Objectives Erectile dysfunction (ED) affects up to 53.4% of men aged 30–80 years. In this study, we aimed to examine the association between homocysteine (HCY), vitamin B12 (B12), folic acid (FA) and ED.
Design Cross-sectional study.
Setting Guangxi, China.
Participants A total of 1381 participants who completed questionnaires were included, between September 2009 and December 2009.
Measures ED was evaluated by the International Index of Erectile Function scores. Also, the values of HCY, B12 and FA were acquired. Then, regression and between-group analyses were performed.
Results No association between FA and ED was found. Significant correlations between HCY and ED were found—the relationships between these two parameters were most notable in men aged over 60 years and in men living alone (bachelors or bachelorhood). B12 levels were higher in men with ED (718.53±234.37 pg/mL vs 688.74±229.68, p=0.015). Using multinomial logistic regression analyses, B12 levels were related to mild ED (multivariate adjusted analysis: OR 1.620, 95% CI 1.141 to 2.300, p=0.007), especially among men aged 40–49 years (OR 2.907, 95% CI 1.402 to 6.026, p=0.004).
Conclusions We report, for the first time, a relationship between B12 levels and ED. We also found specific cohorts of men for whom the relationship between HCY levels and ED is most prominent. Further studies are required to elucidate the mechanisms underlying these relationships—these may ultimately result in new therapies for ED.
- erectile dysfunction
- Vitamin B12
- folic acid
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Strengths and limitations of this study
Our study is a cross-sectional study, mainly based on the large Fangchenggang Area Male Health and Examination Survey project, including a total of 4303 men.
This study includes comprehensive analyses of baseline, linear and logistic regression, and multinomial logistic regression.
According to the changes in the homocysteine (HCY), vitamin B12 (B12) and folic acid (FA) levels, and the order of erectile dysfunction (ED) severity, we investigated the associations between HCY, B12, FA and ED.
The study also took into consideration the effects of age, marital status and educational status.
Nevertheless, as a cross-sectional analysis, the exact mechanisms of the relationship between HCY, B12, FA and ED cannot be clearly defined.
Erectile dysfunction (ED) is defined as the inability to acquire and maintain satisfying sexual intercourse with a sufficient erection, affecting up to 53.4% of men aged 30 to 80 years.1 The morbidity increases sharply among men over 40 years of age.2 3 It has been estimated that the prevalence of ED will reach 322 million worldwide by the year 2025.4
Various factors including smoking, hypertension and hyperlipidaemia have been identified to influence the development of ED. Among these factors, the vascular component is dominant.5 6 Moreover, ED may be one of the indicators of cardiovascular disease (CVD).7 Homocysteine (HCY), a CVD-associated factor, was recently defined as an independent risk factor for ED.8 9 HCY is a thiol-containing amino acid, mainly from methionine, with two steps of transformation. First, methionine is catalysed to form S-adenosylmethionine (SAM) by the enzyme SAM synthase. As a major methyl group donor for various methylation reactions, SAM is mainly transformed into S-adenosylhomocysteine (SAH) after loss of the methyl group. In the second step, SAH is hydrolysed by SAH hydrolase to form HCY and adenosine. Biologically, HCY is involved in two pathways, including remethylation (RM) and transsulfuration (TS). In the RM pathway, HCY regenerates methionine by methylenetetrahydrofolate reductase with cofactors of folic acid (FA) and vitamin B12 (B12). In the TS pathway, HCY is catalysed by the cystathione-β-synthase and γ-cystathionase.10 11
FA and B12, as the cofactors of HCY, have also been identified to be associated with ED.12 However, limited studies have been focused on the relevance of their levels to ED. On the basis of previous studies, we hypothesised that there are likely associations between HCY, B12, FA and ED. In order to comprehensively investigate the exact association between HCY, B12, FA and ED, our study is conducted based on the Fangchenggang Area Male Health and Examination Survey (FAMHES) project. Our study may pave the way to the treatment of ED on the basis of the balance among HCY, B12 and FA.
Methods and materials
Population and data collection
FAMHES is a population-based project, which was mainly performed to investigate environmental and genetic factors, as well as their inter-relations. From September 2009 to December 2009, 4303 men coming for routine physical examination at the Medical Center in Fangchenggang First People’s Hospital were enrolled. Then, 3593 participants responded for further interviews (response rate=83.5%).13 No distinct differences were detected between the men who participated in the interviews and those who did not.
