© 1995 American Heart Association, Inc.
Articles |
Redox Status and Protein Binding of Plasma Homocysteine and Other Aminothiols in Patients With Early-Onset Peripheral Vascular Disease
Homocysteine and Peripheral Vascular Disease
From the Department of Pharmacology and Toxicology (M.A.M., A.M.S., P.M.U.) and the Department of Oncology (P.E.L.), University of Bergen, N-5021 Haukeland Hospital, Bergen, Norway, and the Department of Surgery (C.B.), Karolinska Hospital, S-104 01 Stockholm, Sweden.
Correspondence to Per Magne Ueland, MD, Department of Pharmacology and Toxicology, University of Bergen, N-5021 Haukeland Hospital, Bergen, Norway.
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Abstract Elevated total homocysteine (Hcy) in plasma is an independent risk factor for early-onset vascular disease in the coronary, cerebral, and peripheral arteries. Different forms of Hcy, and their relation to other aminothiols in plasma, have not been investigated in patients with vascular disease. We therefore investigated 65 patients (35 men and 30 women) operated on for peripheral arterial disease at <50 years of age and 65 age- and sex-matched control subjects. Total, reduced, oxidized, and protein-bound Hcy, cysteine (Cys), and cysteinylglycine (CysGly) were measured 0 to 11 years (mean, 6 years) postoperatively, in the fasting state, and after a standard methionine loading dose that caused a transient increase in reduced, oxidized, and protein-bound Hcy. All forms of Hcy and Cys, except reduced Cys, were higher in fasting patients than fasting control subjects. A similar difference between the groups was observed after methionine loading. The levels of most Hcy forms both during fasting and after methionine loading were related to smoking, but multivariate analysis showed that the difference between patients and control subjects could not be explained by smoking alone. Notably, reduced Cys and the reduced/total ratio for Cys were significantly higher in control subjects than in patients, both during fasting and after methionine loading. In both groups, the redox status and protein binding of the various aminothiols in plasma were interactive, as demonstrated by positive correlations between their reduced/total ratios and by a decrease in protein-bound Cys when protein-bound Hcy was elevated during methionine loading. Serum folate and to a lesser degree serum cobalamin and vitamin B6 were predictors of oxidized and protein-bound Hcy in some patients and control subject subgroups. Thus, reduced, oxidized, and protein-bound Hcy are elevated and reduced Cys is decreased in patients with peripheral arterial disease. Reduced Hcy acts as a pro-oxidant in vitro and is a possible atherogenic agent, whereas reduced Cys may be a protective agent as a part of the antioxidant defense system. The protein binding and redox status of different plasma aminothiols are interactive in a way suggesting ongoing redox cycling and disulfide exchange reactions. Thus, Hcy is one component in a complex system.
Key Words: risk factor • cysteine • cysteinylglycine • methionine loading • vitamins
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Introduction |
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There is clinical and epidemiological evidence suggesting that elevated levels of homocysteine (Hcy) in blood are associated with vascular disease. Patients with various inborn errors of Hcy metabolism, causing extremely high levels of homocyst(e)ine in blood and urine, ie, homocystinuria, develop premature cardiovascular disease in early adolescence and even in childhood.1 About 2500 cardiovascular patients and a comparable number of control subjects have been investigated in approximately 20 retrospective and two prospective2 3 epidemiological studies. These investigations show that a moderately elevated concentration of Hcy in plasma, so-called hyperhomocysteinemia, is an independent risk factor for premature vascular disease localized to the coronary, cerebral, and peripheral vessels.4 5 6
Hcy is a sulfur amino acid and a product of transmethylation. It is either degraded to cysteine (Cys) or remethylated to methionine. The former reaction is catalyzed by the vitamin B6–dependent enzyme cystathionine ß-synthase (EC 4.2.1.22), whereas in most tissues, remethylation is catalyzed by methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13), which uses methyltetrahydrofolate as the methyl donor and cobalamin as cofactor.7 Thus, Hcy metabolism is linked to the metabolism and function of folates, cobalamin, and vitamin B6, and this explains why deficiencies of these vitamins are common causes of hyperhomocysteinemia.8
In plasma from healthy subjects, most Hcy (70% to 80%) is protein bound, and the remaining is acid-soluble free Hcy.9 Most free Hcy exists as Cys-Hcy mixed disulfide.10 The sum of all Hcy forms in plasma has been termed total Hcy, which is a robust parameter not affected in vitro by disulfide exchange reactions and redistribution between Hcy forms.8 11 Clinical studies on plasma Hcy in patients with cardiovascular disease,5 6 vitamin deficiencies,12 or other disease states8 are usually based on measurements of total Hcy.
