Homocysteine and myocardial infarction

Atherosclerosis, 71 (1988) 221-233 Elsevier Scientific Publishers Ireland,

227 Ltd.

ATH 04140

Homocysteine and myocardial infarction Bo Israelsson

‘, Lars E. Brattstriim

’

and Bjikn L. Hultberg

’

’Department of Medicine, University of Lund, Malmij General Hospital, Malmij (Sweden) and ’ Depariment of Clinical Chemistv, University of Lund University Hospital, Lund (Sweden) (Received 9 September, 1987) (Revised, received 13 January, 1988) (Accepted 21 January, 1988)

Summary

Five (24%) subjects out of a group of 21 men, 48-58 years old (mean 54), who had suffered their first myocardial infarction (MI) before the age of 55 and with a low risk profile vis-a-vis conventional risk factors in a health screening preceding the MI, had abnormally high total plasma homocysteine values in the fasting state when investigated within l-7 years (mean 3) after their MI. The patient group was exactly matched with 36 control subjects for sex, age, diastolic blood pressure, smoking, and serum concentrations of cholesterol and triglycerides. Total plasma homocysteine was negatively correlated to both erythrocyte folate and serum vitamin Bi2, and vitamin concentrations below the median of the normal distribution were found in the five with high plasma homocysteine content, indicating a possible involvement of reduced remethylation of plasma homocysteine to methionine. After methionine loading, in 3 of the patient group (14%) homocysteine levels exceeded mean + 2 SD for controls, which may indicate heterozygosity for homocystinutia. Results are consistent with the hypothesis that a high plasma homocysteine content may be a risk factor for MI.

Key words: Folate; Homocysteine;

Myocardial infarction; Vitamin B,,

Introduction

Although myocardial infarction (MI) is usually associated with one or more of the risk factors, high lipoprotein concentrations, smoking, and hypertension, some patients with myocardial infarction have a low risk profile vis-8% these factors. Family history of coronary disease is also a risk factor for MI [l-3]. Homocystinuria is a herediCorrespondence to: Bo Israelsson, M.D., Department of Medicine, Malmii General Hospital, S-214 01 Malmli, Sweden.

0021-9150/88/$03.50

0 1988 Elsevier Scientific

Publishers

Ireland,

tary disease caused by cystathionine /?-synthase (CBS) deficiency or, in rare cases, due to defects in certain enzymes of folate and vitamin B,, metabolism. In its homozygote form, CBS deficiency is associated with the development of arteriosclerosis and frequent thromboembolic episodes at an early age [4]. Heterozygosity for CBS deficiency is also a possible risk factor; in one recent study it was commonly found among patients with premature peripheral and cerebral occlusive arterial disease, but not among those with coronary artery disease [5]. One study based on Ltd.

228 questionnaires also failed to demonstrate any significant increase in the incidence of heart attacks among obligate heterozygotes for CBS deficiency [6]. In studies in which the degree of heredity for CBS deficiency was not taken into account, a relationship between a high plasma homocysteine content and coronary or arteriosclerotic cerebrovascular disease was found by some [7-lo] but not by all [ll]. In the present study, middle-aged men with a low risk profile vis-a-vis conventional risk factors and who survived their first MI have been investigated for total plasma homocysteine before and after a methionine load. As far as we know, the study is unique inasmuch as the control subjects, who were exactly matched for the conventional risk factors in the years preceding the MI, were also investigated. For the evaluation of the results, a third group of subjects, obligate heterozygotes for CBS deficiency, were also investigated with an analysis of total plasma homocysteine both before and after methionine load.

preceding their MI. The diagnosis of MI was based on criteria previously described [12]. From these in turn were selected those who in the health screening had had low risk profiles vis-a-vis blood pressure and serum content of cholesterol and triglycerides, and had denied having had any symptoms of angina pectoris, claudication, or cerebral vascular disturbances. Finally, the patient group constituted those with the highest number of first degree relatives (parents or siblings) with a history of MI or stroke. In this case, history alone was the criterion for the designation of MI or stroke. Unlike the patient group, controls were selected on the basis of as few first degree relatives as possible with a history of MI or stroke. For each of the patient group, one or two controls were selected of comparable age, sex, smoking habits, diastolic blood pressure, and serum content of cholesterol and triglycerides (Table 1). Serum cholesterol and triglycerides were determined enzymatically, according to Roschlau et al. [13] and with a commercial kit (Boehringer Mannheim GmbH, F.R.G.), respectively. Finally, 15 patients had 2 controls each, and 6 had only one. The matching procedure was done by computer, as all data from the health screening program is kept in a data base. For the purposes of comparison, a third group was selected: 9 male obligate heterozygotes for CBS deficiency, mean age 59 years, also to be

Methods

Subjects Among male patients with their first myocardial infarction (MI) before the age of 55, available from the computerised hospital diagnosis records, were selected those who had participated in a health screening program in the years immediately TABLE

1

CHARACTERISTICS Serum cholesterol,

OF PATIENT triglycerides

AND

CONTROL

and diastolic

GROUPS

blood pressure

AT HEALTH

are expressed

SCREENING

AND

INVESTIGATION

as mean f SD.

