© 1996 American Heart Association, Inc.
Articles |
Low Whole-Blood S-Adenosylmethionine and Correlation Between 5-Methyltetrahydrofolate and Homocysteine in Coronary Artery Disease
From the Metabolic Unit, University Children's Hospital (F.M.T.L., C.P.A., B.F.); University Computer Center (P.P.J.); and the Divisions of Clinical Pharmacology (W.E.H.) and Intensive Care (R.R.), University Hospital, Basel, Switzerland.
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Abstract |
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Abstract Mild elevation of plasma homocysteine is an independent risk factor for vascular disease. We studied the role of 5-methyltetrahydrofolate (5-MTHF), the folate form directly involved in homocysteine metabolism, in contrast to previous studies, which used total folate measurements, in 70 coronary artery disease (CAD) patients and control subjects. We also measured S-adenosylmethionine (SAM), which controls the activity of critical enzymes of homocysteine metabolism. Fasting plasma total homocysteine was elevated (>12.4 µmol/L for women, >13.3 µmol/L for men) in 17% of patients, in accordance with earlier studies. These patients showed lower 5-MTHF (12.4±1.0 nmol/L, mean±SD) than control subjects (24.2±15.0, P<.001), and there was a clear correlation (multiple linear regression analysis: P=.002) of this relevant form of folate with homocysteine. However, 37% of the normohomocysteinemic patients also revealed similarly low 5-MTHF levels, suggesting that a decrease of 5-MTHF does not necessarily cause hyperhomocysteinemia. SAM was significantly decreased in patients (1.4±0.4 µmol/L) compared with control subjects (1.8±0.3, P<.001) but was not correlated to homocysteine or 5-MTHF. The correlation between homocysteine and 5-MTHF that was found in CAD patients but not in control subjects confirms the direct relationship between these compounds in vivo. The new finding of low SAM in patients demands further studies, since it might indicate that low levels pose risk and that SAM might be a protective factor against the development of CAD.
Key Words: 5-methyltetrahydrofolate • coronary artery disease • homocysteine • methylenetetrahydrofolate reductase • S-adenosylmethionine
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Introduction |
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In recent years, a clear association between mild elevations of plasma homocysteine and the occurrence of occlusive vascular disease has been reported, as reviewed by Boushey et al.1 Several studies have shown elevated plasma homocysteine levels, either after methionine loading or fasting, in a significant proportion of patients with CAD,2 3 4 5 6 peripheral arterial occlusion,7 8 9 or cerebral vascular disease,7 10 11 12 pointing to mild hyperhomocysteinemia as an independent risk factor for arteriosclerotic disease. The incidence of elevated homocysteine in different studies ranged from 23% to 47% in peripheral vascular disease and from 0%7 to 30%13 in CAD.
Homocysteine is formed in humans from methionine by irreversible transmethylation reactions involving the adenosylated compounds SAM and S-adenosylhomocysteine. Homocysteine can be catabolized by transsulfuration, the first step catalyzed by the pyridoxal 5-phosphate–dependent CS. Alternatively, it can be remethylated to methionine by 5-MTHF:homocysteine methyltransferase (methionine synthase), which requires vitamin B12 and 5-MTHF, or by betaine:homocysteine methyltransferase.
