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Departments of 1 Cardiology and 2 Medical Microbiology, Wales Heart Research Institute, University of Wales College of Medicine, Cardiff CF14 4XN; 3 University of Wales Institute, Cardiff CF23 6XD; and 4 University of Dundee Medical School, Dundee DD1 9SY, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Endothelial dysfunction reflects reduced nitric oxide (NO) bioavailability due to either reduced production, inactivation of NO, or reduced smooth muscle responsiveness. Oral methionine loading causes acute endothelial dysfunction in healthy subjects and provides a model in which to study mechanisms. Endothelial function was assessed using flow-mediated dilatation (FMD) of the brachial artery in humans. Three markers of oxidative stress were measured ex vivo in venous blood. NO responsiveness was assessed in vascular smooth muscle and platelets. Oral methionine loading induced endothelial dysfunction (FMD decreased from 2.8 ± 0.8 to 0.3 ± 0.3% with methionine and from 2.8 ± 0.8 to 1.3 ± 0.3% with placebo; P < 0.05). No significant changes in measures of plasma oxidative stress or in vascular or platelet sensitivity to submaximal doses of NO donors were detected. These data suggest that oxidative stress is not the mechanism of endothelial dysfunction after oral methionine loading. Furthermore, the preservation of vascular and platelet NO sensitivity makes a signal transduction abnormality unlikely.
endothelium; homocysteine; oxidative stress; signal transduction; platelet aggregation
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ENDOTHELIAL DYSFUNCTION is a key factor in the development of atherosclerosis (30). It is present not only in established cardiovascular disease but also in young individuals at high risk of developing cardiovascular disease (10, 19, 29). However, the mechanisms leading to endothelial dysfunction are complex and poorly understood. The implicated candidates have included the following: 1) reduced nitric oxide (NO) synthesis, 2) oxidative stress causing reduced bioavailable NO, and 3) signal transduction abnormalities. However, these mechanisms may not be so discrete, especially in a chronic situation. For example, oxidative stress can reduce NO synthesis through direct damage to the endothelium or by inducing a signal transduction pathway abnormality. Furthermore, endothelial dysfunction may lead to increased production of reactive oxygen species. Therefore, the dissection of the mechanisms involved is particularly difficult in chronic disease states.
Oral methionine loading has been shown to cause acute transient endothelial dysfunction in association with a rise in plasma homocysteine concentrations (2, 8). This acute model enables detailed investigation of the potential mechanisms of endothelial dysfunction. To examine these mechanisms, we assessed oxidative stress and responsiveness to exogenous NO donors. Measures of oxidative stress included the following: 1) direct measurement of lipid-derived free radicals, 2) indirect assessment of the effects of free radicals on lipid peroxidation, and 3) total antioxidant capacity of plasma. We also assessed vascular smooth muscle responsiveness to a submaximal dose of glyceryl trinitrate (GTN). Decreased sensitivity (of vascular smooth muscle) to exogenous NO has been described in a number of conditions associated with endothelial dysfunction but has been more extensively studied in platelets (11). However, whether this platelet NO resistance is a primary abnormality contributing to the endothelial dysfunction or a secondary response to endothelial dysfunction is unknown. The mechanism of NO resistance appears to involve both oxidative stress (12) and signal transduction abnormalities that are not mediated by free radicals (31). We therefore assessed platelet sensitivity to exogenous NO, both as a marker of oxidative stress and as a marker of NO signal transduction pathways.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Subjects
We studied 19 healthy male volunteers with no risk factors for vascular disease. The Local Research Ethics Committee approved the study and all subjects gave written consent. The study was a prospective randomized double-blind placebo-controlled crossover design. After an overnight fast, subjects attended for a baseline blood test to measure plasma lipid-derived free radicals, lipid peroxidation, total antioxidant capacity of plasma, and platelet aggregation. Brachial artery flow-mediated dilation (FMD) was performed as a measure of shear-related endothelial function. The subjects were then randomized to receive oral L-methionine (0.1 g/kg, Scientific Hospital Supplies) or placebo. Four hours later, these tests were repeated. Heart rate and blood pressure (model HEM-705 CP, Omron) were measured at each time point. Augmentation of central aortic pressure and vascular smooth muscle responsiveness to NO were then assessed using pulse wave analysis. Seven days later, the subjects crossed over to the other limb of the protocol.Assessment of Endothelial Function
Changes in brachial artery diameter in response to reactive hyperemia were measured noninvasively using a high-resolution ultrasonic wall-tracking system (resolution ± 3 µm; Vadirec) as previously described (28). Subjects rested supine with their arm held outstretched on a pneumatic cushion in a temperature-controlled room (21-23°C). Baseline measurements of internal brachial artery diameter and blood pressure were taken after
10 min of supine rest. Blood pressure was measured noninvasively by
using photoplethysmography (Finapres) with a cuff on the middle finger
of the arm being studied. The brachial artery was imaged using a
7.5-MHz transducer.
