Published online before print July 5, 2001, doi: 10.1161/hh1401.093459
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© 2001 American Heart Association, Inc.
Article |
Investigation of Relationship Between Reduced, Oxidized, and Protein-Bound Homocysteine and Vascular Endothelial Function in Healthy Human Subjects
From the National Heart and Lung Institute (J.C.C., J.S.K.) and the Department of Medical Statistics and Evaluation (M.W.), Imperial College School of Medicine, Hammersmith Hospital, London, UK; the Department of Pharmacology (P.M.U., H.R.), University of Bergen, Armauer Hansen Hus, Bergen, Norway; and the MRC Clinical Trials Unit (C.J.D.), London, UK.
Correspondence to Dr J.S. Kooner, MD, FRCP, Consultant Cardiologist, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. E-mail j.kooner{at}ic.ac.uk
Abstract
Abstract—Previous studies investigating homocysteine and vascular disease have relied on total plasma homocysteine as the sole index of homocysteine status. We examined the dynamic relationship between vascular endothelial function and concentrations of total, protein-bound oxidized, free oxidized, and reduced homocysteine to identify the homocysteine form associated with endothelial dysfunction in humans. We investigated 14 healthy volunteers (10 men, 4 women). Brachial artery flow–mediated dilatation was measured at baseline and at 30, 60, 120, 240, and 360 minutes after oral (1) L-methionine (50 mg/kg), (2) L-homocysteine (5 mg/kg), and (3) placebo. Plasma concentrations of total, protein-bound oxidized, free oxidized, and reduced homocysteine were measured at each time point, and nitroglycerin-induced dilatation at was assessed at 0, 120, and 360 minutes. Flow-mediated dilatation fell, and concentrations of total, protein-bound oxidized, free oxidized, and reduced homocysteine increased after oral homocysteine and oral methionine (all P<0.05 for difference in time course compared with placebo). Flow-mediated dilatation showed a reciprocal relationship with reduced homocysteine during both homocysteine and methionine loading. In both loading studies, peak reduction in flow-mediated dilatation coincided with maximal reduced homocysteine concentrations. In contrast, there was no consistent relationship between flow-mediated dilatation and free oxidized homocysteine, protein-bound oxidized homocysteine, or related species. Nitroglycerin-induced dilatation was unchanged by oral homocysteine and oral methionine (P>0.10 compared with placebo). Reduced homocysteine is closely associated with endothelial dysfunction during oral methionine and oral homocysteine loading. Our observations support the hypothesis that reduced homocysteine is the deleterious form of homocysteine for vascular function in vivo and suggest a less important role for other homocysteine species.
Key Words: homocysteine • endothelium • atherosclerosis • redox • nitric oxide
Hyperhomocysteinemia is a major and independent risk factor for vascular disease.1 2 3 4 5 Homocysteine concentrations exceeding the upper limit of normal (15 µmol/L) are identified in almost 30% of patients with coronary heart disease and are associated with a 3-fold increased risk of subsequent coronary heart disease mortality.3 In North American and European white populations, 10% of coronary disease may be attributable to elevated homocysteine.6
Increasing evidence suggests that the adverse vascular effects of homocysteine are mediated through an action on the endothelium. In healthy human subjects, elevated homocysteine concentrations are associated with impaired endothelium-dependent dilatation, an early manifestation of atherosclerosis.7 8 9 10 Conversely, lowering plasma homocysteine concentrations through treatment with B vitamins is associated with improved vascular endothelial function.11 12 13 However, the mechanisms linking homocysteine to endothelial dysfunction are not known.
In plasma, homocysteine exists in reduced, free oxidized, and protein-bound oxidized forms.14 Most previous studies have relied on total plasma homocysteine as the sole index of homocysteine status, and the active form of homocysteine remains to be determined. Recent observations have indicated that during acute hyperhomocysteinemia, maximal endothelial dysfunction precedes peak total plasma homocysteine concentration8 9 ; these observations imply that short-lived species may mediate the biological effects of homocysteine. Further support for this view comes from in vitro studies, which suggest that reduced, but not oxidized, homocysteine promotes endothelial injury15 and reacts with NO to form nitrosohomocysteine,16 which (because of the stability of this nitrosothiol) may decrease the bioactivity of NO.