Since Hcy in blood is rapidly oxidized and is associated with plasma proteins, assessment of its redox status and protein binding in human plasma requires immediate derivatization of the reduced Hcy and separation of the free and bound forms. We have recently developed such a method, which measures reduced, oxidized, and protein-bound Hcy, Cys, and cysteinylglycine (CysGly) in human plasma.13 We have determined these parameters in healthy subjects given a peroral methionine14 or Hcy15 load and in plasma from patients with homocystinuria16 and cobalamin deficiency,17 ie, states characterized by marked elevation of plasma Hcy. These data suggest that the concentration, protein binding, and redox status of Hcy induce secondary effects on redox status of other aminothiols in plasma.
The purpose of the present study was first to uncover possible aberrations in the concentration, redox status, and protein binding of plasma Hcy but also in related aminothiols in a population with early-onset vascular disease. A second objective was to investigate whether the dynamic relations that exist between aminothiol forms under conditions of marked elevation (>30 µmol/L) of plasma Hcy14 15 16 17 also exist in healthy persons with normal plasma Hcy levels and in vascular disease patients consistently reported to have moderately elevated plasma Hcy compared with control subjects.5 6 Assessment of redox status and protein binding of several aminothiols in plasma may create a more differentiated picture of these components as possible risk factors for premature vascular disease. In addition, knowledge of remote effects of hyperhomocysteinemia on the levels and redox status of other aminothiols may guide future research on the mechanisms behind the vascular lesions.
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Methods |
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Patients and Control Subjects
In the period 1979 to 1990, 82 patients had vascular reconstruction for peripheral vascular disease (excluding trauma and emboli) before the age of 50 years at the Department of Surgery of Karolinska Hospital and St Görans Hospital, Stockholm, Sweden. This represents about 3% of the total population admitted to these departments for operation. Of the 82 patients, 9 died (all from cardiovascular disease), 3 emigrated, 3 refused to participate in the study, and 1 could not be traced. The remaining 66 patients were available for follow-up.
We investigated 65 patients, 35 men and 30 women. The median age at the
onset of symptoms was 40 years (range, 20 to 49 years); at surgery, 44
years (range, 21 to 49 years); and at follow-up, 49 years (range, 36 to
62 years). They had infrainguinal lesions (17 patients), suprainguinal
lesions (28 patients), or multilevel disease (12 patients). Four
patients were operated on for abdominal aortic aneurysms, 2 patients
for renal artery stenosis, and 2 patients for carotid artery stenosis.
They were compared with 65 randomly selected age- and sex-matched
control subjects selected from the population register. Characteristics
of patients and control subjects are summarized in Table 1
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The participants provided their written informed consent, and the protocol was approved by the ethics committee at the Karolinska Hospital.
Methionine Loading and Blood Sampling
The subjects recruited to the study were called in and
investigated in groups of 6 to 8 subjects, and the number was usually
equally divided between patients and control subjects. They were
subjected to methionine loading by oral administration of methionine
(100 mg/kg body wt) in 200 mL of orange juice. Blood samples were
collected after overnight fasting before loading and 4 hours after
loading. Absorption of methionine was verified by determination of
methionine18 in the fasting and postload samples. The
values for plasma methionine 4 hours after loading were higher than 164
µmol/L in all subjects.
