Arterial stiffness is an independent determinant of cardiovascular risk (1). Established cardiovascular risk factors, such as hypertension, diabetes mellitus, and cigarette smoking, are associated with increased stiffness at an early stage, before the development of manifest atheroma (2). Arterial stiffening may be due to both structural and functional changes in the large arteries, and increasing evidence suggests that the vascular endothelium may play an important role in the functional regulation of large artery stiffness (3).
Recent epidemiologic evidence indicates that mild to moderate elevation of plasma homocysteine level is an independent risk factor for cardiovascular disease (4,5). Chronic hyperhomocystinemia is also associated with endothelial dysfunction (6), carotid artery thickening (7), and aortic stiffening (8). However, a causal relation between hyperhomocystinemia and atherosclerosis remains to be established, and elevated plasma homocysteine concentration may simply be a marker of vascular damage (9). Nevertheless, acute elevation of plasma homocysteine levels, after methionine loading, does impair endothelial function (10-13). Pretreatment with the water-soluble antioxidant vitamin C ameliorates this effect (11,12), suggesting that methionine loading may impair endothelial function by increasing oxidant stress. This is supported by data indicating that homocysteine increases oxidant stress assessed in vitro (14), although methionine loading does not appear to increase lipid peroxidation in vivo (15).
We hypothesized that acute elevation of plasma homocysteine concentration would lead to arterial stiffening and that concomitant administration of vitamin C would prevent this. The aim of the present study was to test this hypothesis using methionine loading to increase plasma homocysteine levels. This study was conducted in the same cohort of subjects who participated in our previously reported study addressing the effects of vitamin C on arterial stiffness (16).
Eight healthy male volunteers (mean age, 29 years; range, 20-42 years), were recruited from a community volunteer database held at the Western General Hospital, Edinburgh. The study was approved by the local Research Ethics Committee, and written informed consent was obtained from all participants. Individuals with cardiovascular risk factors including diabetes mellitus, hypercholesterolemia (total cholesterol >5 mM) repeated blood pressure readings >160/95 mm Hg, or clinical evidence of cardiovascular disease were excluded. All subjects were nonsmokers and free from medication. The study was conducted in a double-blind, randomized, placebo-controlled (double-dummy) manner, with three visits each separated by an interval of 1 week.
Peripheral blood pressure was recorded in the dominant arm using a validated (17) oscillometric method (HEM-705CP; Omron Corp., Tokyo, Japan). Mean arterial pressure was defined as diastolic pressure plus one third of the pulse pressure. Cardiac index was assessed noninvasively using transthoracic electrical bioimpedance (NCCOM3-R7; BioMed, Irvine, CA), and total peripheral vascular resistance was calculated as mean arterial pressure divided by cardiac index and expressed in arbitrary units (AU).
Pulse wave analysis
Augmentation index (AIx), central (ascending aortic) pressure, and the timing of the reflected pressure wave (TR) were determined using the technique of pulse wave analysis, as described in detail elsewhere (18,19). Pressure waveforms were recorded from the radial artery at the wrist of the nondominant hand by an experienced operator using gentle pressure. After 20 sequential waveforms had been acquired, the integral system software (SCOR version 6.01; PWV Medical, Sydney, Australia) was used to generate an averaged peripheral and corresponding central waveform, from which AIx, central pressure, and TR were determined. All measurements were made in duplicate and mean values used in the subsequent data analysis.
Venous blood (20 ml) was drawn through a cannula inserted in the antecubital fossa of the dominant arm into lithium-heparin tubes and centrifuged immediately at 4°C (4000 rpm for 20 min). The plasma was separated and stored at −80°C for subsequent analysis. Plasma vitamin C was assayed using an established method (20) (Cobas Bio centrifugal analyzer equipped with fluorescence attachment, Hoffman-LaRoche, Basel, Switzerland), and plasma homocysteine concentration determined using an established high-performance liquid chromatography method (21). For each assay, all samples were analyzed as a single batch.