The cause for hyperhomocysteinemia in patients with atheromatosis has by no means been completely elucidated. Several studies have provided evidence for genetic causes. For example, Genest et al14 showed that in 50% of CAD patients the elevated homocysteine was familial. Reduced CS activity was found in cultured fibroblasts of some patients in some studies,7 13 15 but DNA analysis has shown the reduced activity not to be due to a mutation of the structural gene.16 In addition, increased thermolability of MTHFR may cause hyperhomocysteinemia in some patients.17 18 19 Deficiencies of vitamin B12, folate, and pyridoxal phosphate also seem to play a role in the occurrence of hyperhomocysteinemia.20 21 An inverse correlation between plasma homocysteine and folic acid and vitamin B12 concentrations, respectively, has been reported.20 22 Also, treatment with folic acid and vitamin B12 can lower elevated homocysteine levels in vascular disease patients with normal vitamin status.23 24
In spite of these advances, no unifying explanation for elevated homocysteine has been provided in the majority of patients in whom it is detected. Studies in vitro on purified enzymes point to a vital role of SAM, which acts as an allosteric inhibitor of MTHFR25 and activator of CS26 at micromolar concentrations, but little is known about its action in vivo. An interruption of coordinate regulation of homocysteine metabolism by SAM has been proposed.27 According to the proposed theory, conditions that affect remethylation will directly result in elevation of homocysteine and possibly decreased synthesis of SAM, causing lack of sufficient activation of CS, leading to a disturbance of the transsulfuration pathway. Conversely, when the transsulfuration pathway is blocked, synthesis of SAM will increase through remethylation, leading ultimately to feedback inhibition of MTHFR by SAM and consequently reduce further remethylation. Thus, conditions that directly affect one pathway, such as genetic disorders or nutritional factors, will disturb the coordinate feedback regulation of SAM.28
In this study, the relationship of plasma total homocysteine to plasma
levels of related vitamins and metabolites was investigated in CAD
patients. In particular, we have measured the active form of folate,
5-MTHF, which is directly involved in the remethylation reaction, in
contrast to previous studies, which measured total folate by routine
methods. For the first time, SAM in whole blood has been measured. In
addition, after 
1 year, homocysteine and 5-MTHF were determined
again, and the activity of the MTHFR in lymphocytes was assessed in a
subset of patients.
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Methods |
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Patients and Control Subjects
Seventy patients (aged 28 through 79; 14 women, 56 men) with occlusions of 1 to 3 major coronary arteries undergoing percutaneous transluminal coronary angioplasty at the University Hospital Basel were consecutively studied after obtaining written informed consent. Exclusion criteria were the following: food intake <6 hours before blood sampling; supplementation with vitamin B12, B6, or folate; anemia (Hb<100 g/L); systolic blood pressure <100 mm Hg and/or heart rate >100 beats per minute; and acute angina pectoris, unstable angina, or acute myocardial infarction within the past 7 days.
Healthy control subjects (aged 25 through 66) who were nonsmokers, not on regular medication (except women on contraceptives), not on any vitamin substitution, and had no food intake 6 hours before blood sampling were randomly selected from men (n=23) and women (n=22) living in the same city. Biochemical and hematological parameters were determined in each subject to exclude hematological disorders, as well as abnormal liver and kidney function. Subjects with a family history of premature vascular diseases were excluded.
In the follow-up study, three groups each of 10 selected patients were reinvestigated 10 to 14 months later. These groups were assigned as follows: Group I, patients with elevated homocysteine (female >12.4 µmol/L, men >13.3); Group II, patients with normal homocysteine and 5-MTHF <17.5 nmol/L (the highest value in the hyperhomocysteinemic Group I); and Group III, patients with normal homocysteine and 5-MTHF >17.5 nmol/L.
The protocol for this study was approved by the Ethics Committee of the University Hospital Basel.
Sample Preparation
For homocysteine, 5-MTHF and SAM EDTA blood samples were placed
on ice after collection and processed within half an hour. Plasma for
5-MTHF measurement, processed under light protection, was added to 10
mg/mL ascorbic acid. For SAM, whole blood was deproteinized immediately
with an equal amount of 5% perchloric acid solution and thoroughly
mixed. For lymphocytes, blood samples were kept at room temperature
until cells were isolated. All samples were stored at -70°C
until analysis.
HPLC Determination of Homocysteine and 5-MTHF in Plasma and SAM in
Whole Blood
HPLC separation was performed with a Jasco pump (JA-980), a
UNIFLOW 4-channel degasser, and a Jasco (AS-950) or Merck (655A-40)
autosampler, both with cooling. Compounds were detected with a Linear
Instrument fluorescence detector (LC 304) or a Milton Roy
Spectro Monitor 3100 UV detector. Data handling was carried out by the
AXXIOM software package series 747/MK2.
Plasma total homocysteine was measured as previously described by
Vester and Rasmussen,29 with the following modifications.
