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Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young
Clinical Nutrition xxx (2017) 1e7
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Original article
Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young Kondapura Jayadevappa Rudreshkumar a, 1, Vijaya Majumdar a, 1, Dindagur Nagaraja b, Rita Christopher a, * a b
Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences, Bengaluru, India Department of Neurology, National Institute of Mental Health and Neuro Sciences, Bengaluru, India
a r t i c l e i n f o
s u m m a r y
Article history: Received 29 November 2016 Accepted 7 July 2017
Background & aims: The debated vascular risk potential of total homocysteine (tHcy), due to failed clinical trials designed on B vitamin supplementation, raises many possible explanations like the higher risk potential of the deleterious, free form of homocysteine (fHcy) or, the unchecked confounding effects of B-vitamins in tHcy-based association studies. Additionally, the cardiovascular risk probability of altered status of the homocysteine precursor, methionine (tMet) could shed light on the causality of association between tHcy and cardiovascular diseases. Hence, we aimed to evaluate the risk associations of elevated plasma levels of tHcy, fHcy and low levels of tMet with premature, ischemic stroke. Methods: We recruited 171 young, ischemic stroke patients (aged 45 years) and 249 age- and gendermatched healthy controls. Plasma levels of fHcy, tHcy, tMet and vitamin B6 were estimated using HPLC coupled with coulometric electrochemical detection. Plasma levels of vitamin B12 and folate were estimated by radioimmunoassay. Results: Elevated fHcy (>2.9 mmol/L) was independently and strongly associated with the risk of premature, ischemic stroke (OR ¼ 9.62, 95% CI ¼ 3.51e26.40). On the contrary, association between premature ischemic stroke and elevated tHcy (>15.0 mmol/L) was found to attenuate when adjusted for vitamin B6 values (OR ¼ 0.24, 95%, CI ¼ 0.03e1.69). Interestingly, compromised B6-status (<59.2 nmol/l) was found to confer high risk of premature ischemic stroke (OR ¼ 170.80, 95% CI ¼ 58.22e501.06). We could not establish any significant correlation between fHcy and B-vitamin levels (P > 0.05). Low tMet (<13.86 mmol/L) was also not significantly associated with premature, ischemic stroke (OR ¼ 2.53, 95% CI ¼ 0.613e10.38). Conclusion: Our results indicate significant but not-correlated, independent associations of fHcy and vitamin B6 with risk of premature, ischemic stroke. However, the causality of these associations need prospective and large scale validations. Further, our findings highlight the crucial confounding effects of B-vitamins on risk association between tHcy and premature ischemic stroke. © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Keywords: Free homocysteine Total homocysteine Methionine Premature ischemic stroke
1. Introduction Stroke occurring in young adults, which constitutes 10%e14% of total strokes [1,2], has severe socio-economic consequences particularly in developing countries like India. Current understanding of ischemic stroke etiology points towards
* Corresponding author. Department of Neurochemistry, National Institute of Mental Health & Neuro Sciences (NIMHANS), Bengaluru, 560029, Karnataka, India. Fax: þ080 26564830. E-mail addresses: [email protected], [email protected] (R. Christopher). 1 Equal contribution by these authors.
atherosclerosis as a major contributing factor in young individuals [3]. This indicates a pressing need for the development of appropriate preventive strategies against modifiable and controllable, potentially atherogenic, risk factors such as homocysteine. Although there are studies implicating total homocysteine (tHcy) as a risk factor for ischemic stroke in the young [4,5], the potency and direct causative role of homocysteine in cardiovascular diseases has been doubted owing to failed clinical trials on homocysteine-lowering therapies (HLTs) in disease prevention [6e8]. Further, the doubted causality of hyperhomocysteinemia raises the question whether homocysteine is a biomarker or a risk factor for cardiovascular diseases [9].
http://dx.doi.org/10.1016/j.clnu.2017.07.005 0261-5614/© 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Please cite this article in press as: Rudreshkumar KJ, et al., Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.07.005
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K.J. Rudreshkumar et al. / Clinical Nutrition xxx (2017) 1e7
Vitamin B supplementation has been considered the mainstay of HLT [10]. B vitamins, namely, vitamin B6 (pyridoxine/pyridoxalphosphate), vitamin B12, and folate (vitamin B9) play crucial roles in the metabolism of homocysteine and when administered, result in effective lowering of homocysteine [10]. In particular, vitamin B6 and folate have been demonstrated to have regulatory cardiovascular risk potential independent of homocysteine [11,12]. We hypothesize that the debated causality of tHcy for cardiovascular diseases, point towards many possibilities such as confounding effects of B-vitamin deficiencies in tHcy-based association studies [5,13,14]. It is therefore necessary to control the confounding effects of nutritional deficiencies of vitamins B12, B6, and folic acid, when evaluating the cardiovascular risk associated with elevated homocysteine levels [11e14]. Further, the failed clinical trials on tHcy reduction for cardiovascular disease prevention, highlight the unattended higher vascular risk potential of the deleterious, free form of homocysteine (fHcy). Biologically, the potency of tHcy is fractionated into its protein-bound (70%) and the more potent, free, non protein-bound form (fHcy) (30%); the latter includes reduced homocysteine and homocysteine disulfides [15]. It has been demonstrated that the fHcy associates strongly with vascular endothelial dysfunction [16]. Despite its reported high vascular risk potential [16,17], there has been a lack of thorough assessments on the influence of elevated levels of fHcy on cardiovascular risk including ischemic stroke. Mechanistically, the vascular pathogenicity of elevated homocysteine could derive from the intricately linked, impaired systemic methylation reactions [18]. The emerging role of epigenetic mechanisms in the pathophysiology of cardiovascular diseases support this view [19,20]. Altered status of methionine associated with impaired metabolism of homocysteine, has been considered an indicator of the compromised status of methyl metabolism [18]. We hypothesize that investigations on independent vascular risk potential of altered methionine status, could shed light on the causality of association between tHcy and cardiovascular diseases. Hence, based on our hypotheses, we investigated the risk of premature ischemic stroke associated with the crucial metabolites of homocysteine metabolism, tHcy, fHcy and tMet while controlling the confounding effects of B vitamins. 2. Materials and methods 2.1. Ethical approval This study was approved by the Ethics Committee of the National Institute of Mental Health and Neuro Sciences (NIMHANS), Bengaluru, India. Informed consent was obtained from all healthy and diseased volunteers. 2.2. Study participants We investigated 171 young stroke patients aged 45 years, reporting to the Neurological Services of our hospital. Diagnosis of ischemic stroke was affirmed by cranial computed tomography scan and/or magnetic resonance imaging within 24 h of hospital admission. The Glasgow coma scale (GCS) was used to evaluate the conscious state of the stroke patients during admission [21]. Patients were excluded from the study if diagnosed of i) hemorrhagic stroke ii) infantile hemiplegia iii) neuro-infection with resulting infarction iv) disseminated intra-vascular coagulation v)hypothyroidism vi) renal or liver dysfunction vii) malignancy or any other terminal illnesses. A total of 171 patients were studied for duration of 2 years. The control group consisted of 249 healthy volunteers, free from any pre-existing vascular diseases and matched for age, gender, ethnicity and socioeconomic status.
2.3. Anthropometric and biochemical analyses Demographic and anthropometric details, inclusive of variables: age, sex, smoking habits, alcohol use, presence of diabetes mellitus and hypertension were acquired from the study subjects. Fasting venous blood was withdrawn within 7 days of onset of ischemic stroke symptoms. Serum triglycerides (TG), total cholesterol (TC), and high density lipoprotein (HDL)-cholesterol concentrations were estimated. Lipid profile was measured on an automated, random access, clinical chemistry analyzer (Olympus AU640, Olympus Singapore Pvt. Ltd., Singapore) using commercial kits (Beckman Coulter, Inc., Clare, Ireland). Total cholesterol was estimated by enzymatic method based on combined actions of cholesterol esterase and oxidase [22], (assay kits, OSR 6116 and 6216). Triglyceride measurement was based on a series of coupled enzymatic reactions including hydrolysis by a combination of microbial lipases [assay kit, OSR 60118] [23]. HDL-C was estimated by an immune-inhibition method (assay kit, OSR 6187). Concentration of low density lipoprotein (LDL)-cholesterol was determined using Friedewald's formula [24]. Levels of vitamin B12 and folic acid were assayed by radioimmunoassay using commercially procured kits (SimulTRAC-SNB, MP Biomedicals, USA). 2.4. Assessment of plasma tHcy, fHcy, tMet and vitamin B6 by highperformance liquid chromatography Plasma tHcy, fHcy and tMet were measured by reverse-phase high-performance liquid chromatography (HPLC) and coulometric electrochemical detection using a Shimadzu HPLC system (LC10ADVP, Shimadzu Corporation, Japan) provided with an auto sampler (SIL-HTA) and a ESA Coulochem III detector (ESA Ins, Chelmsford, MA) [25]. Chromatographic separation was conducted at a flow rate of 1.0 ml/min and a pressure of 120e140 kg/cm2 (1800e2100 psi), using a reverse phase C18 Supelco column (5 m, 4.6 250 mm, Shimadzu). The active form of vitamin B6 in plasma, pyridoxal 50 phosphate (PLP) was also assayed by reverse-phase HPLC and coulometric electrochemical detection [26]. 2.5. Statistical analysis Data were analyzed using SPSS (V16.0) program. The differences in the distribution between patients and controls, of continuous variables with normal and skewed distributions, were tested by paired ‘t’ test and ManneWhitney U tests, respectively. All P values were 2-sided. Cut offs for elevated tHcy (15.00 mmol/l) and fHcy (2.90 mmol/l) were defined according to previous reports [27]. Low plasma tMet status was defined based on cut off of 13.86 mmol/l (10th percentile values in control population). Cut offs of elevated tHcy/tMet and fHcy/tMet were also based on 10th percentile values in controls (Table 2). Vitamin B6 deficient and suboptimal statuses were defined according to the recommended cut off values of 20 nmol/l [28] and 30 nmol/l [29] respectively. Stroke risks conferred by plasma levels of different total homocysteine, its free form and methionine levels were evaluated by logistic regression analyses. Multivariate analyses were constituted of covariates such as age, gender, smoking, alcohol consumption, hypertension, diabetes mellitus, lipid levels and B vitamin levels (vitamins B6, B12 and folate). Hypertension was characterized as diastolic blood pressure 90 mmHg and/or systolic blood pressure 140 mmHg [30] and/or use of antihypertensive medication. Diabetes was diagnosed on the basis of fasting plasma glucose values >126 mg/dl or the subjects' self-reported history of diabetes or use of anti-diabetic medication [31]. Coefficients of correlation were calculated using Pearson's/Spearmann's correlation analysis. Logistic regression analyses were carried out to test statistical
Please cite this article in press as: Rudreshkumar KJ, et al., Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.07.005
K.J. Rudreshkumar et al. / Clinical Nutrition xxx (2017) 1e7
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calculated to evaluate the cut off for deficient vitamin B6 status with reference to the predictive risk of ischemic stroke in young.
