H2-Enriched Water for Mood, Anxiety, and Nerve FunctionScientific Research

INTRODUCTION

Health and a vibrant life are much craved by everyone. To improve quality of life (QOL), maintain a healthy state, and prevent the onset of various diseases, evaluation of interventional effects for improving QOL is important. The high metabolic rate of the brain results in the generation of disproportionate amounts of reactive oxygen and nitrogen species, leading to increased oxidative stress.¹ Increased oxidative stress and lipid peroxidation initiate a cascade of proinflammatory signals, leading to inflammation. Altered homeostasis of oxidation, inflammation, and protein aggregation has been suggested to contribute to the death of neurons, which is directly related to impairments in various cognitive domains. As such, chronic oxidative stress and inflammation may cause deteriorations in the function of the central nervous system, leading to reductions in QOL. Hydrogen has antioxidant activity and can prevent inflammation.²³⁴ The distribution of hydrogen throughout the brain and body indicates actions both in the central and peripheral nervous systems. Previous clinical studies have shown that hydrogen-rich water (HRW) reduces concentrations of markers of oxidative stress in patients with metabolic syndrome,⁵⁶ improves lipid and glucose metabolism in patients with type 2 diabetes,⁷ improves mitochondrial dysfunction in patients with mitochondrial myopathies, and reduces inflammatory processes in patients with polymyositis/dermatomyositis.⁸ In another study, exercise-induced declines in muscle function among elite athletes were also improved by administering HRW.⁹ Although such findings suggest that HRW may help alleviate symptoms of several diseases and increase the physical performance of athletes, the effects of prolonged HRW ingestion on the QOL of individuals in the general population remain unknown.

Some reports have demonstrated that oxidative stress is associated with QOL in patients with chronic obstructive pulmonary disease and cervical cancer.¹⁰¹¹ During oncological treatment among patients with cervical cancer, antioxidant supplementation was found to be effective in improving QOL.¹¹ In addition, Kang et al.¹² reported that treatment with HRW for patients receiving radiotherapy for liver tumors decreased oxidative stress and improved QOL. Although the association between oxidative stress and QOL in healthy individuals is still unclear, aging, job stress, and cognitive load over the course of several hours in healthy individuals have also been found to induce increases in oxidative stress,^13141516 suggesting that preventing the accumulation of oxidative stress caused by daily stress and daily work may contribute to the maintenance of QOL and amelioration of the effects of aging. Continuous HRW intake might therefore be expected to reduce accumulation of oxidative stress, thus helping to prevent decreases in QOL.

The aim of the present study was to investigate the effects of drinking 600 mL of HRW per day for 4 weeks on the QOL of adult volunteers using questionnaires for sleep, fatigue, mood, anxiety, and depression, an autonomic function test, and a higher cognitive function test.

SUBJECTS AND METHODS

Subjects

Thirty-one adult volunteers between 20 and 49 years old participated in this double-blinded, randomized, placebo-controlled study with a two-way crossover design. Exclusion criteria comprised: history of chronic illness; chronic medication or use of supplemental vitamins; employment in shift work; pregnancy; body mass index ≤ 17 or ≥ 29 kg/m^{2Declaration of Helsinki) and registered to the UMIN Clinical Trials Registry (No. UMIN000022382). The study protocol was approved by the Ethics Committee of Osaka City University Center for Health Science Innovation (OCU-CHSI-IRB No. 4), and all participants provided written informed consent for participation in the study.}

