Plasma homocysteine concentrations and risk of intracerebral hemorrhage: a systematic review and meta-analysis

Intracerebral hemorrhage (ICH) has the highest mortality rate in all strokes. However, controversy still exists concerning the association between plasma homocysteine (Hcy) and ICH. A systematic review and meta-analysis was conducted using Pubmed, Embase, and Web of Science up to April 18, 2017. Standard mean difference (SMD) for mean differences of plasma Hcy levels with 95% confidence intervals (CI) was calculated. Seven studies including 667 ICH patients and 1821 ischemic stroke patients were identified for meta-analysis. Our results showed that Hcy levels in ICH patients were significantly higher than those in healthy controls (SMD = 0.59, 95% CI = 0.51–0.68, P < 0.001); no statistic differences were found in the comparisons of Hcy levels between ICH and ischemic stroke (SMD = −0.03, 95% CI = −0.13–0.06, P > 0.05); further subgroup analysis of ethnicity (Asians: SMD = 0.57, 95% CI = 0.48–0.66, P < 0.001; Caucasians: SMD = 0.77, 95% CI = 0.51–1.02, P < 0.001) and sample size (small samples: SMD = 0.55, 95% CI = 0.30–0.80, P < 0.001; large samples size: SMD = 0.60, 95% CI = 0.51–0.69, P < 0.001) in relation to Hcy levels between ICH and healthy controls did not change these results. In conclusion, Hcy level may be an aggravating factor in atherosclerosis, which is positively associated with high risk of ICH. Race-specific differences between Asians and Caucasians have no impact on the risk of ICH.

higher concentrations of Hcy than that of healthy controls, which implied an underlying mechanism linking plasma Hcy levels with ICH [16][17][18] .
Homocysteine, an intermediate sulfhydryl-containing amino acid, is synthesized by the liver in a reaction involving the removal of the methyl group from methionine 19,20 . High level of Hcy is mainly found in patients with a nutritional deficiency of B vitamins as well as being present in genetic diseases where there is an insufficient level of cystathionine beta-synthase enzyme (CβS) or a mutation inn the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR). Other factors can elevate the level of Hcy i.e. a diet with high methionine content, smoking and a sedentary lifestyle [21][22][23][24] . According to epidemiological evidence, Hcy is considered to be a potential predictor of pathophysiology of neuronal system disorders 25,26 . High level of Hcy induces a neuro-inflammatory process due to its abilities to react with cytokines in the brain, leading to increased permeability of blood-brain barrier (BBB) and impaired integrity of cerebral vessels 27,28 . There is convincing evidence that elevated plasma Hcy level is associated with higher mortality rates from ischemic stroke, cardiovascular diseases and peripheral artery disease [29][30][31][32] . However, it is still controversial whether elevated plasma Hcy level is a risk factor for ICH. Therefore, we performed a systematic overview of retrospective studies to ascertain the association between Hcy and the risk of ICH.

Materials and Methods
Inclusion and Exclusion Criteria. To identify eligible studies investigating the association between plasma Hcy levels and ICH, published studies meeting the following criteria were considered eligible for inclusion: (1) case-control studies written in English; (2) studies which assessed the correlation between Hcy and patients with ICH; (3) the cases were confirmed ICH patients and the controls were healthy people; (4) providing information of plasma Hcy levels in the control group and case group at the onset of ICH. The exclusion criteria were as follows: (1) conflicts with the inclusion criteria; (2) publications with duplication or studies with overlapping data from the same study; (3) abstracts, case report, proceedings, letters, reviews, or meta-analysis.
Literature Search. Databases of Pubmed, Embase, and Web of Science were comprehensively searched for articles up to April 18, 2017. The terms searched were as follows: ("plasma homocysteine" OR homocysteine OR Hcy OR "plasma Hcy") AND ("intracerebral hemorrhage" OR "cerebral hemorrhage" OR "brain hemorrhage" OR "hemorrhagic stroke"). We also screened the reference lists of relevant reviews and included relevant articles in these lists.

Data Extraction and Quality Assessment.
Two reviewers independently extracted the data and information from the finally selected trials according to a standard protocol, and then discussed or consulted the original report to reach a consensus when discrepancies occurred. In the case of eligible studies, the following information was collected: first author, publication year, country, detection method for plasma Hcy, proportion of men, number of participants as well as plasma Hcy levels in the groups of ICH, control and ischemic stroke. Guidelines of preferred reporting items for systematic reviews and meta-analyses (PRISMA) were followed by the study. Quality assessment was conducted according to the Newcastle-Ottawa Scale (NOS) criteria for retrospective studies 33 . A score of up to 9 points was assigned to each study: 4 points for selection, 2 points for comparability, and 3 points for assessment of outcomes. Studies with 6 or more points were considered as high quality, while those with less than 6 were considered to be of suboptimal quality.
Statistical Analysis. Statistical analysis in this meta-analysis was performed with the software Review Manager 5.2 and STATA version 14.0. Standard mean difference (SMD) with 95% confidence intervals (CI) was calculated by random effects model or fixed effects model to measure the differences in plasma Hcy levels between the case group and control group, the significance of which was assessed by utilizing the Z test. I 2 test (25, 50, and 75% represented low, moderate, and high degrees of heterogeneity) was applied to assess the heterogeneity among studies [34][35][36] . Random effects models were applied for the evidence of significant heterogeneity (I 2 test exhibited > 50%); otherwise, the fixed effects model was utilized 34,35,37 . Hcy levels from plasma and serum were used equally. Subgroup analyses by ethnicity and sample size of participants (≥50 or less) were also conducted. Sensitivity analysis was carried out by sequentially omitting one study to examine its influence on the pooled risk estimates. A publication bias was detected by visual inspection of a funnel plot i.e. an asymmetric funnel plot indicated a possible risk of a publication bias.