Patients at health screening

Controls at health screening

Patients at investigation

Controls at investigation

Serum cholesterol (mmol/l) Serum triglycerides (mmol/l) Diastolic blood pressure (mmHg) Smoking (56)

5X0* 0.85 1.34* 0.46 88 *lo 76

5.97 *0.84 1.55 f 0.41 91 +8 75

6.19 f 0.98 1.54*0.64 81 *6 48

Heredity of MI or stroke (W) Heredity of MI c age 60 or stroke < age 70 (W)

71

37

6.19 f 0.79 1.41 kO.65 87 *6 69 _

33

5

0 0 0 0 0

0 0 0 3 6

Angina pectoris (S) Claudication (%) Stroke (W) Beta-blocking agents (4;) Diuretics (49)

_ 67 10 0 48 19

0 0 0 17 6

229 investigated for total plasma homocysteine values both before and after a methionine load. No individuals with diabetes or impaired glucose tolerance were included in any group. Table 1 also shows the characteristics of patients and controls, not only with regard to heredity but also to smoking habits, different symptoms of arteriosclerotic disease, consumption of /?blocking agents and diuretics. The investigation was performed l-7 years (mean 3) after the MI, which in turn occurred O-6 years (mean 2) after the health screening. In the investigation, the patients had had one MI, whereas control subjects still denied any symptoms of angina pectoris, had no anamnesis or electrocardiographic signs of MI, and no claudication, or anamnesis of any cerebrovascular disease. At the time of the investigation the age of the study population was 48-58 years (mean 54). The investigation was performed after the patients’ informed consent and after approval by the Ethics Committee. Subjects were investigated in the morning after an overnight fast. They had been instructed to avoid vitamins for at least 3 weeks, and acetylsalicylic acid for 1 week. If any other medicine was used, they were told to omit the morning dose on the day of the investigation. No subject was on treatment with folic acid or vitamin B,,. Fasting blood samples were taken for determination of plasma homocysteine, methionine, and co-factors for plasma homocysteine metabolism (erythrocyte folate and serum vitamin B,,). Analysis of vitamin $, which is a co-factor for CBS, was not available at the time of the investigation. Loading with L-methionine in orange juice (3.8 g/m* body surface area) was followed by blood sampling after 4 h for the determination of the post-load concentrations of plasma homocysteine and methionine. Total plasma homocysteine Samples for the amino acid assay were collected in evacuated tubes containing 0.01 ml of 0.38 mol/l sodium EDTA per ml of blood. Tubes were centrifuged within 15 min and the plasma stored at - 70 o C until used for analysis. To 1 ml of thawed plasma were added, 0.95 ml of PICO buffer IV A (Pierce Chemical Company, Rockford, IL, U.S.A.), diluted 10 times with distilled

water and pH-adjusted to 7.0 with 3 mol/l LiOH, and 0.05 ml of 12% dithiothreitol in distilled water, and mixed. During incubation at 37’ C for 60 min disulfides such as homocystine, homocysteine-cysteine, and homocysteine-protein were reduced to sulfhydryl amino acids. The proteins were then precipitated by the action of 0.5 ml 10% sulfosalicylic acid for 30 min at room temperature. After centrifugation (2000 x g for 15 mm), the supernatants were again centrifuged for 10 min before the final protein-free supematants (after adjustment of the pH to 2.2, if necessary with 3 mol/l LiOH or 1 mol/l HCl) were analysed on an automatic amino acid analyzer (Biotronic LC 5000, Biotronik GmBH, Munich, West Germany). This technique is identical to our routine method for amino acid analysis except for one extra preparatory step, the reduction of disulfide bonds in whole plasma with dithiothreitol before deproteinization. Reproducibility was good, with a correlation coefficient r = 0.995 when 26 samples were re-analyzed after 8 months at - 20 ’ C. The reduction of disulfide bonds was tested by adding homocystine, 5 and 100 pmol/l, to plasma samples with known concentration of homocysteine, then stored at - 20” C for 2 weeks before analysis. Recovery was 10.5 t_ 0.2 vs. 203.3 + 2.8 pmol/l of homocysteine. Results Of the 21 men with previous MI, 5 had total plasma homocysteine concentrations in the fasting state which were higher (21.9-29.6 pmol/l) than in any of the 36 control subjects (6.6-21.8 pmol/l) (Fig. 1A). The results of these 5 subjects alone also led to a group difference between patients (16.4 + 6.9) and controls (13.5 f 3.6), P < 0.05 (Student’s t-test). In an attempt to find an explanation for this, the results of these 5 patients were compared to the distribution of results among the control subjects, with regard to various aspects. Homocysteine did not seem to be accumulated in the plasma due to renal failure (Fig. 1B). Erythrocyte folate, a vitamin essential in the remethylation of homocysteine to methionine [4], was below the median in all 5 subjects, of whom 2 had subnormal values (Fig. 1C). Serum vitamin Bi2, also an essential co-factor in the remethylation of homo-

