Plasma Homocysteine in the Acute and Convalescent Phases After Stroke
Background and Purpose Stroke patients frequently manifest moderate hyperhomocysteinemia. In most published studies, plasma homocysteine was measured at least 1 month after stroke (or the interval was not reported). To determine whether plasma homocysteine concentrations change in the acute phase, we compared acute-phase values with both convalescent-phase and control values.
Methods Plasma homocysteine concentrations were measured in the acute phase (mean, 2 days after stroke onset) in 162 first-ever stroke patients aged 50 years or more (median, 75 years) and again at a median interval of 583 days (range, 460 to 645 days) after stroke onset in a subgroup of 17 patients, with values for 60 age-matched subjects serving as controls. Twenty of the control subjects were reexamined 2 to 3 years after their initial examination.
Results The median plasma homocysteine concentration was 13.4 μmol/L in the patient group compared with 13.8 μmol/L for control subjects (NS, Mann-Whitney U test) and increased from 11.4 μmol/L in the acute phase to 14.5 μmol/L in the convalescent phase in the subgroup of patients examined twice (P<.01, Wilcoxon signed rank test). In the 20 reexamined control subjects, no significant change over time in plasma homocysteine concentration was found.
Conclusions The post–acute-phase increase in plasma homocysteine may explain why higher values were obtained for stroke patients than for control subjects in previous studies. Possible reasons for the variation in plasma homocysteine concentrations over time are (1) an acute-phase reduction secondary to a decrease in plasma albumin and (2) an increase in plasma homocysteine during the convalescent phase due to modified vitamin intake and/or lifestyle. The timing of plasma homocysteine measurements relative to stroke onset is a factor to be considered in the interpretation of results.
Homocysteine is a sulfhydryl amino acid formed by the demethylation of methionine. Severe hyperhomocysteinemia (levels >100 μmol/L) has been related to early onset of arteriosclerosis and thromboembolic events including stroke,1 and moderate hyperhomocysteinemia (20 to 100 μmol/L) has been suggested to be a vascular risk factor. Previous studies have shown plasma homocysteine concentrations to be increased in 23% to 47% of patients with cerebral or peripheral occlusive arterial disease.1 However, in several studies of stroke patients, the analyses were performed at least 1 month after stroke onset or the time of measurement was not stated (Table 1⇓). Little has been published about possible variations of plasma homocysteine concentrations in the acute phase. The plasma homocysteine concentration is related to those of serum creatinine, cobalamin, folate, and vitamin B-6 (pyridoxal 5′-phosphate).2 3 Values for all these variables may change after stroke if patients change their lifestyle, including dietary habits, with a possible secondary effect on plasma homocysteine concentrations.
We examined plasma homocysteine concentrations in 162 stroke patients in the acute phase and compared the values with those for 60 control subjects. Each patient was assigned to one of four clinical stroke subgroups and underwent computed tomography (CT) of the brain. In a subset of patients (n=17), we repeated the measurements of plasma homocysteine in the convalescent phase. We also reexamined 20 control subjects 2 to 3 years after their initial examination.
Subjects and Methods
Patients and Control Subjects
From a consecutive series of 241 patients with stroke,4 which was defined as an acute focal neurological deficit lasting for more than 24 hours or leading to death with no other cause than cerebrovascular disease, 162 patients aged 50 years or older (150 with cerebral infarction and 12 with intracerebral hemorrhage) participated in this study. The 79 exclusions were due to no CT examination having been performed, death soon after admission, admission to the hospital after the acute phase, treatment exclusively at departments other than the Department of Neurology, or refusal to participate. All patients included were examined with CT of the brain within 15 days after acute stroke onset. Because the initial analysis of plasma homocysteine showed no difference between patients in the acute phase after stroke and control subjects, we reexamined a subgroup of patients with cerebral infarction in the convalescent phase 1 to 2 years after stroke onset. For the reexamination, 20 survivors with acute stroke onset of less than 2 years past were randomly invited to participate. Seventeen of these were included (1 patient with intracerebral hemorrhage, 1 patient 38 years old, and 1 patient with missing data on homocysteine levels were not included).