Study design

We used a double-blinded, placebo-controlled study with a two-way crossover design, as summarized in Figure 1. After admission to the study, participants were randomized in a double-blinded manner to receive HRW in an aluminum pouch (0.8–1.2 ppm of hydrogen, 300 mL/pouch; Melodian Corporation, Yao, Japan) or placebo water (PLW), representing mineral water from the same source (i.e., same components without hydrogen) in an aluminum pouch (0 ppm of hydrogen, 300 mL/pouch; Melodian Corporation) twice a day for 4 weeks. Fifteen participants were administered PLWfirst, and then HRW. The remaining 16 participants were administered HRWfirst, and then PLW. Participants consumed water within 5 minutes twice a day, at breakfast and dinner in their home, and confirmed the water intake at breakfast and dinner in a daily journal for 4 weeks. We assessed the intake rate of water by checking the daily journal every 4 weeks, on the 2^nd and 4^th experimental days. No participants reported any difference in taste between HRW and PLW. Previous studies have reported interventional effects of administering HRW to humans at hydrogen concentrations under 1.3 ppm.⁵¹² We therefore used a similar concentration of 0.8–1.2 ppm in the present study. Absolute volumes (600 mL) of HRW and PLW were provided to participants rather than a volume proportional to body mass, based on previously reported results.⁵⁶⁷¹² The duration of supplementation was set based on previous findings with HRW administration for 2–8 weeks.⁵¹²¹⁷ A 4-week washout period was provided between HRW and PLW administrations based on a previous study.⁸ The day before starting each experiment, participants were told to finish dinner by 21:00, and were required to fast overnight to avoid any influence of diet on concentrations of measured parameters (markers of inflammation and oxidative stress) in blood samples. At 09:00 the next day, participants completed the questionnaires after confirming that they had refrained from drinking alcohol, had finished dinner by 21:00, and had fasted overnight. Autonomic nerve function was measured at 09:30. Cognitive function testing was conducted at 09:45. Blood samples were collected at 10:00. These measurements were performed a total of four times for each participant, before (pre) and after (post) each of the two 4-week administration periods. From 24 hours (the day before the visit day) before each visit for measurements, participants were told to refrain from drinking alcohol or performing strenuous physical activity and to follow their normal diets, drinking habits, and sleeping hours. During the 4-week PLW or HRW administration periods, daily daytime activity (amount of physical exertion) of participants was measured using a pedometer and participants kept a daily journal to record drinking volume and times of PLW or HRW intake, physical condition (e.g., pain, lassitude, and indefinite complaints), sleeping times, etc.

Questionnaire

Severity of fatigue was measured using the Chalder Fatigue Scale (CFS)¹⁸ and a modified version of the Osaka City University Hospital Fatigue Scale.¹⁹ Mood and anxiety were evaluated using the K6 scale.²⁰ Symptoms of depression were measured using the Center for Epidemiologic Studies Depression Scale.²¹ General sleepiness and daytime sleepiness scores were calculated using the Pittsburgh Sleep Quality Index (PSQI)²² and the Epworth Sleepiness Scale,²³ respectively. The reliability and validity of the Japanese versions of these questionnaires have been confirmed.^192425262728

Autonomic function test

Participants underwent simultaneous electrocardiography and photoplethysmography using a Vital Monitor 302 system (Fatigue Science Laboratory, Osaka, Japan) while sitting quietly with their eyes closed for 3 minutes. These data were analyzed using MemCalc software (GMS, Tokyo, Japan). Frequency analyses for R-R interval variation from electrocardiography and a-a interval variation as the second derivative of photoplethysmography (accelerated plethysmography) were performed using the maximum entropy method, which is capable of estimating the power spectrum density from short time series data, and is adequate for examining changes in heart rate variability under different conditions of short duration.²⁹³⁰ The power spectrum resolution was 600 Hz. For frequency analyses, the low-frequency component power (LF) was calculated as the power within a frequency range of 0.04–0.15 Hz, and the high-frequency component power (HF) was calculated as that within a frequency range of 0.15–0.4 Hz. HF is vagally mediated,³¹³²³³ whereas LF originates from a variety of sympathetic and vagal mechanisms.³⁰³⁴ Some review articles³⁵³⁶³⁷ mentioned that LF reflects sympathetic nerve activity and is used as a marker of sympathetic nerve activity in original articles. Before autonomic nerve function testing was conducted for 3 minutes, a practice test was conducted for a period of 1 minute, in accordance with previous studies.³⁸³⁹⁴⁰ The reliability of these tests has been confirmed.⁴¹⁴²