Cysteamine was used as an internal standard. Compounds were separated
on a 4.6x250-mm Nucleosil 120 C18 (5-µm) column with a 4.6x20-mm
guard column filled with the same packing material. Isocratic elution
was performed at a flow rate of 1 mL/min with 0.1 mol/L potassium
dihydrogen phosphate containing 4.5% acetonitrile adjusted to pH 2.1
with 85% orthophosphoric acid. A three-point calibration curve was
performed, using standards added to pooled plasma with each assay
batch. The interassay CV was 6.1% (n=20), the intra-assay CV was
4.5% (n=10), and the detection limit was 1.0 µmol/L in plasma
(signal-to-noise ratio 
5).
Plasma 5-MTHF was determined by the procedure of Leeming et al,30 with fluorescence detection. Samples were handled under light protection and kept on ice. Briefly, frozen plasma samples containing 10 mg/mL ascorbic acid were thawed, and then 300 µL was thoroughly mixed with 100 µL of freshly prepared 10% perchloric acid containing 1% ascorbic acid, centrifuged, and analyzed immediately. Samples were thawed only once to avoid degradation of 5-MTHF. Deproteinized samples were stable for at least 5 hours at 4°C. A volume of 100 µL was injected on the same type of column as described for homocysteine and eluted at a flow rate of 0.75 mL/min. Standard solutions for the calibration curve were prepared in 0.5% ascorbic acid solution and stored in aliquots at -20°C. The interassay CV was 7.1% (n=12), the intra-assay CV was 4.6% (n=10), and the detection limit was 1.0 nmol/L in plasma.
SAM was determined in deproteinized blood samples after thawing and centrifuging for 3 minutes at 4500g.31 The supernatant was filtered through a 0.45-µm HV Millipore filter and analyzed immediately. A volume of 100 µL was injected on a 4.0x200-mm (3-µm) Hypersil ODS C-18 column with a guard column filled with the same packing material. Isocratic elution was performed with 0.1 mol/L sodium acetate buffer containing 5 mmol/L heptanesulfonic acid and 3.95% acetonitrile adjusted to pH 4.55 with glacial acetic acid, at a flow rate of 0.6 mL/min. Samples were stable for at least 8 hours at a temperature below 10°C.
Standards in the range of 0.5 to 5 µmol/L were prepared in 0.4 mol/L perchloric acid and aliquots stored at -20°C. The interassay CV was 7.6% (n=20), the intra-assay CV was 4.1% (n=10), and the detection limit was 0.1 µmol/L in whole blood. The recovery of SAM determined by adding trace amounts of SAM to whole blood before deproteinization was 98%.
Other Procedures
Total Vitamin B12 was determined with a dual
competitive binding assay performed with the Dualcount Solid Phase No
Boil radioassay from Diagnostic Products Corporation.
Routine biochemical tests were performed with a Hitachi model 911
analyzer. Lymphocytes were isolated at room temperature from
whole blood samples as previously reported.32
Assay of MTHFR Activity
This enzyme was assayed according to the method of Rosenblatt
and Erbe,33 with the following modifications.
[14C]5-MTHF was pretreated with dimedone and extracted
with toluene to reduce the blank values. The final assay volume was 100
µL, and extraction of the reaction product was performed with 1
mL toluene. The protein content of the assay was 50 to 250 µg.
Activity was measured without heat (specific activity) and after
heating at 42°C for 10 minutes (heat stable). These conditions for
determining heat lability were selected after investigations were
conducted in control cells at temperatures between 42°C and 46°C
for different times.
Reagents
Tri-n-butylphosphine,
N,N-dimethylformamide,
7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid, and
2-mercaptoethylamine (cysteamine) were purchased from Sigma Chemical
Company; L-homocysteine and 5-MTHF were from Fluka
Chemicals AG; SAM was obtained from Boehringer Mannheim AG; and
5-[14C]methyl tetrahydrofolic acid barium salt was from
Amersham Life Science. All other chemicals were of the highest purity
commercially available.
Statistical Analysis
Unpaired two-group comparisons, using a t test
for measured variables and 
2 test for
contingency tables, respectively, were performed to detect differences
between the respective groups. Multiple regression models were used to
analyze the dependency of homocysteine and SAM from other
measured variables. The association with the relative risk between
quartiles of homocysteine and SAM was expressed as odds ratios.
