(Stroke. 2000;31:2907.)
© 2000 American Heart Association, Inc.
Original Contributions |
Impairment of Cerebrovascular Reactivity by Methionine-Induced Hyperhomocysteinemia and Amelioration by Quinapril Treatment
From the Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China.
Correspondence to Yuan-Teh Lee, MD, Department of Internal Medicine, National Taiwan University Hospital, 7 Chung-Shan South Rd, Taipei 100, Taiwan, Republic of China. E-mail ytlee{at}ha.mc.ntu.edu.tw
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Abstract |
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Background and Purpose—Human studies have shown that methionine-induced hyperhomocysteinemia impairs brachial artery endothelial function via decreasing nitric oxide activity. However, the effect of homocysteine on cerebrovascular reactivity (CVR), which has been reported to be nitric oxide related in experimental and animal studies, remains unclear in humans. Inhibition of angiotensin-converting enzyme may improve nitric oxide–mediated cerebral as well as peripheral endothelial function. The aim of the present study was to investigate the effect of methionine-induced hyperhomocysteinemia on CVR before and after treatment with quinapril, an angiotensin-converting enzyme inhibitor, in healthy adults.
Methods—Plasma homocysteine and CVR were measured at baseline and 4 hours after methionine load (0.1 g/kg body wt) before and after quinapril treatment (10 mg/d for 1 week) in both younger and older groups. CVR was assessed by transcranial Doppler ultrasonography, measuring the percent increase of flow velocity in the middle cerebral artery after brief carotid compression (expressed as transient hyperemic response ratio [THRR]).
Results—Homocysteine levels were significantly increased after methionine load either before or after quinapril treatment in both groups. Before quinapril treatment, postmethionine THRR was preserved in younger adults (24.2±5.3% versus 23.8±6.3% at baseline, P=0.73) and decreased in older adults (12.9±2.2% versus 21.8±4.0% at baseline, P<0.001). After quinapril treatment, postmethionine THRR was preserved in both groups (24.5±5.9% versus 24.0±5.0% at baseline, P=0.42 in younger adults; 20.4±3.9% versus 21.3±3.3% at baseline, P=0.35 in older adults).
Conclusions—Our study suggests that methionine-induced hyperhomocysteinemia may be causally associated with impairment of CVR in older normal subjects.
Key Words: cerebrovascular reactivity • homocysteine • quinapril • ultrasonography, Doppler
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Cerebrovascular reactivity (CVR) is an index of the vasoregulatory capacity of the cerebral vessels, which maintains constant flow in the face of wide fluctuations in perfusion pressure. Impaired CVR therefore may indicate increased risk for ischemic stroke.1 2
Hyperhomocysteinemia is an emerging risk factor for atherosclerosis and consequent ischemic stroke.3 4 5 In animal models, high homocysteine concentrations (1 mmol/L) may cause nitric oxide–related impairment of CVR in rats.6 In humans, mild to moderate hyperhomocysteinemia (15 to 30 µmol/L) has been associated with nitric oxide–mediated impairment of brachial artery vasodilation,7 8 9 which has been reported to be age related.10 However, the effect of homocysteine on CVR has not been studied in humans.
We hypothesized that elevated blood homocysteine concentrations may cause impaired CVR in humans and that the effect of hyperhomocysteinemia might be ameliorated by therapy with angiotensin-converting enzyme (ACE) inhibitors, which have been shown to improve nitric oxide–mediated endothelial vasodilation of coronary and brachial arteries in humans.11 12
The specific objectives of this study were (1) to investigate the effect of methionine-induced hyperhomocysteinemia on CVR and (2) to evaluate the influence of an ACE inhibitor, quinapril, on CVR in methionine-induced hyperhomocysteinemia. Since aging is associated with reduced nitric oxide activity,10 we prospectively defined 2 age groups for inclusion in the study.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects and Study Design
To consider the effect of aging, we prospectively defined 2 age groups for inclusion in this study. The younger adults were defined as those between 21 and 40 years of age and the older adults as those between 55 and 70 years of age.10 We recruited 24 healthy Chinese adults (20 men, 4 women; aged 21 to 70 years) from hospital staff and community volunteers. Subjects were included only if they were clinically well without family or personal history of premature vascular disease, hypertension, diabetes mellitus, and hyperlipidemia and had no carotid stenosis by B-mode imaging. They were all nonsmokers and not taking regular medications. All subjects gave written informed consent, and the local ethics committee approved the study. The study design is summarized in the Figure
.
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After an overnight fast (10 to 14 hours), venous blood samples were drawn from all volunteers to measure the concentrations of homocysteine, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, and glucose. Supine blood pressure was measured for all subjects, and 10 minutes later all persons had a noninvasive ultrasound study of the middle cerebral artery (MCA) to evaluate CVR. After the ultrasound study, an oral methionine loading test with L-methionine (0.1 g/kg body wt) mixed in orange juice was given. Four hours later, blood sampling for homocysteine and the ultrasound study were performed again. During the 4 hours, only low-methionine nutrients were allowed. After the above procedure, quinapril (Parke-Davis GMBH) was given orally for 1 week (10 mg in the morning daily). After the completion of quinapril administration, the above procedure was repeated on the morning of the next day.
