Elevated homocysteine levels and risk of cardiovascular and all-cause mortality: a meta-analysis of prospective studies

Elevated homocysteine levels and risk of cardiovascular and all-cause mortality: a meta-analysis of prospective studies 血浆同型半胱氨酸水平升高与心血管疾病及各种死亡风险因子的meta分析 first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:jszjfanyu@163.com †E-mail:jszjfanyu@163.com jszjfanyu@163.com Cancer Institute, the Affiliated People’s Hospital, Jiangsu University, Zhenjiang 212002, China Cancer Institute, the Affiliated People’s Hospital, Jiangsu University, Zhenjiang 212002, China Objective: 2 To investigate whether elevated homocysteine levels were a predictor of subsequent coronary heart disease (CHD) mortality, cardiovascular mortality or all-cause mortality in the general population by a meta-analysis. Methods: 2 In a systematic search conducted in the databases of PubMed and Embase prior to October 2013, we identified relevant prospective observational studies evaluating the association between baseline homocysteine levels and CHD mortality, cardiovascular or all-cause mortality in the general population. Pooled adjust risk ratio (RR) and corresponding 95% confidence interval (CI) were calculated separately for categorical risk estimates and continuous risk estimates. Results: 2 Twelve studies with 23 623 subjects were included in the meta-analysis. Comparing the highest to lowest homocysteine level categories, CHD mortality increased by 66% (RR 1.66; 95% CI 1.12–2.47; P =0.012), cardiovascular mortality increased by 68% (RR 1.68; 95% CI 1.04–2.70; P =0.033), and all-cause mortality increased by 93% (RR 1.93; 95% CI 1.54–2.43; P < 0.001). Moreover, for each 5 μmol/L homocysteine increment, the pooled RR was 1.52 (95% CI 1.26–1.84; P < 0.001) for CHD mortality, 1.32 (95% CI 1.08–1.61; P =0.006) for cardiovascular mortality, and 1.27 (95% CI 1.03–1.55; P =0.023) for all-cause mortality. Conclusions: 2 Elevated homocysteine levels are an independent predictor for subsequent cardiovascular mortality or all-cause mortality, and the risks were more pronounced among elderly persons. 目的 2 探讨普通人群血浆同型半胱氨酸水平升高是否能独立预测死亡风险。 创新点 2 通过汇总12篇前瞻性研究，定量分析血浆同型半胱氨酸水平升高与心血管疾病及各种死亡风险因子的关系。 方法 2 通过检索PubMed和Embase数据库，收集普通人群中血浆同型半胱氨酸水平与死亡关系的前瞻性观察性研究，采用STATA统计软件进行meta分析。 结论 2 血浆同型半胱氨酸水平升高是心血管疾病及各种原因死亡风险增加的一个独立预测因素，在老年人群中尤为明显。 Homocysteine Coronary heart disease Cardiovascular mortality All-cause mortality Meta-analysis 同型半胱氨酸 冠状动脉疾病 心血管死亡 全因性死亡 meta分析 Introduction 1 Coronary heart disease (CHD) and cardiovascular mortality represent significant health and social problems in the world. The commonly recognized risk factors, including hyperlipidemia, hypertension, obesity, diabetes, and smoking, have been identified as mortality risk assessments in the general population. However, these characteristics do not fully explain all the mortality risks. Early detection of new risk factors would be beneficial for predicting death and reducing mortality. Homocysteine (Hcy) is a sulfur-containing amino acid produced during catabolism of the essential amino acid methionine. Elevated serum Hcy levels have been implicated with the risks of CHD and cardiovascular disease (CVD). The prevalence estimates of hyperhomocysteinemia ( > 14 mmol/L) vary between 5% and 30% in the general population (Selhub et al., 1993 1993 ; Nygård et al., 1995 1995 ). Some epidemiologic studies (Hoogeveen et al., 2000 2000 ; Vollset et al., 2001 2001 ; Blacher et al., 2002 2002 ; Gonzalez et al., 2007 2007 ; Cui et al., 2008 2008 ; Dangour et al., 2008 2008 ; Waśkiewicz et al., 2012 2012 ; Wong et al., 2012 2012 ) but not all (Stehouwer et al., 1998 1998 ; Kark et al., 1999 1999 ; de Bree et al., 2003 2003 ; Swart et al., 2012 2012 ) indicated that the elevated Hcy represents a risk factor for total death, CHD death, or cardiovascular mortality in the general population; additionally, this incremental increase in risk was pronounced in high risk subjects (Nygård et al., 1997 1997 ; Zylberstein et al., 2004 2004 ; Rossi