From the University of Pittsburgh, Department of Epidemiology,
Pittsburgh, Pa.
The significance of
any association between cardiovascular disease and
circulating homocysteine concentrations is attracting considerable
attention. The normal activities of the transsulfuration and
remethylation pathways maintain intracellular homocysteine levels
within a narrow range, and the controlled release of homocysteine into
blood results in blood measurements that provide an accurate index of
homocysteine status. In the circulation, homocysteine is rapidly
oxidized, and very little homocysteine remains in the reduced form. The
majority of homocysteine forms a disulfide bridge with protein, and
some reacts either with itself to produce homocystine or with cysteine
to form the mixed disulfide
cysteine-homocysteine.1 Most analytical
procedures include a reduction step and do not distinguish between the
reduced and various oxidized forms of homocysteine; thus, the analyte
measured is referred to as homocyst(e)ine. The normal range is unclear
but may fall between 5 and 15 µmol/L.
Analyses of homocysteine usually involve fasting samples
of either serum or plasma. The concentrations are higher in serum, and
increases of
A complicating aspect of homocysteine metabolism for
cardiovascular studies is that homocysteine
concentrations may increase after a myocardial infarction or a stroke.
Critically, data are not available for samples obtained before and
after an event. However, analysis of samples obtained at the
time of a myocardial infarction and up to 180 days later indicated an
increase in homocysteine concentration from 12.9±0.9 to 15.3±1.1
µmol/L.4 Similarly, samples collected within 2
days of a stroke and up to 645 days later exhibited a rise in
homocysteine concentration from 11.4 to 14.5
µmol/L.5
In vitro, a very wide range of effects have been attributed to
homocysteine.6 These include direct damage to
endothelial cells, flawed platelet activity,
elevated procoagulant activity, increased collagen synthesis, and
enhanced proliferation of smooth muscle cells. Biochemically, it has
been proposed that homocysteine modifies eicosanoid
metabolism, promotes translocation of protein kinase C from
cell nuclei and cytoplasm to the cell membranes, and induces
c-fos and c-myb activity.7
Many of these observations are related to the pathogenesis of
cardiovascular disease. General concerns about the
results, however, involve whether the effects are specific for
homocysteine and occur at concentrations found in the majority of
cardiovascular patients. Cysteine, which is also a
sulfur-containing amino acid, is present in blood at far higher
concentrations than homocysteine. In vitro, experiments are typically
performed at homocysteine concentrations of
In animal experiments, the feeding of an atherogenic-type diet to
cynomolgus monkeys produces both
hypercholesterolemia and
hyperhomocystinemia.8 However, the subsequent
reduction of homocysteine levels by vitamin B supplementation was not
associated with an improvement in endothelial function.
The available mouse models of atherosclerosis may
provide a better method to directly test whether moderately elevated
homocysteine levels or reduced folic acid concentrations cause
atherosclerotic lesions and whether the process can be reversed by
supplementation with folic acid or vitamins B6
and B12.
In studies of human patients, the results of the numerous case-control
studies comparing subjects with myocardial infarction, stroke, and
peripheral vascular disease with control subjects are
consistent with the hypothesis that higher levels of
homocysteine and lower blood levels of folic acid,
B12, or B6 are found more
frequently in cases than in controls.9 There is,
however, no evidence that moderately high levels of homocysteine
(generally found in these case-control studies or in prospective human
studies) are a direct cause of atherosclerosis.
The inconsistent results of genetic studies of homocysteine
metabolism involving moderate elevations of homocysteine
and risk of cardiovascular disease are
puzzling.10 We would expect that individuals with
the genetic disorders of homocysteine metabolism who were
subjected to moderately elevated homocysteine levels for long periods
of time (ie, from an early age) would have an increased risk of
vascular disease. The results of the current
Atherosclerosis Risk in Communities (ARIC) study are
consistent with those of other studies that fail to document an
association between mutations in homocysteine-metabolizing enzymes and
risk of vascular disease.
Schwartz et al11 recently studied 79 women <45
years old with coronary heart disease and control subjects
selected in a population-based study in the state of Washington. Case
patients had higher mean homocysteine levels, with a 2-fold relative
risk versus control subjects and lower folic acid concentration in
their blood. Among control subjects, 12.7% were homozygous for the
MTHFRT677 allele compared with 10% of the
case patients. Those with this allele had higher plasma
homocysteine levels, and the differences in homocysteine levels between
those with and without the MTHFRT677 allele
were similar to the differences in homocysteine levels between the
cases and controls.