Biochemical Analysis
Blood was routinely collected into three evacuated tubes
containing either monobromobimane (mBrB) or N-ethylmaleimide
(NEM) as thiol-derivatizing reagent or no additions. The blood was
immediately centrifuged at 10 000g for 1 minute at room
temperature to remove blood cells.
From the analysis of blood collected in a solution containing mBrB we obtained reduced thiols, analysis of blood collected into NEM gave the oxidized forms, and total amount of thiol components was assayed in nontreated plasma. The protein-bound form is calculated by subtracting reduced and free oxidized forms from the total amount.
Details on the construction and performance of these assays are described elsewhere.13
Plasma samples were frozen and stored at -70°C until analysis. Vitamin B6 was measured as pyridoxal 5'-phosphate with an enzymic method.19 Serum cobalamin and serum folate were measured by SimulTRAC-SNB Radioassay Kit from Becton Dickinson. Serum cholesterol and triglycerides were measured with an enzymic colorimetric assay (Boehringer-Mannheim automated analyses for Hitachi system 717, Diagnostica). HDL cholesterol was measured after lipoproteins containing apolipoprotein B were precipitated with phosphotungstate/magnesium chloride. LDL cholesterol was calculated according to Friedewald's formula.20
Statistical Analysis
The SYSTAT statistical program, version 5.2, for
Macintosh computer (Systat, Inc) was used for data analysis. All
parameters were tested for goodness of fit to normal distribution as
raw data or after logarithmic transformation with a Q-Q
plot.21 Total, reduced, oxidized, and protein-bound Hcy,
the reduced/total Hcy ratio, triglycerides, cobalamin, and folate were
found to be best fit to a log-normal distribution. The other parameters
were analyzed as nontransformed data. The influence of disease, gender,
and smoking on aminothiol forms was tested for in a multivariate model
(MANOVA), which estimated possible interactions between the variables.
Protein-bound Cys before and after methionine loading was evaluated by paired t test and serum lipids in patients and control subjects were compared by two-sample t test.
We determined the correlation between vitamins and various forms of Hcy using univariate and stepwise multivariate regression analysis.
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Results |
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Population Characteristics
At follow-up, triglycerides were higher and HDL cholesterol was lower in both male and female patients compared with the respective control groups (P<.05). Total cholesterol and LDL cholesterol were significantly higher only in female patients compared with female control subjects (P<.02).
Among the patients, 63 had a history of smoking and 35 were current
smokers, whereas the corresponding numbers for control subjects were 37
and 19. Seven of the patients had diabetes compared with only 1 female
in the control group. Fifteen patients and 8 control subjects had
hypertension (Table 1
).
Aminothiols in Patients Versus Control Subjects
In fasting subjects, all Hcy forms (total Hcy, reduced, oxidized,
and protein-bound forms) were significantly higher in patients compared
with control subjects. Similarly, total and protein-bound Cys were
significantly higher in the patient group, whereas reduced Cys was
higher in control subjects compared with the patients. There were no
sex differences (Tables 2 and 3).
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After methionine loading, all Hcy forms increased threefold to
sevenfold. The largest increase was observed for the reduced form
(Table 2). The differences in aminothiol levels between patients and
control subjects resembled that observed after fasting. All forms of
Hcy and Cys, except reduced Cys, were higher in patients than in
control subjects. Notably, reduced Cys after loading was higher in
control subjects than in patients, in both sexes (Tables 2 and 3).
The postload values showed some sex differences. The difference in
total and protein-bound Hcy between patients and control subjects was
less in women (6% to 9%) than in men (40%), and there was no
significant difference in reduced Hcy between female patients and
control subjects. Furthermore, reduced Cys was significantly higher in
women than in men (Tables 2 and 3).
Total CysGly (Table 2) was not significantly different
(P>.05) between patients and control subjects.
The most notable findings described above (Tables 2 and 3) are
illustrated in the cumulative frequency distribution curves
presented in Fig 1. The whole frequency distribution
curves for total Hcy and total Cys during fasting and after loading in
men, and the curves for total Hcy for women, are displaced to the right
in patients relative to control subjects. The lower panels of Fig 1
demonstrate the low levels of reduced Cys in both male and female
patients, during fasting and after methionine loading, compared with
control subjects.