Subjects were required to abstain from alcohol and caffeine for the 24 hours preceding each visit, and all studies were conducted after an overnight fast in a quiet, temperature-controlled room (22 ± 2°C). After 45 min of supine rest, duplicate measurements were made of blood pressure and cardiac index. Central haemodynamics were then determined in duplicate using pulse wave analysis. After this, venous blood was drawn for determination of plasma vitamin C and homocysteine concentrations, and subjects then received oral doses of either 100 mg/kg methionine (Norton Healthcare Ltd., London, U.K.), 100 mg/kg methionine plus 2 g of dispersible vitamin C (Roche Consumer Health, Welwyn Garden City, U.K.), or matching placebos. Drugs were dissolved in diluting cordial, and subjects received a low-protein snack 2 hours after dosing. All haemodynamic and biochemical measurements were repeated 6 hours after dosing, when plasma homocysteine levels were predicted to be maximally elevated (11,13).
Data are presented as means ± SEM, or means (confidence limits), unless otherwise stated, and raw data were analyzed using paired Student's t tests. Repeated measures ANOVA (SPSS version 9.0 for personal computer, SPSS Inc., Chicago, IL, U.S.A.) was used to identify differences in the response between treatments. Results were considered significant at p < 0.05.
Plasma vitamin C concentrations increased significantly after oral administration of vitamin C and methionine (45 ± 11.2 vs. 92 ± 11 μM; P < 0.001) but not after methionine alone (44 ± 8 vs. 48 ± 8 μM; p = 0.4) or placebo (52 ± 12 vs. 55 ± 14 μM; p = 0.6). The average baseline plasma vitamin C concentrations were all above the accepted level for deficiency (17 μM) (22). Plasma homocysteine levels increased significantly after administration of methionine alone (11.5 ± 1.6 vs. 28.7 ± 4.4 μM; p < 0.001) and in combination with vitamin C (11.3 ± 1.3 vs. 29.0 ± 5.0 μM; p = 0.002). The 6-hour post-dose values were not significantly different (p = 0.8). There was no significant change in plasma homocysteine concentration after placebo administration (11.6 ± 1.3 vs. 10.7 ± 1.0 μM; p = 0.2).
Baseline values for AIx and TR did not vary significantly between the three treatment periods. After administration of placebo there was no significant change in AIx (mean change, 0.1% [−4.7 to 4.8%]), TR (−2 ms [−7 to 4 ms]), peripheral mean arterial pressure (−2 mm Hg ([−8 to 2 mm Hg]), heart rate (4 beats/min [−1 to 8 beats/min]), or total peripheral vascular resistance (−1.5 AU [−7.2 to 1.7 AU]), but cardiac index increased significantly (0.4 l/min · m−2 [0.12 to 0.7 l/min · m−2]), as shown in Table 1. Similarly, there was no change in any hemodynamic parameter after methionine except for cardiac index, which increased significantly (Table 1). Six hours after combined methionine and vitamin C ingestion there was a significant fall in AIx of 10.5 ± 3.2% (p = 0.02), and an increase in cardiac index of 0.6 ± 0.2 l/min · m−2 (p < 0.01). However, was no change in peripheral or central mean pressure, central pulse pressure, or heart rate, although total peripheral vascular resistance decreased significantly by 4.6 ± 1.9 AU (p < 0.01). When the responses to methionine were compared directly with the response to placebo, there was no significant change in any parameter. However, compared with the placebo phase, there was a significant reduction in AIx after combined methionine and vitamin C dosing (10.5 ± 3.2%; vs. 0.1 ± 3.3% after placebo; p < 0.01). The hemodynamic changes are summarized in Table 1 and Figure 1.