In a related study, deJong et al12 reported that,
among 450 siblings of 167 young patients with vascular disease, they
observed that
The overall results from prospective studies such as ARIC are
inconclusive.13 Some show a positive relationship
between coronary artery disease and homocysteine levels and an
inverse correlation with folic acid, B12, or
B6 concentrations. Other well-designed studies
with equally large numbers of cases and power show no
consistent relationship between homocysteine levels and the
risk of disease.
In ARIC, only plasma pyridoxal 5'-phosphate was consistently
associated with a lower risk of cardiovascular disease.
There was, as noted, no relation between dietary intake of either folic
acid, B12, B6, or vitamin
supplements and risk of cardiovascular disease. The
higher serum levels of the vitamins are almost certainly related to
vitamin supplementation rather than strictly dietary intake.
Unfortunately, the ARIC study has limited information regarding
specific vitamin intake. No detailed information was provided about
selection bias for vitamin supplement use. We know, however, that
vitamin supplement users are healthier and better educated and have
fewer cardiovascular risk factors and therefore would
have lower risk of cardiovascular disease. Thus, the
lower risk associated with pyridoxal 5'-phosphate may be causal or a
measure of selection for lower cardiovascular risk.
What, then, are the possible associations between homocysteine, B
vitamins, and vascular disease?
1. The null hypothesis is that there is no causal association between
moderately elevated homocysteine levels and risk of vascular disease or
atherosclerosis. The causal pathway could be that
vascular disease results in an increase in homocysteine levels. This
hypothesis would be consistent with the results of numerous
case-control studies. The cases would have higher homocysteine levels
because they have more atherosclerosis and vascular
disease, not because homocysteine caused the vascular disease. How,
then, could vascular disease cause an elevation of homocysteine levels?
We know that vascular disease is an inflammatory process. Individuals
with vascular disease have elevated levels of inflammatory markers,
such as acute-phase proteins (ie, C-reactive
protein),14 adhesion
molecules,15 and sedimentation
rate.16 It is unlikely that these markers are
causal, but they probably relate to the inflammatory process of
vascular disease. Folic acid is important in DNA synthesis.
Inflammation is associated with increased mitotic activity. Could the
inflammatory process result in an increased demand for folic acid and
secondary elevation of homocysteine levels, especially in those
individuals with low folic acid intake or those who have a specific
genetic abnormality of homocysteine metabolism?
The results of studies of subclinical vascular disease have shown that
higher homocysteine levels are associated with greater carotid artery
intimal-medial wall thickness and plaque,17 and
the extent of coronary artery disease on angiography would also
be consistent with the null hypothesis. Individuals with more
atherosclerotic burden would have more inflammation, greater demand for
folic acid, and higher homocysteine levels. Thus, we would anticipate
that individuals with extensive subclinical vascular disease would have
both elevated levels of inflammatory markers and raised homocysteine
concentrations associated with low folic acid,
B12, or B6 levels. The ARIC
study, for example, showed a positive association between carotid
artery intimal-medial thickness and homocysteine
levels17 but, as noted, no consistent
relationship for risk of clinical cardiovascular
disease. The association of homocysteine and subclinical vascular
disease appears to be stronger among participants with
hypertension.17 Possibly, homocysteine has
effects on vascular disease independent of
atherosclerosis.
The conclusions of longitudinal studies with regard to homocysteine
levels and the risk of cardiovascular disease could be
influenced by the varied prevalence of subclinical
atherosclerosis among the subjects. Levels of
homocysteine would be higher in clinical cases, who had more extreme
subclinical disease at time of blood draw, than in noncases. It is
important, in prospective studies, to look at the relationship between
homocysteine levels and folic acid in relation to age as well as the
extent of subclinical vascular disease. There is some evidence that
homocysteine levels are positively correlated with inflammatory
markers.
The Cardiovascular Health Study is currently evaluating
homocysteine levels and risk of cardiovascular disease.
This longitudinal study is similar to the ARIC study and also includes
measures of subclinical disease and markers of inflammation.
