The study protocol conforms to the principles of the Declaration of Helsinki and was approved by the Institutional Ethical Committee of Maulana Azad Medical College and associated Lok Nayak Hospital, New Delhi, India. Written informed consent was obtained from all subjects prior to the participation. COPD patients attending the chest clinic and Medicine Outpatient department were recruited from August 2011 to February 2013, whereas asymptomatic healthy controls, irrespective of smoking habits, were among the patient’s attendants visiting the hospital. Asymptomatic controls were defined as persons over the age of 40 years without any known medical illness and who were symptom free.

Lung function tests were performed in all subjects using standardized methods according to the American Thoracic Society guidelines [21,22]. All study participants were examined by a study physician who performed a structured interview of the medical history including exacerbations, co-morbidities, smoking and medication use. Clinical diagnosis of COPD and inclusion criteria were: (a) post-bronchodilator FEV1/FVC<70%; (b) reversibility of FVC or FEV1, induced by β-agonist (200mg salbutamol) <12% or 200 ml; (c) no exacerbation within the last 12 weeks before recruitment. Patients were excluded if they had a diagnosis or history of asthma and conditions known to affect serum levels of Hcy, including coronary heart disease and metabolic syndrome.

Patients were subjected to complete history and physical exami-nation and particulars of the patients such as name, age, sex, pack years of smoking etc., were noted in a pre-structured performa. Baseline spirometry, chest X-ray and ECG were done. A series of hematological and biochemical investigations i.e., hemogram, liver function test, kidney function test, fasting blood sugar and lipid profile were carried out. Anthropometric measurements (height, weight) were also done to calculate Body Mass Index (BMI). All patients were given folic acid therapy (5mg daily) for duration of six weeks. Patients were followed up and repeat plasma Hcy and FEV1 were measured after six week of folic acid supplementation [Table/Fig-1].

[Table/Fig-1]:

Study flow chart.

Blood Sampling: Subjects were asked to fast overnight from 21:00 the night before, after which venous blood samples were collected and serum was separated immediately and stored at -20°C until analysis. Levels of plasma Hcy were analyzed using an ELISA [(The Axis^® Homocysteine Enzyme Immunoassay (EIA) kit 9 (United Kingdom)] in the Department of Biochemistry, Maulana Azad Medical College, New Delhi, India. All the measurements were carried out strictly according to the manufacturer’s instructions.

All statistical analyses were performed by SPSS 18.0 for Windows (IBM, Chicago, IL). Variables with skewed distributions were log transformed before further analysis. All continuous data were presented as mean ± standard deviation (SD), whereas categorical data were presented as frequency and percent. Comparisons of characteristics between groups were performed by unpaired Student’s t or Chi-square tests when appropriate. Pearson’s correlation adjusting for age, BMI, pack-years smoked, and current smoking status was used to analyze the correlations between homocysteine and lung function. The level of significance was set as p<0.05.

Patient’s Characteristics at the baseline: A total of 50 COPD patients and 30 healthy controls were enrolled in this study. All the demographic data and baseline characteristics of the study subjects are summarized in [Table/Fig-2]. Out of 50 COPD patients 86% were male while 14% were female, whereas in control group male and female were 43.3% and 56.7% respectively. The mean BMI of the COPD patients was 21.3±4.26 with range from 16 to 29.8. More than 80% of patients had BMI <25kg/m² at the time of enrollment. There were no differences with respect to the age and BMI between the COPD patients and the controls. Among 50 COPD patients 76% were smokers while 24% non-smokers. There were significant reductions in FEV1% predicted and FEV1/FVC ratio compared with healthy controls. Meanwhile, plasma level of tHcy was significantly elevated in COPD patients compared with healthy controls (27.42±23.89 vs. 15.21±15.71, p<0.001) [Table/Fig-2].