All participants were asked to provide blood samples between 8:00 and 11:00, after fasting for at least 8 hours (overnight). Then, these blood samples were transported to the Department of Clinical Laboratory at the First Affiliated Hospital of Guangxi Medical University in Nanning within 2–3 hours, where they were centrifuged within 15–25 min and stored at −80°C. Serum B12 and FA were detected with electrochemiluminescence immunoassays, while serum HCY was measured with enzymatic cycling methods.
Then, all the participants were invited to complete a comprehensive questionnaire. This process was performed by the trained investigators using a standardised protocol with a face-to-face interview. Essential information (eg, age, sex, smoking and drinking) was collected, and complete physical examinations (eg, height, weight, waistline and hipline) were performed. Smoking status and alcohol consumption were defined as Yes or No. Marital status was classified into living together (married or cohabitation without marriage) and alone (bachelors or bachelorhood). Meanwhile, according to the years of education, three groups could be defined (0–6 years, primary education; 7–12 years, intermediate education; and ≥13 years, superior education). In the physical examination, body weight with thin clothing and height without shoes were measured. Then, body mass index (BMI) was calculated with the formula of weight/(height)2. Waist circumference was measured at the midpoint between the inferior costal margin and the superior iliac crest in the midaxillary line. The hipline was defined as the maximum circumference over the buttocks. The waist:hip ratio (WHR) was calculated as waist circumference/hipline. These processes above including initial examination (including height, weight, waistline and hipline), further interviews (essential information, such as age, sex, smoking and drinking, etc) and blood collection were performed on the same days coherently. The flow of participants’ collection is shown in online supplementary figure S1.
Patient and public involvement
Patients and the public were not involved in the development of the research question and design or recruitment of this study.
ED definition and grouping
In this study, the International Index of Erectile Function (IIEF-5) was applied to define ED.14 The IIEF-5 system has five questions, which mainly cover the conditions of erection confidence, erection firmness, maintenance ability, maintenance frequency and satisfaction, with the scores ranging from 5 to 25. Each question has six selections. According to the orders of answers, the scores are defined as 0–5. Then, participants can be divided into ED (IIEF-5 ≤21) and non-ED (IIEF-5 >21) groups. According to the symptoms, ED can also be classified into five groups: none (IIEF-5 score 22–25), mild (17–21), moderate (12–16) and severe symptoms (5–11).13 15 In addition, HCY level can also be divided into normal (HCY 5–15 µmol/L) and hyperhomocysteinaemia (HCY >15 µmol/L).16
In order to acquire the eligible participants for this study, we developed rigorous exclusion criteria: (1) incomplete data for the individual information and IIEF-5 score; (2) incomplete data for HCY, B12 and FA or refused to provide the blood samples; (3) with diseases such as CVDs, inflammatory/immune diseases and kinds of cancers, which might influence the levels of HCY, B12 and FA (online supplementary table S1); and (4) currently taking drugs that might affect HCY, B12 and FA levels, such as vitamins, antidiabetic medicines, non-steroidal anti-inflammatory drugs, antibiotics, cimetidine or glucocorticoids (online supplementary table S1). Then, 1381 participants were included for further analyses. The flow for screening the eligible participants is shown in online supplementary figure S2.
Before analysis, HCY, B12 and FA levels were tested for Gaussian distribution with the Shapiro-Wilks test. If data were not Gaussian in distribution, they were logarithmically transformed in order to ensure the approximate Gaussian distribution. Based on the 22 IIEF-5 scores, two groups were defined (ED and non-ED), and Student’s t-test and χ2 test were applied in the baseline analysis. Then, linear and logistic regression analyses were used, with the IIEF-5 scores and binary variable (ED or non-ED) as the dependent factors, respectively. Three adjusted models were used: unadjusted, age-adjusted and multivariate adjusted. In the multivariate-adjusted model, the covariates were as follows: age, smoking status, alcohol consumption, BMI and WHR. Among them, BMI and WHR are the indexes applied to estimate obesity. However, BMI tends to evaluate body fatness but has a weak ability to differentiate fatness as central or visceral.17 Alternatively, WHR is said to be more effective in reflecting the visceral fat and central adiposity but is not suitable for an estimation of body fat.17 18 Additionally, the predictive effects of BMI and WHR in diseases are different.19 20 So, in our study, these two obesity indexes were treated as the covariates.