Reactive hyperemia was produced after releasing a pediatric sphygmomanometer wrist cuff, which had been inflated to 50 mmHg above systolic pressure for 5 min. The change in brachial artery diameter between baseline and 60 s after cuff release (FMD) was used as a measure of shear-related endothelial function. Endothelium-independent changes were assessed in the same way after 400 µg of sublingual GTN. Within-subject and between-subject coefficients of variation for this operator in our laboratory are 19 and 25%, respectively.
Markers of Oxidative Stress
Thiobarbituric acid-reactive substances. Markers of lipid peroxidation, called thiorbabituric acid-reactive substances (TBARS), were measured in plasma using a previously described fluorometric technique (36). In brief, plasma or a standard solution of malondialdehyde (MDA) was added to thiobarbituric acid in acetic acid (29 mmol/l; pH 2.4) and heated for 1 h at 95°C. After the samples cooled, HCl (5 mmol/l; pH 1.6) was added and the reaction mixture was extracted with n-butanol. The fluorescence of the butanol layer was measured at wavelengths of 525 nm for excitation and 547 nm for emission. The calibration curve was prepared with MDA standards of 0-0.5 nmol/tube. Coefficients of variation for within and between runs were 4 and 7%, respectively. Each sample was run in triplicate.
Spin-trapping of lipid-derived free radicals by electron
paramagnetic resonance spectroscopy.
In 10 of the subjects, we used the technique of ex vivo spin-trapping
and electron paramagnetic resonance (EPR) spectroscopy to measure
short-lived lipid-derived free radicals in venous blood samples
(1, 6, 14). Venous blood was drawn directly into two
vacutainer bottles containing the spin-trap reagent
-phenyltertbutylnitrone (Sigma). The samples were immediately
centrifuged, and the resultant spin-trap adducts extracted from plasma
into toluene. After taking to dryness the spin adduct was then
dissolved in 100 µl of chloroform and put into sealed Pasteur
pipettes for EPR analysis. Analysis was performed using a Bruker EMX
x-band EPR spectrometer operating at 9.5 GHz. EPR spectral
peak heights, recorded in arbitrary units, were taken as a measure of
spin adduct concentration. Paired samples were taken for each time
point, and the mean of the two samples was taken. In our laboratory,
the coefficient of variation in young healthy subjects is 24%. This
relatively large coefficient of variation is because healthy subjects
have a low basal level of free radicals and a high-EPR spectrometer
gain is required to detect these small amounts.