Cognitive function test

Since previous studies have revealed that a switching attention task is useful for evaluating reduced performance under fatigue conditions,⁴³⁴⁴⁴⁵ we used task E of the modified advanced trail making test (mATMT) as a switching attention task for evaluating executive function.⁴⁶⁴⁷ Circles with numbers (from 1 to 13) or kana (Japanese phonograms, 12 different letters) were shown in random locations on a screen, and participants were required to use a computer mouse to alternately touch the numbers and kana; this task thus required switching attention. When participants touched a target circle, it remained in the same position, but its color changed from black to yellow. Participants were instructed to perform the task as quickly and correctly as possible, and continuously performed this task for 5 minutes. We evaluated three indices of task performance: the total count of correct responses (number of correctly touched numbers and letters); the total count of errors (number of incorrectly touched numbers and letters); and the motivational response (reaction time from a finished trial to the next trial). Based on our previous study,⁴⁷ before participants performed task E of the mATMT on each experimental day, they practiced for a period of 1 minute. The reliability of this test has been confirmed.⁴³⁴⁴

Blood sample analyses

Blood samples were collected from the brachial vein. The amount of blood sampled was 13 mL per experimental day. We thus collected blood samples on four occasions (once per experimental day) in the study. Blood samples for serum analyses were centrifuged at 1,470 × g for 5 minutes at 4°C. The concentration of high-sensitivity C-reactive protein (hs-CRP) in each serum sample was assessed by particle-enhanced immunonephelometry using a BNII analyzer (BN II ProSpec; Siemens, Munich, Germany). Oxidative activity in each serum sample was assessed with the reactive oxygen metabolites-derived compounds (d-ROMs) test (Diacron International, Grosseto, Italy), while anti-oxidative activity was measured with the biological anti-oxidant potential (BAP) test (Diacron International) using a JCABM1650 automated analyzer (JEOL, Tokyo, Japan).⁴⁸ The concentrations of ROMs are expressed in Carratelli units (1 CARR U = 0.08 mg of hydrogen peroxide/dL).⁴⁹ The oxidative stress index (OSI) was calculated using the following formula: OSI = C × (d-ROMs/BAP), where C denotes a coefficient for standardization to set the mean OSI in healthy individuals at 1.0 (C = 8.85).⁴⁵ All supernatants were stored at -80°C until analyzed. Assays for hs-CRP were performed at LSI Medience Corporation (Tokyo, Japan) and those for serum d-ROMs and BAP were performed at Yamaguchi University Graduate School of Medicine.

Daily daytime activity and daily journal

Daily daytime activity, representing the expenditure of calories and amount of physical activity (METs × time) was recorded using an Active style Pro HJA-350IT pedometer (OMRON, Kyoto, Japan). A daily journal was kept for 4 weeks, and included information on fatigue (based on a visual analogue scale from 0, representing “no fatigue”, to 100, representing “total exhaustion”) just after waking up and before bedtime, sleeping times, physical condition (1, good; 2, normal; or 3, bad), and special events (if the day was different from a usual day: 1, no; or 2, yes). We carefully checked the daily journal every four weeks, on the 2^nd, 3^rd, and 4^th experimental days.

Statistical analyses

First, we tested the normality (parametric or non-parametric distributions) of each measured parameter using the Kolmogorov-Smirnov test. Values are presented as the mean ± standard deviation or median and interquartile range based on the results of Kolmogorov-Smirnov test. The Wilcoxon signed-rank test for non-parametric parameters and paired t-test for differences between HRW and PLW administrations after two-way repeated-measurement analysis of variance for parametric parameters were conducted. If significant changes were observed by comparisons within each condition (pre- vs. post-HRW; pre- vs. post-PLW) or between post-treatment values (post-HRW vs. post-PLW), then we compared change ratios between post-HRW/pre-HRW and post-PLW/pre-PLW using the Wilcoxon signed-rank test or paired t-test. All P values were two-tailed, and those less than 0.05 were considered statistically significant. Statistical analyses were performed using IBM SPSS Statistical Package version 20.0 (IBM, Armonk, NY, USA).

RESULTS

General results

During the study, we excluded five participants from data analyses due to symptoms of hay fever, prolonged medication use because of a cold, insufficient intake of HRW or PLW intake (≥ 85%), or a frequency of special events ≤ 15% as recorded in the daily diary. We thus analyzed data from a total of 26 participants (13 females, 13 males; mean age, 34.4 ± 9.9 years; mean body mass index, 21.5 ± 2.6 kg/m²). No side, order, and carry-over effects were observed from the oral administrations of HRW and PLW in any participant.