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We found a significant positive correlation between total Hcy
during fasting and after methionine loading both in patients
(R=.68, P<.001) and control subjects
(R=.56, P<.001), and the postload values did not
discriminate better between the two groups than the fasting levels did
(Table 2, Fig 1). However, fasting and postload values did not give
overlapping results. When the upper limits of the 95% confidence
interval of the individual observations of control subjects were taken
as cutoff points, 8 of 35 male patients had a normal fasting level and
elevated postload total Hcy, and 4 of 35 had an elevated fasting level
and normal postload level. The corresponding fractions for female
patients were 0/30 and 3/30, respectively.
Smoking
We have previously investigated total Hcy in 58 subjects of this
patient population and found that total fasting Hcy was higher in
smokers than in nonsmokers.22 Data presented here show
that this could be ascribed to elevation of both the protein-bound and
oxidized forms. In addition, we could also demonstrate that the
postload levels of all Hcy forms were significantly higher in smokers
than in nonsmokers. Smoking did not influence plasma Cys forms (Table 3). Notably, while the number of smokers was somewhat higher among
patients than control subjects (Table 1), smoking and disease
independently influenced plasma aminothiols (Table 3).
Protein Binding of Aminothiols
There was a positive correlation between protein-bound Hcy and
protein-bound Cys in fasting male (r=.58, P=.001)
and female (r=.47, P=.005) control subjects
whereas only a trend toward a positive relation was found in fasting
male (r=.30, P=.09) and female (r=.18,
P=.35) patients (Fig 2).
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, 
) and after
methionine loading (
, 
). The
lines connect the data points for each individual and indicate the
increase in protein-bound Hcy and decline in protein-bound Cys after
methionine loading.
In female as well as male patients and control subjects, protein-bound
Hcy increased and protein-bound Cys decreased (P<.001)
after methionine loading (Table 2). In almost every individual within
all four subgroups, methionine loading caused a marked drop in
protein-bound Cys (Fig. 2).
Reduced Aminothiols and Redox Status
In both patients and control subjects, reduced Hcy was low during
fasting and increased markedly after methionine loading (Table 2).
After methionine loading, reduced Hcy was positively correlated to
total Hcy in male (r=.36, P<.05) and female
(r=.38, P<.05) control subjects and male
(r=.54, P<.005) and female (r=.77,
P<.001) patients (data not shown).
We investigated the relation between the reduced/total ratio (the
fraction of the total amount that exists in the reduced form) for Hcy,
Cys, and CysGly in patients and control subjects after methionine
loading. In both groups, we found a significant linear relation between
the reduced/total ratio for Hcy and Cys; the relation between the
reduced/total ratio for Hcy and CysGly was weaker. The ratios for Cys
and CysGly also correlated. These relations for patients are shown in
Fig 3. Essentially the same results were obtained for
the control subjects (data not shown).
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Correlations With Vitamins
None of the patients or control subjects had serum cobalamin below
normal (<120 nmol/L), and 10 patients and no control subjects had
serum folate below normal (<5 nmol/L).
A dietary assessment was done with respect to vitamin intake at follow-up. Eight of 65 patients and 9 of 61 control subjects had vitamin supplementation. However, the levels of total fasting Hcy and total fasting Cys were not different in the subjects taking and not taking vitamins (data not shown).
We tested for the relation between various forms of Hcy and serum
folate, serum cobalamin, and vitamin B6 using regression
analysis. Serum folate was negatively correlated with total and
oxidized Hcy in all four groups except in female control subjects and
was negatively correlated with protein-bound Hcy in male patients.
Serum cobalamin and vitamin B6 showed a negative
correlation only with oxidized Hcy, and this relation was confined to
male patients (cobalamin) and to male patients and female control
subjects (vitamin B6) (Table 4).
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Discussion |
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Study Design
This is a case-control study including most patients operated on for early-onset peripheral vascular disease in two hospitals in Stockholm over a 12-year period.