Then, multinomial logistic regression analysis was also performed to discover the potential association between HCY, B12, FA and ED, along with the order of severity of ED or the changes in the HCY, B12 and FA levels quartile (Q1<25%, 25%≤Q2≤50%, 50%<Q3≤75%, Q4>75%). Additionally, considering the non-negligible influences of age on the risk of ED, we also grouped the participants on the basis of age (<40, 40–49, 50–59 and ≥60 years). The Bernoulli correction was applied, with the significant threshold of p value <0.0125 (=0.05/4 tests) for multinomial logistic regression analysis. Additionally, according to the groups of marital status and educational status, logistic regression analyses were also conducted. In these analyses, the missing data were deleted. All statistical tests were two-tailed, which were performed with SPSS V.16.0 software. The threshold for significance was p value <0.05.
In the baseline analysis, based on IIEF-5, the ED and non-ED groups were defined. In line with previous studies, the age of the ED group (37.99±10.75 years) was older than the non-ED group (34.18±8.47 years, p<0.001). Meanwhile, B12 levels were significantly higher in the ED group (p=0.015). However, no significant difference was shown for HCY levels, and the proportion of hyperhomocysteinaemia was higher in the ED group (43.02%) than that in the non-ED group (37.52%, p=0.037). In addition, the proportion of alcohol consumption (p=0.032) and educational status (p<0.001) were also identified to have a statistically significant difference in the two groups (table 1).
Signal for the association between HCY and ED
While we discovered no significant association between HCY levels and ED in the comprehensive analyses (tables 2–5), a slight association of HCY with ED was observed in the participants grouped by age, especially in the old men (age ≥60) (online supplementary table S2). Similar relevance was confirmed in marital status (alone, unadjusted severe ED: OR 4.385, 95% CI 1.070 to 17.974, p=0.040; age-adjusted severe ED: OR 5.085, 95% CI 1.195 to 21.636, p=0.028) (online supplementary table S3).
In the latter analysis, HCY was divided into normal (HCY 5–15 µmol/L) and hyperhomocysteinaemia (HCY >15 µmol/L). The significant association between HCY and ED seemed to be more prominent in men living alone (age-adjusted severe ED: OR 2.448, 95% CI 1.046 to 5.733, p=0.039) (online supplementary table S3).
B12 level is significantly associated with ED
To investigate the association between ED and B12, we applied linear and logistic regression analyses, resulting in no significant association for B12 in the linear regression analysis (in which IIEF-5 scores were treated as the dependent factor). For the binary logistic regression (the status of ED evaluated by IIEF-5 was treated as the dependent factor), B12 was identified to be associated with ED in the unadjusted model (OR 1.438, 95% CI 1.070 to 1.933, p=0.016). However, the association signal diminished in other adjusted models (table 3). We next investigated the relationship between B12 and ED based on the severity grades of ED. Interestingly, the positive correlation between B12 and ED was further confirmed, especially among men with mild ED (unadjusted: OR 1.694, 95% CI 1.207 to 2.376, p=0.002; age-adjusted: OR 1.596, 95% CI 1.135 to 2.244, p=0.007; multivariate adjusted: OR 1.620, 95% CI 1.141 to 2.300, p=0.007) (table 4). Subsequently, the levels of B12 were divided into quartiles. The result showed that B12 might be significantly associated with ED, especially at the higher levels (unadjusted: Q2: OR 0.917, p=0.569; Q3: OR 0.988, p=0.939; Q4: OR 1.452, p=0.015; and p for trend <0.001) (table 5).
After adjusting age for the above analyses, the significant association between B12 and ED diminished (tables 2 and 3 and table 5), suggesting that age cannot be excluded while investigating the relationship between B12 and ED. We thus grouped the participants into four age groups (ages <40, 40–49, 50–59 and ≥60 years). Our results showed that the significant correlations between B12 and mild ED (IIEF-5=17–21) mainly presented in the age group 40–49 years (OR 2.907, 95% CI 1.402 to 6.026, p=0.004) (online supplementary table S2).
