Metformin Treatment and Homocysteine: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

The aim of this systematic review is to assess whether metformin could change the concentration of serum homocysteine (Hcy) with and without simultaneous supplementation of B-group vitamins or folic acid. A literature search was conducted in PubMed, EmBase, and Cochrane Central Register of Controlled Trials (CENTRAL) to identify randomized controlled trials (RCTs) reporting the concentration of serum Hcy in metformin-treated adults. Meta-analysis was applied to assess the association between metformin and the changes of Hcy concentration. Twelve publications were included in this study. In the overall analysis, metformin administration was not statistically associated with the change of Hcy when compared with the control treatment (mean difference (MD), 0.40 μmol/L; 95% confidence interval (CI), −0.07~0.87 μmol/L, p = 0.10). In the subgroup analysis, metformin was significantly associated with an increased concentration of Hcy in the absence of exogenous supplementation of folic acid or B-group vitamins (MD, 2.02 μmol/L; 95% CI, 1.37~2.67 μmol/L, p < 0.00001), but with a decreased concentration of serum Hcy in the presence of these exogenous supplementations (MD, −0.74 μmol/L; 95% CI, −1.19~−0.30 μmol/L, p = 0.001). Therefore, although the overall effect of metformin on the concentration of serum Hcy was neutral, our results suggested that metformin could increase the concentration of Hcy when exogenous B-group vitamins or folic acid supplementation was not given.


Introduction
Metformin, a first-line drug for type 2 diabetes mellitus (T2DM) recommended by most guidelines of diabetes, is also widely used in patients with polycystic ovary syndrome (PCOS), pre-diabetes, and other diseases involving insulin resistance [1,2]. However, Vitamin B 12 deficiency was noted to be a potential disadvantage of metformin by the latest American Diabetes Association (ADA) guidelines [1].
A previous meta-analysis demonstrated that metformin treatment was associated with a decreased concentration of serum Vitamin B 12 in a dose-dependent manner [3].
Homocysteine (Hcy) is a key component in the one-carbon pathway of methionine metabolism, which plays a dominant role in DNA methylation. The accumulation of Hcy, known as hyperhomocysteinemia (HHcy), is often resulted from Vitamin B 12 deficiency [4], and is associated with an increased risk of cardiovascular diseases, cognitive impairment, cancer, chronic renal failure and other chronic diseases [4][5][6][7][8][9][10][11]. However, no consensus was reached on whether metformin could induce Hcy elevation. This systematic review aimed to assess the association between and secondary outcomes of each included study and strategies of intervention were summarized in Supplementary Materials Tables S3 and S4.  resistance. * These two analyses were short-term and long-term outcomes of the same trial, respectively. The longterm follow-up data (de Jager, 2010 [28]) were included in the quantitative analysis.  I/C, intervention/control group; BMI, body mass index; CHD, coronary heart disease; PCOS, polycystic ovary syndrome; T2DM, type 2 diabetes mellitus; IGT, impaired glucose tolerance; NA, not available; IR, insulin resistance. * These two analyses were short-term and long-term outcomes of the same trial, respectively. The long-term follow-up data (de Jager, 2010 [28]) were included in the quantitative analysis.  HPLC, High pressure liquid chromatography; NS, Not significant; CLI, Chemiluminescence immunoassay; FPI, Fluorescence polarization immunoassay. 1 The average daily intake of metformin was 1707 mg at week 4, 1759 mg at week 12, and 1741 mg at week 40; 2 This article included two independent RCTs; 3 These two analyses were short-term and long-term outcomes of the same trial, respectively. The long-term follow-up data (de Jager, 2010 [28]) were included in the quantitative analysis; 4 Each patient in this group was given his or her maximally tolerated daily dose (one, two, or three tablets of 850 mg) during the trial. The actual mean dose in the metformin-treated group was 2050 mg/day; 5,6 These are 2 × 2 factorial designed trials with four treatment arms in each trial; 7 This study did not report the exact duration of follow-up; 8 The average daily intake of metformin was 2000 mg, and that of repaglinide was 3 mg; 9 The average daily intake of metformin was 2500 mg, and that of glimepiride was 3 mg; 10 Each patient in this group was given his or her maximally tolerated daily dose during the trial (15 mg/day in six patients, 30 mg/day in twelve patients, and 45 mg/day in one patient).
Potential publication bias was suspected from the funnel graph analyses, which was presented in Supplementary Materials Figure S4.