The control group comprised 60 age- and sex-matched subjects without stroke or transient ischemic attack who were randomly selected as described earlier.5 To detect a possible change over time of plasma homocysteine concentrations in the control group, 20 randomly selected control subjects were also reexamined 2 to 3 years after their first examination.
The study was approved by the ethics committee of the University of Lund. Informed consent to participate was given by all subjects (or relatives if the patients were unable to communicate).
Clinical subtyping of cerebral infarction was based on the Oxford Community Stroke Project classification,6 as described earlier.4 Subtypes included (1) total anterior circulation infarcts: both cortical and subcortical symptoms from anterior and middle cerebral artery territory; (2) partial anterior circulation infarcts: more restricted and predominantly cortical symptoms from the same arterial territories; (3) lacunar infarcts: lacunar syndromes in anterior, middle, or posterior cerebral or vertebrobasilar artery territories, including sensorimotor lacunar syndrome; and (4) posterior circulation infarcts: vertebrobasilar or posterior cerebral artery symptoms. Cortical involvement of the cerebral lesion was considered present if the patient manifested total or partial anterior circulation infarct syndrome.
Hypertension, diabetes mellitus, and heart disease were considered to be present if the patient was receiving medical treatment for these diseases at the time of investigation. In addition, heart disease was considered to be present if the patient had previously received medical or surgical treatment for heart disease.
Brain Imaging and Carotid Artery and Heart Examinations
All patients underwent CT of the brain within 15 days after stroke onset. All control subjects were examined with CT or magnetic resonance imaging of the brain.5 Sonography of carotid arteries was performed in 157 patients and all control subjects.7 Echocardiography of the heart was performed in 142 patients and all control subjects.7
Blood samples were taken on days 1 through 18 after acute stroke onset (mean and median, day 2). Ninety percent (146/162) of these blood samples were taken on days 1 through 4. Both patients and control subjects were allowed at least 10 minutes of recumbent rest before blood sampling. The 17 patients reexamined in the convalescent phase (range, 460 to 645 days; median, 583 days after acute stroke onset) were allowed approximately 10 minutes of rest in a sitting position before venipuncture. A fasting venous EDTA blood sample was taken in the morning, put on ice, centrifuged within 1 hour, and frozen. The total plasma homocysteine concentration was measured with a Kontron high-performance liquid chromatograph (system 400, Kontron Instruments AG). The validity of this method has been described earlier.8 Serum creatinine, serum cobalamin, and blood folate concentrations were determined with standard laboratory procedures.
As plasma homocysteine concentrations were not uniformly distributed, median values and the Mann-Whitney U test were used for comparison of patient and control groups. Differences between patients and control subjects for nominal scale variables were assessed with the χ2 test. The Wilcoxon signed rank test was used for comparison of acute- and convalescent-phase plasma homocysteine concentrations. Spearman’s ρ was used to test for correlation between continuous variables. The Kruskal-Wallis ANOVA was used for comparison of cerebral infarction subtype and continuous variables without normal distribution (eg, plasma homocysteine). A value of P<.05 was considered significant. Stepwise logistic regression analysis (with spss software) was used to detect any difference in homocysteine concentrations between patients and control subjects; to compare patients with cerebral infarction manifesting cortical symptoms (ie, total or partial anterior circulation infarcts) with those having lacunar infarcts; and to test for correlation between the occurrence of carotid artery or heart disease and either the plasma homocysteine concentration, age, the presence of hypertension, or smoking.
Stroke Patients Versus Control Subjects
The patient and control groups did not differ significantly in median age: 75 (range, 51 to 98) versus 72.5 (range, 51 to 95) years (NS, Mann-Whitney U test). Demographic data for patients and control subjects are shown in Table 2⇓.