Preliminary results to be presented at the Second International
Conference on Homocysteine by Schwartz et al (unpublished data, 1998)
show that higher homocysteine levels were significantly related to the
risk of myocardial infarction and coronary heart disease deaths
but not to stroke. Again, consistent with the ARIC study, there
was no relationship between the MTHFR genotype and
the risk of coronary heart disease or stroke. This study
includes predominantly older individuals, age
Case-control studies (no matter how well designed or how large) cannot
provide information with regard to this null hypothesis and probably
should be abandoned in future studies of homocysteine, vitamins, and
disease. The association of homocysteine levels and, perhaps, B
vitamins should be investigated in other inflammation-related diseases,
and the effects of various therapies that moderate the inflammatory
process should be examined in relation to homocysteine blood
levels.
2. A decrease in folic acid or B12 and
B6 is the primary cause of the increased risk of
vascular disease. Elevated levels of homocysteine may just be a marker
of low vitamin levels but not be important in the causal pathway of the
disease. Treatment with folic acid would decrease the risk of vascular
disease and concurrently reduce homocysteine
levels.18 However, the decline in homocysteine is
not necessarily beneficial. A high sedimentation rate is associated
with the risk of vascular disease. Aspirin will reduce the risk of
myocardial infarction and possibly also reduce the sedimentation rate.
It is unlikely that the high sedimentation rate and its reduction is
the primary benefit of aspirin in terms of reducing vascular
disease.
The reduction in stroke reported in the Linxian County, China, Vitamin
Trial might be consistent with the benefits of folic acid,
B12, or
B6.19
3. High homocysteine levels are directly related to development of
atherosclerosis. The equivocal results of the
prospective studies may be due to small sample sizes and power,
measurement error for homocysteine, and perhaps problems of storage of
samples for long periods of time (especially in nested case-control
studies). Nevertheless, there is no experimental evidence from animal
studies indicating that moderately elevated homocysteine is a "cause
of atherosclerosis."
Currently, there is no consistent evidence that blood
concentrations of homocysteine or of folic acid are related to the
population levels of atherosclerosis. In general, there
is a fairly high correlation between homocysteine levels in populations
and elevated LDL cholesterol. Alfthan et
al20 noted a positive correlation of homocysteine
levels with cardiovascular disease mortality rates, but
the European Concerted Action Project found no consistent
geographic trend among 9 countries.21
A comparison of the prevalence of atherosclerosis in
populations that are discordant (ie, have high or low
cholesterol levels and high or low homocysteine levels) and
the extent of atherosclerosis or
cardiovascular disease might be useful, especially in
relation to intake of B vitamins and perhaps genetic polymorphism.
It will be interesting to see whether there are populations in which
there are high homocysteine levels at the population level but low LDL
cholesterol levels and whether, in these populations, there
is evidence of extensive atherosclerosis. To date, no
population data have been presented to support such an
association.
4. Homocysteine levels are related to the risk of vascular disease
independently of any effect on the development of
atherosclerosis. There are at least 2
possibilities:
A. High homocysteine levels could be associated with an enhancement of
inflammatory processes, with the stability of the atherosclerotic
plaque, and with increased risk of clinical disease, given subclinical
atherosclerotic disease. A high homocysteine level would be additive to
other risk factors for atherosclerosis and subclinical
disease. The strong association of homocysteine levels and risk of
clinical disease among higher-risk populations, such as hypertensives
and diabetics, would support this hypothesis. Longitudinal studies,
such as ARIC and perhaps the Cardiovascular Health
Study, could test the hypothesis that individuals with subclinical
disease and with high homocysteine levels would have an increased risk
of clinical disease independently of other inflammatory markers and
risk factors. Other studies could evaluate the relation of plaque
morphology and changes in plaque characteristics to homocysteine
levels.
B. Higher homocysteine levels could also be related to increased risk
of thrombosis and subsequent clinical disease. Experiments to
characterize the effects of homocysteine include the infusion of
homocysteine into animals. The results appear to be species dependent,
but, particularly in baboons, the infusions result in damage to the
vascular endothelium and reduced platelet survival.
