Hydrogen-rich saline improves non-organic erectile dysfunction (ED
Combined therapy with sildenafil and hydrogen-rich saline for the
treatment of non-organic erectile dysfunction in low responders to
Min Fan, 1, † Xianlin Xu, 1, * Qianfeng Zhuang, 1, † Xi Qian , 2 Jipu Liu, 3 Xiaozhou He 1, *
Department of Urology, the Third Affiliated Hospital of Soochow University,
Changzhou 213000, Jiangsu Province, China;. 2 Department of Animal Science,
University of Vermont, 121 Terrill Building, 570 Main Street, Burlington, VT 05405,
USA. 3 Department of Urology, the People's Hospital of Huiyang District,Changan St,
Huizhou 516200, Guangdong Province, China
These authors contributed equally to this work
* Correspondence: Xiaozhou He, Department of Urology, the Third Affiliated Hospital of
Soochow University, Jiangsu Changzhou 213003, China, email: [email protected],
Tel: 86-519-68871251, Fax: 86-519- 86621235; and Xianlin Xu, Department of Urology,
the Third Affiliated Hospital of Soochow University, Jiangsu Changzhou 213003, China,
email: [email protected], Tel: 86-519-68871251, Fax: 86-519- 86621235
Running title: Treatment of Non-organic ED
Objective: The study aimed to investigate the effects of hydrogen-rich water (HRS) on
non-organic erectile dysfunction in patients who were low responders to sildenafil.
Methods: Patients were administered sildenafil for three months and assigned to two
groups: treatment group (HRS) or sham group. The experimental groups were given
either oral HRS or saline twice daily (900 ml/d) for 12 consecutive weeks. Serum was
collected before and after treatment to detect oxidative stress. Two markers of oxidative
stress were detected - SOD (superoxide dismutase) and MDA (malondialdehyde). IIEF
(International Index of Erectile Function questionnaire)-5 scores were evaluated to
determine erectile function. Erectile function in low responders to sildenafil was defined
as IIEF-5 less than 21.
Results: 25 patients were included in the study, and 12 were allocated into the HRS
treatment group, while 13 were assigned to the sham group. After 12 weeks of treatment,
mean IIEF-5 scores improved significantly for men who received HRS compared with
those in sham group. SOD activity increased significantly, while MDA decreased in the
HRS group compared to the sham group.
Conclusion: HRS therapy enhanced the efficacy of sildenafil in restoring erectile function
in low responders to sildenafil, possibly by reducing oxidative stress and increasing
antioxidative enzyme activity.
Key words: Hydrogen rich water; Oxidative stress; Erectile dysfunction
Erectile dysfunction (ED) is defined as the recurrent difficulty in developing or
maintaining an erection suitable for satisfactory sexual performance.
treatment of ED has a profound impact on quality of life[2,3]. By 2025, the population in
Westernized countries affected by ED will be over 320 million, which is double the
prevalence seen 15 years ago. Therefore, there is a need to reevaluate ED therapeutic
strategies. The current first-line monotherapy are PDE-5 inhibitors. However, 11-44%
of patients do not respond to these inhibitors [2,5]. Furthermore, the recommended
second-line alternatives (such as penile injection therapy with vasodilators, vacuum pump
therapy, or penile implants) tend to be more intrusive. Therefore, additional therapies are
Nitrous oxide (NO), a vasodilator produced by the endothelium and nitrergic neurons in
the penis, regulates penile vascular tone by directing relaxation of corporeal smooth
muscles. As a highly reactive free radical, NO interacts with superoxide anion to form
peroxynitrite, leading to endothelial dysfunction. This may explain sildenafil resistance.
Decreased ROS via antioxidant treatment might be useful in the reduction of NO and
enhance the effectiveness of PDE-5 inhibitors. Some studies have found that, compared
to PDE5 inhibitors alone, a combination of PDE5 inhibitors and an adjunctive antioxidant
agent was more effective in treating diabetic ED. Moreover, the combination of
vitamin E and sildenafil has been shown to improve erectile function.
In 2007, Ohsawa discovered that hydrogen gas was associated with antioxidant and
antiapoptotic properties, which could selectively neutralize hydroxyl and peroxynitrite
radicals. In human subjects, these characteristics protect the brain against
ischemia-reperfusion injury and stroke. Our previous studies have demonstrated the
protective effects of hydrogen-rich saline (HRS) against erectile dysfunction in a diabetic
animal model. We aimed to evaluate whether treatment with HRS improved erectile
function in patients with an unsatisfactory response to sildenafil monotherapy.
Materials and Methods
Participants comprised patients who met the following criteria: age 25-75 years, presently
in a stable relationship, normal sexual desire, normal blood testosterone, normal
nocturnal penile tumescence and rigidity (evaluated by NPTR-RigiScan), normal
dynamic duplex ultrasound, and a IIEF-5 (International Index of Erectile Function
questionnaire) score of less than 21 after use of sildenafil. Patients were recruited via
physician referral and media advertisements. However, those who had significant
cardiovascular disease, uncontrolled diabetes mellitus, neurological and psychiatric
disorders, previous genitourinary surgery, or a history of intolerance to alpha-blockers
were excluded. Based upon the outcomes of these diagnostic tests, we prescribed a limit
to ED as “nonorganic” or “without an identifiable organic etiology”. We could not
determine if these patients were suffering from psychogenic ED, because no
psychometric tests were performed; however, none of the patients had psychiatric
disorders or were on psychotropic medications.