The median plasma homocysteine concentration was 13.4 μmol/L for the patient group and 13.8 μmol/L for the control group (NS). The 90th percentile for homocysteine in the control group was 20.1 μmol/L. Thirty (18.5%) of the patients had homocysteine concentrations above this level (χ2 test, NS; compared with control subjects). If only control subjects with levels of serum creatinine <120 μmol/L, serum cobalamin >150 pmol/L, and blood folate >125 nmol/L were included (n=45), the median plasma homocysteine concentration was 12.8 μmol/L, and the 90th percentile was 18.3 μmol/L; however, there was no significant difference between this subgroup of control subjects and stroke patients (Mann-Whitney U test). The distribution of patients and control subjects according to the level of plasma homocysteine concentrations is shown in Fig 1⇓. Although there was no overall significant difference between patients and control subjects, 15% of the patients had plasma homocysteine concentrations above 22 μmol/L compared with only 5% of the control subjects (Fig 1⇓) (P<.05, χ2 test). To ascertain whether patients differed from control subjects regarding vascular risk factors, stepwise logistic regression analysis of the data was performed. Compared with control subjects, the patient group was characterized by higher frequencies of diabetes mellitus (P<.01), atrial fibrillation (P=.0001), other major cardioembolic risk factors on echocardiography (P<.05), and by carotid artery stenosis ≥50% (P<.05), but the groups did not differ significantly in plasma homocysteine concentrations, age, sex, history of hypertension, current smoking, or the concentrations of cobalamin, folate, and creatinine.
Reexamination of Patients With Cerebral Infarction and Control Subjects
In the subgroup of 17 patients (median age, 69 years; 13 men) reexamined at a median interval of 583 days (range, 460 to 645 days) after stroke onset, the median plasma homocysteine concentration increased significantly from its acute-phase level of 11.4 to 14.5 μmol/L (P<.01, Wilcoxon signed rank test). The individual changes between acute- and convalescent-phase values are shown in Fig 2⇓. Spearman’s ρ for correlation between acute- and convalescent-phase values was 0.55 (P=.03). The patients’ convalescent-phase plasma homocysteine concentrations did not differ significantly from control values (Mann-Whitney U test). There was no significant difference between the patients’ acute- and convalescent-phase values for creatinine (median, 83 versus 85 μmol/L), cobalamin (median, 262 versus 276 pmol/L), or folate (median, 330 versus 300 nmol/L) concentrations. In the 20 control subjects (median age, 65 years; 14 men) reexamined after 2 to 3 years, the median plasma homocysteine concentration was 12.7 μmol/L at baseline and 12.6 μmol/L at reexamination (NS, Wilcoxon signed rank test). The individual values are shown in Fig 2⇓.
Cerebral Infarction Versus Intracerebral Hemorrhage
The cerebral infarction subgroup (n=150) did not differ from the intracerebral hemorrhage subgroup (n=12) in median homocysteine concentrations (13.3 versus 14.2 μmol/L), age, or concentrations of creatinine and cobalamin, although their blood folate concentrations were slightly higher (median, 337 versus 281 nmol/L; P<.05).
Cerebral Infarction Subgroups
The stroke subgroups differed significantly in median plasma homocysteine concentrations (P=.02) (Table 3⇓), with the level being highest in the total anterior circulation infarction subgroup. However, median age was greater in this subgroup (81 years) than in the other subgroups (68 to 76 years) (P=.0001). The subgroups did not differ significantly in concentrations of creatinine, cobalamin, or folate. Stepwise logistic regression analysis yielded no independent correlation between plasma homocysteine concentrations and infarct type (cortical versus lacunar) or the degree of carotid artery stenosis (<80% versus ≥80%).
Level of Plasma Homocysteine in Relation to Other Vascular Disease Risk Factors
As shown in Table 4⇓, no significant relationship existed between the homocysteine concentration and the presence/absence of carotid artery stenosis or major cardioembolic risk factors other than atrial fibrillation (AF). However, although plasma homocysteine concentrations were higher in patients with atrial fibrillation (P<.05), stepwise logistic regression analysis with age as a factor showed atrial fibrillation to be not independently correlated to the level of the plasma homocysteine concentration. Multiple regression analysis showed the homocysteine concentration to be independently correlated to age (P=.0001) and to the concentrations of creatinine (P=.0001) and folate (P<.01) but not to that of cobalamin. The median homocysteine concentration was greater in the ≥85-year-old age group (15.8 μmol/L, n=32) than in the 55- to 64-year-old age group (10.7 μmol/L, n=24). Compared with patients with homocysteine concentrations below the 90th percentile of the control subjects, the 30 patients with concentrations above the 90th percentile differed significantly by being older (P<.01), having higher creatinine values (P<.01), and having lower cobalamin (P<.01) and folate (P<.001) values.
