Beneficial effects of molecular hydrogen (H₂) in various experimental models of human diseases and in many clinical studies was documented. H₂ can be administered by various ways, as a gas inhalation, drinking of H₂-enriched water, or taking a H₂-dissolved bath as well as in saline infusions. As antioxidant selectively scavenges hydroxyl and peroxynitrite radicals, decreases oxidative stress. However, theH₂ effect on antioxidant–coenzyme Q information is lacking. This pilot study found protective effects of H₂ on coenzyme Q₉ in plasma, myocardial tissues and mitochondria of rats. Our results can contribute to the explanation of a new beneficial mechanism of H₂ on a part of antioxidant protection in organism.

Keywords: molecular hydrogen, myocardium, coenzyme Q_9, rat

In the last decade positive the effect of H₂was documented in several experimental and clinical studies. In the in preventive and therapeutic treatment of various diseases as cancer, cardiovascular system, central nervous system, metabolic syndrome, respiratory system diseases beneficial effects of H₂ have been observed.^1,2 H₂ can be administrated by various ways, as a gas inhalation, by saline injection, by drinking hydrogen rich water, by injection or eye-dropping of H₂-dissolved saline or taking H₂-rich water bath.^3,4 Due to the low molecular weight H₂ rapidly diffuses into tissues and cells.^5,6 As an antioxidant H₂selectively scavenges hydroxyl radical (^.OH) and peroxynitrite radical (ONOO^-) to reduce oxidative stress. Further identified H₂modes of action are anti-inflammatory and anti-apoptotic effects, regulation of gene expression,^7-10 protection of antioxidant enzyme (superoxide dismutase) and regulation of antioxidant defence.¹¹ The effect of H₂ on other antioxidantin organism - coenzyme Q(a key component for mitochondrial bioenergetics) - remains unknown. We hypothesized that the oral intake of molecular hydrogen rich water may protectcoenzyme Q₉(CoQ₉) concentration in rats.

In the study male Wistar rats were included, 3 months aged, with body weight 200g. Number of rats in each group was 5. Three control groups were fed with pure water. Three H₂ groups were fed with H₂ rich water by gastric tube 3x3ml daily for 2 days (CH₂-2); for 2 weeks gavage and 4 weeks without H₂ (CH₂-45/2); 45 days with H₂ gavage (CH₂-45). The animal experiments were in compliance with the Ethics Commitee of the Institute for Heart Research, Slovak Academy of Sciences and protocols approved by the State Veterinary and Food Administration of the Slovak Republic (permit No. 4091/16-221). Male Wistar rats used in this study were purchased from the Department of Toxicology and Laboratory Animals Breeding, Slovak Academy of Sciences, housed and bred under standard environmental conditions (12h light/dark cycle, ambient temperature 22 – 24 °C and 45 – 65% humidity) in the Institute for Heart Research, Slovak Academy of Sciences. Food and water were available during the whole experiment ad libitum.

Isolation of mitochondria

Mitochondria from heart muscle were isolated by differential centrifugation.¹² Isolated solution contained 180mmol/l KCl, 4mmol/l EDTA, 20mmol TRIS and 0.1% of albumin with the addition of Nagarse 2.5mg/g of the tissue. Sedimented mitochondria were washed twice without albumin. Mitochondrial proteins were estimated spectrofotometrically.¹³

Determination of coenzyme Q₉

CoQ_9-TOTAL (ubiquinone – oxidized form + ubiquinol – reduced form) was determined in plasma, CoQ_9-OX (oxidized form) in myocardial tissue and mitochondria by HPLC method with UV detection. For CoQ₉ extraction hexane/ethanol (5/2 v/v) was used, organic phase was collected, evaporated under nitrogen, the residue dissolved in ethanol and injected into column SGX C18 7µm (Tessek). The mobile phase consisted of methanol/acetonitrile/ethanol (6/2/2 v/v/v, Merck). Coenzyme Q was detected at 275nm using external standards (Sigma).^14,15 The results were evaluated using unpaired Student´s t-test, p<0.05 were considered statistically significant.