Table 1 Distribution of covariates between ischemic stroke patients and controls. Covariates
Ischemic stroke patients (n ¼ 171)
Controls (n ¼ 249)
P value
Age (years) Males, n (%) Smoking, n (%) Alcohol use, n (%) Hypertensives, n (%) Diabetics, n (%) TC, (mmol/L) TG, (mmol/L) LDL-C, (mmol/L) HDL-C, (mmol/L) tHcy, (mmol/L) fHcy, (mmol/L) tMet, (mmol/L) Elevated tHcy (>15.00 mmol/L), n (%) Elevated fHcy (>2.90 mmol/L), n (%) Low tMet (<13.86 mmol/L), n (%) Vitamin B12, pmol/L Vitamin B6, nmol/L Folate, nmol/L
27.70 ± 10.63 130 (76.02) 56 (32.75) 42 (24.56) 7 (4.09) 7 (4.09) 5.43 ± 2.56 1.57 ± 1.35 3.56 ± 1.20 0.91 ± 1.30 20.79 ± 1.77 3.68 ± 0.14 21.59 ± 8.86 77 (45.03)
27.02 ± 6.01 170 (68.27) 20 (8.03) 39 (15.66) 2 (0.80) 1 (0.40) 4.57 ± 1.45 1.37 ± 0.50 2.80 ± 1.50 1.10 ± 0.99 11.10 ± 0.23 1.99 ± 0.05 24.80 ± 9.86 29 (11.65)
0.445 0.100 <0.001 <0.001 0.035 0.009 <0.001 0.033 0.041 0.060 <0.001 <0.001 <0.001 <0.001
103 (60.23)
38 (15.26)
<0.001
40 (23.39)
25 (10.04)
<0.001
315.50 ± 18.73 42.52 ± 11.96 13.99 ± 0.81
475.09 ± 17.90 68.29 ± 9.03 20.02 ± 0.81
<0.001 <0.001 <0.001
3. Results
Abbreviations: High density lipoprotein cholesterol, (HDL)-C; low density lipoprotein cholesterol, LDL-C; total homocysteine, tHcy; free homocysteine, fHcy; total methionine, tMet; Total cholesterol, TC; triglycerides, TG.
association between altered levels of tHcy, fHcy and tMet and premature ischemic stroke. Various multivariate models were generated, the first model was the crude unadjusted model. The second model, model II, was adjusted for the following confounders; age, gender, hypertension, diabetes mellitus, smoking, alcohol drinking, lipid levels. Further, model III was subcategorized into IIIa/IIIb/IIIc, based on the respective differential adjustment for variables, tMet/tHcy/fHcy levels respectively. Subsequently, models IVa/IVb/IVc were additionally adjusted for vitamin B12 and folic acid levels. Models Va/Vb/Vcwere generated by additional adjustment for vitamin B6 levels. Similarly, for evaluation of risk of ischemic stroke in the young, conferred by compromised vitamin B6 levels, various models were generated for logistic regression analyses. Model I was the crude unadjusted model. The second model, model II, was adjusted for age, gender, hypertension, diabetes mellitus, smoking, alcohol drinking, lipid levels; Models III, was additionally adjusted for vitamin B12 and folic acid levels; Model IVa/IVb/IVc was subcategorized for differential adjustment for tMet/tHcy/fHcy levels respectively. The receiver operating characeteristic (ROC) curve analysis was carried out and area under the curve (AUC) was
Majority of patients (n ¼ 138, 80.70%) had GCS of 10 and above at admission indicating their neurological status. None of the patients had GCS of 3, and 19.30% (n ¼ 33) patients had GCS of 4e9 on admission. Demographic and clinical details of premature ischemic stroke patients and control subjects are shown in Table 1. Proportion of male subjects was higher in patients as well in controls, 76.02% and 68.27%, respectively. Similarly, distribution of age between patients (27.70 ± 10.63 years) and controls (27.02 ± 6.01 years) was also not significantly different (P ¼ 0.445) (Table 1). Conventional vascular risk factors, viz., smoking, alcohol use, hypertension and diabetes, were more prevalent in patients as compared to the control group (Table 1). Young ischemic stroke patients were also found to have significantly altered lipid profile as compared to controls; mean levels of TC, TG and LDL-C were found to be significantly elevated in patients as compared to controls (P < 0.05). Levels of HDL-C were also lower in patients as compared to controls, and this differential distribution was of borderline statistical significance (P ¼ 0.06). Mean plasma levels of tHcy and fHcy were found to be higher in patients as compared to controls (tHcy, 20.79 ± 1.77 mmol/L vs. 11.10 ± 0.23 mmol/L, P < 0.0001, respectively, and fHcy, 3.68 ± 0.14 vs. 1.99 ± 0.05 mmol/L, P < 0.0001, respectively). Plasma levels of tMet were significantly lower in ischemic stroke patients as compared to controls (21.59 ± 8.86 vs. 24.80 ± 9.86 mmol/L, P < 0.0001). Plasma levels of B-vitamins namely, B6, B12 and folic acid were found to be lower in patients as compared to controls (Table 1). In controls, tHcy levels were found to correlate significantly with folate (r ¼ 0.126, P ¼ 0.047), vitamin B6 (r ¼ 0.154, P ¼ 0.015), and tMet levels (r ¼ 0.209, P ¼ 0.001) (Fig. 1). Correlation between plasma tHcy and vitamin B12 levels was found to be at borderline significance (r ¼ 0.122, P ¼ 0.05). Plasma fHcy levels were found to correlate significantly with tMet levels (r ¼ 0.153, P ¼ 0.016) in controls (Fig. 1). However, in controls, no significant correlations could be established between plasma fHcy and any of the vitamin B levels; folate (r ¼ 0.063, P ¼ 0.319), vitamin B6 (r ¼ 0.081, P ¼ 0.205) and vitamin B12 (r ¼ 0.023, P ¼ 0.723) (Table 2). Similarly, no correlation could be established between any of the studied vitamins and between vitamins and tMet levels (Fig. 1). In patients, plasma tHcy levels were found to correlate significantly with vitamin B6 (r ¼ 0.143, P ¼ 0.024), folate (r ¼ 0.354, P ¼ 0.031) and correlation between tHcy and vitamin B12 was found to be at borderline significance (r ¼ 0.142,
Table 2 Odds ratios (ORs) for risk of ischemic stroke in young for plasma levels of tHcy, fHcy and tMet. Variable
Odds Ratio (95% CI), P value Model II
Model IIIa/IIIb/IIIc
Model IV(IVa/IVb/IVc)
Model V (Va/Vb/Vc)
6.44 (3.92e10.56), P < 0.0001 8.41 (5.30e13.34), P < 0.0001 2.74 (1.59e4.71), P < 0.0001
5.99 (3.54e10.15), P < 0.0001 9.26 (5.62e15.26), P < 0.0001 2.65 (1.49e4.72), P ¼ 0.001
a
a
a
5.91 (3.54e9.89), P < 0.0001 8.24 (4.88e13.92), P < 0.0001
5.22 (3.04e8.97), P < 0.0001 8.23 (4.75e14.27), P < 0.0001
Model I tHcy (>15.00 vs. 15.00 mmol/L) fHcy (>2.90 vs. 2.90 mmol/L) tMet (<13.86 vs. 13.86 mmol/L)
tHcy/tMet (>0.89 vs. 0.89) fHcy/tMet (>0.17 vs. 0.17)
6.17 (3.62e10.54), P < 0.0001 a 9.55 (5.75e15.87), P < 0.0001 b 2.69 (1.48e4.91), P ¼ 0.033 c 1.89 (0.96e3.73), P ¼ 0.066 e e
5.51 (3.16e9.62), P < 0.0001 a 10.38 (6.02e17.90), P < 0.0001 b 2.53 (1.33e4.81), P ¼ 0.005 c 1.78 (0.86e3.70), P ¼ 0.119 4.42 (2.51e7.76), P < 0.0001 9.28 (5.10e16.90), P < 0.0001
