The Effect of Adjuvant Therapy with Molecular Hydrogen on Endogenous Coenzyme Q10 Levels and Platelet Mitochondrial Bioenergetics in Patients with Non-Alcoholic Fatty Liver Disease

Abstract

Molecular hydrogen (H₂) has been recognized as a novel medical gas with antioxidant and anti-inflammatory effects. Non-alcoholic fatty liver disease (NAFLD) is a liver pathology with increased fat accumulation in liver tissue caused by factors other than alcohol consumption. Platelet mitochondrial function is considered to reflect systemic mitochondrial health. We studied the effect of adjuvant therapy with hydrogen-rich water (HRW) on coenzyme Q₁₀ (CoQ₁₀) content and platelet mitochondrial bioenergetics in patients with NAFLD. A total of 30 patients with NAFLD and 15 healthy volunteers were included in this clinical trial. A total of 17 patients (H₂ group) drank water three × 330 mL/day with tablets producing HRW (>4 mg/L H₂) for 8 weeks, and 13 patients (P group) drank water with placebo tablets producing CO₂. The concentration of CoQ_10-TOTAL was determined by the HPLC method, the parameter of oxidative stress, thiobarbituric acid reactive substances (TBARS), by the spectrophotometric method, and mitochondrial bioenergetics in platelets isolated from whole blood by high-resolution respirometry. The patients with NAFLD had lower concentrations of CoQ_10-TOTAL in the blood, plasma, and platelets vs. the control group. Mitochondrial CI-linked LEAK respiration was higher, and CI-linked oxidative phosphorylation (OXPHOS) and CII-linked electron transfer (ET) capacities were lower vs. the control group. Plasma TBARS concentrations were higher in the H₂ group. After 8 weeks of adjuvant therapy with HRW, the concentration of CoQ₁₀ in platelets increased, plasma TBARS decreased, and the efficiency of OXPHOS improved, while in the P group, the changes were non-significant. Long-term supplementation with HRW could be a promising strategy for the acceleration of health recovery in patients with NAFLD. The application of H₂ appears to be a new treatment strategy for targeted therapy of mitochondrial disorders. Additional and longer-term studies are needed to confirm and elucidate the exact mechanisms of the mitochondria-targeted effects of H₂ therapy in patients with NAFLD.

Keywords: coenzyme Q10; mitochondria; molecular hydrogen; oxidative phosphorylation; platelets.

Figure 1

Bioenergetics in platelet mitochondria at the beginning of the study. (A) Parameters of mitochondrial respiration and ATP production in platelets of patients with NAFLD and the healthy volunteers expressed as flux control ratio. (B) Parameters of mitochondrial respiration and ATP production in platelets of the hydrogen (H₂) and placebo (P) groups of patients with NAFLD at the beginning of the study expressed as O₂ flow (pmol/s/10⁶ cells). The bars show mean ± standard error of mean (sem). The evaluated respiratory capacities are marked according to the titration steps in the substrate–uncoupler–inhibitor titration (SUIT) reference protocol 1 [27] and correspond to following respiratory states: ce—routine respiration of intact cells; Dig—residual oxygen consumption (ROX) after permeabilization with digitonin; 1PM—LEAK respiration with CI-linked substrates pyruvate and malate; 2D—CI-linked OXPHOS capacity (associated with ATP production); 2D; c—CI-linked OXPHOS capacity after addition of cytochrome c as a test for integrity of outer mitochondrial membrane; 3U—CI-linked electron transfer (ET) capacity with pyruvate and malate; 4G—CI-linked ET capacity with pyruvate, malate, and glutamate; 5S—CI&II-linked ET capacity; 6Rot—CII-linked ET capacity; 7Ama—ROX after inhibition of mitochondrial CIII. CI—respiration related to mitochondrial CI activity; CI&II—respiration related to mitochondrial CI and CII activity; CII—respiration related to mitochondrial CII activity. LEAK—non-phosphorylating state of respiration; OXPHOS—the capacity of oxidative phosphorylation; ET—the capacity of electron transfer. * p < 0.05 vs. the control group.

Figure 2

Bioenergetics in platelet mitochondria in the groups of NAFLD patients at the beginning and end of the study. (A,C) Parameters of mitochondrial respiration and ATP production in platelets of H₂ and P groups of patients with NAFLD expressed as O₂ flow (pmol/s/10⁶ cells). (B,D) Parameters of mitochondrial respiration and ATP production in platelets of H₂ and P groups of patients with NAFLD expressed as flux control ratio. The bars show mean ± standard error of mean (sem). The evaluated respiratory capacities are marked according to the titration steps in the substrate–uncoupler–inhibitor titration (SUIT) reference protocol 1 [27] and correspond to following respiratory states: ce—routine respiration of intact cells; Dig—residual oxygen consumption (ROX) after permeabilization with digitonin; 1PM—LEAK respiration with CI-linked substrates pyruvate and malate; 2D—CI-linked OXPHOS capacity (associated with ATP production); 2D; c—CI-linked OXPHOS capacity after addition of cytochrome c as a test for integrity of outer mitochondrial membrane; 3U—CI-linked electron transfer (ET) capacity with pyruvate and malate; 4G—CI-linked ET capacity with pyruvate, malate, and glutamate; 5S—CI&II-linked ET capacity; 6Rot—CII-linked ET capacity; 7Ama—ROX after inhibition of mitochondrial CIII. H₂ before, H₂ after—the group of patients with NAFLD before and after 8-week adjuvant therapy with HRW; P before, P after—the group of patients with NAFLD before and after 8 weeks of taking placebo. CI—respiration related to mitochondrial CI activity; CI&II—respiration related to mitochondrial CI and CII activity; CII—respiration related to mitochondrial CII activity. + p < 0.05, ++ p < 0.01 vs. the same group at the beginning of the study.

Figure 3

P-L control efficiency in platelet mitochondria in the control group, NAFLD patients, and groups H₂ and P of NAFLD patients at the beginning and the end of the study. P-L control efficiency was calculated from parameters of mitochondrial respiration as (2D-1PM)/2D (for parameters of mitochondrial respiration refer to Figure 1). * p < 0.05, ** p < 0.01 vs. the control group; + p < 0.05 vs. the same group at the beginning of the study. The values (%) above the bars show the relative change vs. baseline values in each group.

Figure 4

Coenzyme Q_10-TOTAL concentration in platelets (PLTs) (A), blood (B), and plasma (C). Control—the control group; H₂ before, H₂ after—the group of patients with NAFLD before and after 8-week adjuvant therapy with HRW; P before, P after—the group of patients with NAFLD before and after 8 weeks of taking placebo. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group; + p < 0.05 vs. the same group at the beginning of the study. The values (%) above the bars show the relative change vs. baseline values in each group.

Figure 5

The correlation between CII-linked ET capacity and concentration of CoQ₁₀ in platelets. H₂—the group of patients with NAFLD before and after adjuvant treatment with HRW; P—the group of patients with NAFLD before and after treatment with placebo; r—Pearson correlation coefficient; p—the p-value.

Figure 6

The concentration α-tocopherol in platelets (A) and plasma (B); the concentration of β-ca-rotene in plasma (C); and the concentration of thiobarbituric acid reactive substances (TBARS) in plasma (D). Control—the control group; H₂ before, H₂ after—the group of patients with NAFLD before and after 8-week adjuvant therapy with HRW; P before, P after—the group of patients with NAFLD before and after 8 weeks of taking placebo. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group; + p < 0.05, ++ p < 0.01 vs. the same group at the beginning of the study. The values (%) above the bars show the relative change vs. baseline values in each group.