230 A

C

160

600 r

,x : 100

E lb

w

ul

Patients

0

Controls

0-

t

o-

Fig. 1. Total plasma homocysteine in the fasting state, comparison between patients (0) and controls (0). (A) Frames f 2 SD for controls. Serum creatinine, (B) erythrocyte folate, (C) and serum vitamin B,, (D) are compared between with the highest plasma homocysteine levels and the distribution of controls.

‘asting

state

Pattents

with myocardial infarction

Post load

Fasting

state

Post load

Heterozygotes for cystathlonine p-synthase deficiency

Fig. 2. Total plasma homocysteine of the patients and obligate heterozygotes for cystathionine /3-synthase deficiency, in the fasting state and after loading with methionine 3.8 g/m2. Frames indicate mean + 2 SD for control subjects.

indicate mean the 5 subjects

cysteine to methionine [4], was within the range of normal distribution, but all 5 patients had values below the median (Fig. 1D). Calculated for the total population, 21 patients and 36 control subjects, there was a positive correlation between plasma homocysteine in the fasting state and serum creatinine (r = 0.36, P < 0.01) and a negative correlation between the amino acid level and erythrocyte folate (r = -0.35, P -c0.01) and serum vitamin B,, (r = -0.34, P < 0.01). After the methionine load, 3 of the 5 patients with the highest total plasma homocysteine in the fasting state also reached levels beyond the mean + 2 SD for the 36 control subjects. One subject had an exceptional post-load level of 97.1 pmol/l (Fig. 2). Plasma methionine 4 h after loading 3.8 g/m* was 549-980 pmol/l except for 1 control who reached 1383 pmol/l. The post-load methionine concentration for the five patients with the highest total plasma homocysteine levels both in the fasting state and post-load did not exceed the mean + 2 SD for the controls. Fig. 2 also illustrates total plasma homocysteine of the 9 heterozygotes for CBS deficiency both before and after a methionine load. In the fasting state, only one man reached total plasma homocysteine higher than those of the controls and 4 had post-load levels higher than the mean + 2 SD of the normal subjects. The 5 patients denoted l-5 had post-load levels corresponding well with those of the heterozygotes.

231 Discussion

With the special form of selection used by us in order to minimize the role of established risk factors for MI, except for heredity for MI and/or stroke, which we tried to maximize, we found that among 21 men with MI before 55 years of age 5 subjects (24%) had abnormally higher levels of total plasma homocysteine in the fasting state than any of the control subjects. This finding is in agreement with that of Kang et al. [9], who found significantly elevated fasting levels of total plasma homocysteine in 173 male patients with angiographically verified coronary artery disease compared to 93 male control subjects with normal angiograms. Three of the 5 subjects (14%) also responded abnormally to methionine loading with post-load plasma homocysteine exceeding the mean + 2 SD for controls. Wilcken et al. [7], who were the first to study the possibility of homocysteine being a risk factor for coronary artery disease, found, post-methionine loading, an increased plasma homocysteine-cysteine mixed disulfide that could be considered abnormal in 7 of 25 male patients (28%) with this disease, but only in one of 22 controls (5%). In a more recent study Wilcken et al. [ll] could not reproduce these results, but of 20 men with coronary artery disease 2 patients, who were identical twins, had clearly abnormal plasma levels of total non-protein-bound homocysteine both in the fasting state and after methionine loading. Interestingly, both had subnormal or low normal serum levels of folate and vitamin B,,, though erythrocyte folate was normal. Folic acid substitution restored homocysteine levels to normal both in the fasting state and post-load. More recently, Beers et al. [5] found that the total amount of non-protein-bound homocysteine in the plasma was normal after methionine loading in all of 22 men and 3 women studied who had had myocardial infarction. However, in a later study Murphy-Chutorian et al. [lo] reported intolerance to methionine loadings with abnormal post-load levels of homocysteine in 16 of 99 male patients (16%) with angiographically verified coronary artery disease compared to one of 39 angiographitally normal controls (2%). Levels of folate and vitamin B,, were not measured in these studies. Increased plasma homocysteine in the fasting