Adjustments for sex, age, BMI, cholesterol, and
triglycerides were calculated as estimates, using a logit
model. This analysis was performed by the PROC PROBIT with the
SAS/STAT software package.34 Values of P<.05
were considered to indicate statistical significance. Unless indicated
otherwise, values are expressed as mean±SD.
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Results |
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Two patients with severe renal failure, with creatinine values >800 µmol/L, were excluded from the study, since it is known that hyperhomocysteinemia is present in such patients from the early stage of renal failure and increases as creatinine clearance declines.35
Comparison Between Cases and Control Subjects
The individual values of homocysteine, 5-MTHF, and SAM are shown
in Figs 1
and 2. Mean biochemical
parameters and demographic data are shown in Table 1. Since the concentrations of cholesterol,
triglyceride, creatinine, total homocysteine,
5-MTHF, SAM, and vitamin B12 were not normally distributed,
the natural logarithms were used in the statistical
analyses.
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Plasma total homocysteine was significantly elevated in CAD patients
compared with the control group (female: 9.3±3.0 µmol/L versus
7.3±1.9, P<.05; male: 11.0±4.3 versus 8.0±2.0,
P<.001 in patients and control subjects, respectively). The
upper limit of the normal range of homocysteine was 12.4 µmol/L for
women and 13.3 µmol/L for men. The normal range was defined by the
95% tolerance interval (with 
=.05), ie, the interval that contains
at least 95% of the population, with a probability of 1-. These
intervals were calculated with the tolerance factor obtained from
scientific tables.36 Twelve patients (1 female, 11 male)
had total homocysteine levels above this limit and were considered
abnormal. No significant differences in homocysteine, 5-MTHF, SAM, and
vitamin B12 levels or biochemical parameters
were found between sexes in the control group or the patient group,
except for creatinine and estimated creatinine
clearance.37 5-MTHF and SAM were both significantly lower
in patients than in control subjects. Vitamin B12 values
were similar in both groups. There were also no significant differences
between cases and control subjects for biochemical
parameters such as cholesterol,
triglycerides, glucose, creatinine, and
estimated creatinine clearance when gender was considered.
However, significant differences were found for BMI and age. When
adjusted for age and BMI, significant differences for homocysteine,
5-MTHF, and SAM still remained between patients and control subjects
(P<.002, P<.02, and P<.01,
respectively).
Dependency of Total Homocysteine on Other
Parameters
Multiple regression analysis was performed to reveal a
possible dependency of homocysteine on any of the other
parameters. It showed a significant relation of
homocysteine only with 5-MTHF in the patient group (Table 2). The relationship between 5-MTHF and homocysteine in
the individual CAD patients is shown in Fig 1
. 5-MTHF levels were
clearly lower (12.4±1.0 versus 21.2±9.2 nmol/L) in patients with
elevated homocysteine levels than in those with normal homocysteine
levels. However, about 40% of the patients with normal homocysteine
levels had 5-MTHF levels <17.5 nmol/L, which was the highest value in
the hyperhomocysteinemic group.
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To ascertain the role of homocysteine as a risk factor in this group of
patients, the odds ratios were calculated for cases and control
subjects per quartile increase of homocysteine after adjustment for
sex, age, BMI, cholesterol, and triglycerides
(Table 3). This method of data analysis confirms
that the prevalence of CAD is higher for homocysteine values within the
highest quartile.
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A 2 test for contingency tables for smoking and
hypertension revealed no significant difference between patients with
normal and those with elevated homocysteine levels.
Dependency of SAM on Other Parameters
Since there was a marked difference of SAM values between control
subjects and patients (Fig 2), multiple regression analysis for
SAM and the other variables was performed. SAM concentrations were
independent of any of the parameters measured in patients
and were correlated only with cholesterol in the control
group (P<.05). The odds ratios for cases and control
subjects per quartile increase of SAM when adjusted for sex, age, BMI,
cholesterol, and triglycerides showed a higher
prevalence of CAD within the lowest quartile of SAM (Table 3).
Follow-up Study
Homocysteine and 5-MTHF values measured initially and between 10
and 14 months later in patients divided into three groups, as defined
in the "Methods" section are shown in Table 4. No
significant difference in homocysteine or 5-MTHF concentrations was
observed between the first and second measurements in any of the three
patient subgroups, and there was a significant correlation between
initial and follow-up values for both homocysteine and 5-MTHF
(r=.82, P<.001 and r=.68,
P<.001, respectively).