0.24 (0.03e1.69), P ¼ 0.152 a 9.62 (3.51e26.40), P < 0.0001 b 3.58 (1.05e12.16), P ¼ 0.041 c 2.52 (0.61e10.38), P ¼ 0.200 1.01 (0.29e3.51), P ¼ 0.985 12.81 (4.29e38.25), P < 0.0001
Abbreviations: tHcy, total homocysteine; fHcy, free homocysteine; tMet, total methionine. Model I, unadjusted; Model II, adjusted for age, gender, hypertension, diabetes mellitus, smoking, alcohol drinking, lipid levels; model III, subcategorized into IIIa/IIIb/IIIc, depending upon the respective differential adjustment for variables, tMet/tHcy/fHcy;Models IVa/IVb/IVc, additionally adjusted for vitamin B12 and folate levels; Models Va/ Vb/Vc, further adjusted for vitamin B6 levels
Please cite this article in press as: Rudreshkumar KJ, et al., Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.07.005
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Fig. 1. Scatter-plot matrix for tHcy, fHcy, tMet, vitamin B12, folate, and vitamin B6 representing the pairwise associations between each of these variables. Correlation analyses of the pairwise relationships were estimated. Circles represent patients and triangles represent controls. In controls, Pearson's/Spearman's pairwise correlations for tHcy were as follows: vs. fHcy: 0.523 (P < 0.05),vs. tMet: 0.209 (P < 0.05), vs. B6: 0.154 (P < 0.05), vs. B12: 0.122 (NS), vs. Folate: 0.126 (P < 0.05). In patients, pairwise correlations for tHcy vs. fHcy: 0.122 (NS), vs. tMet: 0.077 (NS), vs. B6: ¼ 0.143 (P < 0.05), vs. B12: 0.142 (BS), and vs. folate: 0.354 (P < 0.05). In controls, pairwise correlations for fHcy were as follows: vs. tMet: 0.153 (P < 0.05), B6: 0.081 (NS), vs. B12: 0.023 (NS), vs. Folate: 0.063 (NS). In patients, pairwise correlations for fHcy vs. tMet: 0.048 (NS), vs. B6: 0.044 (NS), vs. B12: 0.138(NS), and vs. folate: 0.08 (NS). In controls, pairwise correlations for tMet were as follows: vs. B6: 0.093 (NS), vs. B12: 0.087 (NS), vs. Folate: 0.038 (NS). In patients, pairwise correlations for tMet vs. B6: 0.022 (NS), vs. B12: 0.035 (NS), vs. Folate: 0.052 (NS). NS: non significant, BS: borderline significant.
P ¼ 0.06). However, correlations between plasma tHcy and tMet (r ¼ 0.077, P ¼ 0.319) were lost in patients. In patients, plasma fHcy levels were also not found to correlate significantly with tMet levels (r ¼ 0.048, P ¼ 0.532). No significant correlations could be established between plasma fHcy and any of the vitamin B levels in patients as well; folate (r ¼ 0.08, P ¼ 0.281), vitamin B6 (r ¼ 0.044, P ¼ 0.569) and vitamin B12 (r ¼ 0.138, P ¼ 0.071)
(Table 2). Further, we could establish significant correlation between fHcy and tHcy levels in controls (r ¼ 0.523, P ¼ 0.001), however, the correlation was lost in patients (r ¼ 0.122, P ¼ 0.113). We performed logistic regression analyses to assess associations between elevated levels of total and free levels of homocysteine, and their precursor methionine, with the risk of premature ischemic stroke (Table 2). When adjusted for
Please cite this article in press as: Rudreshkumar KJ, et al., Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.07.005
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conventional risk factors (Model IVa), elevated tHcy levels (>15 mmol/L) were found to predict 5.5-fold risk (OR ¼ 5.51, 95% CI ¼ 3.16e9.62, P < 0.0001) of ischemic stroke in young subjects (Table 2). However, additional adjustment for vitamin B6 abrogated the association (OR ¼ 0.24, 95% CI ¼ 0.03e1.69, P ¼ 0.152) (Table 2). On the contrary, elevated levels of fHcy (>2.9 mmol/L) were found to independently predict the risk of stroke in young, when adjusted for all the studied covariates including vitamin B6 levels (adjusted OR ¼ 9.62, 95% CI ¼ 3.51e26.40, P < 0.0001) (Table 2). The association between low tMet levels (<13.86 mmol/ L) and ischemic stroke was established to be independent of conventional risk factors, vitamin B levels as well as tHcy levels, model bV (OR ¼ 3.58, 95% CI ¼ 1.05e12.16, P ¼ 0.041). However when the logistic regression model was adjusted for fHcy levels, the risk of ischemic stroke was found to attenuate, model cV (OR ¼ 2.52, 95% CI ¼ 0.61e10.38, P ¼ 0.200) (Table 2). Secondarily, we analyzed the association between elevated ratios of tHcy/tMet and fHcy/tMet with risk of ischemic stroke in young. In completely adjusted logistic regression model, the elevated values of tHcy/tMet ratio were not found to independently predict the risk of ischemic stroke (OR ¼ 1.01, 95% CI ¼ 0.29e3.51, P ¼ 0.985) (Table 2). On the contrary, elevated fHcy/tMet ratio was found to significantly and independently predict the risk of ischemic stroke (OR ¼ 12.81, 95% CI ¼ 4.29e38.25, P < 0.0001); notably, the association was found to be negatively confounded by B6 levels (Table 2, Model V). Since adjustment with vitamin B6 levels attenuated the association between tHcy levels and ischemic stroke, we attempted to further clarify the independent risk potential of low vitamin B6 for premature ischemic stroke. There was a marked difference in levels of B6 between patients and controls (42.52 ± 11.96 nmol/L vs. 68.29 ± 9.03 nmol/L, P < 0.001, respectively) (Table 1). However, when the widely used cut off of 20 nmol/l [28] was used to define vitamin B6 deficiency, none of the controls and only 3 (1.75%) stroke patients were found to be deficient. When categorized by serum values of 20e30 nmol/l for suboptimal status of B6 [29], we observed 18 (10.53%) patients to have suboptimal status of the vitamin as compared to none of controls. Due to lack of distribution of deficient and suboptimum values of B6 in controls, odds ratio of the ischemic stroke risk conferred by low B6 could not be calculated. ROC curve analysis was done to evaluate the cut off for deficient vitamin B6 status with reference to the predictive risk of ischemic stroke in young. The findings of ROC curve analysis indicated 59.2 nmol/l (sensitivity, 89.00%, specificity, 91.80%) as the best cut off for defining low vitamin B6; area under the curve (AUC ¼ 0.969, 95% CI-0.95e0.99) (Supplementary Fig. 1). Using the cut-off of 59.2 nmol/L, we redefined low/compromised vitamin B6 status and analyzed the association between vitamin B6 levels and ischemic stroke in the young. We found very strong, ~170-fold, independent risk of premature ischemic stroke (OR ¼ 170.80, 95% CI ¼ 58.22e501.06, P < 0.0001) conferred by low vitamin B6 levels (Table 3).