Questionnaire results

Results from the questionnaires are summarized in Table 1. No questionnaire scores at baseline (pre) showed any significant differences between HRW and PLW administration groups. With HRW administration, scores for K6, CFS, and PSQI were significantly decreased after the 4-week administration period. In addition, the change ratio (post/pre) for K6 score was significantly lower in the HRW administration group than in the PLW administration group (Figure 2). No significant changes were seen in any other questionnaire scores (modified version of the Osaka City University Hospital Fatigue Scale, Center for Epidemiologic Studies Depression Scale or Epworth Sleepiness Scale) after HRW administration and no significant changes in any of the scores were seen after PLW administration. Likewise, these scores did not differ significantly between HRW and PLW after administration.

Autonomic function results

Results for the autonomic nerve function are summarized in Table 1. LF, HF, and LF/HF ratio at baseline (pre) did not differ significantly between HRW and PLW administrations, indicating similar autonomic nerve function in the two groups before water intake. Although the HF and LF/HF ratio were not significantly affected by 4-week administrations of HRW or PLW, LF after HRW administration was significantly lower than that after PLW administration. The change ratio (post/pre) for LF was also significantly lower in the HRW administration group than in the PLW administration group (Figure 2).

Cognitive function results

Results for the cognitive function test are shown in Table 1. Motivational response and total counts of correct responses and errors at baseline (pre) did not differ significantly between HRW and PLW administrations, indicating similar cognitive function between groups before water intake. Motivational response after HRW administration was significantly faster than that before HRW administration. The change ratio (post/pre) for motivational response was not significantly different in the HRW administration group than in the PLW administration group. No significant differences in motivational response, total counts of correct responses, or errors after water administration were seen between HRW- and PLW-administered conditions.

Blood sample results

No significant differences were seen in any blood parameters (hs-CRP, d-ROMs, BAP, and OSI) before HRW or PLW administration (Table 1), indicating the comparability of the two groups before water intake. After HRW and PLW administrations, we again found no significant differences in these blood parameters.

Daily daytime activity and daily journal results

The daily expenditure of calories and amount of physical activity during the 4-week administration periods did not differ significantly between HRW and PLW administration conditions (Table 2). Similarly, visual analogue scale scores for fatigue just after waking and before bedtime, sleeping times, physical condition, and counts of special events were comparable between HRW and PLW administration conditions (Table 2), indicating that living habits were successfully controlled during the experimental period in the two groups.

DISCUSSION

The present findings suggest that HRW administration for 4 weeks may have improved the QOL of adult volunteers in terms of improved mood and anxiety and reduced activity of the sympathetic nervous system at rest.

In terms of associations between hydrogen and the central nervous system, a report by Ohsawa et al.⁴ was the first to demonstrate that molecular hydrogen acts, at least in part, as an anti-oxidant as it binds to hydroxyl ions produced in central nervous system injuries. Previous studies have proposed that HRW administration has neuroprotective effects⁵⁰ and anti-aging effects on periodontal oxidative damage in healthy aged rats.⁵¹ In a rat model of Alzheimer’s disease, hydrogen-rich saline prevented neuroinflammation and oxidative stress, and improved memory function.⁵² In terms of the association between HRW and QOL, only one study reported that HRW administration for 6 weeks improved QOL scores in patients treated with radiotherapy for liver tumors.¹² Although reports on the effects of HRW administration in healthy populations have not been accumulated, job stress¹⁴¹⁵ and acute fatigue caused by mental and physical loading for several hours¹⁶⁵³ have been shown to enhance oxidative stress. As for physical fatigue, in order to alleviate acute physical fatigue in healthy volunteers not including athletes, we have previously demonstrated that treatment with antioxidant supplements is effective.⁵⁴⁵⁵⁵⁶ The present study provided new findings that HRW affects not only physical condition but also mental conditions such as mood, anxiety, and autonomic nerve function. One of the advantages of HRW is the ability to cross the blood-brain barrier, offering high potential to reduce oxidative stress in the brain. A previous study in rats found that levels of malondialdehyde, a marker of oxidative stress, were around 4.8-fold higher in the brain than in the blood (plasma).⁵⁷ These results suggest that HRW may be effective for reducing accumulated oxidative stress in the brain in daily life, potentially contributing to the maintenance of central nervous system activity and preventing decreases in QOL.