et al., 2006 2006 ; Naess et al., 2013 2013 ). Furthermore, two conducted studies (Bostom et al., 1999 1999 ; Colon Lopez et al., 2008 2008 ) were difficult to compare because the Hcy cut-off values varied. Therefore, the associations of elevated Hcy levels and mortality risk remained conflicting. Given that the results of these studies have not yet been quantitatively evaluated, a meta-analysis would help to classify this issue. Here, we present a meta-analysis of prospective studies to determine the magnitude of Hcy levels and CHD, cardiovascular and all-cause mortality in the general population. Materials and methods 1 Search strategy 2 The meta-analysis was conducted according to the checklist of the meta-analysis of observational studies in epidemiology (Stroup et al., 2000 2000 ). The literature search was conducted in PubMed and Embase databases published prior to October 2013. Potentially relevant studies included the following keywords: mortality/death, coronary heart disease death/mortality, and cardiovascular death/mortality, in combination with hyperhomocysteinemia or homocysteine, prospective, and follow-up. Moreover, the reference lists of the relevant publications were manually searched to identify additional studies. Study selection 2 Studies included in the meta-analysis should meet the following criteria: (1) prospective observational study was conducted in the general population; (2) the exposure was plasma or serum Hcy; (3) the outcome was all-cause mortality or CHD and cardiovascular mortality; (4) provided at least the age-adjusted risk ratio (RR), hazard ratio (HR), or odds ratio (OR) with their confidence intervals (CIs) for categorical risk estimates and continuous risk estimates. The outcome measures included CHD death, cardiovascular death (defined as death from myocardial infarction, heart failure, stroke or arrhythmia), and all-cause mortality. Mortality at the end of follow-up was obtained from the official death certificates, medical records, or personal interviews. Studies were excluded if they met the following criteria: (1) subjects were in a highly selected disease group; (2) the design was a retrospective study, review, and/or case report; (3) only provided a risk estimate with no means by which to calculate the 95% CI or unadjusted OR or RR was reported. Data extraction and quality assessment 2 From eligible studies, two authors (Peng H.Y. and Xu J.) independently extracted the data. Any disagreement was settled by discussion. We did not contact authors to request additional information when data were not available. The following items were extracted from the included papers: first author, year of publication, study design, country, sample size, gender, age, Hcy comparison, death assessment, mortality events, RR or OR with its 95% CI, duration of follow-up, and statistical adjustments for confounding factors. The quality of all the included studies was assessed by the following aspects according to the Newcastle-Ottawa scale (NOS) (Wells et al., 2013 2013 ): selection of study groups, comparability of the study groups, and reporting of important outcomes. Using the scale, we assigned a number of stars based on the answer options of each study. A maximum of two stars was assigned for comparability. The total NOS star count ranges 0–9. A score of ≤5 indicated a high risk of bias. Statistical analysis 2 All statistical analysis was performed using the STATA statistical software (Version 12.0, Stata Corp LP, College Station). P < 0.05 was considered statistically significant. Since nested case-control and cohort studies are all prospective studies, for the purpose of meta-analysis, ORs, and HRs were considered as approximations of RRs. We used the highest versus the lowest category as the measure of the relationships between Hcy levels and risk estimates. In addition, we also pooled risk estimates for per 5 μmol/L increment serum Hcy levels. Homogeneity among studies was determined using the Cochrane Q statistic ( P < 0.10 