Sensitivity Analysis and Publication bias. Sensitivity analysis performed by omitting one individual
study sequentially showed that no single study had any obvious influence on the pooled mean difference (Fig. 4). Since the funnel plot for overall analysis was symmetrical (Fig. 5), there was no obvious risk of publication bias in the meta-analysis.

Discussion
As far as we are aware, there has been no relevant systematic review of case-control studies carried out to explore whether plasma Hcy are associated with the risk of ICH. Seven eligible studies with a total of 667 ICH patients were included into our meta-analysis 15,[50][51][52][53][54][55] . In comparison with a single study, the meta-analysis makes it possible to have a more accurate estimate and evaluation of the impact of Hcy levels on ICH. The findings emerging from comprehensive meta-analysis confirmed that patients with ICH exhibited prominently higher plasma Hcy levels than healthy controls, and this association could not be affected by study quality, participant characteristics and publication bias.
The precise mechanism of Hcy on the susceptibility of ICH remains unresolved. However, our meta-analysis supported that the pathogenesis of ICH likely involves some factor elevating Hcy levels. Experimental studies have verified multiple biological responses triggered by Hcy during the process of atherosclerosis, for example, Hcy inhibits glutathione peroxidase to stimulate the proliferation of smooth muscle cells, by which endothelial nitric oxide synthase (eNOS) can be suppressed via the asymmetric dimethyl-L-arginine (ADMA) pathway, leading to endothelial dysfunction and vascular spasms 26,[56][57][58] . In addition, Hcy enhances the release of arachidonic acid from platelets to generate reactive oxygen species (ROS), leading to the deposition of calcium and lipid in the endothelial wall; these changes degrade arterial elasticity and accelerate the process of atherosclerosis 21,59-61 . In the present results, the comparisons of plasma Hcy levels between ICH and ischemic stroke showed no significant difference. This implied that the plasma Hcy level was likely identified as an aggravating factor in atherosclerosis,  www.nature.com/scientificreports/ owing to which the pathophysiology of endothelial degeneration and necrosis of vessel walls could elevate the risk of ischemic stroke and cerebral hemorrhage 62 . However, we are unable to rule out the possibility that the high levels of Hcy may be attributable to the acute stress reaction evoked by stroke, as all blood samples of plasma Hcy were obtained at the onset of ICH rather than before its occurrence. There is one prospective study in which participants were invited to donate a fasting blood sample for future research; this showed that plasma Hcy levels were significantly higher in patients who would develop ICH compared to their healthy controls 49 . Another study reported that Hcy levels initially decreased within 24 hours and then gradually raised within 48 hours in patients with hemorrhagic stroke, whereas differences in mean Hcy levels at 0, 24 and 48 hours post stroke were insignificant compared with mean Hcy levels in controls 54 . Accordingly, it is speculated that Hcy levels have been already elevated before the onset of ICH and that their levels could not be affected by acute stress reaction after a stroke. Further studies will be needed to assess the influence of the acute stress reaction during follow-up on the association between Hcy and ICH.
Consistent with the overall results, subgroup analysis on either ethnicity or sample size confirmed that plasma Hcy levels of ICH patients were significantly higher than those of healthy controls. Preview study revealed that  Africans had apparently higher risk of ICH compared with Caucasians, suggesting that race played an important role in the ICH risk pattern 63 . Interestingly, in the present study, the subgroup analysis based on ethnicity in relation to plasma Hcy levels between Asians and Caucasians had no statistic differences, which implied that race-specific differences exerted no effect on the results of our meta-analysis. The reason for this inconsistency might be that race-specific differences between Africans and Caucasians had a profound impact on the  risk of ICH rather than race-specific differences between Asians and Caucasians in our study. Although our meta-analysis revealed no statistical heterogeneity, there can also be clinical heterogeneity or methodological heterogeneity between studies. Some possible sources of clinical heterogeneity such as inclusion and exclusion criteria, intervention measures, defining indicators may be the differences. Other sources of methodology heterogeneity such as experimental design, bias risk and outcome integrity may also affect the results.
Limitations. Several potential limitations of this meta-analysis must be acknowledged. All included studies followed a retrospective design, which may be more subjected to bias and artifact than prospective studies. The different detection methods employed for plasma Hcy levels may have sensitivity and reliability issues. We were unable to analyze the effect of the acute stress reaction of ICH on plasma Hcy levels since there were insufficient data about Hcy levels before the onset of ICH. Although the result of the funnel plot did not suggest any publication bias, there is the possibility of journals publishing only positive findings as only published data were utilized in the meta-analysis. Additionally, the relatively limited number of studies and the fact that most of patients were Asians, may also influence the sensitivity of statistical analysis. Further randomized controlled trials and larger numbers of participants will be necessary to confirm these findings.

Conclusions
Our results of meta-analysis provide support for the hypothesis that elevated level of Hcy in blood is positively associated with the incidence of ICH. Hcy level may be an aggravating factor in atherosclerosis which contributes to ICH. There is no impact of race-specific differences between Asians and Caucasians on the risk of ICH. More clinical trials with more comprehensive data, using larger sample sizes and better design, will be needed to validate our results.