In the present study, mood and anxiety levels improved after HRW administration. These negative emotions are also known to be involved in conditions related to oxidative stress; social phobia,⁵⁸⁵⁹ depression,⁶⁰ anxiety,⁶¹⁶² and other neuropsychiatric disorders⁶³ have been shown to be associated with increased oxidative stress. Neuroinflammation is also related to fatigue, mood, anxiety, and sleep.^64656667 In older mice, HRW administration succeeded in suppressing depression-like behaviors.⁶⁸ These findings suggest that administration of HRW for 4 weeks may be effective for controlling such negative emotions by reducing oxidative stress and inflammation of the central nervous system. Increasing evidence suggests that oxidative stress and inflammation in neurons are involved in the pathological manifestations of many neurological and neuropsychiatric disorders, and HRW administration may thus help alleviate the symptoms of these disorders. Previous study revealed that oxidative stress of the brain causes cognitive and motivational deficits in a mouse model of neuropsychiatric disorder (schizophrenia).⁶⁹ In the present study, motivational response of cognitive function test was improved by prolonged HRW intake, suggesting that a reduction of oxidative stress in the brain by the intake of HRW may increase motivational performance of cognitive task.

Stressors can enhance sympathetic hyperactivity, promote oxidative stress, and boost pro-inflammatory cytokine production.⁷⁰⁷¹⁷² Autonomic nerve function is thus closely associated with oxidative stress and inflammation. Attenuation of sympathetic nervous system activity during the resting state in adult volunteers may therefore be the result of decreases in inflammation and oxidative stress as an effect of prolonged HRW administration. However, the lack of changes in oxidative stress markers noted in the present study after HRW intake for 4 weeks could be due to the low severity of oxidative stress in the participants. Actually, serum d-ROMs (307.1 ± 49.4 CARR U) and BAP (2,549 ± 194 µM) concentrations at the first measurement point in the present study were within normal ranges based on the results of serum d-ROMs (286.9 ± 100.2 CARR U) and BAP (2,541 ± 122 µM) concentrations measured in 312 healthy participants in our previous study.⁴⁸ However, levels of oxidative stress fluctuate depending on daily work load and stress. In addition, the rat study by García-Niño et al.⁵⁷ that found malondialdehyde levels around 4.8-fold higher in the brain than in plasma indicate that oxidative stress in the brain is more severe. Daily administration of HRW for 4 weeks may thus contribute to attenuation of and prevention from the cumulative oxidative stress in the brain. Mood, anxiety, and autonomic nerve function could thus potentially be improved. Although the range of sympathetic nerve activity in the present study considers to be normal based on our previous studies,⁷³⁷⁴ sympathetic nerve activity also fluctuates depending on daily work load and stress.³⁵ Therefore, lower sympathetic nerve activity of resting state may contribute to suppress an excessive increase in sympathetic nerve activity after the daily work load and stress.

We conducted this study with a limited number of participants. Before our results can be generalized, studies involving larger numbers of participants are essential.

Although we mainly examined the effects of HRW on the central nervous system, we did not directly evaluate the dynamics of inflammation and oxidation in the brain. Neuroimaging studies using positron emission tomography and magnetic resonance imaging are thus underway in our laboratory to identify the mechanisms underlying the effects of HRW intake on the central nervous system that can improve QOL.

In conclusion, HRW administration for 4 weeks in adult volunteers improved mood, anxiety, and autonomic nerve function, suggesting that HRW administration may offer an effective method to reinforce QOL and maintain good health. In a further study, we will try to identify the effects of HRW administration in participants with ongoing stress or chronic fatigue.

Acknowledgments

We would like to thank Ms. Mika Furusawa for her excellent technical assistances and Forte Science Communications for editorial help with this manuscript.