To the best of our knowledge, this is the first study performed to ascertain whether plasma homocysteine concentrations in stroke patients differ between the acute and convalescent phases. In the 17 patients reexamined in the convalescent phase, plasma homocysteine values were significantly higher than those in the acute phase. In contrast, the median plasma homocysteine concentration did not change significantly over time in the 20 reexamined control subjects. In the patient series as a whole (n=162), acute-phase homocysteine concentrations were similar to values for the control group (n=60) without stroke or transient ischemic attack; although the frequency of plasma homocysteine concentrations ≥22 μmol/L was significantly greater among acute-phase stroke patients than among control subjects (P<.05) (Fig 1⇑), when the 90th percentile of control values was used as a cutoff level, there was no significant difference between stroke patients and control subjects. The relationships found between the plasma homocysteine concentration in stroke patients and both age and the concentrations of creatinine and folate are in accord with findings of earlier studies by both our group9 and others.2 3 We found a high plasma homocysteine concentration to be significantly correlated to the presence of atrial fibrillation, and plasma homocysteine values were significantly higher in the total anterior circulation infarct subgroup than in the other infarct subgroups, although in both cases the statistical significance disappeared when the data were subjected to stepwise logistic regression analysis with correction for age.
Present Findings in Relation to Those of Earlier Studies
The lack of a significant difference in plasma homocysteine concentrations between the present acute stroke patients and control subjects is in contrast to findings of most earlier studies, in which patients manifested higher homocysteine concentrations than did control subjects.10 11 12 13 14 15 This discrepancy may be due to the fact that in some earlier stroke studies plasma homocysteine was measured in the convalescent phase and not the acute phase (Table 1⇑). Another possible explanation is that the patients in our study were older overall (median age, 75 years) than those in several earlier studies that included only patients 60 years or younger (Table 1⇑). A stronger association between plasma homocysteine levels and risk of ischemic stroke among younger (<61 years) than among older subjects has recently been reported.16 In one study,17 plasma homocysteine concentrations were reported to be higher in acute stroke patients than in the control group; however, the control subjects were younger (mean age, 61 years versus 67 years for stroke patients), and all control subjects had normal serum creatinine concentrations (both age and the serum creatinine concentration are related to the levels of the homocysteine concentration). In a recent prospective study, no significant association between elevated plasma homocysteine levels and risk of ischemic stroke was found,16 which may support the hypothesis that plasma homocysteine concentrations change after stroke onset (see below).
Change in Plasma Homocysteine Concentrations Over Time
There are at least two possible explanations for the finding that plasma homocysteine concentrations were not increased in the acute phase after stroke but were in the convalescent phase.
First, the acute situation with its accompanying stress may cause a transient decrease in the plasma homocysteine concentrations. In patients with an acute inflammatory reaction, serum albumin concentrations are known to be lowered; because albumin is the main binding protein for plasma homocysteine,1 this decrease in albumin may cause a reduction in the total plasma homocysteine concentration. The acute phase of stroke may also give rise to oxidative stress with production of oxidative oxygen radicals and possibly a subsequent change in the elimination rate of thiols, including homocysteine.18 19
Second, plasma homocysteine may increase after the acute phase of stroke, perhaps due to changes in vitamin intake or other lifestyle factors or to impaired renal function. Vitamin status may be a determinant of the homocysteine concentration,2 and deficiencies of cobalamin, folate, and vitamin B-6 are common in the elderly.3 Low folate concentrations have been found to be associated with an increased risk of coronary artery disease, an effect suggested to be mediated by change in the plasma homocysteine concentration.20 Patients with premature vascular disease may have disorders of the methionine/homocysteine metabolism.21 Blockage of one of the pathways for homocysteine metabolism may result in the impairment of the other metabolic pathway.22 Although the patients in our study had no significant differences between acute and convalescent concentrations of creatinine, cobalamin, and folate, it is possible that some of the patients may have had altered plasma homocysteine concentrations after stroke because of changes in vitamin status or renal function.