In COPD group 16% had normal Hcy level while, 68% moderate, 14% intermediate, and 2% had severe hyperhomocysteinemia. On the other hand among the control group 60% had normal Hcy levels and 36.6% moderate, 3.34 %intermediate, none had severe hyperhomocysteinemia. Those patients who were smokers had higher homocysteine levels with mean 28.3±26.7μmol/L as compared to non-smokers 24.6±11.17μmol/L (p-value=0.64). Also, the mean FEV1 was lower (0.99±0.48) as compared to non- smokers (1.06±0.43) (p-value=0.66).

Homocysteine, lung function and BMI after six weeks of folic acid supplementation: In our study folic acid treatment did not have significant affect on pulmonary function parameters in COPD patients. The mean FEV1 value before and after supplementation were 1.14±0.60 and 1.08±0.56 respectively with p-value=0.534. The difference was statistically non-significant. On the other hand, the mean Hcy values decreased significantly from 27.42±23.89μmol/L to 15.2±15.71μmol/L after folic acid supplementation respectively (p-value<0.001) [Table/Fig-3].

At the baseline and after six weeks of folic supplementation Hcy exhibited a weak and non-significant correlation with FEV1 (r=0.125, p=0.43 and r=0.188 p=0.23, respectively), and BMI also weakly correlated with FEV1 at the baseline and after folic acid supplementation (r=0.073, p=0.64 and r=0.085, p=0.56) in crude analyses. The correlation was non-significant [Table/Fig-4]. However, a significant correlation was observed between BMI and Hcy at the baseline and after six weeks of folic acid supplementation (r=0.283, p=0.04 and r=0.340, p=0.02 respectively). We also found a strong positive and significant correlation between plasma Hcy levels at the baseline and after six weeks of folic acid supplementation (r=0.840 p=0.001) [Table/Fig-4,5].

[Table/Fig-5]:

Total homocysteine levels at the baseline and after six weeks of folic acid supplementation in the COPD group.

A significant reduction was observed in plasma tHcy levels after six weeks of folic acid supplementation (p<0.001).

The possible pathogenic mechanism of elevated concentrations of Hcy in lung disease remains unclear. Elevated concentrations of Hcy and decreased concentrations of folate may have other pro-inflammatory properties in the COPD patients but their amelioration through folic acid supplementation is likely to influence concentrations of Hcy in such patients.

The possible pathogenic mechanism remains unsolved and convincing evidence to support a causal relationship between elevated concentrations of Hcy and risk of developing COPD is absent. In this study, we observed a marked reduction of elevated Hcy concentrations through daily folic acid supplementation for six weeks duration however; it did not affect the pulmonary function parameters in COPD patients. Our data strongly suggests that hyperhomocysteinemia may result from poor folic acid status in COPD patients which are recognized co-factors in tHcy degradation [5]. To the best of our knowledge this was the first study which looked into the status of folic acid supplementation and its effect on hyperhomocysteinemia and severity of disease in COPD patients in Indian population.

In our study mean baseline values of tHcy were highly variable with range from 9 to 150. The mean value of Hcy in COPD patients was 27.42±23.89μmol/L as compared to control which had a mean of 15.21±15.71 μmol/L (p-value=0.001). Anderson et al., investigated the difference in plasma Hcy in 19 patients with COPD and in 29 healthy subjects [15]. They assessed the influence of pro-oxidant activity on the metabolism of Hcy and found a higher level of tHcy in COPD patients than controls. They observed a decreased concentration of reduced glutathione and a decreased ratio of reduced/total glutathione in COPD patients compared to the healthy individuals. They speculated a possible effect of extracellular pro-oxidant activity on the concentration of total plasma Hcy in patients with COPD. Also, previous studies done by Kai S, et al., (12μmole/L vs. 9.8μmole/ L), Seemungal et al., (12μmole/L vs. 8.2μmole/L) and Fimognari et al., (13.9μmole/L vs. not available) had reported plasma tHcy to be higher in COPD patients than in controls [8,16,17]. The Hcy levels in the COPD patients (27.4±23.85μmol/L) were higher in our study as compared to the above mentioned studies. Also, all of the above studies were relatively small, cross-sectional and done on COPD outpatients and involved a control arm of asymptomatic subjects.