was considered indicative of statistically significant heterogeneity) and I 2 statistic (values of more than 50% as significant heterogeneity) (Higgins et al., 2003 2003 ). When there was no evidence of significant heterogeneity, effect sizes were determined using a fixed-effects model, otherwise we used a random effects model. Begg’s rank correlation test (Begg and Mazumdar, 1994 1994 ) and Egger’s linear regression test (Egger et al., 1997 1997 ) at P < 0.10 were employed to assess potential publication bias. A sensitivity analysis was carried out by sequentially omitting individual studies at each turn with the metaninf command in STATA. Results 1 Literature search 2 The detailed steps of the study selection are presented in Fig. 1 1 . Through the initial literature search, a total of 960 potentially relevant publications were identified. After screening the abstracts or titles and reviewing the full texts, 12 studies (Stehouwer et al., 1998 1998 ; Kark et al., 1999 1999 ; Hoogeveen et al., 2000 2000 ; Vollset et al., 2001 2001 ; Blacher et al., 2002 2002 ; de Bree et al., 2003 2003 ; Gonzalez et al., 2007 2007 ; Cui et al., 2008 2008 ; Dangour et al., 2008 2008 ; Swart et al., 2012 2012 ; Waśkiewicz et al., 2012 2012 ; Wong et al., 2012 2012 ) met our inclusion criteria. Study characteristics and quality assessment 2 The characteristics of the papers in the meta-analysis are listed in Table 1 1 . All studies were published between 1998 and 2012. In total, the studies included 23 623 participants. Most participants were represented from the elderly population except for two studies (de Bree et al., 2003 2003 ; Waśkiewicz et al., 2012 2012 ), which included a wide range population. The follow-up duration across studies ranged from 4.1 to 14 years. The sample size ranged from 264 to 7165. Most of the studies were conducted in predominantly European countries. The NOS for assessing the quality of the included studies are listed in Table 2 2 , and the scores ranged from 5 to 7. Data are presented as number (percentage of female) Data are presented as range or mean±standard deviation Highest tertile vs. lowest tertile Highest quintile vs. lowest quintile Data for per 5 μmol/L increment Hcy: homocysteine; NL: the Netherlands; AU: Australia; M: male; F: female; BMI: body mass index; RR: relative risk; HR: hazard ratio; OR: odds ratio; SBP: systolic blood pressure; DBP: diastolic blood pressure; MI: myocardial infarction; HDL: high-density lipoprotein; TC: total cholesterol; CHD: coronary heart disease; IHD: ischemic heart disease; CVD: cardiovascular disease; hs-CRP: high-sensitivity C-reactive protein Study Country Subject * * Age ** ** (year) Hcy comparison (μmol/L) Outcome assessment HR or OR (95% CI) Follow-up (year) Adjustment for covariates Stehouwer et al. ( 1998 1998 ) NL 878 (0%) 64–84 > 17 vs. < 12 ▲ ▲ ICD-9 codes 410–414 CHD death: 1.58 (0.93–2.69) 10 Age, BMI, SBP, TC, HDL, DB, and smoking Kark et al. ( 1999 1999 ) Israel 1788 (54.8%) ≥50 > 14.7 vs. < 8.52 ☆ ☆ ICD-9 codes 390–459 Total death: 1.97 (1.31–2.98); 3.45 (1.54–7.70) M; 1.53 (0.91–2.58) F 9–11 Age, SBP, serum glucose, health status, and serum creatinine Hoogeveen al. ( 2000 2000 ) NL 811 (50.7%) 50–75 Per 5 μmol/L increment ICD 9 codes 390–459 Total death: 1.26 (1.02–1.55); CVD death: 1.45 (1.01–2.08) 5 Age, sex, current smoking, DB, hypertension, serum albumin, hypercholesterolemia, and glycated hemoglobin Vollset et al. ( 2001 2001 ) Norway 4766 (47.5%) 65–67 ≥20.4 vs. ≤8.9 ☆ ☆ Per 5 μmol/L increment CVD death: ICD-9 Total death: 3.56 (1.92–6.60); CVD death: 3.48 (1.38–8.79); Total death: 1.49 (1.28–1.72) ■ ■ ; CVD death: 1.50 (1.21–1.85) ■ ■ ; CHD death: 1.46 (1.14–1.87) ■ ■ 4.1 Age, sex, TC, SBP, DBP, smoking, BMI, and physical activity (cardiovascular disease risk status at baseline total death) Blacher et al. ( 2002 2002 ) France 264 (18.6%) 65±11 (case) 66±13 (control) > 12.97 vs. < 9.76 ▲ ▲ Mortality records of National Statistics CVD death: 1.06 (1.01–1.10) 14 