Oxygen-radical absorbance capacity. Antioxidant capacity in plasma was assessed using the oxygen-radical absorbance capacity (ORAC) method (7). This method utilizes allophycocyanine (APC; a fluorescent protein), which loses fluorescence when damaged by oxygen radicals. Copper sulfate and hydrogen peroxide (30%) were used to generate hydroxyl radicals, and APC (9.6 µM) was added in phosphate-buffered saline (pH 7.0). Total plasma ORAC was measured by assessing the buffering capacity of 2 µl of plasma on this reaction. Proteins in the plasma sample were then precipitated with the use of a saturated ammonium sulfate solution. The samples were centrifuged at 100,000 g for 10 min at 4°C, and the supernatants were stored on ice until assayed. This enabled separate assessment of nonprotein antioxidants (vitamin E and vitamin C) versus soluble protein antioxidants (ceruloplasmin, albumin, and superoxide dismutase), respectively. The excitation fluorescence was read at 598 nm and emission was read at 651 nm by using a 96-well plate reader. The area under the kinetic curve was proportional to the ORAC of the sample. Total serum ORAC is the sum of the supernatant ORAC and the protein-soluble ORAC. To correct for small differences in analytical instrument, sensitivity, reagents, or other assay conditions, the final result is expressed with reference to a known amount of standard antioxidant (either vitamin C or the spin-trap dimethyl pyrolidine-1-oxide). The sensitivity and repeatability of this method were similar to that published previously (7), with coefficients of variation within a run as <2% and between runs as ~5%. Positive and negative standards were run for each set of eight samples.
Pulse Wave Analysis
Pulse wave analysis was performed at the radial artery by using aplanation tonometry (37). A high-fidelity pressure transducer (model SPC-301, Millar Instruments) connected to a computer with specialized software (SCOR version 5.03, PWV Medical) was used to record radial artery pressure waveforms. A central aortic pressure waveform was then derived by using a validated transfer function. Early wave reflection from the periphery can augment the central pressure; the degree of augmentation of central aortic pressure by the reflected waveform is expressed as a percentage of the total pulse pressure and referred to as the augmentation index (AIx). The reproducibility of the technique has been described elsewhere (15, 37). Once satisfactory recordings were obtained, 50 µg of GTN was administered sublingually to assess vascular NO sensitivity. The measurements were then repeated 4 min later.Assessment of NO Responsiveness
Vascular smooth muscle responsiveness was assessed in response to both a submaximal and maximal dose of GTN. First, the ability of a submaximal dose of GTN (50 µg) to cause a drop in AIx was measured using aplanation tonometry as described above. Second, the vasodilator effect of 400 µg of GTN on brachial arterial diameter was measured using the wall-tracking device as described above. We also evaluated the degree of inhibition of platelet aggregation by NO donors ex vivo in whole blood. These studies permit detection of more subtle abnormalities of NO responsiveness, due either to oxidative stress or to signal transduction abnormalities.Platelet Aggregometry
Venous blood (8 ml) was obtained and stored in a tube containing 3.8% trisodium citrate at room temperature. The blood was allowed to settle for 10 min and checked to ensure no visible clot had formed. Whole blood aggregation was examined using a dual chamber impedance aggregometer (model 560, Chronolog; Haverston, PA) as previously described (11, 12). Tests were performed at 37°C and with a stirring speed of 900 rpm. We diluted 450-µl samples of whole blood with 500 µl saline and prewarmed for 5 min at 37°C with a further 10 µl of saline or 10 µl GTN or sodium nitroprusside (final concentration of 10 and 100 µmol/l, respectively). Aggregation was monitored continually for 7 min, and responses were recorded onto a personal computer with the use of AggroLink software. Inhibition of aggregation was evaluated as a percentage of comparing the extent of maximal aggregation in the presence and in the absence of the anti-aggregatory agent studied. Therefore, 100% inhibition means that aggregation is completely abolished.Statistical Analysis
Results were expressed as means ± SE. Statistical analysis was carried out by using statistical software (version 10.1, SPSS; Surrey, UK). Identification of significant differences was performed by using three-way ANOVA (factors were subject period and treatment). Point estimates and 95% confidence intervals (CI) were given. A P value of <0.05 was considered to be statistically significant. Changes in brachial artery diameter were expressed as percent change in diameter of the artery from the baseline obtained just before increasing flow (FMD) or GTN.| |
RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Mean age of the subjects was 35 yr (range 18-68). Body mass index was 25.8 ± 0.7 kg/m2; fasting total cholesterol (4.7 ± 0.2 mmol/l), triglycerides (1.4 ± 0.3 mmol/l), glucose (5.0 ± 0.1 mmol/l), and folate (8.5 ± 1.0 µg/l) were all in the normal range. Heart rate was slightly slower on the day subjects received methionine (61 ± 3 vs. 68 ± 3 beats/min; P < 0.05) but blood pressure (BP) was similar (systolic BP 122 ± 3 vs. 126 ± 4 mmHg).