5
4. Discussion The vascular risk potential of homocysteine, a non-protein aamino acid that serves as an intermediate in methionine metabolism, has been recognized for decades. Elevation of tHcy has been ascribed to its impaired metabolism [4,5]. In the present study, elevation in tHcy and fHcy levels was observed along with reduced levels of B vitamins and tMet, in young ischemic stroke patients, indicative of their impaired homocysteine metabolism. In controls, tHcy levels were found to significantly inversely correlate with B6, folic acid levels and tMet levels. Our findings support the established and widely accepted view on the metabolism of homocysteine [32], which commences from its precursor methionine and diverges into two intersecting pathways: remethylation to methionine, requiring folate and vitamin B12; and trans-sulfuration to cystathionine, vitamin B6. Our findings strengthen the roles of vitamins B6 and folate, as strong determinants of tHcy [33,34]. The borderline significant correlation between B12 and tHcy is in line with the previously reported controversial status of this relation [33e35]. Available findings suggest that vitamin B12 might not crucially modulate physiological homocysteine status [34]. Unlike controls, in patients, correlation between plasma tHcy and tMet levels was absent. This indicates that low plasma status of tMet in young ischemic stroke patients is primarily influenced by their reduced dietary intake of methionine rather than homocysteine metabolism [18]. The causality of association between tHcy and cardiovascular diseases has been debated for long, owing to the failed clinical trials on HLT [6,8]. The failed trials point towards the “not thoroughly explored” high cardiovascular risk potential of the non-protein bound, free-fraction of homocysteine, fHcy [16,17]. We investigated if elevated fHcy could confer a more pronounced risk of premature ischemic stroke, compared to elevated tHcy. This is the first study to establish a strong independent [~10-fold (adjusted OR ¼ 9.62, 95% CI ¼ 3.51e26.40)] risk association between elevated levels of fHcy (>2.9 mmol/L) and premature, ischemic stroke. This tenfold association was more robust as compared to the previously reported associations of elevated tHcy with stroke [36e38]. Our finding supports the previous report on acute coronary syndrome (ACS) [17]. Authors reported fHcy as a better predictor of recurrence of cardiovascular events in ACS patients, compared to insignificant predictive value of elevated tHcy [17]. In the present study, elevated tHcy (>15.0 mmol/L)was found to confer ~5-fold risk of ischemic stroke (OR ¼ 5.51, 95% CI ¼ 3.16e9.62, P < 0.0001) in multivariate model. However, the association was lost when the model was further adjusted by vitamin B6 (OR ¼ 0.22, 95% CI ¼ 0.03e1.55, p ¼ 0.12). Vitamin B6 is an important nutritional co-factor in the trans-sulfuration pathway of homocysteine metabolism [32]. Independent of its coenzyme functions, the vitamin also exhibits various other biological attributes as anti-oxidant and antiinflammatory molecule [39,40]. The observed confounding effect of vitamin B6 on risk association between tHcy and ischemic stroke, indicates that high tHcy status of patients could be a mere
Table 3 Odds ratios (ORs) for risk of ischemic stroke in young for plasma levels of vitamin B6. Variable
Vitamin B6 (<59.2 vs. 59.2 mmol/L)
Odds Ratio (95% CI), P value Model I
Model II
Model III
Model IVa/IVa/IVc
92.21 (46.85e181.47), P < 0.0001
147.43 (63.13e344.26), P < 0.0001
138.08 (58.44e326.24), P < 0.0001
a
129.66 (53.54e314.02), P < 0.0001 b 144.23 (59.88e347.40), P < 0.0001 c 170.80 (58.22e501.06), P < 0.0001
Model I, unadjusted; Model II, adjusted for age, gender, hypertension, diabetes mellitus, smoking, alcohol drinking, lipid levels; Models III, additionally adjusted for vitamin B12 and folic acid levels; Model IVa/IVb/IVc further adjusted for tMet/tHcy/fHcy levels respectively. Represents the odds ratio obtained after adjustment with fHcy levels, the most significant confounding variable of the study.
Please cite this article in press as: Rudreshkumar KJ, et al., Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.07.005
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K.J. Rudreshkumar et al. / Clinical Nutrition xxx (2017) 1e7
reflection of the pathogenic, low/sub-optimum vitamin B6 status. We hypothesized an independent premature ischemic stroke risk potential of the vitamin. Only 1.75% of young patients were B6 deficient and 10.53% had sub-optimal status based upon the respective cut offs of 20 and 30 nmol/l. None of controls were deficient in vitamin B6, therefore, OR for risk association with ischemic stroke could not be calculated. Surprisingly, ROC-curve analysis indicated a strikingly higher cut-off value of 59.2 nmol/L of vitamin B6, for prediction of the risk of premature ischemic stroke. Vitamin B6 levels, <59.2 nmol/l were found to independently predispose young individuals to ~170-fold risk of ischemic stroke (OR ¼ 170.80, 95% CI ¼ 58.22e501.06, P < 0.0001). This finding raises the possibility of a higher cutoff of vitamin B6 for defining the adequate vascular health and identification of another treatable and controllable risk factor for prevention of ischemic stroke in young individuals. Consistent with previous report of Oijen et al., which stated weak correlation between plasma fHcy and tHcy levels in acute coronary syndrome patients [17], we observed lack of correlation between fHcy and tHcy levels in patients (r ¼ 0.122, p ¼ 0.113). This indicates that tHcy measurements might not reflect the correct invivo status of the biologically more deleterious, fHcy, in patients and hence, explains the futility of clinical trials in patients targeted for reductions in tHcy [6e8]. We also observed lack of correlation between fHcy levels and any of the studied B vitamins. This indicates that the plasma status of non-protein bound fHcy is not modulated by B vitamins and hence, might not respond to HLT, implemented by B-vitamin supplementation. This further resolves the documented failures of clinical trials on vitamin B supplementation. Our finding supports the postulate by Ebbing et al. [8] stating “the detrimental fraction of homocysteine, might not respond to B vitamin supplementation”. However, contrary to our finding, a link between B vitamin supplementation, improved endothelium-dependent dilatation and reductions in fHcy concentration has been suggested previously [16]. Hence, the relationship between fHcy and B vitamins, needs thorough large-scale investigations. Further, elevated tHcy level could be a mere marker of altered methylation status, which primarily contributes to the pathogenesis of ischemic stroke. Impaired homocysteine metabolism is associated with reduced methionine status, and a concomitant reduction in Ado Met levels, and altered global DNA methylation status, as evident in vascular diseases [18e20]. We investigated the independent premature-ischemic stroke risk potential of altered status of methionine, the homocysteine precursor, and marker of global methylation status. No independent association could be established between low tMet status and risk of ischemic stroke (OR ¼ 2.52, 95% CI ¼ 0.61e10.38, P ¼ 0.200); rather, the association was found to be confounded/mediated by fHcy levels. This further strengthens the possibility of a significant contribution of fHcy in ischemic stroke etiology. We extrapolated the finding to plausible mechanistic link of tMet and fHcy with the detrimental metabolite of homocysteine, Hcy-thiolactone (Supplementary Fig. 2). Hcythiolactone is formed by methionyl-tRNA synthetase in an errorediting reaction in protein biosynthesis, by mistaken selection of Hcy in place of Met [41]. We tested the risk of ischemic stroke in young conferred by elevated tHcy/tMet and fHcy/tMet ratios, as markers of Hcy-thiolactone status. Mechanistically, homocysteinethiolactone formation is favored by low tMet levels and elevated homocysteine levels consequent of impaired homocysteine metabolism [41]. As well evident impaired homocysteine metabolism might involve inhibition of crucial enzymes of Hcy-metabolism i.e., either B6-dependent cystathionine b-synthase or B12- and folatedependent methionine synthase, respectively. Elevated tHcy/tMet ratio was not an independent risk factor for premature ischemic
stroke. Elevated fHcy/tMet ratio was found to have a high risk potential (OR ¼ 12.81, 95% CI ¼ 4.29e38.25, P < 0.0001), however, the association was negatively confounded by vitamin B6. Notably, the negative confounding effect of B6 indicates towards an intricate role of reduced tMet status in Hcy-thiolactone formation (Supplementary Fig. 2). Due to impaired trans-sulfuration, we expect an above normal status of tMet in vitamin B6 compromised individuals. Hence, despite their elevated fHcy, Hcy-thiolactone formation might not be favored in B6-compromised individuals due to their above-normal tMet status. Hypothetically, in such individuals, elevated fHcy could lead to vascular damage via pathogenic mechanisms other than Hcy-thiolactone formation. Findings of the present study are limited by its cross-sectional design, small sample size, and lack of direct assessment of Hcythiolactone levels and markers of atherosclerosis such as carotid intima-media thickness. We conclude that elevated fHcy and compromised vitamin B6 status could serve as strong risk factors for premature ischemic stroke, however, the causality of these associations need prospective and large scale validations. Further, our findings highlight the crucial confounding effects of B-vitamins on risk association between tHcy and premature-ischemic stroke. Statement of authorship Kondapura Jayadevappa Rudresh Kumar- Data collection, data analysis and data interpretation. Vijaya Majumdar- Data analysis, data interpretation and manuscript writing. Dindagur NagarajaStudy design, supervision of research work. Rita Christopher- Study design, supervision of research work, data interpretation, review of the manuscript. All authors read and approved the final manuscript. Conflict of interest None declared. Acknowledgment and funding This work was supported by financial assistance from the Department of Biotechnology (DBT), Government of India, New Delhi. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.clnu.2017.07.005. References [1] Ji R, Schwamm LH, Pervez MA, Singhal AB. Ischemicstroke and transient ischemic attack in young adults: risk factors, diagnostic yield, neuroimaging, and thrombolysis. JAMA Neurol 2013;70:51e7. [2] Wasay M, Khatri IA, Kaul S. Stroke in South Asian countries. Nat Rev Neurol 2014;10:135e43. [3] Griffiths D, Sturm J. Epidemiology and etiology of young stroke. Stroke Res Treat 2011;2011:209370. [4] Bos MJ, van Goor ML, Koudstaal PJ, Dippel DW. Plasma homocysteine is a risk factor for recurrent vascular events in young patients with an ischemicstroke or TIA. J Neurol 2005;252:332e7. [5] Biswas A, Ranjan R, Meena A, Akhter MS, Yadav BK, Munisamy M, et al. Homocystine levels, polymorphisms and the risk of ischemic stroke in young Asian Indians. J Stroke Cerebrovasc Dis 2009 MareApr;18(2):103e10. http:// dx.doi.org/10.1016/j.jstrokecerebrovasdis.2008.09.014. [6] Lonn E, Yusuf S, Arnold MJ, Sheridan P, Pogue J, Micks M, et al. Heart Outcomes Prevention Evaluation (HOPE) 2 Investigators. Homocysteine lowering with folic acid and B vitamins in vascular disease. N Engl J Med 2006 Apr 13;354(15):1567e77. [7] Bønaa KH, Njølstad I, Ueland PM, Schirmer H, Tverdal A, Steigen T, et al., NORVIT Trial Investigators. Homocysteine lowering and cardiovascular events after acute myocardial infarction. N Engl J Med 2006 Apr 13;354(15):1578e88.