In the present study, we tested the hypothesis that the reduced form, but not the free oxidized or protein-bound oxidized form, of homocysteine is associated with vascular endothelial dysfunction during hyperhomocysteinemia in humans.
Materials and Methods
Subjects
We investigated 14 healthy volunteers (10 men, 4
women; mean age 29 [range 19 to 37] years). All subjects were
normotensive, with normal serum cholesterol and with no
previous history of diabetes or vascular disease. Five of the 14
subjects were cigarette smokers. All subjects abstained from smoking on
the day of the study. None were taking medications. The study was
approved by the local ethics committee, and all subjects gave informed
written consent to participate.
Methods
For each subject, we measured brachial artery
vascular responses at baseline and at 30, 60, 120, 240, and 360
minutes after oral (1)
L-methionine (50 mg/kg),
(2) L-homocysteine (5
mg/kg), and (3) placebo (fruit juice only). Flow-mediated
(endothelium-dependent) dilatation was measured at each
time point, but nitroglycerin (NTG)-induced
(endothelium-independent) dilatation was measured only
at 0, 120, and 360 minutes to allow sufficient time for recovery before
the next set of vascular measurements. Oral homocysteine and oral
methionine (the metabolic precursor of homocysteine,
Figure 1
) were given as separate metabolic
challenges at least 14 days apart. Both oral homocysteine and oral
methionine were expected to increase homocysteine concentrations.
However, in contrast to the almost immediate rise in plasma
homocysteine levels expected after oral
homocysteine,17 oral
methionine increases homocysteine concentrations over 4 to 8
hours.18 We examined the
differing time courses and magnitudes of change in homocysteine species
after these two challenges to identify the homocysteine form associated
with endothelial dysfunction in humans.
|
Plasma concentrations of reduced homocysteine, free oxidized homocysteine, protein-bound oxidized homocysteine, total homocysteine, methionine, cystathionine, and cysteine were measured at each time point. Additional samples for fasting glucose, total cholesterol, HDL cholesterol, triglycerides, red blood cell folate, and serum vitamin B12 were collected for each subject at baseline. Vascular studies were performed on separate days and in random order.
Methionine and Homocysteine
Administration
L-Homocysteine
was administered in its reduced form. Reduced
L-homocysteine was
generated by incubation of
L-homocysteine thiolactone
(Sigma Chemical Co) with 5N NaOH for 5 minutes
at room
temperature.17 19
The resultant solution was then neutralized by the addition of HCl,
diluted to 200 mL in fruit juice, and administered orally to the
subject within 15 minutes. Oral methionine was administered as
L-methionine
(Sigma) in diluted fruit
juice.
Brachial Artery Diameter
Brachial artery vascular responses were measured
using a 7.0-MHz linear array transducer, an
Acuson 128XP/10 system, and a high-resolution
ultrasonic vessel wall tracking system (Vadirec, Ingenious
Systems) as previously
described.7 8 In
brief, the brachial artery was scanned longitudinally, and a clear
section of the vessel was identified. The M-mode cursor was then placed
over this point at right angles to the vessel wall. A 5-second segment
of the A-mode signal was then routed to the wall tracking system
designed to track vessel wall movement on a beat-to-beat basis.
Arterial diameter was calculated from the distance between
opposite lumen-arterial interfaces, as identified by manual
selection of the maximal change in recorded radiofrequency
amplitude. After the baseline resting scan, a pneumatic cuff placed at
the level of the mid forearm was inflated to 300 mm Hg for 4.5
minutes. Flow-mediated dilatation was determined from a second scan,
performed 55 to 65 seconds after cuff deflation. Fifteen minutes was
allowed for vessel recovery, after which time a further baseline scan
was performed. NTG (400 µg) was then administered, and 4 minutes
later, a fourth scan of the brachial artery was undertaken to provide
NTG-induced dilatation. Vascular data were stored digitally to enable
offline measurement of vessel diameter by two independent observers
unaware of the subject’s clinical details and the type and stage of
the study. The technique for measurement of brachial artery
flow–mediated dilatation is reproducible in our laboratory. The
intraindividual between-day coefficient of variation for flow-mediated
dilatation is 3%, which compares favorably with that in other
centers.20 Flow-mediated
dilatation of conduit arteries is endothelium dependent
and largely mediated by
NO.21
Biochemical Measurements
Plasma samples for homocysteine, methionine,
cystathionine, and cysteine were collected with each measurement of
flow-mediated
dilatation.17 18 22
Blood was collected into two heparinized tubes. One sample was
immediately centrifuged at
10 000g for 1 minute,
deproteinized with sulfosalicylic acid, and then recentrifuged.