Metformin and Homocysteine
The results of the overall analysis showed that metformin did not have a statistically
Potential publication bias was suspected from the funnel graph analyses, which was presented in Supplementary Materials Figure S4.

Metformin and Homocysteine
The results of the overall analysis showed that metformin did not have a statistically  Subgroup analyses were conducted according to gender, disease type, dosage of metformin, background treatment, pre-study treatment, control treatment, duration of follow-up, change of Vitamin B12 concentration, and assay method of Hcy. Detailed results were presented in Supplementary Materials Table S6. It was demonstrated that the administration of metformin was associated with a significant reduction of serum Hcy among young female patients with PCOS. In addition, subgroup analysis based on the dosage of metformin showed that higher dosages of Subgroup analyses were conducted according to gender, disease type, dosage of metformin, background treatment, pre-study treatment, control treatment, duration of follow-up, change of Vitamin B 12 concentration, and assay method of Hcy. Detailed results were presented in Supplementary Materials Table S6. It was demonstrated that the administration of metformin was associated with a significant reduction of serum Hcy among young female patients with PCOS. In addition, subgroup analysis based on the dosage of metformin showed that higher dosages of metformin (≥2000 mg daily) were associated with an elevation of serum Hcy, when compared with dosages less than 2000 mg daily (MD, 1.07 µmol/L; 95% CI, −0.17~2.30 µmol/L, p = 0.09).

Adverse Events
Five studies reported that more patients in the metformin group suffered gastrointestinal side effects when compared with those in the control [27,28,30,33,35]. Also, five papers did not provide any information about adverse events [26,29,31,32,34]. No death was reported. Details were shown in Supplementary Materials Table S7.

Discussion
Our study did not find significant association between metformin treatment and the change of serum Hcy concentration in the overall population. However, the subgroup analyses noted that metformin administration was associated with elevation of Hcy in the patients without supplementation of folic acid or B-group vitamins, which indicated that metformin might induce HHcy in the absence of exogenous folic acid or B-group vitamins supplementation.
Hcy is a sulfur amino acid with a free sulfhydryl group as the final metabolite of methionine and Vitamin B 12 serves as a cofactor in the degeneration of Hcy to methionine. The insufficiency in Vitamin B 12 results in the accumulation of Hcy, which is known as HHcy. HHcy is a well-established risk factor for cardiovascular diseases, cognitive impairment, and chronic renal failure [4][5][6][7][8][9], moreover, Hcy has been found to be an independent predictor of all-cause and vascular mortality [38,39]. Metformin has been demonstrated to be associated with reduction of serum Vitamin B 12 concentration [3,40]. It has been shown that metformin could induce Vitamin B 12 malabsorption by enhancing bacterial overgrowth, altering bacterial flora in enteric canal, and binding to the Vitamin B 12 -intrinsic factor (IF). This malabsorption ultimately leads to a reduction of serum Vitamin B 12 [41][42][43][44][45]. Hence, some researchers were calling attention to the monitoring of Vitamin B 12 concentration in the diabetic patients treated with metformin, and suggested Vitamin B 12 supplementation could be considered in patients with Vitamin B 12 deficiency [3,46,47], although some authors still doubt the clinical significance of this reduction [48,49]. A recent clinical trial suggested that the metformin-associated reduction of the serum Vitamin B 12 was due to the increased transportation and utility of Vitamin B 12 by cells stimulated by metformin [50]. One of our subgroup analyses showed that metformin raised serum Hcy in the patients without folic acid or Vitamin B 12 supplementation, but reduced Hcy when folic acid or Vitamin B 12 was supplemented, indicating that metformin-associated Vitamin B 12 reduction might be responsible for Hcy elevation, and exogenous folic acid and Vitamin B 12 may rescue the methionine metabolic disturbance in metformin-treated patients. Considering Hcy as an important biomarker of a series of diseases and the few adverse effects of folic acid and Vitamin B 12 , exogenous supplementation of these two vitamins could be necessary for metformin-treated patients, which is consistent with the recommendation of regular Vitamin B 12 supplementation in the current American Association of Clinical Endocrinologists (AACE) guideline [51]. However, this recommendation had not yet been supported by well-designed randomized trials.
Our subgroup analyses also demonstrated that, the administration of metformin might cause a significant reduction of serum Hcy in young women with PCOS. However, it must be noted that, most of the young women enrolled received exogenous folic acid or B-group vitamins supplementation, and hence the observed Hcy reduction might be partially caused by the effects of exogenous folic acid or B-group vitamins. Meanwhile, estrogen, progestin, and age may also have some effects on the concentration of Hcy [5,8]. Experimental studies are required to further explain this difference.
The increase of serum Hcy concentration in the metformin-treated patients was confirmed by a series of observational studies [16][17][18]20,25,[52][53][54][55][56]. These studies indicated that metformin was associated with an elevated concentration of serum Hcy compared with control treatment. Moreover, in Yilmaz's trial [20], where all the included patients were young women with PCOS but without Vitamin B 12 or folic acid supplementation, Hcy was found to be elevated, while Vitamin B 12 was reduced in the metformin-treated patients. In addition, Carlsen and colleagues [27] noticed that Hcy was reduced in pregnant women but not in infertile women. No explanation has been established currently, but further investigations on the pseudo reduction of Vitamin B 12 during pregnancy and the effect of estrogen and progestin on the concentration of Hcy might help us better understand the underlying mechanism. In Carlsen's and Kilic's trials [27,29], all participants received exogenous folic acid or B-group vitamins and, interestingly, metformin-treated patients had a lower concentration of Hcy compared with controls. A possible explanation was that exogenous folic acid or B-group vitamins might counteract the reduction of the Vitamin B 12 absorption caused by metformin [46]. In Schachter's trial [32], no matter whether the patients were treated with metformin or not, the reduction of Hcy in the patients receiving both Vitamin B 12 and folic acid was greater than that in the patients receiving folic acid only (metformin-treated: −0.18 versus −0.12 µmol/L; metformin-untreated: −0.32 versus −0.07 µmol/L). It indicated that Vitamin B 12 was critical in reducing serum Hcy, which could be explained by the vital role of Vitamin B 12 in the metabolism of methionine [5].
Our study has several limitations. Firstly, the heterogeneity among the included studies was significant. Although subgroup analyses were conducted to explore possible sources of heterogeneity, factors such as weight, age, gender, and race might still influence the results of our study. Particularly, the dosage and the follow-up duration of included studies varied largely, although subgroup analyses did not find significant effects of these factors on the results. Secondly, in our subgroup analysis concerning exogenous B-group vitamins or folic acid supplementation, most patients receiving exogenous B-group vitamins or folic acid were diagnosed with PCOS or infertility, which could induce some potential biases. Further studies are required to demonstrate the interaction between metformin and B-group vitamins in patients with PCOS or infertility. Thirdly, long-term outcomes such as mortality and cardiovascular events were not studied in our analysis. Finally, the strength of the pooled results was restricted by the generally high risk of bias of included studies.