Please cite this article in press as: Rudreshkumar KJ, et al., Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.07.005
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Original article
Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young Kondapura Jayadevappa Rudreshkumar a, 1, Vijaya Majumdar a, 1, Dindagur Nagaraja b, Rita Christopher a, * a b
Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences, Bengaluru, India Department of Neurology, National Institute of Mental Health and Neuro Sciences, Bengaluru, India
a r t i c l e i n f o
s u m m a r y
Article history: Received 29 November 2016 Accepted 7 July 2017
Background & aims: The debated vascular risk potential of total homocysteine (tHcy), due to failed clinical trials designed on B vitamin supplementation, raises many possible explanations like the higher risk potential of the deleterious, free form of homocysteine (fHcy) or, the unchecked confounding effects of B-vitamins in tHcy-based association studies. Additionally, the cardiovascular risk probability of altered status of the homocysteine precursor, methionine (tMet) could shed light on the causality of association between tHcy and cardiovascular diseases. Hence, we aimed to evaluate the risk associations of elevated plasma levels of tHcy, fHcy and low levels of tMet with premature, ischemic stroke. Methods: We recruited 171 young, ischemic stroke patients (aged 45 years) and 249 age- and gendermatched healthy controls. Plasma levels of fHcy, tHcy, tMet and vitamin B6 were estimated using HPLC coupled with coulometric electrochemical detection. Plasma levels of vitamin B12 and folate were estimated by radioimmunoassay. Results: Elevated fHcy (>2.9 mmol/L) was independently and strongly associated with the risk of premature, ischemic stroke (OR ¼ 9.62, 95% CI ¼ 3.51e26.40). On the contrary, association between premature ischemic stroke and elevated tHcy (>15.0 mmol/L) was found to attenuate when adjusted for vitamin B6 values (OR ¼ 0.24, 95%, CI ¼ 0.03e1.69). Interestingly, compromised B6-status (<59.2 nmol/l) was found to confer high risk of premature ischemic stroke (OR ¼ 170.80, 95% CI ¼ 58.22e501.06). We could not establish any significant correlation between fHcy and B-vitamin levels (P > 0.05). Low tMet (<13.86 mmol/L) was also not significantly associated with premature, ischemic stroke (OR ¼ 2.53, 95% CI ¼ 0.613e10.38). Conclusion: Our results indicate significant but not-correlated, independent associations of fHcy and vitamin B6 with risk of premature, ischemic stroke. However, the causality of these associations need prospective and large scale validations. Further, our findings highlight the crucial confounding effects of B-vitamins on risk association between tHcy and premature ischemic stroke. © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Keywords: Free homocysteine Total homocysteine Methionine Premature ischemic stroke
1. Introduction Stroke occurring in young adults, which constitutes 10%e14% of total strokes [1,2], has severe socio-economic consequences particularly in developing countries like India. Current understanding of ischemic stroke etiology points towards
* Corresponding author. Department of Neurochemistry, National Institute of Mental Health & Neuro Sciences (NIMHANS), Bengaluru, 560029, Karnataka, India. Fax: þ080 26564830. E-mail addresses: [email protected], [email protected] (R. Christopher). 1 Equal contribution by these authors.
atherosclerosis as a major contributing factor in young individuals [3]. This indicates a pressing need for the development of appropriate preventive strategies against modifiable and controllable, potentially atherogenic, risk factors such as homocysteine. Although there are studies implicating total homocysteine (tHcy) as a risk factor for ischemic stroke in the young [4,5], the potency and direct causative role of homocysteine in cardiovascular diseases has been doubted owing to failed clinical trials on homocysteine-lowering therapies (HLTs) in disease prevention [6e8]. Further, the doubted causality of hyperhomocysteinemia raises the question whether homocysteine is a biomarker or a risk factor for cardiovascular diseases [9].
http://dx.doi.org/10.1016/j.clnu.2017.07.005 0261-5614/© 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
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Vitamin B supplementation has been considered the mainstay of HLT [10]. B vitamins, namely, vitamin B6 (pyridoxine/pyridoxalphosphate), vitamin B12, and folate (vitamin B9) play crucial roles in the metabolism of homocysteine and when administered, result in effective lowering of homocysteine [10]. In particular, vitamin B6 and folate have been demonstrated to have regulatory cardiovascular risk potential independent of homocysteine [11,12]. We hypothesize that the debated causality of tHcy for cardiovascular diseases, point towards many possibilities such as confounding effects of B-vitamin deficiencies in tHcy-based association studies [5,13,14]. It is therefore necessary to control the confounding effects of nutritional deficiencies of vitamins B12, B6, and folic acid, when evaluating the cardiovascular risk associated with elevated homocysteine levels [11e14]. Further, the failed clinical trials on tHcy reduction for cardiovascular disease prevention, highlight the unattended higher vascular risk potential of the deleterious, free form of homocysteine (fHcy). Biologically, the potency of tHcy is fractionated into its protein-bound (70%) and the more potent, free, non protein-bound form (fHcy) (30%); the latter includes reduced homocysteine and homocysteine disulfides [15]. It has been demonstrated that the fHcy associates strongly with vascular endothelial dysfunction [16]. Despite its reported high vascular risk potential [16,17], there has been a lack of thorough assessments on the influence of elevated levels of fHcy on cardiovascular risk including ischemic stroke. Mechanistically, the vascular pathogenicity of elevated homocysteine could derive from the intricately linked, impaired systemic methylation reactions [18]. The emerging role of epigenetic mechanisms in the pathophysiology of cardiovascular diseases support this view [19,20]. Altered status of methionine associated with impaired metabolism of homocysteine, has been considered an indicator of the compromised status of methyl metabolism [18]. We hypothesize that investigations on independent vascular risk potential of altered methionine status, could shed light on the causality of association between tHcy and cardiovascular diseases. Hence, based on our hypotheses, we investigated the risk of premature ischemic stroke associated with the crucial metabolites of homocysteine metabolism, tHcy, fHcy and tMet while controlling the confounding effects of B vitamins. 2. Materials and methods 2.1. Ethical approval This study was approved by the Ethics Committee of the National Institute of Mental Health and Neuro Sciences (NIMHANS), Bengaluru, India. Informed consent was obtained from all healthy and diseased volunteers. 2.2. Study participants We investigated 171 young stroke patients aged 45 years, reporting to the Neurological Services of our hospital. Diagnosis of ischemic stroke was affirmed by cranial computed tomography scan and/or magnetic resonance imaging within 24 h of hospital admission. The Glasgow coma scale (GCS) was used to evaluate the conscious state of the stroke patients during admission [21]. Patients were excluded from the study if diagnosed of i) hemorrhagic stroke ii) infantile hemiplegia iii) neuro-infection with resulting infarction iv) disseminated intra-vascular coagulation v)hypothyroidism vi) renal or liver dysfunction vii) malignancy or any other terminal illnesses. A total of 171 patients were studied for duration of 2 years. The control group consisted of 249 healthy volunteers, free from any pre-existing vascular diseases and matched for age, gender, ethnicity and socioeconomic status.
2.3. Anthropometric and biochemical analyses Demographic and anthropometric details, inclusive of variables: age, sex, smoking habits, alcohol use, presence of diabetes mellitus and hypertension were acquired from the study subjects. Fasting venous blood was withdrawn within 7 days of onset of ischemic stroke symptoms. Serum triglycerides (TG), total cholesterol (TC), and high density lipoprotein (HDL)-cholesterol concentrations were estimated. Lipid profile was measured on an automated, random access, clinical chemistry analyzer (Olympus AU640, Olympus Singapore Pvt. Ltd., Singapore) using commercial kits (Beckman Coulter, Inc., Clare, Ireland). Total cholesterol was estimated by enzymatic method based on combined actions of cholesterol esterase and oxidase [22], (assay kits, OSR 6116 and 6216). Triglyceride measurement was based on a series of coupled enzymatic reactions including hydrolysis by a combination of microbial lipases [assay kit, OSR 60118] [23]. HDL-C was estimated by an immune-inhibition method (assay kit, OSR 6187). Concentration of low density lipoprotein (LDL)-cholesterol was determined using Friedewald's formula [24]. Levels of vitamin B12 and folic acid were assayed by radioimmunoassay using commercially procured kits (SimulTRAC-SNB, MP Biomedicals, USA). 2.4. Assessment of plasma tHcy, fHcy, tMet and vitamin B6 by highperformance liquid chromatography Plasma tHcy, fHcy and tMet were measured by reverse-phase high-performance liquid chromatography (HPLC) and coulometric electrochemical detection using a Shimadzu HPLC system (LC10ADVP, Shimadzu Corporation, Japan) provided with an auto sampler (SIL-HTA) and a ESA Coulochem III detector (ESA Ins, Chelmsford, MA) [25]. Chromatographic separation was conducted at a flow rate of 1.0 ml/min and a pressure of 120e140 kg/cm2 (1800e2100 psi), using a reverse phase C18 Supelco column (5 m, 4.6 250 mm, Shimadzu). The active form of vitamin B6 in plasma, pyridoxal 50 phosphate (PLP) was also assayed by reverse-phase HPLC and coulometric electrochemical detection [26]. 2.5. Statistical analysis Data were analyzed using SPSS (V16.0) program. The differences in the distribution between patients and controls, of continuous variables with normal and skewed distributions, were tested by paired ‘t’ test and ManneWhitney U tests, respectively. All P values were 2-sided. Cut offs for elevated tHcy (15.00 mmol/l) and fHcy (2.90 mmol/l) were defined according to previous reports [27]. Low plasma tMet status was defined based on cut off of 13.86 mmol/l (10th percentile values in control population). Cut offs of elevated tHcy/tMet and fHcy/tMet were also based on 10th percentile values in controls (Table 2). Vitamin B6 deficient and suboptimal statuses were defined according to the recommended cut off values of 20 nmol/l [28] and 30 nmol/l [29] respectively. Stroke risks conferred by plasma levels of different total homocysteine, its free form and methionine levels were evaluated by logistic regression analyses. Multivariate analyses were constituted of covariates such as age, gender, smoking, alcohol consumption, hypertension, diabetes mellitus, lipid levels and B vitamin levels (vitamins B6, B12 and folate). Hypertension was characterized as diastolic blood pressure 90 mmHg and/or systolic blood pressure 140 mmHg [30] and/or use of antihypertensive medication. Diabetes was diagnosed on the basis of fasting plasma glucose values >126 mg/dl or the subjects' self-reported history of diabetes or use of anti-diabetic medication [31]. Coefficients of correlation were calculated using Pearson's/Spearmann's correlation analysis. Logistic regression analyses were carried out to test statistical
Please cite this article in press as: Rudreshkumar KJ, et al., Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.07.005
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calculated to evaluate the cut off for deficient vitamin B6 status with reference to the predictive risk of ischemic stroke in young.