Pulse wave analysis provides a noninvasive method for deriving central arterial pressure waveforms, from which AIx and the timing of the reflected pressure wave (TR) can be determined as indices of arterial stiffness. Augmentation index is dependent on three main factors: the pulse wave velocity, the site of wave reflection, and the amplitude of the reflected pressure wave (23). Stiffening of the smaller muscular arteries increases the amplitude of the reflected wave and shifts the site of wave reflection proximally, whereas stiffening of the larger arteries increases the pulse wave velocity. Together, these effects result in a larger reflected wave reaching the ascending aorta earlier, and augmentation of central systolic pressure. Therefore, AIx provides a composite measure of systemic arterial stiffness. As previously described (24), TR affords a measure of the aortic pulse wave velocity and, thus, aortic stiffness.
The main findings of the present study are that acute elevation of plasma homocysteine concentration, produced by methionine loading, was not associated with any significant change in arterial stiffness as assessed by both AIx and TR. Conversely, combined administration of vitamin C and methionine resulted in a similar reduction in AIx to that which we have previously reported after a single oral dose of 2 g of vitamin C alone (16). Besides supporting our original observations regarding the reduction in arterial stiffness after oral vitamin C administration, the present data indicate that elevation of plasma homocysteine levels does not ameliorate this effect.
In contrast, chronic hyperhomocystinemia is associated with stiffening of the large arteries, as assessed by the aortic pulse wave velocity (8), and impaired endothelial function (6). However, such associations do not prove a causal relation between homocysteine and atherosclerosis: elevated plasma homocysteine level may simply reflect vascular damage (9). Nevertheless, most but not all (25) studies have demonstrated impaired vasomotor endothelial function of both resistance (11) and conduit vessels (10,12,13) after acute elevation of plasma homocysteine concentration by methionine loading. Despite raising plasma homocysteine to a similar level, and one that is consistent with mild hyperhomocystinemia, we were unable to demonstrate any change in arterial stiffness. Indeed, our data are supported by a preliminary report showing that aortic pulse wave velocity does not change after oral methionine loading (26).
In the present study, we elected to assess changes in arterial stiffness only once, 6 hours after methionine loading, because previous studies have demonstrated that plasma homocysteine levels are maximally elevated at between 6 and 8 hours after dosing (11,13). Therefore, we may have missed changes in arterial stiffness occurring either before or after this time point. Nevertheless, plasma homocysteine concentration was significantly elevated in all subjects at 6 hours (mean elevation, 151 ± 12%; p < 0.001 vs. placebo phase). Moreover, others have demonstrated clear endothelial dysfunction 6 to 8 hours after oral methionine loading (11,13). The present study was designed to investigate the effects of acute elevation of homocysteine levels on arterial stiffness and, therefore, we cannot exclude the possibility that chronic elevation of homocysteine, in otherwise healthy subjects, would result in arterial stiffening. Indeed, arterial stiffening may be more likely with chronic elevation of plasma homocysteine concentration, as a consequence of both functional and structural changes within vessel walls.
In conclusion, acute elevation of plasma homocysteine level after methionine loading did not result in an alteration of arterial stiffness in healthy subjects. Administration of vitamin C and methionine resulted in a similar reduction in arterial stiffness to that previously reported with vitamin C alone.
Acknowledgment: The Sir Stanley & Lady Davidson Fund, the Urquhart Trust, and the High Blood Pressure Foundation jointly supported this study. Professor Webb is currently the recipient of a Research Leave Fellowship from the Wellcome Trust (WT 0526330). We would also like to thank Mr. N. Johnston, Ms. S. McCall, and Ms. M. Millar, for technical assistance, and Dr. R. Riemersma for advice.
