdehyde, one of the most frequently used indicators of lipid peroxidation, was decreased significantly (Supplemental Figure 1A), and the activity of superoxide dismutase, which act as an antioxidant and protects cellular components from being oxidized by reactive oxygen species, was increased by H2 (Supplemental Figure 1B). Moreover, the activity of paraoxonase-1 (PON-1), an antioxidant enzyme associated with HDL, was markedly increased in both plasma and HDL3 fractions by H2 water (Supplemental Figure 1, C and D). In addition, intake of H2 water decreased the plasma levels of inflammatory biomarkers, including TNF-α (P < .05) and IL-6 (P = .0723) (Supplemental Figure 1, E and F).

Discussion

In the present study, H2 increased the plasma pre-β1-HDL level and enhanced HDL functions, including the ability to stimulate ABCA1-dependent efflux of reverse cholesterol transport (RCT), the ability to protect against LDL oxidation, the ability to inhibit ox-LDL–induced inflammation and the ability to protect endothelial cell apoptosis in a double-blinded, randomized, and placebo-controlled trial. In addition, the effective rates of H2 to lower plasma TC and LDL-C were increased, and H2 treatment significantly decreased plasma apoB100 levels compared with those for the placebo control group, suggesting that H2 might have beneficial lipid-lowering effects. This finding highlights the potential of H2 for the regression of hypercholesterolemia and atherosclerosis.

HDLs are known to protect against the development of atherosclerosis and are widely documented as a “negative risk factor” for coronary heart disease (26). Although H2 did not change HDL-C levels compared with that for the control group, cholesterol efflux was enhanced via ABCA1. We considered the possibility that H2 altered the HDL composition and analyzed pre-β1-HDL and apoM levels. We found that H2 significantly increased the level of pre-β1 HDL, which has a higher cholesterol efflux capacity. Although the previous report showed that ischemic heart disease (IHD) was associated with high pre-β1-HDL concentrations and low LCAT levels (27), it is possible that the high pre-β1-HDL concentration in patients with IHD was induced by the disease itself and might have a protective effect during the pathophysiological process of IHD.

LCAT is one of the major modulators of plasma HDL-C and plays a central role in RCT by esterifying free cholesterol from the surface of HDL, thus contributing to the conversion of nascent pre-β1-HDL to α-migrating mature HDL (28). Therefore, it is possible that H2 reduces LCAT activity and increases the nascent HDL component. However, in this study, H2 treatment increased pre-β1-HDL without any changes in LCAT activity. We know that both hepatic lipase and endothelial lipase have effects on the composition of HDL particles in addition to LCAT. Also, endothelial lipase expression resulted in the generation of small pre-β-HDL particles in wild-type mice (29), and hepatic lipase induced the formation of pre-β1-HDL from triacylglycerol-rich HDL2 (30). Thus, it is possible that H2 may affect the activities of hepatic lipase and endothelial lipase, changing the composition of HDL and increasing pre-β-HDL. Further animal studies are needed to explore this possibility. In addition, it has been reported that pre-β-HDL and pre-β-HDL formation are positively associated with apoM in humans (31, 32). We observed increased plasma apoM levels and decreased phospholipid content in HDL3 in the present study, which may support the finding that H2 increases small lipid-poor pre-β-HDL particles. Future experiments on the effects of H2 on apoM synthesis and metabolism in the liver and plasma will elucidate this possibility. Collectively, our results indicate that H2 might improve HDL function by regulating HDL composition and enzymatic activities, as supported by the following observations. First, H2 increased plasma small pre-β HDL and enhanced pre-β-HDL–dependent cholesterol efflux via ABCA1. Second, H2 treatment decreased phospholipid content in HDL3. Third, the level of plasma apoM that mainly exists in HDL and plays roles in the HDL function in antiatherogenesis (31, 33) was increased by H2. Fourth, H2 treatment increased the plasma activities of PON-1, an HDL-associated lactonase that protects against macrophage-mediated LDL oxidation (34). To further elucidate the molecular mechanisms by which H2 influences HDL functions, the lipidomics of HDL and the alterations of nucleotides in HDL including microRNAs will be studied in future experiments.