Determination of redox status of three aminothiols in plasma is a cumbersome procedure requiring immediate sample processing13 ; stored plasma cannot be used. This strictly limits the number of both patients and control subjects included in this study. Because of limited recruitment of patients, we had to collect patients with a history of early-onset vascular disease, and follow-up and blood sampling were 0 to 11 years (mean, 6 years) after surgery. This time interval may introduce an error, because plasma Hcy changes with age23 and possibly because of altered lifestyle, in particular, vitamin intake. However, the change in plasma Hcy over a 10-year period is limited (about 10%) and not significant.23 Furthermore, in both healthy subjects and patients with cardiovascular disease, there is a highly significant correlation between plasma Hcy in fresh samples and old plasma samples from the same individuals stored for 6 to 16 years.24 Only 8 patients were regularly taking vitamin supplements and the plasma Hcy was not significantly lower in this subgroup.
The patients were compared with age- and sex-matched control subjects
selected from the population register. Since age and sex in addition to
vascular disease are known to correlate with the plasma Hcy
levels,5 these parameters were included in the statistical
model (Table 3). We also tested for smoking and its interactions (Table 3), because smoking is a particularly strong risk factor for peripheral
vascular disease25 and has been found to influence plasma
Hcy in two recent studies.26 27 The small size of the
sample matrix did not justify the inclusion of additional dimensions
such as serum lipids, diabetes, and hypertension, which in most studies
have been found not to influence plasma Hcy level.5
Normal Hcy Values and Definition of Hyperhomocysteinemia
The mean values for total Hcy in healthy men (11.7 µmol/L) and
women (11.2 µmol/L) reported in the present work (Table 2) equal
the mean total Hcy (11.58 µmol/L) reported for a large population
(n=3000) of healthy men aged 40 to 42 years8 and normal
values published by others.23
There is some variability of total Hcy relative to age and sex, and the normal range for total Hcy has been somewhat arbitrarily set at 5 to 15 µmol/L.8 Kang et al6 suggested the term hyperhomocysteinemia for total Hcy above normal and defined moderate hyperhomocysteinemia as levels up to 30 µmol/L. These are the levels often encountered in subjects without known defects in Hcy metabolism and in patients with premature cardiovascular disease.5 Intermediate hyperhomocysteinemia, defined as levels between 30 and 100 µmol/L, is often present in patients with cobalamin or folate deficiencies, whereas severe hyperhomocysteinemia, defined as >100 µmol/L, is usually confined to patients with inborn errors of Hcy metabolism, ie, homocystinuria.8
Aminothiol Forms in Patients and Control Subjects
The present investigation demonstrates that fasting total Hcy
is 34% to 42% higher in female and male patients with early-onset
peripheral vascular disease than in matched control subjects, a
difference that is highly significant. Similar differences were found
after a methionine loading test (Tables 2 and 3, Fig 1). These data are
consistent with the results from several epidemiological studies,
showing that the amount of total plasma Hcy in a population with
cardiovascular disease is about 30% higher than in healthy
subjects.5 6
In some early works28 29 30 31 and one recent study32 the Cys-Hcy mixed disulfide, which corresponds to free oxidized Hcy in the present work, was measured and found to be elevated in plasma from patients with cardiovascular disease. However, the different Hcy forms in plasma from patients with hyperhomocysteinemia and cardiovascular disease have not been investigated previously.
Table 2 shows that all Hcy forms were increased in fasting patients
compared with control subjects. Particularly in male patients, reduced
Hcy was markedly elevated. A similar difference between patients and
control subjects was observed after methionine loading, except that the
amount of reduced Hcy in female patients equaled that found in female
control subjects.
We also measured different forms of plasma Cys and CysGly and found
that total, protein-bound, and oxidized Cys were significantly higher
in both female and male patients compared with control subjects, both
during fasting and after methionine loading (Tables 2 and 3, Fig 1).
This finding agrees with the results of a Japanese study33
showing that total and free Cys (and Hcy) in plasma were elevated in 45
patients with cerebral infarction.