Age, sex, DB, creatinine, BMI, SBP, DBP, CRP, smoking, lipid profile, folate, and alcohol consumption de Bree et al. ( 2003 2003 ) NL 630 (39.9%) 20–59 ≥19.2 vs. < 11 ▲ ▲ ICD-9 codes 410-/414 or ICD-10 codes I20-/I25 CHD death: 1.14 (0.50–2.61) M; 2.04 (0.48-8.62) F 10.3 Age, study center, HDL cholesterol, TC, hypertension, creatinine, and smoking Gonzalez et al. ( 2007 2007 ) Spain 215 (59.1%) 75.1±6.5 ≥16.7 vs. < 8.7 ☆ ☆ Personal interview Total death: 2.297 (1.021–5.169) 4.3 Age, sex, smoking, BMI, and cognitive score Dangour et al. ( 2008 2008 ) UK 853 (50.9%) ≥75 19.4 vs. 9.8 ☆ ☆ CVD death: ICD-9 codes 390–459 or ICD-10 codes I1–I99 Total death: 2.20 (1.76–2.75) CVD death: 1.96 (1.39–2.78) 7.6 Age, sex, diabetes, history of CVD, cancer, smoking, alcohol consumption, physical activity, homocysteine and/or folate and/or vitamin B 12 Cui et al. ( 2008 2008 ) Japan 888 (64.9%) 40–79 ≥15.3 vs. ≤10.5 ☆ ☆ Per 5 μmol/L increment ICD-9 and ICD-10 CVD death: 1.68 (1.02–2.77); CHD death: 3.40 (1.17–9.86); CVD death: 1.15 (1.00–1.32) ■ ■ ; CHD death: 2.01 (1.21–3.35) ■ ■ 10 Age, sex, community, BMI, total and HDL-cholesterol, ethanol intake, DB, smoking, history of hypertension, and year of serum storage Swart et al. ( 2012 2012 ) NL 1117 (51.4%) 75.1±6.4 ≥17.57 vs. ≤11.96 ☆ ☆ (M); ≥15.64 vs. ≤10.35 ☆ ☆ (F) Death certificate Total death: 1.14 (0.77–1.67) M; 1.91 (1.22–3.00) F 11 Age, education, region, BMI, smoking, creatinine, alcohol consumption, physical activity, and serum vitamin B 12 Waśkiewicz et al. ( 2012 2012 ) Poland 7165 20–74 > 10.5 vs. < 8.2 ▲ ▲ CVD death: ICD-10 codes I00–I99 Total death: 1.766 (1.197–2.605); CVD death: 1.937 (1.051–3.569) 5.4 Age, sex, BMI, TC, smoking, hypertension, glucose, and hs-CRP Wong et al. ( 2012 2012 ) AU 4248 (0%) 70–88 Per 5 μmol/L increment Medical records of Western Australian Data Linkage System Total death: 1.11 (1.07–1.16) 5.1±1.3 Age, CVD, living circumstance, smoking, diabetes, hypertension, dyslipidemia, education, Charlson comorbidity index, renal function, and frailty status Using the Newcastle-Ottawa scale (NOS) (Wells et al., 2013 2013 ), the number of stars based on the answer options of each study was assigned A maximum of two stars was assigned for comparability. The total NOS star count ranges 0–9. A score of ≤5 indicated a high risk of bias. A1: representativeness of the exposed cohort; A2: selection of non-exposed cohort; A3: ascertainment of exposure; A4: demonstration that outcome was not present at study start; A5: comparability of cohorts on the basis of the design or analysis; A6: assessment of outcome; A7: enough follow-up periods; A8: lost to follow-up acceptable (reported <10%) Study A1 A2 A3 A4 A5 A6 A7 A8 Overall score Stehouwer et al. ( 1998 1998 ) * * * * * * * 7 Kark et al. ( 1999 1999 ) * * * * * * * * 8 Hoogeveen et al. ( 2000 2000 ) * * * * * * * 7 Vollset et al. ( 2001 2001 ) * * * * * * 6 Blacher et al. ( 2002 2002 ) * * * * * * 6 de Bree et al. ( 2003 2003 ) * * * * * * * * 8 Gonzalez et al. ( 2007 2007 ) * * * * * 5 Dangour et al. ( 2008 2008 ) * * * ** * * * 8 Cui et al. ( 2008 2008 ) * * * * * * 6 Swart et al. ( 2012 2012 ) * * * * ** * * * 9 Waśkiewicz et al. ( 2012 2012 ) * * * * * * * 8 Wong et al. ( 2012 2012 ) * * * * * 5 All-cause mortality 2 Six studies (Kark et al., 1999 1999 ; Vollset et al., 2001 2001 ; Gonzalez et al., 2007 2007 ; Dangour et al., 2008 2008 ; Swart et al., 2012 2012 ; Waśkiewicz et al., 2012 2012 ) reported an outcome on all-cause mortality on the highest versus lowest Hcy categories. The total number of participants included in this meta-analysis was 15 904 with 1936 death events. As shown in the part 1 of Fig. 2 2 , there was a statistically significant heterogeneity among the studies ( I 2 =52.3%, P =0.05), and the random effects model was used to pool the results. Overall, elevated serum Hcy levels were associated with a greater risk of all-cause mortality (RR 1.93; 95% CI 1.54–2.43; P < 0.001). Publication bias was not indicated in Begg’s rank correlation test ( P =0.368) or