The median plasma homocysteine concentrations in the 20 control subjects reexamined 2 to 3 years after the first examination did not change significantly. This is in accordance with earlier results from our group that have shown that plasma homocysteine concentrations do not change during the convalescent phase in patients14 or over time in control subjects.23
In our study, there was no correlation (Spearman’s correlation coefficient or multiple regression) between the homocysteine levels in stroke patients and time of blood sampling in the acute phase. Because the majority (90%) of the patients were examined on days 1 through 4, this indicates that plasma homocysteine concentrations do not increase during the first 4 days after acute stroke onset.
About 70% of plasma homocysteine is protein bound.1 Serum albumin concentrations are approximately 9% higher in standing than in recumbent subjects.24 As at reexamination, blood was sampled when the patients had been sitting at rest for 10 to 12 minutes; their plasma homocysteine concentrations might have been as much as 5% to 6% higher than if blood had been sampled with the patients in the recumbent position. However, even if the results are corrected for this possible source of error, the difference between acute- and convalescent-phase values remains significant (P<.01).
The initial aim of our study was to compare patients with acute stroke with control subjects. When the results were analyzed and no differences between patients and control subjects were found, we examined the hypothesis that plasma homocysteine concentrations may vary with time in stroke patients. However, at this stage approximately 1 to 2 years had passed after the acute stroke onset, explaining the wide time interval between acute- and convalescent-phase measurements. The plasma homocysteine levels increased from the acute to the convalescent phase after stroke in the small sample of patients examined twice. The best method to address the hypothesis that plasma homocysteine concentrations change with time after stroke would be to perform replicate measurements at frequent and specified intervals after stroke onset in a large number of patients.
Homocysteine and Atherosclerosis
A moderate increase in the plasma homocysteine concentration has been reported to be associated with a risk of subsequent myocardial infarction.25 An increased plasma homocysteine concentration has also been found to be associated with carotid artery intimal-medial wall thickening.26 Several pathogenetic mechanisms have been suggested to explain how increased homocysteine concentrations may cause atherosclerosis and vascular disease:27 (1) endothelial cell damage in the vessel wall with a subsequent increase in platelet adhesiveness; (2) promotion of vascular smooth muscle cell growth and an inhibitory effect on endothelial cell growth28 (3) modifying of blood clotting factors with a consequent increase in tendency to thrombosis; and (4) adverse effects on lipid metabolism. Even though we found no difference in plasma homocysteine concentrations between patients with stroke in the acute stage after stroke and control subjects, increased plasma homocysteine concentration measured at other times may be an indicator of cerebrovascular disease.
Stroke patients in the acute phase and control subjects had similar plasma homocysteine concentrations, but the homocysteine concentrations of the stroke patients increased significantly after the acute phase. This may explain why previous studies performed in the convalescent phase have shown homocysteine concentrations to be higher in stroke patients than in control subjects. There are two possible explanations of these findings: (1) acute stress may cause a decrease of plasma albumin, and the cerebral tissue damage may cause an increased production of oxidative oxygen radicals, with a secondary reduction of plasma homocysteine levels; and (2) plasma homocysteine concentrations may increase in the convalescent phase after stroke because of changes in vitamin intake or other lifestyle factors. The timing of plasma homocysteine measurements relative to stroke onset is a factor to be taken into consideration in the interpretation of results.
This study was supported by the Rut and Erik Hardebo Donation Fund, the Elsa Schmitz Foundation, the 1987 Foundation for Stroke Research, the Medical Faculty of the University of Lund, and the Swedish Association for the Neurologically Disabled (NHR). Statistical advice was given by Eva Kelty of Clinical Data Care AB.
- Received October 29, 1994.
- Revision received February 9, 1995.
- Accepted February 17, 1995.
- Copyright © 1995 by American Heart Association
Ueland PM, Refsum H, Brattström L. Plasma homocysteine and cardiovascular disease. In: Francis RB Jr, ed. Atherosclerotic Cardiovascular Disease, Hemostasis, and Endothelial Function. New York, NY: Marcel Dekker Inc; 1992.
Joosten E, van den Berg A, Riezler R, Naurath HJ, Lindenbaum J, Stabler SP, Allen RH. Metabolic evidence that deficiencies of vitamin B-12 (cobalamin), folate, and vitamin B-6 occur commonly in elderly people. Am J Clin Nutr. 1993;58:468-476.