Reduced Cys (and accordingly the reduced/total ratio) was the only
aminothiol component in plasma that was significantly higher in control
subjects than in patients. This was a consistent finding in men as well
as women and was found in fasting as well as after methionine loading
(Tables 2 and 3, Fig 1). The low level of reduced Cys and the low
reduction state of Cys in patients with peripheral vascular disease may
reflect impaired redox thiol status in at least some of these patients.
Conceivably, other thiols may be involved, as recently demonstrated by
low sulfhydryl reactivity of albumin in patients with coronary artery
disease.34
Redox Status and Protein Binding of Aminothiols
We have previously studied the dynamic relation existing between
the reduced, oxidized, and protein-bound forms of various aminothiols
in human plasma. These studies included healthy subjects with a
transiently increased plasma Hcy due to a methionine14 or
Hcy15 load, 8 homocystinuric patients,16 and
13 patients with hyperhomocysteinemia due to cobalamin
deficiency.17 These clinical data demonstrated that
reduced Hcy is low under physiological conditions but increases as a
function of total Hcy, especially at high (>100 µmol/L) levels; that
alterations in the redox status of Hcy affected the redox status of
other aminothiol components in plasma; and finally, that high levels of
Hcy displace Cys from the binding site in plasma.14 15 16 17
One objective of the present work was to investigate whether these relations between plasma aminothiols observed under conditions of intermediate and severe hyperhomocysteinemia14 15 16 17 also exist in patients with vascular disease having moderate hyperhomocysteinemia (15 to 30 µmol/L) during fasting and in healthy subjects with normal Hcy level. Knowledge about secondary effects on other aminothiol components in plasma may point to future directions of research on processes responsible for the vascular lesions in patients with hyperhomocysteinemia.
First, we showed that reduced Hcy increased as a function of
total Hcy in both patients and control subjects (data not shown).
Second, we could demonstrate a positive correlation between the
reduced/total ratio for Hcy and Cys, between the ratio for Hcy and
CysGly, and between the ratio for Cys and CysGly in both control
subjects (data not shown) and patients (Fig 3). For a particular
aminothiol component, this ratio represents the fraction of the
total amount (sum of reduced, oxidized, and protein-bound) existing in
the reduced form and is a measure of its redox status in plasma. The
positive correlation between the ratios suggests interaction between
these aminothiol forms through redox reaction and thiol-disulfide
exchange. Thus, altered redox status of plasma Hcy is not an isolated
event but affects the redox status of related aminothiol components.
The positive correlation between protein-bound Hcy and protein-bound
Cys in the fasting state may reflect that both Hcy and Cys are products
of the transsulfuration pathway.7 The marked increase in
bound Hcy after methionine loading caused a drop in bound Cys in most
patients and control subjects (Fig 2). Similarly, in patients with
intermediate and severe hyperhomocysteinemia, a negative
correlation between protein-bound Hcy and Cys has been
found.16 17 35 These relations between protein binding of
Hcy and Cys may be explained by displacement of bound Cys by Hcy. This
explanation is supported by the presence in plasma of
saturable35 binding sites that preferentially interact
with Hcy.35 36 37
Sex
It has been suggested that efficient methionine metabolism in
premenopausal women offers protection against cardiovascular
diseases.38 This hypothesis was based on the findings of
lower levels of fasting and postload Hcy-Cys mixed disulfide
(corresponding to oxidized Hcy in the present work) in
premenopausal women than in postmenopausal women and men.
Marked sex-related differences in Hcy levels38 have
recently been contested by Andersson et al.23 Notably,
they also observed that about 30% of healthy postmenopausal women
responded to methionine loading with distinctly higher values than men
and premenopausal women. We made similar observations in the
present study. Total fasting Hcy in men was only marginally higher
than in women (Table 2). Furthermore, female control subjects had
higher postload total Hcy than men (Table 1), and 5 (age, 46 to 60
years) out of 31 female control subjects and no male control subjects
had postload protein-bound Hcy higher than 34 µmol/L (Fig 2).