The supernatant was used for measurement of reduced homocysteine, free
homocysteine (the sum of reduced and free oxidized homocysteine),
cysteine, cystathionine, and methionine concentrations. The second,
untreated plasma sample was used for measurement of total homocysteine
and cysteine. Plasma concentrations of total homocysteine, free
homocysteine, methionine, and cystathionine were determined by
high-pressure liquid
chromatography.17 18 22 23 24
Reduced homocysteine was determined by mass spectroscopy with
deuterated homocysteine as an internal standard by using methodology
validated as described by Nexo et
al.25 Concentrations of
protein-bound oxidized homocysteine were calculated by subtracting free
homocysteine from total homocysteine. Levels of free oxidized
homocysteine were calculated by subtracting reduced homocysteine from
free homocysteine. For each subject, samples were analyzed in
one batch. The within-assay coefficients of variation for measurement
of total, free, and reduced homocysteine were 2%, 2%, and 11%,
respectively.
Lipid profiles were determined by using an Olympus AU800 multichannel analyzer; vitamin B12 and red blood cell folate by were determined by MEIA (Abbott IMX system).
Data Processing and Statistical
Analysis
Data were analyzed by using Genstat release 4
and STATA release 6 statistical packages. Continuous data are expressed
as mean±SD. Split-unit ANOVA with treatment (oral homocysteine, oral
methionine, and oral placebo) and time (0, 30, 60, 120, 240, and 360
minutes) used as factors examined the effects of the administration of
oral homocysteine and oral methionine on flow-mediated dilatation,
NTG-induced dilatation, and concentrations of plasma thiols. The effect
of treatment was assessed relative to the variation between study days
within subjects. The effect of time and the interaction between
treatment and time were assessed relative to the variation within study
days. Pairwise comparison of the three treatments was then performed by
using modified t tests and the
appropriate pooled estimate of the SD from ANOVA. The
P values obtained for the three
multiple comparisons were Bonferroni-adjusted by multiplying each
unadjusted P value by a factor
of 3. The P values
presented are from the interaction between treatment and time
and are intended to investigate whether the time course of the response
differs between treatments. Logarithmic transformation was necessary
for reduced, free oxidized, protein-bound oxidized, and total
homocysteine and for methionine, because these had positively skewed
distributions. In addition, Wilcoxon matched-pairs signed rank
tests were performed to compare the time of peak reduction in
flow-mediated dilatation with the time for the maximum change in
concentration of each biochemical species with the use of summary
measures for each subject on each study day. Univariate
regression analysis was conducted to investigate the
relationship between flow-mediated dilatation and concentrations of
plasma thiols and methionine. Because repeated measures were made on
each participant, robust "sandwich" estimates were made of the
between-subject standard errors in the regression analyses.
This technique adjusts for any within-subject
correlations.26 Statistical
significance was inferred at a value of
P<0.05.
Results
The baseline clinical and biochemical measurements of
participants are summarized in
Table 1. All subjects had normal blood pressure, fasting
blood glucose, lipid profile, serum folate, and vitamin
B12 levels.
|
Vascular Responses After Oral
Homocysteine and Oral Methionine
Flow-mediated dilatation was reduced during both
homocysteine and methionine loading
(P=0.009 and
P=0.03, respectively, for
difference in time course compared with placebo). Impaired
flow-mediated dilatation was evident by 30 minutes, was maximal between
60 and 120 minutes, and showed partial recovery by 360 minutes after
both oral homocysteine and oral methionine
(Figure 2). The mean reduction in flow-mediated dilatation
was similar during homocysteine and methionine loading, with no
evidence of a different time course
(P=0.54).