Table 1 Distribution of covariates between ischemic stroke patients and controls. Covariates
Ischemic stroke patients (n ¼ 171)
Controls (n ¼ 249)
P value
Age (years) Males, n (%) Smoking, n (%) Alcohol use, n (%) Hypertensives, n (%) Diabetics, n (%) TC, (mmol/L) TG, (mmol/L) LDL-C, (mmol/L) HDL-C, (mmol/L) tHcy, (mmol/L) fHcy, (mmol/L) tMet, (mmol/L) Elevated tHcy (>15.00 mmol/L), n (%) Elevated fHcy (>2.90 mmol/L), n (%) Low tMet (<13.86 mmol/L), n (%) Vitamin B12, pmol/L Vitamin B6, nmol/L Folate, nmol/L
27.70 ± 10.63 130 (76.02) 56 (32.75) 42 (24.56) 7 (4.09) 7 (4.09) 5.43 ± 2.56 1.57 ± 1.35 3.56 ± 1.20 0.91 ± 1.30 20.79 ± 1.77 3.68 ± 0.14 21.59 ± 8.86 77 (45.03)
27.02 ± 6.01 170 (68.27) 20 (8.03) 39 (15.66) 2 (0.80) 1 (0.40) 4.57 ± 1.45 1.37 ± 0.50 2.80 ± 1.50 1.10 ± 0.99 11.10 ± 0.23 1.99 ± 0.05 24.80 ± 9.86 29 (11.65)
0.445 0.100 <0.001 <0.001 0.035 0.009 <0.001 0.033 0.041 0.060 <0.001 <0.001 <0.001 <0.001
103 (60.23)
38 (15.26)
<0.001
40 (23.39)
25 (10.04)
<0.001
315.50 ± 18.73 42.52 ± 11.96 13.99 ± 0.81
475.09 ± 17.90 68.29 ± 9.03 20.02 ± 0.81
<0.001 <0.001 <0.001
3. Results
Abbreviations: High density lipoprotein cholesterol, (HDL)-C; low density lipoprotein cholesterol, LDL-C; total homocysteine, tHcy; free homocysteine, fHcy; total methionine, tMet; Total cholesterol, TC; triglycerides, TG.
association between altered levels of tHcy, fHcy and tMet and premature ischemic stroke. Various multivariate models were generated, the first model was the crude unadjusted model. The second model, model II, was adjusted for the following confounders; age, gender, hypertension, diabetes mellitus, smoking, alcohol drinking, lipid levels. Further, model III was subcategorized into IIIa/IIIb/IIIc, based on the respective differential adjustment for variables, tMet/tHcy/fHcy levels respectively. Subsequently, models IVa/IVb/IVc were additionally adjusted for vitamin B12 and folic acid levels. Models Va/Vb/Vcwere generated by additional adjustment for vitamin B6 levels. Similarly, for evaluation of risk of ischemic stroke in the young, conferred by compromised vitamin B6 levels, various models were generated for logistic regression analyses. Model I was the crude unadjusted model. The second model, model II, was adjusted for age, gender, hypertension, diabetes mellitus, smoking, alcohol drinking, lipid levels; Models III, was additionally adjusted for vitamin B12 and folic acid levels; Model IVa/IVb/IVc was subcategorized for differential adjustment for tMet/tHcy/fHcy levels respectively. The receiver operating characeteristic (ROC) curve analysis was carried out and area under the curve (AUC) was
Majority of patients (n ¼ 138, 80.70%) had GCS of 10 and above at admission indicating their neurological status. None of the patients had GCS of 3, and 19.30% (n ¼ 33) patients had GCS of 4e9 on admission. Demographic and clinical details of premature ischemic stroke patients and control subjects are shown in Table 1. Proportion of male subjects was higher in patients as well in controls, 76.02% and 68.27%, respectively. Similarly, distribution of age between patients (27.70 ± 10.63 years) and controls (27.02 ± 6.01 years) was also not significantly different (P ¼ 0.445) (Table 1). Conventional vascular risk factors, viz., smoking, alcohol use, hypertension and diabetes, were more prevalent in patients as compared to the control group (Table 1). Young ischemic stroke patients were also found to have significantly altered lipid profile as compared to controls; mean levels of TC, TG and LDL-C were found to be significantly elevated in patients as compared to controls (P < 0.05). Levels of HDL-C were also lower in patients as compared to controls, and this differential distribution was of borderline statistical significance (P ¼ 0.06). Mean plasma levels of tHcy and fHcy were found to be higher in patients as compared to controls (tHcy, 20.79 ± 1.77 mmol/L vs. 11.10 ± 0.23 mmol/L, P < 0.0001, respectively, and fHcy, 3.68 ± 0.14 vs. 1.99 ± 0.05 mmol/L, P < 0.0001, respectively). Plasma levels of tMet were significantly lower in ischemic stroke patients as compared to controls (21.59 ± 8.86 vs. 24.80 ± 9.86 mmol/L, P < 0.0001). Plasma levels of B-vitamins namely, B6, B12 and folic acid were found to be lower in patients as compared to controls (Table 1). In controls, tHcy levels were found to correlate significantly with folate (r ¼ 0.126, P ¼ 0.047), vitamin B6 (r ¼ 0.154, P ¼ 0.015), and tMet levels (r ¼ 0.209, P ¼ 0.001) (Fig. 1). Correlation between plasma tHcy and vitamin B12 levels was found to be at borderline significance (r ¼ 0.122, P ¼ 0.05). Plasma fHcy levels were found to correlate significantly with tMet levels (r ¼ 0.153, P ¼ 0.016) in controls (Fig. 1). However, in controls, no significant correlations could be established between plasma fHcy and any of the vitamin B levels; folate (r ¼ 0.063, P ¼ 0.319), vitamin B6 (r ¼ 0.081, P ¼ 0.205) and vitamin B12 (r ¼ 0.023, P ¼ 0.723) (Table 2). Similarly, no correlation could be established between any of the studied vitamins and between vitamins and tMet levels (Fig. 1). In patients, plasma tHcy levels were found to correlate significantly with vitamin B6 (r ¼ 0.143, P ¼ 0.024), folate (r ¼ 0.354, P ¼ 0.031) and correlation between tHcy and vitamin B12 was found to be at borderline significance (r ¼ 0.142,
Table 2 Odds ratios (ORs) for risk of ischemic stroke in young for plasma levels of tHcy, fHcy and tMet. Variable
Odds Ratio (95% CI), P value Model II
Model IIIa/IIIb/IIIc
Model IV(IVa/IVb/IVc)
Model V (Va/Vb/Vc)
6.44 (3.92e10.56), P < 0.0001 8.41 (5.30e13.34), P < 0.0001 2.74 (1.59e4.71), P < 0.0001
5.99 (3.54e10.15), P < 0.0001 9.26 (5.62e15.26), P < 0.0001 2.65 (1.49e4.72), P ¼ 0.001
a
a
a
5.91 (3.54e9.89), P < 0.0001 8.24 (4.88e13.92), P < 0.0001
5.22 (3.04e8.97), P < 0.0001 8.23 (4.75e14.27), P < 0.0001
Model I tHcy (>15.00 vs. 15.00 mmol/L) fHcy (>2.90 vs. 2.90 mmol/L) tMet (<13.86 vs. 13.86 mmol/L)
tHcy/tMet (>0.89 vs. 0.89) fHcy/tMet (>0.17 vs. 0.17)
6.17 (3.62e10.54), P < 0.0001 a 9.55 (5.75e15.87), P < 0.0001 b 2.69 (1.48e4.91), P ¼ 0.033 c 1.89 (0.96e3.73), P ¼ 0.066 e e
5.51 (3.16e9.62), P < 0.0001 a 10.38 (6.02e17.90), P < 0.0001 b 2.53 (1.33e4.81), P ¼ 0.005 c 1.78 (0.86e3.70), P ¼ 0.119 4.42 (2.51e7.76), P < 0.0001 9.28 (5.10e16.90), P < 0.0001