Laboratory Assays
Venous blood samples were collected into tubes containing EDTA.
Samples were centrifuged within 30 minutes at 2000 rpm for 10
minutes. The plasma was then separated and stored at -70°C until
analysis. Total homocysteine concentrations were measured by
fluorescence polarization immunoassay (Abbott IMx
System).10 13 The coefficients of variation were within
5.2%. Lipid concentrations were determined by the Eppendorf EPOS 5060
analyzer.
Ultrasound Studies
CVR was assessed by transcranial Doppler
ultrasonography, measuring the percent increase of flow velocity in the
MCA after carotid compression for 10 seconds (expressed as transient
hyperemic response ratio [THRR]). Ultrasound measurements
were performed according to the method described by Smielewski et
al.14 Briefly, each volunteer was studied in the left
lateral decubitus position with the head resting on a pillow. The right
MCA was identified from the right temporal area by recognition of the
characteristic waveform and typical flow velocity at a depth from 55 to
60 mm, with the use of a 2- to 4-MHz probe (Hewlett-Packard SONOS
5500). Marks were then placed on the subjects’ temporal regions to
permit the best penetration of the ultrasound signal onto the MCA and
achieve the constant and highest MCA flow velocity throughout the
study. During the study, the probe was handheld with carotid
compression done by the same examiner, and a second individual
manipulated recordings of flow velocity. Systolic flow
velocity (FVS) of the MCA was continuously recorded before, during,
and after compression. In this small-group study, we performed carotid
compression carefully to avoid the influence of compression magnitude
fluctuations on THRR, either within or between subjects. Carotid
compression was only accepted when a sudden and maximal decrease in
flow velocity was achieved at the onset of compression; otherwise, the
compression was terminated and repeated 60 seconds later. In the
younger group, carotid compression was done once in 9 and twice in 3
subjects (mean, 1.3 trials); the transient increase of velocity after
compression lasted for 5 seconds on average. In the older group,
carotid compression was done once in 7 subjects, twice in 4 subjects,
and 3 times in 1 subject (mean, 1.5 trials); the transient increase of
velocity lasted for 4 seconds on average. Basal FVS was then calculated
using the average value of FVS from 5 heart cycles preceding the
compression. Hyperemic FVS was calculated using the average FVS
value of 2 heart cycles after the compression release with exception of
the first cycle. THRR was then obtained according to the following
formula:
THRR=(FVShyperemia-FVSbasal)/FVSbasal.
The compression ratio (CR) measuring the magnitude of decrease in flow
velocity during compression was defined as follows:
CR=(FVSbasal-FVScompression)/FVSbasal.
In our laboratory, 2 independent examiners performed the measurements.
The intraobserver and interobserver variations were 3.2% and 4.6%,
respectively.
Statistical Analysis
Continuous data were expressed as mean±SD value. For the
continuous data, comparisons between the younger and older groups were
analyzed by Student’s t test; comparisons between
premethionine and postmethionine load or prequinapril and postquinapril
treatment in the same group were accomplished by paired t
test. The 
2 test was used for comparison
between the binary groups. P<0.05 was considered
significant.
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Results |
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The younger persons had an average age of 31±4 years. For the older adults, the average age was 62±3 years. There were 10 men and 2 women in each group. Other baseline characteristics, such as height, weight, body mass index, glucose, cholesterol, and triglycerides, were similar in both groups (Table 1
).
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Plasma homocysteine and ultrasound measurements at baseline and 4 hours
after methionine load before quinapril treatment are summarized in
Table 2. After methionine load,
homocysteine levels were significantly increased in both groups.
Postmethionine THRR was preserved in the younger group and decreased in
the older group in comparison with that at baseline in each group.
Between the 2 groups, homocysteine levels were not significantly
different at baseline and 4 hours. Basal FVS in older adults was lower
than that in younger adults either at baseline (P=0.002) or
4 hours (P=0.002). In the older group, postmethionine THRR
(12.9±2.2%) was significantly lower than that in the younger group
(24.2±5.3%) (P<0.001).
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After quinapril treatment, blood pressure, which was measured 24 hours
after quinapril discontinuation, was not significantly changed in both
groups (Table 3). Plasma homocysteine and
ultrasound measurements at baseline and 4 hours after methionine load
after quinapril treatment are summarized in Table 4. After methionine load, THRR was
preserved in both groups. Between the 2 groups, homocysteine levels and
THRR, either at baseline or 4 hours after methionine load, were not
significantly different. Basal FVS in older adults was lower than that
in younger adults either at baseline (P=0.005) or 4 hours
(P=0.002).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The principal findings of the present study are (1) methionine- induced mild to moderate hyperhomocysteinemia impairs CVR in older but not younger healthy adults and (2) quinapril treatment ameliorates impairment of CVR in older healthy adults after methionine load.