Our baseline analysis discovered different proportions of educational status in the ED and non-ED groups. In order to discuss the influences of marital and educational status in the relevance to ED and B12, we further performed between-group analyses. Similar to previous results, B12 was also identified to be associated with mild ED, even after multivariate adjustment (marital status, living together: OR 1.501, 95% CI 1.035 to 2.175, p=0.032; alone: OR 3.449, 95% CI 1.113 to 10.692, p=0.032; and educational status, intermediate: OR 1.858, 95% CI 1.214 to 2.845, p=0.004) (online supplementary table S3).
ED is a common disorder affecting a large number of men.1–4 Recent studies suggest HCY may be an independent risk factor for ED.8 9 In order to test this association, we conducted the current study based on the larger population-based FAMHES project. We confirmed that HCY is significantly associated with ED, especially severe ED. Moreover, B12 may also be relevant to mild ED. In contrast, we observed no significant association between FA and ED in our study.
HCY was reported to be associated with many diseases and health conditions, such as psychological disorders,21 22 lipid profiles,23 renal impairment24 and inflammatory/immune factors.25 Moreover, HCY is also identified to be a useful marker for CVD.26 27 Meanwhile, ED could be a potentially predictive factor for cardiovascular and other chronic diseases.28 Based on the relevance, it was assumed that HCY might be a risk factor for ED.8 9 Consistent with this, we revealed that HCY was significantly associated with ED, especially severe ED. The main mechanism might be that HCY could influence endothelial dysfunction and nitric oxide (NO) diffusion. Previously, in vitro and in vivo studies discovered that HCY could be a toxin for the vasculature by inducing endothelial dysfunction.29 Additionally, NO is mainly involved in vascular dilation, smooth muscle relaxation (including genitourinary smooth muscle) and permitting penile erection.30 31 Studies showed that increased HCY could inhibit NO synthase, probably influencing the production of NO, and the development of ED.32 So, on the basis of these relevance, we could understand the risk effect of HCY on ED. Additionally, the status of living alone for men would also influence this association, hinting the pathogenesis of psychological factors for ED.
B12 is also known as cobalamin. Similar to FA, it is an important cofactor in methionine synthesis and HCY metabolism.33 Although previous studies identified that FA might be a potential protective factor for ED,34 no significant association has been detected. In contrast to B12, HCY has been found to protect against ED.35 Our study also identifies the potential association between B12 and ED, though ED tends to have high levels of B12 (ED: 718.53±234.37, non-ED: 688.74±229.68, p=0.015). Meanwhile, the significant association between B12 and ED was more prominent for mild ED at higher B12 levels. There are two possible explanations. First, our results suggest that the function of B12 in ED might be dose dependent. Excessive B12 levels would increase the risk of mild ED with some unclear mechanisms. Second, increased B12 might provide negative feedback for this disease. At the beginning of the disease, defence mechanisms are triggered. As a potential protective factor, the absorption of B12 is enhanced. Combining the limited reports, our study can also propose that B12 is significantly associated with ED. As for the exact effects of B12 on ED, further studies are needed, which might pave the way for the treatment of ED with B12 in the future.
Our study verified the previous conclusions that HCY could increase the risk of ED. However, some limitations still need to be noted: (1) this study is a cross-sectional analysis, which just reflects the status of specific time points and populations; (2) there are limited numbers of participants with primary educational status. So, the results need to be examined further; (3) although we have identified a significant association between B12 and ED, the exact mechanisms and effects were unclear until now; and (4) after multiple testing, no positive association can be detected, suggesting that our results might be unstable. So, further studies will be needed.
ED is one of the most common male diseases. This study was conducted in order to discover the functions of HCY, B12 and FA in ED. Our results confirmed the positive correlations of HCY and ED. Meanwhile, B12 was also likely to be significantly associated with ED. Further studies with larger cohorts of participants should be focused on the potential mechanisms and therapeutic effects of B12 on ED.
YC and JL contributed equally.
Contributors YC, JieL, ZM and JC participated in participants’ collection, field investigation, design, writing and modification of all the paper. YC and JieL took part in the statistical analysis. ZM and JC provided important advice for this paper. TL, JiaL, JinL and G-HW provide efforts in the processes of modification.
Funding This study was funded by National Natural Science Foundation of China (81770759, 81370857), and Innovation Project of Guangxi Graduate Education (YCBZ2017037) .
Competing interests None declared.
Ethics approval This study was approved by the Medical Ethics Committee of Guangxi Medical University.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement The data for this study are available in the supplementary materials. Further questions could be sent to ZM (email@example.com) and JC (firstname.lastname@example.org).
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