This was a randomized, double-blinded, parallel group trial with two treatment arms
(hydrogen-rich saline, sham), and was reviewed and approved by the Ethics Committee
of the Third Affiliated Hospital of Soochow University. All subjects provided written and
verbal informed consent. Following screening and enrollment, the study began with a
four-week run-in period in which patients were instructed to use sildenafil (100 mg) prior
to all sexual activity, and counseled on the most effective use of sildenafil (use on an
empty stomach, appropriate sexual stimulation, appropriate timing). After the run-in
period, baseline testing was conducted, and those scoring less than 21 on the IIEF
questionnaire were randomized to 3-month treatment with HRS or matching saline, along
with continuation of adjunctive sildenafil. Patients consumed 450-500 ml of
hydrogen-rich saline or physiological saline twice a day to achieve a total minimum
consumption of 900 ml. All participants were instructed to have intercourse once or twice
a week for the 3-month duration. At the end of combined treatment, IIEF-5 scores were
used to assess the clinical improvement in erectile function. Other anti-oxidant treatment
was discontinued within one week of study commencement. Fasting venous blood (4 ml)
was collected in the morning before and after the 12-week treatment period. After the
centrifugation of venous blood at 3000 rpm for 5 minutes, serum was collected and stored
at -70º C for further use.
International Index of Erectile Function questionnaire
The International Index of Erectile Function (IIEF) is a well validated and widely used
multi-dimensional self-reported instrument for the assessment of male sexual function.
The full version of the IIEF consists of 15 questions measuring several domains of
male sexual function (i.e. erectile function, orgasmic function, sexual desire, intercourse
satisfaction, and overall satisfaction). One specific segment of the full IIEF was used to
measure erectile function, namely an abridged five-item version (IIEF-5). In the current
study, we chose to measure only the erectile function components, and thus limited our
data analysis to the IIEF-5 as described in detail below.
The IIEF-5, a proven component of the overall IIEF questionnaire, was used to screen
patients for study inclusion. It comprises five specific questions that assess erectile
function from the full version of the IIEF (questions 2, 4, 5, 7, and 15). The maximum
score for the IIEF-5 is 25, and a cutoff of less than 21 was considered as ED. After the
run-in period with administration of sildenafil (100 mg), only men who scored less than
21 on the IIEF-5 were included in the study.
Measurement of serum oxidative stress
Serum levels of malondialdehyde (MDA), a marker for oxidative stress, were determined
by a spectrophotometric measurement of thiobarbituric acid-reactive substances
(TBARS), following the manufacturer’s instructions (Nanjing Jiancheng Biochemistry,
China). Superoxide dismutase (SOD), which acts as antioxidant and protects cellular
components from oxidation by reactive oxygen species, was measured by a commercial
kit (Nanjing Jiancheng Biochemistry, China), according to the manufacturer’s
Statistical analyses were employed using GraphPad Prism 5.0 software (GraphPad
Software, Inc, San Diego, USA). Descriptive statistics were presented as mean ± standard
deviation (SD) for IIEF scores. We performed Student’s t-tests to compare the same
group of patients (pre vs. post) and unpaired T tests between the two treatment groups
(HRS vs. sham). A p-value less than 0.05 was considered statistically significant.
A total of 35 men completed the study (HRS, n=12; sham, n=13). As summarized in
Table 1, no significant differences were observed between groups in age (HRS: 60.2±3.0
years; sham: 57.1±5.8 years) or body mass index (HRS: 26.2±5.3 kg/m2; sham:
27.14±3.84 kg/m2). There were five subjects with type 2 diabetes (3 sham group, 2 HRS
group). 6 subjects were found to smoke and were evenly distributed across groups (3 in
each group). Using results of the IIEF-5 questionnaire, we found that HRS significantly
improved the symptoms of ED compared with the sham group, (19.58±4.60 vs.
11.33±2.01; p<0.01) (Figure 1). Three months later, it was found that IIEF-5 score
increased significantly after HRS treatment (pre: 9.75±3.84 vs. post; 19.58±4.60; p<0.05).
Notably, the score also increased in the sham group after 3-month treatment, but the
difference was not found to be statistically significant (pre: 9.41±3.57 vs. post:
As shown in Table 2, SOD activity was found significantly increased (pre: 65.55±22.25
vs. post: 121.80±21.44; p<0.01) and MDA concentration decreased dramatically (pre:
7.8±2.03 vs. post: 4.1±1.75; p<0.01) after hydrogen treatment. In the HRS group, SOD
activity increased significantly (121.80±21.44 vs. 68.64±12.28) and MDA concentration
(6.86±2.25 vs. 4.1±1.75; p<0.01) decreased dramatically compared with the sham group
The current study indicated that treatment with HRS combined with sildenafil
significantly improved the symptoms of ED and substantially attenuated oxidative stress
in sildenafil refractory patients. To our knowledge, this is the first reported clinical trial
demonstrating the effectiveness of HRS for treating low responders to a PDE-5 inhibitor.