In humans, methionine-induced mild to moderate hyperhomocysteinemia may impair brachial artery endothelial function via decreasing nitric oxide activity,7 8 9 especially in older but not in younger adults.10 In this study we also found that impairment of CVR in response to carotid compression was age related (mainly in older adults) in acute hyperhomocysteinemia. Our data suggest that methionine challenge could exhaust age-related reduction of nitric oxide activity, which is already compromised but not physiologically evident under resting conditions. Methionine-induced hyperhomocysteinemia might provide a way to monitor relative levels of cerebral endothelial nitric oxide activity. Although CVR in response to acute elevation of homocysteine concentrations was intact in our younger adults, whether longer exposure of the endothelium of cerebral arteries to hyperhomocysteinemia leads to reduction of nitric oxide activity in younger adults requires further investigation.
In our study subjects, the age-related decline of basal FVS was in accordance with that reported by Grolimund and Seiler.15 This finding may result from an age-related reduction of cerebral blood flow.16 In addition, this study showed no significant difference of CVR before methionine load in different age groups. This result was similar to that reported by Matteis et al17 and Kastrup et al,18 who showed no age difference of CVR in response to hypercapnia in men. In young adults, the preserved CVR after a stress test for nitric oxide activity was also reported by White et al,19 who revealed that cerebral blood flow in response to hypercapnia was not attenuated by nitric oxide synthase inhibitor in healthy young adults.
Administration of ACE inhibitors has been reported to reduce the incidence of atherosclerotic diseases, including stroke.20 ACE inhibition, either via suppression of angiotensin II production, inhibition of bradykinin breakdown, or other mechanisms, may improve endothelial nitric oxide–mediated vasodilation of coronary and brachial arteries in humans.11 12 In humans, the effect of ACE inhibition on nitric oxide–mediated CVR is rarely explored.21 Démolis et al21 evaluated the basal cerebral blood flow (as assessed by mean flow velocity of the MCA) and CVR (as assessed by measuring the percent increase of mean flow velocity in the MCA after intravenous acetazolamide administration) using a single dose (20 mg) of lisinopril in 8 male healthy volunteers (aged 22 to 28 years). They found that the basal cerebral blood flow was not influenced by lisinopril treatment, which is similar to our finding by quinapril treatment. However, CVR was increased after lisinopril but not after quinapril treatment in young adults. Whether the inconsistency results from the disparity of study design needs further investigation. In addition, our data indicate that quinapril preserves CVR in older healthy adults after methionine load and suggest that quinapril treatment might ameliorate the detrimental effect of homocysteine on nitric oxide activity in older adults.
In this study we measured the changes of blood flow velocity in the MCA in response to carotid compression. The carotid compression may lead to a reduction of perfusion pressure in the ipsilateral MCA territory with consequent compensatory vasodilation of cortical MCA branch vessels,22 23 and thus a transient velocity increase in the MCA after release of carotid compression. The compensatory vasodilation of MCA branch vessels due to pressure reduction has been reported to be associated with nitric oxide in an experimental study.24 The diameter of large cerebral arteries such as the MCA, however, remains relatively constant during a reduction of blood pressure.25 26 Therefore, the changes of flow velocity in the MCA might correlate with the changes of cerebral blood flow.27 This noninvasive transient hyperemic response test is simple, safe, and reproducible at the bedside and could serve as a marker of CVR.
Study Limitations
This study was nonrandomized, not placebo controlled, and lacked
blinding of the operators. A randomized, placebo-controlled, crossover
study would be the optimal design for demonstrating the effects of
hyperhomocysteinemia and ACE inhibition on CVR. The evaluation of CVR,
especially that in response to hypercapnia, has become a widely used
tool in clinical assessments, including the risk of ischemic
stroke.1 2 In this study the use of carotid compression
instead of hypercapnia stimulation to assess CVR was based on the fact
that reduction of perfusion pressure and hypercapnia have been
associated with nitric oxide in experimental and animal
studies24 28 and that carotid compression is simple and
reproducible. However, discrepancies may exist if the different methods
are used. Whether the assessment of CVR using carotid compression
provides a useful index of ischemic stroke needs further
validation.
In conclusion, this is the first study to elucidate the direct relationship between changes of homocysteine concentrations and CVR before and after an ACE inhibitor treatment. Our data indicate that methionine-induced hyperhomocysteinemia causes age-related impairment of CVR (mainly in older adults), which could be ameliorated by quinapril treatment.
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Acknowledgments |
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This study was supported by a grant from the University Research Fund (1500–39-1539).
Received April 17, 2000; revision received August 16, 2000; accepted August 16, 2000.
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References |
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