Results of studies in homocystinuric patients, although not unanimous,
suggest impairment of platelet activity and of the clotting
cascade. The association of homocysteine levels and thrombosis could be
related to platelet function, thrombin generation, or
fibrinolysis. The investigation of such associations in
human observational studies is difficult. Several of the large, ongoing
longitudinal studies of cardiovascular disease, such as
CHS and ARIC, have relatively high-quality measures of thrombosis and
fibrinolysis and should look at the associations of
these data with homocysteine and B vitamin levels.
At present, the totality of evidence does not refute or support any
of the above-mentioned hypotheses. More case-control studies will be of
little or no value. Essays describing the wonders of folic acid and
folic acid supplementation and reduction of homocysteine levels to
prevent heart attacks may be counterproductive by offering yet
another false promise to the public, who may become less responsive to
more proven methods of reducing coronary heart disease (such as
lowering LDL cholesterol, ceasing smoking, and controlling
high blood pressure).
The test of the above hypotheses requires good human clinical trials
and more animal experimental studies. It is very unfortunate that it
takes so many years to move from a hypothesis based on observational
case-control studies to prospective longitudinal studies and then to
important clinical trials. This time process needs to be substantially
shortened to bring proven therapies to public health and clinical
utility and to discard promising but relatively ineffective therapies.
Clearly, too much time is spent on reproducing results of observational
studies that have similar limitations.
Secondary prevention trials require smaller sample sizes and probably
shorter follow-up time than primary prevention. If secondary prevention
trials show a direct benefit of lowering homocysteine with vitamin
supplements on the risk of cardiovascular disease, then
the causal hypothesis would be greatly strengthened, and the much
larger and long-term primary prevention trials may not be necessary.
Trials using intermediate vascular end points (such as changes in
carotid artery intimal-medial thickness, plaque, coronary
calcification) may also be of some value. If the secondary prevention
trials are negative, it may force us either to accept surrogate
subclinical end points or to wait a longer period of time for the
results of the primary prevention trials.
It would be pleasant to be able to put folic acid in a hamburger bun
and enjoy half a pound of a juicy high-fat hamburger without worrying
about high LDL cholesterol, coronary
atherosclerosis, and thrombosis. Carrots were not the
panacea. Some hope that putting folic acid in bread will be the next
great public health advance for cardiovascular
disease.
Footnotes
Reprint requests to Lewis H. Kuller, MD, DrPH, University of Pittsburgh, GSPH, Department of Epidemiology, 130 DeSoto St, Pittsburgh, PA 15261.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
References
© 1998 American Heart Association, Inc.
Editorials
Homocysteine, Vitamins, and Cardiovascular Disease
Key Words: Editorials • cardiovascular diseases • homocysteine
10% have been reported in the postprandial
stage.2 Homocysteine levels also increase with
age and are higher in men than in women. A variety of disease states
and medications modify homocysteine concentrations, and notably,
impaired renal function may greatly increase homocysteine
levels.3 Measurement of homocysteine should avoid
blood samples that have been stored at room temperature, because red
blood cells may release homocysteine, causing an artifactual increase
in extracellular homocysteine concentrations. 
100 µmol/L, levels
that are found only in the rare individuals homozygous for
cystathionine ß-synthase deficiency. Furthermore, the results are
compared with those obtained from tissue suspended in homocysteine-free
buffer, a concentration never observed in vivo. In contrast,
epidemiological studies showing a significant correlation between
homocysteine levels and cardiovascular disease tend to
report homocysteine concentrations among patients of 11 to 16
µmol/L, only 3 µmol/L higher than the corresponding control
group. Although researchers are attempting to mimic experimentally in a
matter of a few hours or days what may occur naturally over many years,
it is questionable whether a metabolite can be raised 10-fold above
normal concentrations without causing some derangement of
metabolism. 28% of the siblings had hyperhomocystinemia as
estimated by either fasting measurements or postmethionine loading.
They then compared measures of subclinical
atherosclerosis, either peripheral,
coronary, or carotid, among the siblings with or without
elevated homocysteine levels. There was no relationship between levels
of homocysteine and subclinical vascular disease or with the genetic
polymorphisms of the MTHFR gene. The primary
determinants of high risk of subclinical vascular disease among the
siblings of the probands with premature coronary disease were
smoking, high blood pressure, and high cholesterol
levels. 65 years, with a heavy
burden of subclinical disease, inflammatory markers, and measures of
clotting and thrombosis. Further analysis may provide important
information regarding the association between homocysteine and
disease.
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