Egger’s linear regression test ( P =0.937). As shown in Fig. 3 3 , in the subgroup analysis based on the gender of the two studies (Kark et al., 1999 1999 ; Swart et al., 2012 2012 ), women with elevated Hcy levels increased the risk of all-cause mortality (RR 1.74; 95% CI 1.24–2.44; P =0.001); in contrast, no statistical significance was found for the men (RR1.87; 95% CI 0.64–5.50; P =0.255). A significant dose-response association, between serum Hcy levels and the risk of all-cause mortality, was noted in three studies (Hoogeveen et al., 2000 2000 ; Vollset et al., 2001 2001 ; Wong et al., 2012 2012 ). The total number of participants included in this meta-analysis was 9825, with 1179 death events. As shown in the part 2 of Fig. 2 2 , there was a statistically significant heterogeneity among the studies ( I 2 =86.8%, P =0.001), the random effects model was used to pool the results. The pooled RR of all-cause mortality for the per 5 μmol/L increment serum Hcy was 1.27 (95% CI 1.03–1.33; P =0.023). Cardiovascular mortality 2 Four studies (Vollset et al., 2001 2001 ; Blacher et al., 2002 2002 ; Cui et al., 2008 2008 ; Dangour et al., 2008 2008 ) reported outcomes on cardiovascular mortality on the highest versus lowest Hcy levels categories. The total number of participants, included in this meta-analysis, was 6771 with 860 cardiovascular death events. As shown in the part 1 of Fig. 4 4 , there was a statistically significant heterogeneity among the studies ( I 2 =85.9%, P =0.000), the random effects model was used to pool the results. Overall, elevated serum Hcy levels were significantly associated with an increased cardiovascular mortality risk (RR 1.68; 95% CI 1.04–2.70; P =0.033). Publication bias was observed in Begg’s rank correlation test ( P =0.734), but not in the Egger’s linear regression test ( P =0.039). A significant dose-response association between the serum Hcy levels and risk of cardiovascular mortality was indicated in the three studies (Hoogeveen et al., 2000 2000 ; Vollset et al., 2001 2001 ; Cui et al., 2008 2008 ). The total number of participants included in this meta-analysis was 6465, with 641 cardiovascular death events. As shown in the part 2 of Fig. 4 4 , there was a statistically significant heterogeneity among the studies ( I 2 =58.6%, P =0.089), the random effects model was used to pool the results. The pooled RR of cardiovascular mortality for the per 5 μmol/L increment serum Hcy level was 1.32 (95% CI 1.08–1.61; P =0.006). CHD mortality 2 Three studies (Stehouwer et al., 1998 1998 ; de Bree et al., 2003 2003 ; Cui et al., 2008 2008 ) reported the outcome on CHD mortality in the highest versus lowest Hcy level categories. The total number of participants included in this meta-analysis was 2396 with 334 CHD death events. As shown in the part 1 of Fig. 5 5 , there was no significant heterogeneity among the studies ( I 2 =0%, P =0.45), the fixed-effects model was used to pool the results. Overall, elevated serum Hcy levels were significantly associated with an increased CHD mortality risk (RR 1.66; 95% CI 1.12–2.47; P =0.012). However, after we excluded one study (Cui et al., 2008 2008 ), the sensitivity analysis only showed some trend, this positive association was not statistically significant (RR 1.48; 95% CI 0.97–2.27; P =0.071). A significant dose-response association between serum Hcy levels and the risk of CHD mortality was indicated in three studies (Hoogeveen et al., 2000 2000 ; Vollset et al., 2001 2001 ; Cui et al., 2008 2008 ). The total number of participants included in this meta-analysis was 6207, with 344 CHD death events. As shown in the part 2 of Fig. 5 5 , there was no significant heterogeneity among the studies ( I 2 =0%, P =0.516), the fixed-effects model was used to pool the results. The pooled RR of CHD mortality for per 5 μmol/L increment Hcy was 1.52 (95% CI 1.26–1.84; P < 0.001). Discussion 1 The findings of our current meta-analysis found that the subjects increased by 93% the risk of all-cause