Endothelial Function
There was a greater fall in FMD after oral methionine loading (from 2.8 ± 0.8 to 0.3 ± 0.3%) compared with placebo (which was associated with a fall from 2.8 ± 0.8 to 1.3 ± 0.3%) (P < 0.05). Baseline diameters were similar at each measurement (4,330 ± 170, 4,270 ± 150, 4,380 ± 160, and 4,280 ± 140 µm; P > 0.05). The absolute change in diameter with increased flow was 90 ± 30 µm after placebo and 10 ± 30 µm after methionine (P < 0.05).Pulse Wave Analysis
Augmentation was significantly higher 4 h after methionine (7.6 ± 3.3%) than after placebo (0.1 ± 3.6%; P < 0.01), although no baseline measurements were performed.Oxidative Stress
Markers of lipid peroxidation (TBARS) were similar at baseline on each visit (Table 1). TBARS did not change significantly after methionine (baseline, 0.46 ± 0.05 arbitrary units vs. 4 h, 0.53 ± 0.07 arbitrary units; P > 0.05) or after placebo (baseline, 0.46 ± 0.06 vs. 4 h, 0.46 ± 0.05 arbitrary units; P > 0.05) (95% CI
0.07 to 0.19).
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EPR signal intensity (representing lipid-derived free radical
concentration) did not change significantly after oral methionine loading (baseline, 7,398 ± 1,862 vs. 4 h, 6,949 ± 1,721 arbitrary units; P > 0.05) or placebo (baseline,
6,308 ± 1,348 vs. 4 h, 7,794 ± 2,235 arbitrary units;
P > 0.05) (95% CI 5,011 to 899).
Total plasma antioxidant capacity was similar at baseline on both
occasions (Table 1). Total ORAC did not significantly change after oral
methionine (2,847 ± 347 vs. 2,592 ± 214 arbitrary units; P > 0.05) or placebo (2,447 ± 311 vs. 2,738 ± 309 arbitrary units; P > 0.05) (95% CI 919 to
715). The changes from baseline for lipid-derived free radicals, TBARS,
and total ORAC did not differ significantly between the methionine and
placebo limbs. The relative contribution of the protein versus the
nonprotein component of ORAC did not alter significantly with
methionine (Table 1).
Nitrate Sensitivity
The fall in augmentation in response to a 50 µg dose of GTN was similar after methionine versus placebo (drop in AIx 10.5 ± 2.7 vs. 9.4 ± 2.5%; P > 0.05). Dilatation of the brachial artery in response to 400 µg GTN was similar after methionine vs. placebo (% dilatation with GTN at baseline was 9.4 ± 1.2 and 7.7 ± 1.4% after placebo compared with 10.2 ± 1.0 before and 8.5 ± 0.7% after methionine; P > 0.05). Platelet aggregation in response to ADP was similar on both study days and was unchanged by methionine. The ability of the direct NO donor SNP and the indirect donor GTN to inhibit aggregation was not impaired by methionine (Table 1).| |
DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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This study provides important insights into the mechanism of acute endothelial dysfunction in the oral methionine loading model in humans. We found no evidence of significantly increased oxidative stress in plasma, and preserved responsiveness to NO in both vascular smooth muscle and platelets, thereby suggesting impaired NO synthesis or release as the mechanism. These observations may have important implications for our understanding of endothelial dysfunction in disease states.
