Hydrogen-rich water decreases serum LDL-cholesterol levels and improves HDL function in patients with potential metabolic syndrome

Serum LDL and HDL are particles composed of a variety of lipids and protein components; thus, it is necessary to clarify which content of the lipoprotein could be affected by H₂ treatment. Consistent with the difference observed for LDL-C, apoB100, the major protein on LDL, was significantly decreased by consumption of H₂ water in patients with potential metabolic syndrome (Fig. 4). The apoE protein, which is mainly present in VLDL and LDL particles, was also lowered with the treatment of H₂ (Fig. 4). ApoAI, the major protein on HDL, was not altered after intake of H₂ water (Fig. 4), which is consistent with the changes observed for HDL-C. These data suggested that H₂ could decrease the expression of the major protein constituents of LDL and VLDL.

HDL is known to undergo dramatic modification in structure and composition under the concerted actions of inflammation and oxidative stress (14, 15). Recent evidence indicates that H₂ acts as a therapeutic medical gas in a variety of disease models by exerting antioxidant and anti-inflammatory effects (3, 25, 26). Therefore, it is possible that administration of H₂ might improve the functional quality of HDL particle. First, H₂ water supplementation tended to decrease the MDA content in HDL3 (Fig. 5A), suggesting that H₂ improves the oxidation of HDL particle.

Second, the biological effect of H₂ on the antioxidative functionality of HDL was tested, namely, the protection of LDL particles from oxidation. As shown in Fig. 5B, H₂ treatment significantly inhibited the formation of TBARS.

Third, the effect of H₂ on the anti-inflammatory properties of HDL was tested, including protection of cytokine-induced monocyte adhesion to endothelial cells and stimulation of endothelial nitric oxide production, which has been suggested as an important endothelial-atheroprotective effect of HDL. As shown in Fig. 5C, after incubation of HUVECs for 6 h with TNF-α, adhesion of monocytes to HUVECs was significantly increased, and preincubation of HUVECs with HDL3 isolated from patients after 10 weeks of drinking H₂ water (HDL-H₂-A) markedly reduced TNF-α-induced adhesion of monocyte to HUVECs compared with those of HDL3 isolated at baseline (0 week) (HDL-H₂-B). These data suggest that the anti-inflammatory function of HDL was improved by H₂ water. We also determined the effects of HDL-H₂-A compared with HDL-H₂-B on endothelial nitric oxide production. Unfortunately, the nitric oxide production was not altered significantly after intake of H₂ water (data not shown), which might be attributable to the sensitivity of Griess method.

Fourth, the ability of the isolated HDL particles to elicit efflux from cholesterol-loaded macrophages was tested. As shown in Fig. 5D, HDL particles isolated from the serum after H₂ treatment exhibited dramatically higher efflux properties compared with the HDL particles isolated from the serum before H₂ treatment, indicating that the cholesterol efflux ability mediated by HDL particles was increased by H₂.

Finally, the biological effect of H₂ on the antiapoptotic functionality of HDL was determined. As shown in Fig. 5E, H₂ treatment significantly inhibited TNF-α induced endothelial cell apoptosis. These data indicate that dyslipidemia-injured HDL functions, including the ability to protect against LDL oxidation, the ability to inhibit cytokine-induced monocyte adhesion to endothelial cells, the ability to stimulate cholesterol efflux from macrophage foam cells, and the ability to protect endothelial cell apoptosis, were markedly improved by administration of H₂ water in patients with potential metabolic syndrome.

The oxidation of LDL plays an important role in atherogenesis and may influence lipid metabolism. In this study, we determined the effects of H₂ water on the oxidation of LDL and LDL-mediated inflammation. As shown in Fig. 6A, the MDA content of the isolated LDL was reduced by H₂ treatment, suggesting that H₂ reduces the oxidation of LDL. To determine LDL-mediated inflammation, 100 μg protein/ml of the isolated LDL was added to the cultured RAW264.7 macrophages and bone marrow-derived macrophages for 24 h. As shown in Fig. 6B, C, the secretion of TNF-α and IL-6 by macrophages was reduced by H₂ treatment. Furthermore, we tested the effects of H₂ on LDL-induced monocyte adhesion to endothelial cells. As shown in Fig. 6D, consumption of H₂ water significantly decreased LDL-induced monocyte adhesion to endothelial cells. These data reveal that H₂ reduces LDL-mediated inflammatory properties in culture.

In a previous study, we found that H₂ has beneficial lipid-lowering effects in high-fat diet-fed Syrian golden hamsters (6). However, it remains unknown whether H₂ has effects on lipid and lipoprotein metabolism in humans. The key finding of our present study is that the novel antioxidant chemical element H₂ seems to alleviate lipid metabolism disorder, including hyperlipidemia and defective HDL function, in patients with potential metabolic syndrome. Our results were not consistent with that of human studies in type 2 diabetes mellitus, as they showed significant decreases in modified LDL-C levels and no effect on total and LDL-C (3). The inconsistency might be attributable to the difference in pathological conditions, the dosage of administration, or the intervention period. Despite this possibility, the discrepancy might be explained by the fact that modified LDL, like oxidized LDL, largely existed in the hyperlipidemia model, which was used in our study.

Subanalysis was conducted on 20 subjects, and we found the serum TC levels of 17 subjects with hyperlipidemia (TC > 5.18 mmol/l) was decreased by intake of H₂ water for 10 weeks, and the serum TC levels of the remaining 3 subjects without hyperlipidemia (TC < 5.18 mmol/l) was not significantly altered by H₂ treatment, although the TC level of 1 subject was slightly increased after treatment (Table 2, patient 14). The data gave us a clue that H₂ might exert a lipid-lowering effect on the patients with hyperlipidemia but have little effect on the population without hyperlipidemia.