0.24 (0.03e1.69), P ¼ 0.152 a 9.62 (3.51e26.40), P < 0.0001 b 3.58 (1.05e12.16), P ¼ 0.041 c 2.52 (0.61e10.38), P ¼ 0.200 1.01 (0.29e3.51), P ¼ 0.985 12.81 (4.29e38.25), P < 0.0001
Abbreviations: tHcy, total homocysteine; fHcy, free homocysteine; tMet, total methionine. Model I, unadjusted; Model II, adjusted for age, gender, hypertension, diabetes mellitus, smoking, alcohol drinking, lipid levels; model III, subcategorized into IIIa/IIIb/IIIc, depending upon the respective differential adjustment for variables, tMet/tHcy/fHcy;Models IVa/IVb/IVc, additionally adjusted for vitamin B12 and folate levels; Models Va/ Vb/Vc, further adjusted for vitamin B6 levels
Please cite this article in press as: Rudreshkumar KJ, et al., Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.07.005
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K.J. Rudreshkumar et al. / Clinical Nutrition xxx (2017) 1e7
Fig. 1. Scatter-plot matrix for tHcy, fHcy, tMet, vitamin B12, folate, and vitamin B6 representing the pairwise associations between each of these variables. Correlation analyses of the pairwise relationships were estimated. Circles represent patients and triangles represent controls. In controls, Pearson's/Spearman's pairwise correlations for tHcy were as follows: vs. fHcy: 0.523 (P < 0.05),vs. tMet: 0.209 (P < 0.05), vs. B6: 0.154 (P < 0.05), vs. B12: 0.122 (NS), vs. Folate: 0.126 (P < 0.05). In patients, pairwise correlations for tHcy vs. fHcy: 0.122 (NS), vs. tMet: 0.077 (NS), vs. B6: ¼ 0.143 (P < 0.05), vs. B12: 0.142 (BS), and vs. folate: 0.354 (P < 0.05). In controls, pairwise correlations for fHcy were as follows: vs. tMet: 0.153 (P < 0.05), B6: 0.081 (NS), vs. B12: 0.023 (NS), vs. Folate: 0.063 (NS). In patients, pairwise correlations for fHcy vs. tMet: 0.048 (NS), vs. B6: 0.044 (NS), vs. B12: 0.138(NS), and vs. folate: 0.08 (NS). In controls, pairwise correlations for tMet were as follows: vs. B6: 0.093 (NS), vs. B12: 0.087 (NS), vs. Folate: 0.038 (NS). In patients, pairwise correlations for tMet vs. B6: 0.022 (NS), vs. B12: 0.035 (NS), vs. Folate: 0.052 (NS). NS: non significant, BS: borderline significant.
P ¼ 0.06). However, correlations between plasma tHcy and tMet (r ¼ 0.077, P ¼ 0.319) were lost in patients. In patients, plasma fHcy levels were also not found to correlate significantly with tMet levels (r ¼ 0.048, P ¼ 0.532). No significant correlations could be established between plasma fHcy and any of the vitamin B levels in patients as well; folate (r ¼ 0.08, P ¼ 0.281), vitamin B6 (r ¼ 0.044, P ¼ 0.569) and vitamin B12 (r ¼ 0.138, P ¼ 0.071)
(Table 2). Further, we could establish significant correlation between fHcy and tHcy levels in controls (r ¼ 0.523, P ¼ 0.001), however, the correlation was lost in patients (r ¼ 0.122, P ¼ 0.113). We performed logistic regression analyses to assess associations between elevated levels of total and free levels of homocysteine, and their precursor methionine, with the risk of premature ischemic stroke (Table 2). When adjusted for
Please cite this article in press as: Rudreshkumar KJ, et al., Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.07.005
K.J. Rudreshkumar et al. / Clinical Nutrition xxx (2017) 1e7
conventional risk factors (Model IVa), elevated tHcy levels (>15 mmol/L) were found to predict 5.5-fold risk (OR ¼ 5.51, 95% CI ¼ 3.16e9.62, P < 0.0001) of ischemic stroke in young subjects (Table 2). However, additional adjustment for vitamin B6 abrogated the association (OR ¼ 0.24, 95% CI ¼ 0.03e1.69, P ¼ 0.152) (Table 2). On the contrary, elevated levels of fHcy (>2.9 mmol/L) were found to independently predict the risk of stroke in young, when adjusted for all the studied covariates including vitamin B6 levels (adjusted OR ¼ 9.62, 95% CI ¼ 3.51e26.40, P < 0.0001) (Table 2). The association between low tMet levels (<13.86 mmol/ L) and ischemic stroke was established to be independent of conventional risk factors, vitamin B levels as well as tHcy levels, model bV (OR ¼ 3.58, 95% CI ¼ 1.05e12.16, P ¼ 0.041). However when the logistic regression model was adjusted for fHcy levels, the risk of ischemic stroke was found to attenuate, model cV (OR ¼ 2.52, 95% CI ¼ 0.61e10.38, P ¼ 0.200) (Table 2). Secondarily, we analyzed the association between elevated ratios of tHcy/tMet and fHcy/tMet with risk of ischemic stroke in young. In completely adjusted logistic regression model, the elevated values of tHcy/tMet ratio were not found to independently predict the risk of ischemic stroke (OR ¼ 1.01, 95% CI ¼ 0.29e3.51, P ¼ 0.985) (Table 2). On the contrary, elevated fHcy/tMet ratio was found to significantly and independently predict the risk of ischemic stroke (OR ¼ 12.81, 95% CI ¼ 4.29e38.25, P < 0.0001); notably, the association was found to be negatively confounded by B6 levels (Table 2, Model V). Since adjustment with vitamin B6 levels attenuated the association between tHcy levels and ischemic stroke, we attempted to further clarify the independent risk potential of low vitamin B6 for premature ischemic stroke. There was a marked difference in levels of B6 between patients and controls (42.52 ± 11.96 nmol/L vs. 68.29 ± 9.03 nmol/L, P < 0.001, respectively) (Table 1). However, when the widely used cut off of 20 nmol/l [28] was used to define vitamin B6 deficiency, none of the controls and only 3 (1.75%) stroke patients were found to be deficient. When categorized by serum values of 20e30 nmol/l for suboptimal status of B6 [29], we observed 18 (10.53%) patients to have suboptimal status of the vitamin as compared to none of controls. Due to lack of distribution of deficient and suboptimum values of B6 in controls, odds ratio of the ischemic stroke risk conferred by low B6 could not be calculated. ROC curve analysis was done to evaluate the cut off for deficient vitamin B6 status with reference to the predictive risk of ischemic stroke in young. The findings of ROC curve analysis indicated 59.2 nmol/l (sensitivity, 89.00%, specificity, 91.80%) as the best cut off for defining low vitamin B6; area under the curve (AUC ¼ 0.969, 95% CI-0.95e0.99) (Supplementary Fig. 1). Using the cut-off of 59.2 nmol/L, we redefined low/compromised vitamin B6 status and analyzed the association between vitamin B6 levels and ischemic stroke in the young. We found very strong, ~170-fold, independent risk of premature ischemic stroke (OR ¼ 170.80, 95% CI ¼ 58.22e501.06, P < 0.0001) conferred by low vitamin B6 levels (Table 3).