Sildenafil is a PDE-5 inhibitors that acts on the smooth muscle of the corpus cavernosum
and can dramatically reduce the concentration of calcium ions in cytosol. This drug is
now considered first-line monotherapy for ED. However, a significant number of patients
have been identified as non-responders to sildenafil[13,14] . In light of our experience,
most sildenafil-refractory patients with non-organic ED respond well to adjunctive HRS.
Therefore, we believe that sildenafil refractory patients, prior to starting invasive
second-line treatment, should be offered adjunctive HRS. HRS enhanced the therapeutic
effect of the PDE5 inhibitor in our study, which supports our initial hypothesis that a
reactive oxygen scavenger may enhance PDE5 inhibitor efficacy by prolonging NO
half-life, as well as other potential physiologic effects. Reports of a relationship between
hydrogen in osteoblast and macrophage activity have been previously published[15,16].
Molecular hydrogen has a selective property for hydroxyl radical and can penetrate
biomembranes, making it a effective potential antioxidant. Detrimental hydroxyl
radicals and peroxynitrite can be selectively reduced by hydrogen, while steady-state
levels of nitric oxide cannot be decreased. Hydrogen also increases intracellular cyclic
AMP, and leads to relaxation of cavernosal smooth muscle cells. Cyclic AMP may also
have a positive feedback effect on the nitric oxide-cyclic GMP pathway, which increases
the concentration of cyclic GMP and leads to the clinical efficacy of sildenafil. Notably,
our results are in accordance with the finding that vitamin E enhances the efficacy of
PDE5 inhibitors. In addition, hydrogen increases eNOS protein expression in rat
corpus cavernosum and bone tissue, as described in our previous work. Thus, we
speculate that HRS may restore endothelial-derived NO vasodilation and erectile function
by increasing eNOS activity, cGMP levels, and corporal smooth muscle relaxation.
In the current study, we compared serum levels of MDA and SOD between experimental
groups. We found that MDA, one of the end products of lipid peroxidation, was
significantly decreased in the HRS group after 12-week treatment. MA El-Latif
found that high levels of MDA show a strong correlation with severity of ED in human
subjects. Data from the current study confirm previous findings of a relationship between
MDA and severity of ED. Another target, SOD, an antioxidant enzyme, has an important
role in protecting against free radicals by reducing ROS levels. We demonstrated that
SOD activity was significantly increased after HRS therapy. These findings are consistent
with those of Khan et al. On one hand, in diabetic patients, increased levels of superoxide
anion convert NO to peroxynitrite, which diverts NO away from the erectogenic pathway.
On the other hand, addition of superoxide dismutase strengthens NO-mediated muscle
relaxation. Clinical studies have revealed that consuming hydrogen-rich water
reduces oxidative stress markers in patients with diabetes mellitus, metabolic syndrome,
muscular disease, and acute erythematous skin diseases. As a supplement,
our study has addressed the role of HRS therapy in improving ED symptoms in patients
who are refractory to sildenafil. The results of the present study provide further evidence
for clinical application of HRS therapy in men with ED.
It should be noted that the size of our recruited sample is small and the findings
should be interpreted appropriately. Another limitation of our study is that the influence
of confounding factors may not be fully considered since we have only compared BMI
and age between groups while other factors such as smoking, alcohol addiction and
diabetes mellitus may affect erectile function. In addition, the results of the current study
may not be representative of the general population with ED, since the population we
recruited consists of low responders to sildenafil.
In conclusion, HRS reduces oxidative stress and improved symptoms of ED in men with
an unsatisfactory response to sildenafil therapy alone. Hydrogen may be a new
therapeutic option in the treatment of ED. Additional, long-term studies with larger
sample sizes are warranted to confirm the effectiveness of using a PDE-5 inhibitor
combined with HRS in the treatment of patients’ refractory to sildenafil therapy.
There are no competing interests to declare. The authors are alone responsible for the
content and writing of this paper.
FM, XLX, XJS and XZH conceived the study design, collected the clinical data from
patients, and drafted the manuscript. QFZ and JNY, performed the biochemical
experiments. JPL participated in collecting the clinical data from patients. XQ
participated in the interpretation of the data. FM performed the statistical analysis of the
data. All authors have read and approved the final version of the manuscript.
We are also grateful to patients for their important contribution to this study. This project
was financially support in part by the Medical Science and Technique Foundation of
Jiangsu Province, PR China (No. BL2012045). All authors have read the journal's policy
on conflicts of interest and have none to declare.
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Figure 1. Effects of HRS on IIEF-5 score in men with erectile dysfunction.
(IIEF-5=International Index of Erectile Function-5). *p<0.05 vs. hydrogen pre group.
vs. sham post group. # #p>0.05 vs. sham pre group.
Table 1. BMI and age between groups ( x ±s)
Table 2. Oxidative stress parameters before and after treatment between groups ( x ±s)
Note: ap<0.05 vs. hydrogen pre, bp<0.05 vs. sham