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MTHFR activity in lymphocytes, measured without preheating, was lower in the patient group (n=25, 12.6±8.5 nmol·mg protein-1·h-1) than in control subjects (n=10, 22.3±6.7). The level of activity remaining after preheating was below the lowest control value in seven of the patients. When the three subgroups are considered individually, increased thermolability was observed in three of the nine patients with elevated homocysteine and low 5-MTHF (Group I), in none of those with normal homocysteine and low 5-MTHF (Group II, n=7) and in four of those with normal 5-MTHF and homocysteine values (Group III, n=9).
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Discussion |
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This study confirms previous reports3 4 5 21 of elevated homocysteine levels in a significant proportion of patients with CAD. Seventeen percent of patients in this study showed homocysteine levels above the upper limit of the control values, even when sex was considered in calculating the normal range and in the statistical analysis, which was necessary because only 20% of the patient group were female compared with 50% in the control group. Consistent with other studies,22 we showed an association of the occurrence of CAD with elevated homocysteine concentrations, even after adjustment for sex, age, and other risk factors. The lack of the expected gradual increase5 of the relative risk of homocysteine levels (Table 3
) may be due to the rather
healthy control group, which is insufficiently matched with the patient
group. It must be borne in mind that with this study design, calculated
risk factors must be interpreted with caution, since the possibility
that the disease may alter homocysteine or vitamin levels cannot be
excluded. However, a recent prospective study showed that high levels
of homocysteine occur before the disease.4 The finding of
similar homocysteine values in patients after an interval of 1 year
supports the idea that this parameter plays a role in the
disease process and is not just altered by the disease itself. It
confirms stable values over time in this type of disease pathology, as
also reported in studies of control subjects and patients with
different forms of vascular disease.38 39 In this study we found significantly lower 5-MTHF levels in patients than in control subjects, supporting a previous study in which significantly lower total folate levels were found in CAD patients compared with control subjects22 and in contrast to the finding of Hopkins et al,6 who found no difference.
Furthermore, there was a significant relationship between homocysteine
and 5-MTHF in the patient group, as indicated by the multiple
regression analysis (Table 2). Interestingly, this
analysis showed no significant correlation between these two
related metabolites in the control subjects, in contrast to other
studies that reported a correlation between homocysteine and total
folate in control subjects also.6 40 For the first time,
we have determined 5-MTHF itself in relation to mild
hyperhomocysteinemia. 5-MTHF constitutes about 90% of total
folate41 in plasma of healthy, normally nourished humans,
which was confirmed by Leeming et al30 using a specific
HPLC method. In erythrocytes, however, reported 5-MTHF-polyglutamate
content varies between 100% of the total folyl-polyglutamates
(measured by microbiological and radioisotope assays41 )
and only about 40%, measured by the HPLC method of Leeming.
Additionally, relative proportions of different folate forms may differ
in some disease states, particularly when a disruption of folate
metabolism exists, as for example in patients with the
thermolabile MTHFR mutation, in which reduced extracellular and
elevated intracellular folate was reported.42 In addition,
the proportion of 5-MTHF in relation to 10-formyltetrahydrofolate may
be lower in some disease states in which cell division is accelerated,
for example, in leukemia or polyarthritis patients.41
Therefore, the measurement of the active substrate directly involved in
one of the main enzyme reactions that removes homocysteine would seem
to be logical in the investigation of factors related to
hyperhomocysteinemia. Using this approach, we have found a clear
correlation between plasma 5-MTHF and total homocysteine in these
patients with CAD. Even though none of the patients were deficient in
folate,6 the finding of relatively low plasma 5-MTHF
values in all patients with increased total homocysteine (compared with
the whole group of patients) indicates a direct relationship between
these two metabolites. Thus, low levels of 5-MTHF may lead directly to
elevated homocysteine owing to lack of sufficient substrate for normal
function of methionine synthase. However, 37% of all patients who show
similarly low 5-MTHF concentrations have normal homocysteine levels,
indicating that 5-MTHF levels do not predict homocysteine concentration
in all cases. Furthermore, it cannot be excluded that high homocysteine
itself may lead to low plasma levels of 5-MTHF owing to extra loading
of the methionine synthase system. As found for homocysteine, plasma
5-MTHF did not vary substantially when measured in two samples
collected about a year apart. This finding suggests that 5-MTHF levels
remain stable over such a period of time despite possible changes of
diet or lifestyle, which might be expected after recovering from a
cardiac event.