Lindgren A, Norrving B, Rudling O, Johansson BB. Comparison of clinical and neuroradiological findings in first-ever stroke: a population-based study. Stroke. 1994;25:1371-1377.
Lindgren A, Roijer A, Rudling O, Norrving B, Larsson E-M, Eskilsson J, Wallin L, Olsson B, Johansson BB. Cerebral lesions on magnetic resonance imaging, heart disease, and vascular risk factors in subjects without stroke: a population-based study. Stroke. 1994;25:929-934.
Lindgren A, Roijer A, Norrving B, Wallin L, Eskilsson J, Johansson BB. Carotid artery and heart disease in subtypes of cerebral infarction. Stroke. 1994;25:2356-2362.
Andersson A, Isaksson A, Brattström L, Hultberg B. Homocysteine and other thiols determined in plasma by HPLC and thiol-specific postcolumn derivatization. Clin Chem. 1993;39:1590-1597.
Brattström LE, Hardebo JE, Hultberg BL. Moderate homocysteinemia: a possible risk factor for arteriosclerotic cerebrovascular disease. Stroke. 1984;15:1012-1016.
Malinow MR, Kang SS, Taylor LM, Wong PWK, Coull B, Inahara T, Mukerjee D, Sexton G, Upson B. Prevalence of hyperhomocyst(e)inemia in patients with peripheral arterial occlusive disease. Circulation. 1989;79:1180-1188.
Mereau-Richard C, Muller JP, Faivre E, Ardouin P, Rousseaux L. Total plasma homocysteine determination in subjects with premature cerebral vascular disease. Clin Chem. 1991;37:126. Letter.
Verhoef P, Hennekens CH, Malinow MR, Kok FJ, Willett WC, Stampfer MJ. A prospective study of plasma homocyst(e)ine and risk of ischemic stroke. Stroke. 1994;25:1924-1930.
Coull BM, Malinow MR, Beamer N, Sexton G, Nordt F, de Garmo P. Elevated plasma homocyst(e)ine concentration as a possible independent risk factor for stroke. Stroke. 1990:21;572-576.
Banks MF, Stipanuk MH. The utilization of N-acetylcysteine and 2-oxothiazolidine-4-carboxylate by rat hepatocytes is limited by their rate of uptake and conversion of cysteine. J Nutr. 1994;124:378-387.
Pancharuniti N, Lewis CA, Sauberlich HE, Perkins LL, Go RCP, Alvarez JO, Macaluso M, Acton RT, Copeland RB, Cousins AL, Gore TB, Cornwell PE, Roseman JM. Plasma homocyst(e)ine, folate, and vitamin B-12 concentrations and risk for early-onset coronary artery disease. Am J Clin Nutr. 1994;59:940-948.
Dudman NPB, Wilcken DEL, Wang J, Lynch JF, Macey D, Lundberg P. Disordered methionine/homocysteine metabolism in premature vascular disease: its occurrence, cofactor therapy, and enzymology. Arterioscler Thromb. 1993;13:1253-1260.
Selhub J, Miller JW. The pathogenesis of homocysteinemia: interruption of the coordinate regulation by S-adenosylmethionine of the remethylation and transsulfuration of homocysteine. Am J Clin Nutr. 1992;55:131-138.
Young DS, Bermes EW. Specimen collection and processing: sources of biological variation. In: Tietz NW, ed. Fundamentals of Clinical Chemistry. Philadelphia, Pa: WB Saunders Co; 1987:266-286.
Malinow MR, Nieto FJ, Szklo M, Chambless LE, Bond G. Carotid artery intimal-medial wall thickening and plasma homocyst(e)ine in asymptomatic adults: the Atherosclerosis Risk in Communities Study. Circulation. 1993;87:1107-1113.
Wu LL, Wu J, Hunt SC, James BC, Vincent GM, Williams RR, Hopkins PN. Plasma homocyst(e)ine as a risk factor for early familial coronary artery disease. Clin Chem. 1994;40:552-561.
Tsai J-C, Perrella MA, Yoshizumi M, Hsieh C-M, Haber E, Schlegel R, Lee M-E. Promotion of vascular smooth muscle cell growth by homocysteine: a link to atherosclerosis. Proc Natl Acad Sci U S A. 1994;91:6369-6373.