mortality, 68% the risk of cardiovascular mortality, and 66% CHD mortality comparing the highest versus lowest Hcy levels. Furthermore, the dose-response analysis showed that an increment of 5 μmol/L Hcy increased by 27% the risk of all-cause mortality, 32% the risk of cardiovascular mortality, and 52% CHD mortality. Total Hcy levels were higher in men than in women and increased with age (Selhub et al., 1993 1993 ). Studies from the Framingham study showed that the prevalence of high Hcy was 29.3% for the entire cohort and over 40% for individuals aged 80 years and older (Selhub, 2006 2006 ), although elevated Hcy levels appeared to increase the CHD mortality for categorical risk estimates. However, the sensitivity analysis only showed some trend, the risk of CHD mortality was not statistically significant (RR1.48; 95% CI 0.97–2.27) comparing the highest to lowest Hcy. These results could be partly explained by the age of the subjects included in the studies who were relatively young, and their ages might have affected the findings of the pooled estimates. Elevated Hcy levels were not a strong risk factor for mortality in relatively young subjects (de Bree et al., 2003 2003 ). Subgroup analysis based on gender indicated that women with elevated Hcy levels increased the risk of all-cause mortality (RR 1.74; 95% CI 1.24–2.44); in contrast, this positive association was not statistically significant among men (RR 1.87; 95% CI 0.64–5.50). The present results suggest that the relationship between Hcy levels and all-cause mortality appears to be more pronounced in women. However, due to the limited number of studies, the results might be not robust enough to draw a conclusion. Therefore, whether men or women with elevated Hcy had more risk of mortality is still unclear. The mechanisms underlying Hcy levels and risk of mortality have not yet been elucidated. Experimental studies suggest that higher Hcy caused endothelial dysfunction (Celermajer et al., 1993 1993 ), platelet activation and thrombus formation (Dionisio et al., 2010 2010 ). Meta-analysis of prospective cohort studies demonstrated that hyperhomocysteinemia was associated with cardiovascular disease (Bautista et al., 2002 2002 ), cognitive decline (Ho et al., 2011 2011 ), and fracture (Yang et al., 2012 2012 ). All above diseases increased death in elderly persons. Our meta-analysis had several strengths. First, the present analysis included only prospective observational studies, which greatly reduced the selection bias. Moreover, according to the NOS, all the included studies belonged to a high quality class. Second, this meta-analysis only included multivariable adjusted OR or RR. Third, the robustness of the Hcy level as a predictor of mortality was significantly reinforced by per 5 μmol/L increment Hcy as a continuous variable. Several potential limitations of this study should be mentioned. First, Hcy levels are influenced by a complex interaction of environmental and genetic factors, such as nutrition, lifestyle, renal function and hormonal factors. However, the included study did not adjust some important confounding factors, such as folate and vitamin B 12 levels. Additionally, there were variant adjusted confounding factors from the individual studies. Second, the ages of the subjects included in most of the studies were elderly; so the positive association between Hcy levels and death risk could not be generalized to younger persons. Third, four studies (Stehouwer et al., 1998 1998 ; Kark et al., 1999 1999 ; de Bree et al., 2003 2003 ; Dangour et al., 2008 2008 ) measured Hcy levels based on non-fasting blood samples and this might underestimate the associations with mortality due to regression dilution bias. Fourth, the number of studies was small for subgroup analysis, which might exert bias. Finally, most of the studies were from European populations, additional studies in Asian populations should be investigated to generalize our findings. 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