Endothelial dysfunction is believed to play a key role in the initiation and progression of atherosclerosis (30). Endothelial dysfunction is characterized by decreased bioavailability of NO, and this can occur through a number of different mechanisms. Recently, attention has focused particularly on the role of increased oxidative stress, which is believed to play a crucial role either by reducing bioavailable NO (through conversion to peroxynitrite), or by direct damage to the endothelium or both. The acute endothelial dysfunction produced by oral methionine loading is thought to be mediated by its conversion to homocysteine (2). Chronic hyperhomocysteinemia is associated with an increased atherosclerotic risk (24), and with increased oxidative stress (25). Furthermore, in vitro studies have suggested that homocysteine may acutely induce oxidative stress by auto-oxidation of the sulfadryl group of homocysteine to generate hydrogen peroxide (33), by decreasing intracellular antioxidant defenses (glutathione and glutathione peroxidase) (35), and by direct cytotoxic effects (in cultured endothelial cells) (4). However, the homocysteine concentrations achieved were an order of magnitude higher than those that induce endothelial dysfunction after oral methionine loading in humans.
Previous observations (8, 16, 27) that the endothelial dysfunction can be prevented by pretreatment with the antioxidants vitamin C or vitamin E suggest that oxidative stress may be important. In contrast, a recent study (9) reported no evidence of increased oxidative stress after oral methionine loading in humans, although this study can be criticized for two reasons. First, there was no placebo group. Second, the measures of oxidative stress employed (P-selectin and phosphatidylcholine hydroperoxide) were neither specific nor sensitive for oxidative stress.
We have confirmed that oral methionine loading produces endothelial dysfunction, as assessed by flow-mediated dilatation. In addition, we have demonstrated increased augmentation of central aortic pressure after methionine. This is in keeping with other studies (2, 8, 18, 27) on methionine loading, although this effect is not seen in all studies (9). The endothelial dysfunction occurs predominantly in an older population, which may explain why some studies (9) have not demonstrated endothelial dysfunction or an increase in arterial stiffness. There is increasing evidence that the vascular endothelium plays an important role in regulating large artery distensibility (28), and we have shown in patients with growth hormone deficiency that impaired endothelial responses to flow are associated with increased AIx (32). AIx is determined, at least in part, by NO, as infusion of an inhibitor of NO synthase (NOS) increases AIx for a given blood pressure (38). Therefore, both these findings suggest reduced bioavailability of NO to vascular smooth muscle. In this study, we went on to assess possible mechanisms that might have caused this.
We employed three measures of oxidative stress, and each measure
assessed different key levels of the oxidative process. Direct measurement of free radicals is difficult due to their short half-life. We have recently reported and validated the use of ex vivo
spin-trapping of second-generation free radicals by EPR spectroscopy.
In chronic heart failure (13), after a fatty meal in
diabetics (14), or after exercise in normal subjects
(1), there is a significant increase in these radicals. In
one study, exhaustive exercise caused a rise in these radicals from
0.05 ± 0.02 to 0.19 ± 0.03, which was prevented by
pretreatment with vitamin C (1). The EPR technique in the
present study is much more sensitive than the one used in the earlier
study, and this change would equate to an increase of about 14,000 units in the present study. This would have been detected by the
technique used in the present study (95% CI 5,011 to 899). While
there was a slight increase in TBARS after methionine in this study
(from 0.46 ± 0.05 to 0.53 ± 0.07), this is very small
compared with the biologically significant differences in oxidative
stress (TBARS) between normal subjects (0.394 ± 0.03) and those
subjects with dilated cardiomyopathy (0.624 ± 0.07)
(39). Our study would have been able to detect such an
increase in oxidative stress (95% CI 0.07 to 0.19). TBARS are
longer-lived products of lipid peroxidation that are increased in
several situations associated with increased oxidative stress (such as
heart failure and coronary artery disease) but are known to be
nonspecific (22). Given the significant impairment in endothelial function with methionine, we would have expected a change
in each of the parameters of oxidative stress that we measured if this
was the mechanism of the endothelial dysfunction. Our findings are
consistent with a recent study (34) in rat skeletal muscle
arterioles in which neither superoxide dismutase nor catalase (potent
free radical scavengers) ameliorated the endothelial dysfunction associated with diet-induced hyperhomocysteinemia.