In our study, 82% patients had moderate to intermediate while 2% patients had severe hyperhomocysteinemia among the COPD group as compared to control who had 39.94% (moderate to severe). The Hcy levels were more elevated in COPD stage III-IV (84%) as compared to stage I-II (16%). Similar to our findings, Seemungal et al., also found tHcy was elevated in male COPD patients when compared with female COPD patients, subjects with low FEV1/FVC ratio and low FEV1 % predicted [16]. This study also showed tHcy was higher in patients with GOLD stages III/IV disease as compared with Stages I/II suggesting a role in COPD pathogenesis.

In the current study, we observed a significant decrease in the mean Hcy values from 27.42±23.89 to 15.21±15.71/ L after six weeks folic acid supplementation (p-value<0.001). In cases, relative percent improvement in tHcy among smokers was 39.4±42.5μmol/ L as compared to non-smokers 43.9±36.12μmol/ L (p-value=0.77) after intervention. The mean FEV1 value before and after intervention were 1.14±0.60 and 1.08±0.56 respectively with a non-significant p-value of 0.654. The relative percent improvement in FEV1 in those who were smokers had a mean value of 11.8±33.94 as compared to non-smoker 17.9±26.7 with p-value 0.618 (non-significant). In our study, the mean plasma Hcy declined following six weeks supplementation of folic acid but it was not found to be associated with severity of disease as measured by FEV1. The failure to achieve improvement in FEV1 in our study may be due to the shorter period of supplementation (Six weeks).

Folic acid is the most effective vitamin B to reduce elevated concentrations of Hcy, being responsible for a reduction of approximately 25%. All the previously done studies have found that low folic acid is related to hyperhomocysteinemia [23,24]. A recent case-control study was conducted in central Japan by Fumi Hirayama et al., [25] found that an inverse association was evident between dietary folate intake and the prevalence of breathlessness for adults, together with a significant dose response-relationship for the COPD risk. Moreover, they speculated that increased folate intake might be beneficial to lung function. However, in our study, we did not find any significant change in the lung function. The potential reason which explains the poor folic acid status in COPD patients is unclear. However it is well recognized that in COPD nutritional abnormalities especially of vitamin B group, alternation in caloric intake and decreased BMI complicates the disease [26]. Hence, our results which showed significant decrease in tHcy levels in COPD patients following folic acid supplementation may reflect the underlying micro-nutritional deficiencies that lead to hyperhomocysteinemia in majority of COPD patients. However, whether a dietary folate approach or supplemental folic acid may determine the success of lowering Hcy concentrations still remains unclear.

Our study has certain limitations. First being sample size which is relatively small therefore the study may have lacked statistical power. Second, we did not measure the baseline folic acid status in cases and controls and thirdly patients were on standard treatment of COPD, therefore many patients were receiving inhaled steroids and their effect on plasma homocysteine levels is not well understood.

Homocysteine is a ubiquitous amino acid, elevation of which is associated with several diseases as diverse as thrombotic disorders and lung diseases. A strong link between cardiac disease and Hcy levels is already established. The cause and effects of Hcy elevation in COPD are unknown but studies so far available suggest that Hcy is related to COPD pathogenesis and is likely to be associated with alterations in the redox pathway leading to oxidative stress in COPD. However, for now, COPD patients with an elevated Hcy should be screened for cardiac disease and more closely monitored for evidence of a faster decline in lung function. The usage of folate supplements to improve lung function and treatment of COPD and breathlessness symptom should also be investigated in future clinical studies in different population. Possible protective effect of folic acid, director via lowering of Hcy concentrations, may be due to a decrease in inflammation. Lowering of homocysteine concentrations may affect other inflammatory markers, which may affect other pathways leading to COPD. Further prospective cohort studies are recommended to confirm the observed protective effects in other populations and to determine whether increased intakes can improve the mortality rate due to COPD.