5
4. Discussion The vascular risk potential of homocysteine, a non-protein aamino acid that serves as an intermediate in methionine metabolism, has been recognized for decades. Elevation of tHcy has been ascribed to its impaired metabolism [4,5]. In the present study, elevation in tHcy and fHcy levels was observed along with reduced levels of B vitamins and tMet, in young ischemic stroke patients, indicative of their impaired homocysteine metabolism. In controls, tHcy levels were found to significantly inversely correlate with B6, folic acid levels and tMet levels. Our findings support the established and widely accepted view on the metabolism of homocysteine [32], which commences from its precursor methionine and diverges into two intersecting pathways: remethylation to methionine, requiring folate and vitamin B12; and trans-sulfuration to cystathionine, vitamin B6. Our findings strengthen the roles of vitamins B6 and folate, as strong determinants of tHcy [33,34]. The borderline significant correlation between B12 and tHcy is in line with the previously reported controversial status of this relation [33e35]. Available findings suggest that vitamin B12 might not crucially modulate physiological homocysteine status [34]. Unlike controls, in patients, correlation between plasma tHcy and tMet levels was absent. This indicates that low plasma status of tMet in young ischemic stroke patients is primarily influenced by their reduced dietary intake of methionine rather than homocysteine metabolism [18]. The causality of association between tHcy and cardiovascular diseases has been debated for long, owing to the failed clinical trials on HLT [6,8]. The failed trials point towards the “not thoroughly explored” high cardiovascular risk potential of the non-protein bound, free-fraction of homocysteine, fHcy [16,17]. We investigated if elevated fHcy could confer a more pronounced risk of premature ischemic stroke, compared to elevated tHcy. This is the first study to establish a strong independent [~10-fold (adjusted OR ¼ 9.62, 95% CI ¼ 3.51e26.40)] risk association between elevated levels of fHcy (>2.9 mmol/L) and premature, ischemic stroke. This tenfold association was more robust as compared to the previously reported associations of elevated tHcy with stroke [36e38]. Our finding supports the previous report on acute coronary syndrome (ACS) [17]. Authors reported fHcy as a better predictor of recurrence of cardiovascular events in ACS patients, compared to insignificant predictive value of elevated tHcy [17]. In the present study, elevated tHcy (>15.0 mmol/L)was found to confer ~5-fold risk of ischemic stroke (OR ¼ 5.51, 95% CI ¼ 3.16e9.62, P < 0.0001) in multivariate model. However, the association was lost when the model was further adjusted by vitamin B6 (OR ¼ 0.22, 95% CI ¼ 0.03e1.55, p ¼ 0.12). Vitamin B6 is an important nutritional co-factor in the trans-sulfuration pathway of homocysteine metabolism [32]. Independent of its coenzyme functions, the vitamin also exhibits various other biological attributes as anti-oxidant and antiinflammatory molecule [39,40]. The observed confounding effect of vitamin B6 on risk association between tHcy and ischemic stroke, indicates that high tHcy status of patients could be a mere
Table 3 Odds ratios (ORs) for risk of ischemic stroke in young for plasma levels of vitamin B6. Variable
Vitamin B6 (<59.2 vs. 59.2 mmol/L)
Odds Ratio (95% CI), P value Model I
Model II
Model III
Model IVa/IVa/IVc
92.21 (46.85e181.47), P < 0.0001
147.43 (63.13e344.26), P < 0.0001
138.08 (58.44e326.24), P < 0.0001
a
129.66 (53.54e314.02), P < 0.0001 b 144.23 (59.88e347.40), P < 0.0001 c 170.80 (58.22e501.06), P < 0.0001
Model I, unadjusted; Model II, adjusted for age, gender, hypertension, diabetes mellitus, smoking, alcohol drinking, lipid levels; Models III, additionally adjusted for vitamin B12 and folic acid levels; Model IVa/IVb/IVc further adjusted for tMet/tHcy/fHcy levels respectively. Represents the odds ratio obtained after adjustment with fHcy levels, the most significant confounding variable of the study.
Please cite this article in press as: Rudreshkumar KJ, et al., Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.07.005
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reflection of the pathogenic, low/sub-optimum vitamin B6 status. We hypothesized an independent premature ischemic stroke risk potential of the vitamin. Only 1.75% of young patients were B6 deficient and 10.53% had sub-optimal status based upon the respective cut offs of 20 and 30 nmol/l. None of controls were deficient in vitamin B6, therefore, OR for risk association with ischemic stroke could not be calculated. Surprisingly, ROC-curve analysis indicated a strikingly higher cut-off value of 59.2 nmol/L of vitamin B6, for prediction of the risk of premature ischemic stroke. Vitamin B6 levels, <59.2 nmol/l were found to independently predispose young individuals to ~170-fold risk of ischemic stroke (OR ¼ 170.80, 95% CI ¼ 58.22e501.06, P < 0.0001). This finding raises the possibility of a higher cutoff of vitamin B6 for defining the adequate vascular health and identification of another treatable and controllable risk factor for prevention of ischemic stroke in young individuals. Consistent with previous report of Oijen et al., which stated weak correlation between plasma fHcy and tHcy levels in acute coronary syndrome patients [17], we observed lack of correlation between fHcy and tHcy levels in patients (r ¼ 0.122, p ¼ 0.113). This indicates that tHcy measurements might not reflect the correct invivo status of the biologically more deleterious, fHcy, in patients and hence, explains the futility of clinical trials in patients targeted for reductions in tHcy [6e8]. We also observed lack of correlation between fHcy levels and any of the studied B vitamins. This indicates that the plasma status of non-protein bound fHcy is not modulated by B vitamins and hence, might not respond to HLT, implemented by B-vitamin supplementation. This further resolves the documented failures of clinical trials on vitamin B supplementation. Our finding supports the postulate by Ebbing et al. [8] stating “the detrimental fraction of homocysteine, might not respond to B vitamin supplementation”. However, contrary to our finding, a link between B vitamin supplementation, improved endothelium-dependent dilatation and reductions in fHcy concentration has been suggested previously [16]. Hence, the relationship between fHcy and B vitamins, needs thorough large-scale investigations. Further, elevated tHcy level could be a mere marker of altered methylation status, which primarily contributes to the pathogenesis of ischemic stroke. Impaired homocysteine metabolism is associated with reduced methionine status, and a concomitant reduction in Ado Met levels, and altered global DNA methylation status, as evident in vascular diseases [18e20]. We investigated the independent premature-ischemic stroke risk potential of altered status of methionine, the homocysteine precursor, and marker of global methylation status. No independent association could be established between low tMet status and risk of ischemic stroke (OR ¼ 2.52, 95% CI ¼ 0.61e10.38, P ¼ 0.200); rather, the association was found to be confounded/mediated by fHcy levels. This further strengthens the possibility of a significant contribution of fHcy in ischemic stroke etiology. We extrapolated the finding to plausible mechanistic link of tMet and fHcy with the detrimental metabolite of homocysteine, Hcy-thiolactone (Supplementary Fig. 2). Hcythiolactone is formed by methionyl-tRNA synthetase in an errorediting reaction in protein biosynthesis, by mistaken selection of Hcy in place of Met [41]. We tested the risk of ischemic stroke in young conferred by elevated tHcy/tMet and fHcy/tMet ratios, as markers of Hcy-thiolactone status. Mechanistically, homocysteinethiolactone formation is favored by low tMet levels and elevated homocysteine levels consequent of impaired homocysteine metabolism [41]. As well evident impaired homocysteine metabolism might involve inhibition of crucial enzymes of Hcy-metabolism i.e., either B6-dependent cystathionine b-synthase or B12- and folatedependent methionine synthase, respectively. Elevated tHcy/tMet ratio was not an independent risk factor for premature ischemic
stroke. Elevated fHcy/tMet ratio was found to have a high risk potential (OR ¼ 12.81, 95% CI ¼ 4.29e38.25, P < 0.0001), however, the association was negatively confounded by vitamin B6. Notably, the negative confounding effect of B6 indicates towards an intricate role of reduced tMet status in Hcy-thiolactone formation (Supplementary Fig. 2). Due to impaired trans-sulfuration, we expect an above normal status of tMet in vitamin B6 compromised individuals. Hence, despite their elevated fHcy, Hcy-thiolactone formation might not be favored in B6-compromised individuals due to their above-normal tMet status. Hypothetically, in such individuals, elevated fHcy could lead to vascular damage via pathogenic mechanisms other than Hcy-thiolactone formation. Findings of the present study are limited by its cross-sectional design, small sample size, and lack of direct assessment of Hcythiolactone levels and markers of atherosclerosis such as carotid intima-media thickness. We conclude that elevated fHcy and compromised vitamin B6 status could serve as strong risk factors for premature ischemic stroke, however, the causality of these associations need prospective and large scale validations. Further, our findings highlight the crucial confounding effects of B-vitamins on risk association between tHcy and premature-ischemic stroke. Statement of authorship Kondapura Jayadevappa Rudresh Kumar- Data collection, data analysis and data interpretation. Vijaya Majumdar- Data analysis, data interpretation and manuscript writing. Dindagur NagarajaStudy design, supervision of research work. Rita Christopher- Study design, supervision of research work, data interpretation, review of the manuscript. All authors read and approved the final manuscript. Conflict of interest None declared. Acknowledgment and funding This work was supported by financial assistance from the Department of Biotechnology (DBT), Government of India, New Delhi. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.clnu.2017.07.005. References [1] Ji R, Schwamm LH, Pervez MA, Singhal AB. Ischemicstroke and transient ischemic attack in young adults: risk factors, diagnostic yield, neuroimaging, and thrombolysis. JAMA Neurol 2013;70:51e7. [2] Wasay M, Khatri IA, Kaul S. Stroke in South Asian countries. Nat Rev Neurol 2014;10:135e43. [3] Griffiths D, Sturm J. Epidemiology and etiology of young stroke. Stroke Res Treat 2011;2011:209370. [4] Bos MJ, van Goor ML, Koudstaal PJ, Dippel DW. Plasma homocysteine is a risk factor for recurrent vascular events in young patients with an ischemicstroke or TIA. J Neurol 2005;252:332e7. [5] Biswas A, Ranjan R, Meena A, Akhter MS, Yadav BK, Munisamy M, et al. Homocystine levels, polymorphisms and the risk of ischemic stroke in young Asian Indians. J Stroke Cerebrovasc Dis 2009 MareApr;18(2):103e10. http:// dx.doi.org/10.1016/j.jstrokecerebrovasdis.2008.09.014. [6] Lonn E, Yusuf S, Arnold MJ, Sheridan P, Pogue J, Micks M, et al. Heart Outcomes Prevention Evaluation (HOPE) 2 Investigators. Homocysteine lowering with folic acid and B vitamins in vascular disease. N Engl J Med 2006 Apr 13;354(15):1567e77. [7] Bønaa KH, Njølstad I, Ueland PM, Schirmer H, Tverdal A, Steigen T, et al., NORVIT Trial Investigators. Homocysteine lowering and cardiovascular events after acute myocardial infarction. N Engl J Med 2006 Apr 13;354(15):1578e88.
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Please cite this article in press as: Rudreshkumar KJ, et al., Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.07.005