In vitro studies (35) have suggested that homocysteine may acutely impair antioxidant defenses, in contrast to chronic hyperhomocysteinemia, where they appear to be increased (23). We therefore assessed the effects of oral methionine loading on plasma antioxidant capacity in the protein phase versus the nonprotein phase. Total plasma antioxidant capacity was not reduced nor was there any change in the balance between the protein and nonprotein antioxidant capacity. Our observations do not support any acute impairment of plasma antioxidant defenses in this model. A potential limitation of this study is that it does not directly address differences in intra- versus extracellular antioxidant capacity. However, the ORAC methodology used in this study is well established and is widely used as a general indicator of induced antioxidant capacity (7).
Vascular and platelet resistance to exogenous NO donors have been reported in conditions associated with endothelial dysfunction such as ischemic heart disease, hypertension, and in chronic heart failure (10, 26, 19), although not in all studies (21). Available evidence suggests that oxidative stress (reducing bioavailable NO) and signal transduction pathways may both contribute. Most studies using FMD have assessed the maximal responsiveness of vascular smooth muscle to GTN as a measure of endothelium-independent dilatation. However, the maximal response to GTN can be maintained despite a reduction in sensitivity due to a parallel shift in the dose-response curve (3). We have demonstrated that the brachial artery response to a maximal dose of GTN (400 µg) was not impaired by methionine. To investigate whether there was a parallel shift in the dose-response curve due to a more subtle abnormality of NO signaling, we assessed the response to a submaximal (50 µg) dose of GTN on AIx. We found that methionine did not alter this response, suggesting that NO responsiveness in smooth muscle was not impaired by methionine. Similarly, there was no reduction in the antiaggregatory effects of NO on whole blood from subjects after oral methionine loading.
We therefore have convincing data from three measures, which show that oxidative stress is not significantly enhanced by oral methionine loading. In addition, we have shown that vascular smooth muscle and platelet responsiveness to NO are not impaired. These two observations also make oxidative stress unlikely to be the principal mechanism of the endothelial dysfunction. They also make a signal transduction abnormality unlikely. We therefore conclude that an abnormality of NO synthesis is a possible cause of the endothelial dysfunction. The mechanism could relate to the recent finding that asymmetric dimethylarginine, an endogenous inhibitor of NOS, is elevated in monkeys with hyperhomocysteinemia (5) and in humans (16).
Our findings would appear at odds with previous reports that pretreatment with the antioxidant vitamins C and E can prevent the endothelial dysfunction that follows oral methionine loading. It may be that these vitamins have other effects in addition to free radical scavenging. Endothelial NOS is regulated by redox-sensitive signal transduction pathways and transcriptional regulatory networks that therefore might be influenced by these antioxidant vitamins (17, 20). It is possible, therefore, that the protective effect of these vitamins may be via increasing NO synthesis although this remains to be determined. In addition, our methods do not allow us to assess evidence of oxidative stress at a tissue level, and it may be here that the antioxidants have their effect.
In conclusion, we present evidence that the acute endothelial dysfunction after oral methionine loading is neither due to enhanced oxidative stress in plasma nor due to a signal transduction abnormality in vascular smooth muscle. This suggests that impaired NO synthesis may be an important factor in the endothelial dysfunction after oral methionine loading. This has important implications for other conditions that are associated with endothelial dysfunction.
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ACKNOWLEDGEMENTS |
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The authors thank Joan Parton and Peter Gapper for technical assistance.
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FOOTNOTES |
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A. K. Nightingale, J. Morris-Thurgood, P. P. James, and M. P. Frenneaux were supported by the British Heart Foundation.
Address for reprint requests and other correspondence: M. Frenneaux, Dept. of Cardiology, Wales Heart Research Institute, Univ. of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom (E-mail: nightingaleak{at}cardiff.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 July 2000; accepted in final form 31 October 2000.
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METHODS
RESULTS
DISCUSSION
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