Recently, thermolabile MTHFR has been shown to be an independent risk factor for vascular diseases, with an incidence of 17% in CAD patients and 5% in control subjects. In the study by Kang et al17 in 1991, not all patients with this enzyme abnormality showed elevated homocysteine levels, whereas Engbersen et al18 reported increased thermolability of MTHFR in 28% of hyperhomocysteinemic patients with vascular disease but not in patients with normal homocysteine concentrations. Such an abnormality ought to result in reduced tissue levels of the active coenzyme of folate, 5-MTHF. In keeping with this idea, three of nine patients with elevated homocysteine and relatively low 5-MTHF from our study did indeed show increased thermolability of MTHFR. In patients without increased thermolability, another mechanism for elevated homocysteine must exist. The existence in our study of patients with normal homocysteine, relatively high 5-MTHF, and increased thermolability, as found in four of nine subjects, could be explained by a protective increase of 5-MTHF owing to an exogenous factor, such as diet. Furthermore, none of those patients with normal homocysteine and relatively low 5-MTHF exhibited increased thermolability, in keeping with the idea of sufficiently efficient enzyme capacity to prevent hyperhomocysteinemia despite the relatively low 5-MTHF levels.
An important finding is the previously unreported significant difference of SAM values between patients and control subjects. The lack of correlation of SAM with other parameters indicates that its low level in erythrocytes may be independently associated with the development of CAD. It can be speculated that relatively high levels of SAM might act as a protective factor against vascular disease. Such an effect would presumably be insufficient to prevent vascular damage by the extremely high levels of homocysteine in homozygous CS deficiency, in which high levels of SAM occur. Also, whether low levels of SAM in persons with MAT deficiency might lead to a higher prevalence of vascular events cannot be answered because of the rarity of such subjects of sufficiently high age.43 However, it cannot be excluded that medication taken by the patients may lead to these low SAM levels. Only one study44 in rats has addressed this possibility. In that study, no effect of long-term administration of aspirin and acetaminophen on SAM metabolism was found.
SAM is formed in humans by the action of MAT, which exists in various isoenzyme forms.45 Only the MAT-II isoenzyme occurs in erythrocytes, in contrast to liver, which also contains the additional MAT-III.45 Therefore, red cell SAM is probably influenced only by the isoenzyme MAT-II, which cannot adapt immediately to changes in methionine and may therefore not reflect liver metabolism. Also, SAM is thought to be retained within its cell of synthesis,46 and these changes may only reflect red cell metabolism. Nevertheless, this finding of an association between CAD and low SAM levels demonstrated by the odds ratios in these cases and control subjects warrants further investigation and confirmation by long-term studies, especially in different vascular disease patient groups with appropriate control subjects and also accounting for possible influences by drugs.
In summary, we showed that despite a significant correlation between homocysteine and 5-MTHF in CAD patients, low levels of 5-MTHF alone do not conclusively predict elevated homocysteine concentrations. We also demonstrated significantly low SAM levels in both hyperhomocysteinemic and normohomocysteinemic CAD patients, leading to the question of whether SAM plays a role as a protective agent in vascular pathology. Further investigations are needed to confirm our findings in additional vascular disease patients, to exclude any influences by drugs, and to investigate the ratio of SAM to S-adenosylhomocysteine, which requires more sensitive methods for measuring S-adenosylhomocysteine.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants from the Swiss National Science Foundation (No. 32-039439.93), the Treubel Foundation, and the Sandoz Foundation, Basel, Switzerland.
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Footnotes |
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Reprint requests to Brian Fowler, PhD, Metabolic Unit, University Children's Hospital, 4005 Basel, Switzerland.
Received November 7, 1995; accepted February 1, 1996.
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