|
), oral methionine (
), and oral placebo (•) loading. Flow-mediated dilatation was reduced, and concentrations of reduced homocysteine were increased during both homocysteine and methionine loading (all P<0.05 for difference in time course compared with placebo).In contrast, NTG-induced dilatation was not significantly changed after either oral homocysteine (0 minutes, 19.7+4.4%; 120 minutes, 17.6+5.4%; and 360 minutes, 18.9+3.5%) or oral methionine (0 minutes, 19.9+4.6%; 120 minutes, 17.8+4.8%; and 360 minutes, 19.4+5.5%) compared with measurements after placebo (0 minutes, 18.8+3.7%; 120 minutes, 18.2+4.7%; and 360 minutes, 19.5+4.5%) (both P=1.0). Baseline brachial artery diameter and brachial artery flow characteristics were also not significantly influenced by oral homocysteine or oral methionine.
Concentrations of Reduced Homocysteine After
Oral Homocysteine and Oral Methionine
Reduced homocysteine concentrations increased
during both homocysteine and methionine loading compared with placebo
loading (both P<0.001 for
difference in time course compared with placebo). The increase in
reduced homocysteine was evident by 30 minutes, was maximal between 60
and 120 minutes, and showed partial recovery by 360 minutes after both
oral homocysteine and oral methionine
(Figure 2
). The mean rise in reduced homocysteine
concentrations was similar during homocysteine and methionine loading,
although the time course of the responses differed between the two
challenges (P<0.001). In
regression analysis, there was a close inverse relationship
between flow-mediated dilatation and concentrations of reduced
homocysteine
(Table 2). Maximal reduction in flow-mediated dilatation
coincided with peak concentrations of reduced homocysteine during both
oral homocysteine loading and oral methionine loading
(Figures 2 and 3).
|
|
Concentrations of Other Homocysteine
Species
Mean levels of plasma total homocysteine, protein-bound
oxidized homocysteine, and free oxidized homocysteine increased during
homocysteine and methionine loading (all
P<0.001 for difference in time
course compared with placebo,
Figure 4). However, whereas oral homocysteine and oral
methionine had similar effects on flow-mediated dilatation, the
increments in concentrations of total, protein-bound oxidized, and free
oxidized homocysteine were all greater during oral homocysteine
compared with oral methionine loading and followed different time
courses (P<0.001).
|
In regression analysis, there was an inverse
relationship between flow-mediated dilatation and concentrations of
total, protein-bound oxidized, and free oxidized homocysteine
(Table 2). However, whereas the peak levels of homocysteine
species occurred at a time similar to that for the maximal reduction in
flow-mediated dilatation during oral homocysteine loading,
concentrations of total, protein-bound oxidized, and free oxidized
homocysteine were delayed and still rising at 4 hours after oral
methionine, a time at which flow-mediated dilatation was improving
(Figures 3 and 4).
Concentrations of Related Metabolites
Although oral homocysteine and oral methionine had
similar effects on flow-mediated dilatation, these challenges had very
different effects on plasma concentrations of methionine,
cystathionine, and cysteine
(Figures 4 and 5). Plasma methionine was unchanged after oral
homocysteine (P=1.00 for
difference in time course compared with placebo) but increased by
20-fold after oral methionine
(P<0.001 compared with
placebo). Concentrations of plasma-free cysteine fell after oral
homocysteine but increased after oral methionine. Furthermore, there
was no consistent relationship between the time to maximal
reduction in flow-mediated dilatation and the time of peak change in
methionine, cystathionine, or cysteine concentrations. In regression
analysis, methionine and cystathionine were less strongly
related, and free cysteine concentrations were unrelated to
flow-mediated dilatation
(Table 2).
|
Discussion
The results of the present study show that the reduced form of homocysteine is closely associated with vascular endothelial dysfunction induced by both oral methionine and homocysteine loading. Our observations support the hypothesis that reduced homocysteine is the deleterious form of homocysteine for vascular function in vivo and argue against a significant vascular effect for other homocysteine species.