It is well known that high cholesterol level is one of the important risk factors for atherosclerosis. Therefore, the elucidation of the mechanism by which H₂ decreases serum cholesterol will provide solid evidence for the application of H₂ in cardiovascular disease therapy. Serum LDL and VLDL are particles composed of a variety of lipids and protein components; thus, it is necessary to clarify which content of the lipoprotein could be affected by H₂ treatment. First, LDL is a contributing factor to the development of atherosclerosis. ApoB100 is the major protein present in LDL particles, and like LDL-C, the serum apoB level has been positively correlated with risk for atherosclerotic disease. In the present study, we found H₂ treatment not only decreased serum LDL-C levels, but also remarkably lowered apoB100 and apoE protein levels in serum. The data gave us a clue that H₂ might regulate the metabolism of LDL-C by decreasing the synthesis of apoB or increasing the catabolism of LDL-C_. Second, like LDL-C, VLDL-C is considered a type of ‘‘bad’’ cholesterol because elevated levels are associated with an increased risk of coronary artery disease (27). We found that apoB and apoE, the major proteins in VLDL, were lowered by H₂, although cholesterol analysis by FPLC revealed no changes of VLDL-C after H₂ treatment. The inconsistencies in protein and cholesterol levels might be explained by the suspicion that the functional target of H₂ might be apolipoprotein expression, not cholesterol. Taken together, the data indicate that molecular H₂ dissolved in water might have a beneficial regulating effect on lipid abnormality in patients with metabolic syndrome, especially in patients with hyperlipidemia. This effect is partially related to its regulation of lipid and protein contents of LDL and VLDL. Further experiments are needed to identify the mechanisms by which H₂ regulates the lipid and protein contents of lipoprotein particles and improves the serum lipoprotein profile. Previous studies have demonstrated that administration of H₂-rich saline improves insulin sensitivity (3), which, in our view, could partly contribute to the improved lipid metabolism in our study. Furthermore, liver cells sense the reduced levels of hepatic intracellular cholesterol and seek to compensate by synthesizing LDL receptors to draw cholesterol out of the circulation (28). Future studies on hepatic HMG-CoA reductase and LDL receptors may elucidate the mechanism by which H₂ acts on lipoprotein regulation.

HDL is known to protect against the development of atherosclerosis and is widely documented as a “negative risk factor” for coronary heart diseases (29). The antiatherogenic activity of HDL is principally attributable to a variety of antioxidative, anti-inflammatory, and antiapoptotic properties and the reverse transport of cholesterol (30). In the present study, we that found H₂ treatment seems to improve the functionality of HDL3 without altering HDL-C serum levels. It is known that the beneficial effects of H₂ on different disease models are mostly dependent on its antioxidative, anti-inflammatory, and antiapoptotic properties (16). Therefore, it is possible that the protective effect of H₂ on HDL function in our study is attributable, at least in part, to the antioxidative and anti-inflammatory properties of H₂. A number of therapeutic strategies are being developed to target HDL-C in an attempt to inhibit the progression or induce regression of atherosclerosis and reduce cardiovascular events. Therefore, the protective effect of H₂ on HDL function in the our study might provide a further evidence for H₂ application in vascular disease therapy. We did not observe any unwanted side effects of H₂, including headache, diarrhea, and vomiting (data not shown), suggesting fewer toxic and adverse effects of H₂.

The oxidation of LDL plays an important role in atherogenesis and may influence lipid metabolism. Our data reveals that H₂ seems to reduce the oxidation of LDL and LDL-mediated inflammation, and they further support our prediction that H₂ might improve lipid metabolism in patients with hyperlipidemia by inhibiting LDL-mediated inflammation. Previous studies have demonstrated that administration of H₂ reduces atherogenesis in apoE^−/− mice (5); in our view, this could partly contribute to the observed protective effects of H₂ on the oxidation of LDL and LDL-mediated inflammation in our study.

Indeed, our data give us a clue that H₂ might have the potential to be used as a novel lipid-regulating agent with the advantage of no toxicity compared with other commonly used lipid-regulating drugs that have side-effects on the liver and kidney. Further understanding of the mechanisms underlying the signaling pathways involved in the ability of H₂ to influence lipid and cellular metabolism is required to fully exploit intake of H₂ gas as a therapeutic strategy.

There are several limitations to the present study. First, the number of subjects recruited is small and it is hard to draw a solid conclusion, particularly having smokers among the subjects. It should, however, be emphasized that we analyzed the data in smokers and nonsmokers separately, and the response to H₂ in smokers seems to be stronger than that in nonsmokers, but the difference did not reach statistical significance. It would be useful to enlarge the sample size and examine the reaction to H₂ in smokers due to the antioxidative property of H₂. Another limitation of the study was that it was not a double-blind, randomized, controlled trial comparing H₂-rich water to placebo and comparing experimental endpoints from H₂-treated metabolic syndrome subjects with those measured in untreated healthy individuals. It is possible that during the 10 weeks of the study, the subjects altered their lifestyles, which could have affected the parameters studied. Therefore, it is difficult for us to draw a solid conclusion, and it limits the conclusions to stating that H₂ treatment appear to regulate lipid metabolism in patients.

In summary, our data show that in vivo administration of H₂ water seems to decrease serum TC and LDL-C levels and improve HDL functions in patients with potential metabolic syndrome, suggesting that H₂ may be used as a newer pharmacological agent to treat or control lipid metabolism disorder.