Moreover, we found that H2 consumption increased the macrophage to HDL RCT by activating cholesterol efflux via ABCA1. In addition, we know that cholesterol ester transfer protein inhibitors markedly increase HDL-C and decrease LDL-C when administered as monotherapy or when administered in combination with statins (35). Based on the results of the present study, the combination of cholesterol ester transfer protein inhibitors with H2 may promote RCT and promote the regression of atherosclerosis via the induction of pre-β-HDL.

Previously, we have reported that H2 decreases plasma LDL-C and apoB levels and improves HDL function in patients with potential metabolic syndrome in a before-after self-controlled study (12), and in the present study, we further confirmed the lipid-regulating effects of H2 in a double-blinded, randomized, and placebo-controlled trial. As for our previous report, we did not observe any unwanted side effects of H2 in the present study, including headache, diarrhea, and vomiting (data not shown). However, our study still has some limitations. First, the study included a limited number of patients, and, therefore, its results should be confirmed by a larger study. Second, we did not perform a dose-dependent experiment; therefore, the possibility that the effect might be stronger with higher doses of H2 water cannot be excluded. Finally, because H2 treatment in the present studies lasted for only 10 weeks, the potential protective effect of long-term H2 treatment on plasma lipid levels and HDL functions in patients with hypercholesterolemia need to be further elucidated.

In conclusion, our results indicate that H2 treatment not only increases plasma pre-β-HDL levels and activates ABCA1-dependent cholesterol efflux from peripheral macrophage cells but also improves other HDL functions including protection against LDL oxidation, inhibition of ox-LDL–induced monocyte adhesion to endothelial cells, and protection of endothelial cells from ox-LDL–induced apoptosis. Moreover, H2 treatment decreases plasma cholesterol levels, plasma oxidative stress, and inflammatory status. Therefore, we conclude that oral administration of H2 water may prevent or delay the development and progression of hypercholesterolemia and atherosclerosis.

Acknowledgments

We thank Beijing Hydrovita Biotechnology Company (HuoLiQingYuan, Beijing, China) for providing H2 water and placebo water and Dr Dawei Zhang for his helpful suggestions in preparation of the revised article.

This research was supported by the Science and Technology Development Program of Shandong Province (2013GSF11830 and 2014GSF118081), the Taishan Scholars Foundation of Shandong Province (200867), the National Natural Science Foundation of China (81170785 and 81200216), and the Promotive Research Fund for Excellent Young and Middle-Aged Scientists of Shandong Province (BS2012YY034).

This study was registered at the International Clinical Trials Registry under Registration No. ChiCTR-IPR-14005548 (http://apps.who.int/trialsearch/Trial2.aspx?TrialID=ChiCTR-IPR-14005548).

Disclosure Summary: The authors have nothing to disclose.

*

G.S. and Q.L. contributed equally to the study.

Abbreviations

     
  • ABCA1

    ATP-binding cassette transporter A1

  •  
  • apo

    apolipoprotein

  •  
  • ASCVD

    atherosclerotic cardiovascular disease

  •  
  • H2

    dihydrogen

  •  
  • HDL

    high-density lipoprotein

  •  
  • HDL-C

    high-density lipoprotein cholesterol

  •  
  • HUVEC

    human umbilical vein endothelial cell

  •  
  • ICAM-1

    intercellular adhesion molecule 1

  •  
  • IHD

    ischemic heart disease

  •  
  • LCAT

    lecithin-cholesterol acyltransferase

  •  
  • LDL

    low-density lipoprotein

  •  
  • LDL-C

    low-density lipoprotein cholesterol

  •  
  • MTT

    3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

  •  
  • ox-LDL

    oxidized low-density lipoprotein

  •  
  • PON-1

    paraoxonase-1

  •  
  • RCT

    reverse cholesterol transport

  •  
  • S1P

    sphingosine-1-phosphate

  •  
  • TC

    total cholesterol

  •  
  • VCAM-1

    vascular cell adhesion molecule 1.

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Supplementary data