The differences between patients and control subjects in the levels of
all Hcy forms after loading were larger in men than in women (Tables 2 and 3), as revealed by a statistical interaction between disease and
sex (Table 3). This is also illustrated for total Hcy in the frequency
distribution graph (Fig 1). Furthermore, both reduced Hcy and reduced
Cys after loading were significantly higher in women than in men
(Tables 2 and 3).
Vitamins
Total, protein-bound, and oxidized Hcy were negatively correlated
with serum folate in most groups, whereas serum cobalamin was
negatively correlated only with oxidized Hcy in male patients and
vitamin B6 with oxidized Hcy in male patients and female
control subjects (Table 4). These data agree with the observations that
serum folate is a strong predictor of plasma Hcy, whereas weaker
correlations with serum cobalamin and vitamin B6 are
occasionally found.39 40 However, the design of the
present study was not optimized to study the relation between Hcy
forms and vitamin status, especially because only a few
vitamin-deficient subjects were included.
Possible Mechanisms
Several mechanisms, involving diverse targets like
lipoprotein(a),41 endothelium anticoagulant
mechanisms,42 43 endothelial cells, coagulation factors,
platelets, and LDL, have been suggested for the vascular damage caused
by elevated Hcy.5 In vitro oxidative modification of
LDL,44 45 inhibition of endothelium anticoagulant
mechanisms,43 and endothelial cell
injury46 47 have been demonstrated in the presence of
reduced Hcy and are believed to be mediated by oxygen-derived free
radicals formed during oxidation of Hcy catalyzed by a redox metal.
Various forms of Hcy should be considered as mediators of the possible atherogenic effect of hyperhomocysteinemia. The fraction associated with plasma protein(s) is probably biologically inactive, and the free forms (reduced and oxidized) are the most likely candidates.
Aminothiols, like Hcy and Cys, may function as pro-oxidants at
low concentration and antioxidants at high
concentration,45 but their redox properties may also be
influenced by the pH45 and the composition48
of the medium in which the reactions take place. In biochemical model
reactions, Hcy, but not Cys, has pro-oxidant potentials, and
physiological levels of Cys even antagonize the oxidative damage by
Hcy.49 Thus, one may speculate whether elevated levels of
reduced Hcy in patients with early-onset peripheral vascular disease
(Table 2) have pro-oxidant effect–causing vascular lesions, whereas
the low level of reduced Cys in these patients (Table 2, Fig 1) may
reflect impaired mechanisms responsible for scavenging reactive oxygen
species.
Reduced Cys is the most abundant low-molecular-weight thiol in plasma,13 and the reduced/total ratio may reflect the overall antioxidant status of plasma.50
Summary and Conclusions
In patients with early-onset peripheral arteriosclerosis, all Hcy
and Cys forms in plasma are elevated above normal, except reduced Cys,
which is consistently lower than in control subjects. An attractive
hypothesis is that reduced Hcy has pro-oxidant properties provoking
vascular lesions, whereas reduced Cys may serve as a protective factor.
Both in patients and in healthy control subjects, the redox status and
the protein binding of different aminothiols in plasma were related in
a manner (Figs 2 and 3) suggesting that the aminothiols undergo redox
cycling and thiol-disulfide exchange reactions. These processes
probably also involve other thiol components and may also interact with
membrane-associated antioxidant systems.51 52 Thus,
altered redox status or concentrations of aminothiols in plasma may
directly or indirectly cause the damage responsible for
arteriosclerosis. Whatever the mechanism, the present work
demonstrates that alteration in total Hcy in patients with vascular
disease may represent a single component in a complex
interactive system.
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Acknowledgments |
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This study was supported by grants from the Norwegian Council on Cardiovascular Diseases, the Norwegian Research Council for Science and the Humanities, and the Swedish Heart-Lung Foundation.
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Footnotes |
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Reprint requests to Dr M. Azam Mansoor, Central Hospital in Rogaland, Division of Clinical Chemistry, Armauer Hansens vei 20, 4003 Stavanger, Norway.
Received July 25, 1994; accepted October 28, 1994.
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