Investigations of the possible mechanisms underlying the association between homocysteine and vascular disease have been limited by the fact that little is known about which homocysteine species exerts vascular effects in vivo. Most previous studies investigating the link between elevated homocysteine concentrations and vascular disease have been based on measurements of total plasma homocysteine,1 2 3 4 7 8 9 10 11 12 because routine laboratory assays do not distinguish between the protein-bound oxidized, free oxidized, and reduced forms of homocysteine.14 22 23 In the present study, we observed an inverse and dynamic relationship between the reduced form of plasma homocysteine and endothelial function. Oral methionine and oral homocysteine induced a rise in reduced homocysteine concentrations and a fall in brachial artery endothelium-dependent dilatation. In both time-course studies, peak concentrations of reduced homocysteine coincided with the maximal reduction in flow-mediated dilatation, and regression analysis showed an inverse relationship between reduced homocysteine concentrations and flow-mediated dilatation. These observations suggest that vascular endothelial dysfunction during hyperhomocysteinemia may be accounted for by the reduced form of homocysteine. Additional independent support for this view comes from physiological studies showing that the effects of homocysteine on vascular endothelial function are mediated by oxidation stress mechanisms7 10 and from in vitro studies demonstrating that it is the reduced, but not the oxidized, form of homocysteine that promotes the generation of oxygen-derived free radicals and reacts with NO.15 16 27
Although a separate effect of free oxidized or protein-bound oxidized homocysteine on endothelial function cannot be excluded, this possibility is unlikely because the relationship between these homocysteine species and flow-mediated dilatation was not consistent between the studies. After oral homocysteine, peak concentrations of total, protein-bound oxidized, and free oxidized homocysteine occurred within 60 minutes, a time similar to that for the maximal reduction in flow-mediated dilatation. In contrast, peak concentrations of total, protein-bound oxidized, and free oxidized homocysteine were delayed and were still rising at 4 hours after oral methionine, a time at which flow-mediated dilatation was improving. A threshold or other nonlinear relationship between homocysteine forms and flow-mediated dilatation is unlikely to account for these findings because previous studies show that the relationship between homocysteine and flow-mediated dilatation is graded and dose dependent.8 A separate effect of cysteine, which has been linked to coronary heart disease in some epidemiological studies,28 is also unlikely because free cysteine concentrations fell during oral homocysteine loading but increased during oral methionine loading. Furthermore, because homocysteine loading was not associated with a significant rise in plasma methionine concentrations, our results exclude a role for methionine or methionine-dependent methylation in the observed vascular responses.
In the present study, there was no constant relationship between total plasma homocysteine concentrations and vascular endothelial function. In particular, we found that during hyperhomocysteinemia induced by oral methionine, maximal reduction in flow-mediated dilatation occurred at 2 hours and then showed improvement by 6 hours, even though concentrations of total homocysteine were still rising. These findings indicate that total plasma homocysteine concentrations may not be a consistent marker of the biological activity of homocysteine in vivo. This important limitation may have obscured the interpretation of the relationship between homocysteine and vascular disease in previous experimental and clinical studies.29 30 31 32 Because the relationship between total and reduced homocysteine concentrations varies between subjects and under differing metabolic conditions,17 18 total homocysteine may only be a marker of reduced homocysteine and vascular risk under certain circumstances. Our results suggest that reduced homocysteine may prove to be a more valid measurement for future epidemiological and experimental studies. This possibility needs to be examined in specific studies using simultaneous measurements of reduced and total homocysteine.
In summary, we have shown that the reduced form of homocysteine is closely associated with vascular endothelial dysfunction, which is associated with elevated homocysteine in humans. Identification of reduced homocysteine as the deleterious form of homocysteine for vascular function in vivo and the resultant changes in cellular redox potential will provide new insight into the mechanisms linking hyperhomocysteinemia and atherosclerosis.
Acknowledgments
This study was supported by British Heart Foundation project grant PG99204 and European Union Commission Demonstration Project Contract No. BMH4-98-3549. We are indebted to Andrew McGregor and Jeff Jean-Marie for their assistance with the vascular studies.
Footnotes
Original received January 2, 2001; revision received May 22, 2001; accepted May 23, 2001.
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