Conclusions
Although there is no significant change of the concentration serum Hcy between metformin-treated and non-biguanide-treated patients in the overall pooled analysis, our subgroup analysis suggested that metformin may induce an elevation of serum Hcy concentration in the absence of B-group vitamins or folic acid supplementation. Nevertheless, given the supplementation of B-group vitamins or folic acid, metformin could even be associated with reduced concentration of serum Hcy. Since HHcy is a risk factor for a series of adverse clinical outcomes, supplementation of B-group vitamins or folic acid might be necessary in metformin-treated patients, regardless of the background diseases. However, further investigations are still required to demonstrate the effects and long-term outcomes of Vitamin B 12 or folic acid supplementation in the metformin-treated patients.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/8/12/798/s1; Table S1: Detailed inclusion and exclusion criteria of each included studies in the meta-analysis, Table S2: Rationale for excluding studies after full-text screening, Table S3: Primary and secondary outcomes of each included study, Table S4: Intervention strategy of each included study, Table S5: Rationale of quality assessment for each included study, Table S6: Summary of subgroup analysis, Table S7: Adverse events, Figure S1: Risk of bias graph: review authors' judgements about each risk of bias item presented as percentages across all included studies, Figure S2: Risk of bias summary: review authors' judgements about each risk of bias item for each included study, Figure S3: Funnel plot of comparisons. Author Contributions: S.L. and J.L. designed this study; Q.Z., Q.L. and K.R. extracted the data; X.S. and L.L. are both biostatisticians from the Chinese Evidence-Based Medicine Center, West China Hospital, Sichuan University, China; X.S. provided methodological guidance of this manuscript as well as reviewed the statistics; L.L. and Q.Z. performed the data analysis and statistical procedure; Q.Z. and S.L. drafted the manuscript and all authors reviewed and revised the manuscript; J.L. is responsible for the integrity of the study as a whole.

Conflicts of Interest:
The authors declare no conflict of interest.