state and after methionine loading may depend on many different factors. Besides homozygote forms of homocysteinurias due to deficiency of CBS or certain enzymes in folate and vitamin B,, metabolism, heterozygosity for CBS deficiency has been recognized. Renal dysfunction may lead to impaired homocysteine excretion and the accumulation of homocysteine in the plasma [14,15], and remethylation of homocysteine to methionine may be hampered because of deficiency of folate or vitamin B,, [4]. Very recently Kang et al. [16] not only found significant homocysteinemia in folatedepleted subjects but also in the majority of patients with low normal levels of serum folate. Homocysteinemia was also more pronounced in those with concomitant vitamin B,, deficiency. Also we have found significantly increased homocysteine levels in vitamin B,, deficient subjects that could be normalized after replenishment [17] and Stabler et al. [18] claim to have found very high homocysteine levels in patients with either deficiency of vitamin B,, or folate. In our study all 5 subjects with previous MI and with increased plasma homocysteine in the fasting state also had subnormal or low normal levels of erythrocyte folate and vitamin B,,. However, since this is a retrospective study nothing is known about the vitamin status at the time of the MI or in the years preceding it. The only reason for increased plasma homocysteine that definitely should have preceded the MI are enzyme aberrations like CBS deficiency. We found that three of the 5 patients with high total plasma homocysteine in the fasting state also had homocysteine levels post-methionine that could suggest heterozygosity for homocysteinuria, using the criterion of Boers (homocysteine post-load higher than mean + 2 SD of controls) [5]. However, like Wilcken et al. [ll], we found a pronounced overlapping in post-methionine results from obligate heterozygotes and controls. Thus, interpretation cannot be so definitive. The possibility exists that more than the 3 previously mentioned subjects with abnormal, post-load homocysteine, or even controls, could be heterozygotes for CBS deficiency. The possibility of homocysteine accumulation due to reduced renal function was estimated by serum creatinine analysis. In one patient (No. 1)

232

serum creatinine content was above the normal range for our laboratory and high within the range of the controls of this study. In view of the reports by Wilcken et al. [14] and Kang et al. [15], who found increased levels of plasma homocysteine in patients with reduced renal function, the increased plasma homocysteine at least in patient No. 1 might be explained in this way. In the present study a positive correlation was found between serum creatinine and plasma homocysteine, supporting this possibility. Vitamin $, which is a co-factor for CBS activity, was not determined in this study. Studies on for homocysteine the importance of this Vitamin metabolism are sparse. In a study by Park and Linkswilder [17] vitamin $ was a more important co-factor for the enzyme cystathionase than for CBS. In an experimental study on 6 subjects with vitamin $ depletion they found that only trace amounts of homocysteine was excreted in the urine while cystathionine was accumulated to a much higher degree and excreted in the urine, depending on impairment of cystathionase activity. We, therefore, assume that the lack of vitamin $ analyses in this study was probably not of great importance in explaining the 24% with high plasma homocysteine in the fasting state and the 14% with increased levels after methionine loading. In a parallel study of homocysteine metabolism in 72 patients with occlusive non-coronary arterial diseases we have found as many as 60% to have subnormal levels of plasma pyridoxal 5-phosphate, but there was no correlation with plasma total homocysteine, either in the fasting state or after methionine loading. However, both erythrocyte folate and vitamin B,, were, as in the present study, strongly and negatively correlated to homocysteine in the fasting state but not after a methionine load (unpublished results). Our results are consistent with the hypothesis that increased homocysteine in plasma is a risk factor for coronary artery disease. The retrospective design makes it impossible to say anything about how important moderate homocysteinemia could have been in the pathogenesis of premature coronary sclerosis or thrombotic complications in MI development. However, this study confirms that moderate homocysteinemia exists in a high proportion of men with low conventional risk

factors for arteriosclerotic disease, who, despite low risk factors, developed MI at an early age. Subnormal or low levels of folate and vitamin B,, seem to have influenced our results most. The hypothesis emerges that deficiency of these co-factors for homocysteine remethylation might, via increased plasma homocysteine, participate in the development of vascular disease. Further studies with a prospective design are needed. If moderately increased plasma homocysteine can be verified as a risk factor for arteriosclerotic disease, simple and safe therapy is available. We have previously found [20] and recently confirmed [21] that all levels, not only high levels, of total plasma homocysteine can be reduced simply by giving a supplement of 5 mg folic acid a day. Acknowledgements

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