1. Blacher J, Asmar R, Djane S, et al. Aortic pulse wave velocity as a marker of cardiovascular risk in hypertensive patients. Hypertension
2. Glasser SP, Arnett DK, McVeigh GE, et al. Vascular compliance and cardiovascular disease: a risk factor or a marker? Am J Hypertens
3. Ramsey MW, Goodfellow J, Jones CJH, et al. Endothelial control of arterial distensibility is impaired in chronic heart failure. Circulation
4. Boushey CJ, Beresford SAA, Omenn GS, et al. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: probable benefits of increasing folic acid intakes. JAMA
5. Graham IM, Daly LE, Refsum HM, et al. Plasma homocysteine as a risk factor for vascular disease: the European Concerted Action Project. JAMA
6. Woo KS, Chook P, Lolin YI, et al. Hyperhomocyst(e)inemia is a risk factor for arterial endothelial dysfunction in humans. Circulation
7. McQuillan BM, Beilby JP, Nidorf M, et al. Hyperhomocysteinemia but not the C677T mutation of methylenetetrahydrofolate reductase is an independent risk determinant of carotid wall thickening: the Perth carotid ultrasound disease assessment study (CUDAS). Circulation
8. Bortolotto LA, Safar ME, Billaud E, et al. Plasma homocysteine, aortic stiffness, and renal function in hypertensive patients. Hypertension
9. Dudman NPB, Chesterman C. An alternative view of homocysteine. Lancet
10. Bellamy MF, McDowell IFW, Ramsey MW, et al. Hyperhomocysteinemia after an oral methionine load acutely impairs endothelial function in healthy adults. Circulation
11. Kanani PM, Sinkey CA, Browning RL, et al. Role of oxidant stress in endothelial dysfunction produced by experimental hyperhomocyst(e)inemia in humans. Circulation
12. Chambers JC, McGregor A, Jean-Marie J, et al. Demonstration of rapid onset vascular endothelial dysfunction after hyperhomocysteinemia: an effect reversible with vitamin C therapy. Circulation
13. Usui M, Matsuoka H, Miyazaki H, et al. Endothelial dysfunction by acute hyperhomocyst(e)inaemia: restoration by folic acid. Clin Sci
14. Loscalzo J. The oxidant stress of hyperhomocyst(e)inemia. J Clin Invest
15. Chao C-L, Kuo T-L, Lee Y-T. Effects of methionine-induced hyperhomocysteinemia on endothelium-dependent vasodilation and oxidative status in healthy adults. Circulation
16. Wilkinson IB, Megson IL, MacCallum H, et al. Oral vitamin C reduces arterial stiffness and platelet aggregation in man. J Cardiovasc Pharmacol
17. O'Brien E, Mee F, Atkins N, Thomas M. Evaluation of three devices for self measurement of blood pressure according to the revised British Hypertension Society Protocol: the Omron HEM-705CP, Philips HP5332, and Nissei DS-175. Blood Press Monitoring
18. Wilkinson IB, Fuchs SA, Jansen IM, et al. The reproducibility of pulse wave velocity and augmentation index measured by pulse wave analysis. J Hypertens
19. Wilkinson IB, MacCallum H, Flint L, et al. The influence of heart rate on augmentation index and central arterial pressure in humans. J Physiol
20. Lee W, Roberts SM, Labb RF. Ascorbic acid determination with an automated enzymatic procedure. Clin Chem
21. Daskalakis I, Lucock MD, Anderson A, et al. Determination of plasma total homocysteine and cysteine using HPLC with fluorescence detection and an ammonium 7-fluoro-2, 1,3-benzoxadiazole-4-sulphonate (SBD-F) derivatization protocol optimized for antioxidant concentration, derivatization reagent concentration, temperature and amazon matrix pH. Biomed Chromatogr
22. Tietz textbook of clinical chemistry.
Burtis CA, et al., eds. Philadelphia: WB Saunders, 1986.
23. Nichols WW, O'Rourke MF. McDonald's blood flow in arteries: theoretical, experimental and clinical principles.
London: Arnold, 1998.
24. Marchais SJ, Guerin AP, Pannier BM, et al. Wave reflections and cardiac hypertrophy in chronic uremia. Hypertension
25. McAuley DF, Hanratty CG, McGurk C, et al. Effect of methionine supplementation on endothelial function, plasma homocysteine, and lipid peroxidation. J Toxicol Clin Toxicol
26. Aziz O, Rajkumar C, Chambers JC, et al. Does acute hyperhomocysteinaemia alter arterial compliance? [abstract]. Clin Sci