Effect of molecular hydrogen on coenzyme Q

CoQ_9-TOTAL concentrations in plasma increased after 2-days of H₂ application by 23.3%, after 45 days (CH₂-45/2) increased by 12.3%, after 45 days (CH₂-45) increased by 31.3%, (p= 0.088) in comparison with control groups. In the tissue and mitochondria of the heart concentration of CoQ_9-OX was evaluated. Two days of H₂ application in myocardial tissue had no effect on CoQ_9-OX concentration. Long term (CH₂-45/2) and (CH₂-45) effect of H₂ application stimulated CoQ_9-OX concentration by 16.6% and by 31.79% respectively, significantly (p<0.035). In isolated mitochondriathe positive effect of H₂ was found after 2 days. CoQ_9-OX concentration increased by 44.4%, in group (CH₂-45/2) by 42.8%. When H₂ was applicated for 45 days (CH₂-45), its effect was lower. CoQ_9-OX concentration increased by 17.1% in comparison with control group (CC-45). CoQ_9-OX concentration in isolated mitochondria were evaluated in %, while some samples were collected due to small quantities of tissues for the mitochondrial isolation (Table 1).

Molecular hydrogen is a colorless and odorless gas, which selectively scavenges hydroxyl and peroxynitrite radicals, but not the same applies for hydrogen peroxide and nitric oxide in cells and tissues.¹⁶ H₂ successful effect in animal models of human disease was documented. The role of H₂ was found in hypoxic post-conditioning, radiation-induced heart injury, mediastinal irradiation in rats, acute cardiac injury, radiation-induced heart disease and changes in microRNA-1, -15b and -21 levels in irradiated rat hearts.^17-22 Beneficial effect of H₂ in clinical medicine was found in various diseases, such as cardiovascular diseases, type 2 diabetes, dyslipidemia, obesity and metabolic syndrome, in vascular health.^23-25 Molecular hydrogen water improved the progression of Parkinson´s disease.²⁶ Molecular hydrogen is a short-live small molecule which is able to diffuse through membranes upon theconcentration gradient.²¹Afterthe H₂ inhalation by rats, its level immediately increases in the myocardial tissue and probably diffusesinto mitochondria.²⁷ H₂ protects antioxidant enzyme – superoxide dismutase.²² Molecular hydrogen effect on other antioxidant – coenzyme Q (CoQ) – is not known up to now.

Coenzyme Q was discovered by Frederick Loring Crane in 1957. Human’s dominant form is CoQ₁₀, rats dominant form is CoQ₉. CoQ₁₀ as a crucial mobile component of the mitochondrial respiratory chain acts in three forms in the„ Q-CYCLE“. CoQ –oxidized form (ubiquinone), CoQH₂ – reduced form (ubiquinol) and CoQ – radical from (ubisemiquinone). The central role of CoQ₁₀ is electrons and protons transfer from Complex I and Complex II to Complex III ofthe mitochondrial respiratory chain. CoQ₁₀ as antioxidant scavenges free oxygen radicals, decreases oxidative stress. Its concentration is changed during semicircadian cycles, every twelve hours has maximum (PEAKS) and minimum (NADIRS) concentration. CoQ has its own biological clock – Q₁₀-CLOCK.^28,29 CoQ₁₀ was found in all the tissues of the body, and its higher concentrations were found in tissues with very active metabolism and energy demands, as heart, brain, kidney and skeletal muscle. Ubiquinol is a lipophilic antioxidant and is capable to recycle and regenerate other antioxidants, as alpha-tocopherol and vitamin C.³⁰ In this study the stimulation of CoQ_9-TOTAL concentrations in plasma and CoQ_9-OX concentration in myocardial tissues and mitochondria after H₂ application in rats were found (Table 1).

To the best of our knowledge, we present the first data showing short and prolonged effect of H₂ on coenzyme Q₉ in plasma, myocardial tissues and mitochondria of rats.³¹ We suppose, that theH₂ application could protect the mitochondrial concentration of Co Q by reducing the concentration of ^.OH. Our results can contribute to the explanation of a new beneficial mechanism of H₂ on a part of the antioxidant protection in organism. Next studies of the effect of molecular hydrogen effect on human mitochondrial function using a non-invasive method in isolated platelets could confirm our pilot results in rats.

This work was supported by grants APVV-15-0376; VEGA 2/0063/18 and VEGA 2/0021/15. We thank for technical assistance to Anna Štetková.

The authors declare that there is no conflict of interest.

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