- Email: [email protected]
Acute Hyperhomocysteinemia Induces Microvascular and Macrovascular Endothelial Dysfunction
Archives of Medical Research 38 (2007) 411e416
ORIGINAL ARTICLE
Acute Hyperhomocysteinemia Induces Microvascular and Macrovascular Endothelial Dysfunction Thomas N. Abahji,a Lars Nill,a Nagatoshi Ide,b Christiane Keller,b Ulrich Hoffmann,a and Norbert Weissa,b a
Department of Vascular Medicine, bSection General Internal Medicine and Metabolic Diseases, Medical PolicliniceCity Campus, University of Munich Medical Center, Munich, Germany Received for publication August 2, 2006; accepted January 4, 2007 (ARCMED-D-06-00327).
Background. Hyperhomocysteinemia (Hhcy) has been shown to induce endothelial dysfunction due to a decrease in bioavailable nitric oxide (NO) by increased vascular oxidant stress. This can be detected as an impairment of endothelium-dependent vasodilation in conductance arteries, like brachial or coronary arteries. The effect of Hhcy on endothelial function (EF) in small resistance vessels that critically determine organ perfusion, however, has not been studied systematically in humans. Therefore, we simultaneously determined macro- and microvascular EF in 11 healthy subjects before and during acute Hhcy induced by an oral methionine challenge. Methods. Macrovascular EF was determined by measuring endothelium-dependent flowmediated vasodilation of the brachial artery by vascular ultrasound and microvascular EF by measuring skin perfusion during iontophoresis of acetylcholine using laser Doppler fluxmetry. Results. Oral methionine significantly increased homocysteine levels by about 5.1-fold. Acute Hhcy leads to a significant decrease in flow-mediated vasodilation of the brachial artery from 8.1 0.5% to 3.6 0.6% and to a significant decrease in the ratio of acetylcholine-stimulated vs. baseline laser Doppler flow in the forearm skin (from 9.2 1.0- to 7.8 1.3-fold). Conclusions. Acute Hhcy impairs macro- as well as microvascular (EF) in humans. Ó 2007 IMSS. Published by Elsevier Inc. Key Words: Homocysteine, Endothelial dysfunction, Nitric oxide, Endothelium-derived hyperpolarizing factor, Microcirculation.
Introduction The pathobiological mechanisms that lead to the atherogenic propensity associated with elevated homocysteine (Hcy) levels suggest that a key target is the vascular endothelium leading to endothelial dysfunction (ED) and structural endothelial injury (1,2). An impairment of the endotheliumdependent regulation of vascular tone in large conductance vessels like the coronary arteries or the brachial artery is an integral component of ED and is indicative of a reduction in the bioavailability of the endothelium-derived signaling
Address reprint requests to: Norbert Weiss, Medical PolicliniceCity Campus, University of Munich Medical Center, Pettenkoferstrasse 8a, D-80336 Munich, Germany; E-mail: [email protected]
molecule nitric oxide (NO) (3,4). This can be detected in chronic hyperhomocysteinemic subjects free of overt cardiovascular disease (5e7) as well as in healthy subjects during acute hyperhomocysteinemia (Hhcy) induced by an oral methionine challenge (8e12). After an oral methionine challenge, the time course of the impairment of endothelium-dependent vasodilation strongly correlates with the time-course of the increase of total homocysteine (Hcy) (13) and especially in the plasma levels of free reduced Hcy (14). This suggests that reduced Hcy may be the form of Hcy that impairs NO bioavailability. In addition to Hcy’s effect on large artery function, recent studies have associated Hhcy with small vessel complications, e.g., with subcortical vascular encephalopathy, a small vessel disease leading to dementia (15), and with
0188-4409/07 $esee front matter. Copyright Ó 2007 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2007.01.004
412
Abahji et al./ Archives of Medical Research 38 (2007) 411e416
microvascular complications in diabetics leading to nephropathy and retinopathy (16). Furthermore, microangiopathy is described as part of the hereditary disorder of homocystinuria (17). Several lines of evidence have indicated that endothelium-derived hyperpolarizing factor (EDHF), and not NO, is a major determinant of vascular tone in microvessels and thereby seems to be a critical determinant of organ perfusion (18). In rats with chronic hyperhomocysteinemia (Hhcy) subsequent to intravenous methionine supplementation, EDHF-mediated vasodilation was impaired, as evaluated as the renal blood flow response to intrarenal acetylcholine during systemic NO synthase and cyclooxygenase inhibition (19). Whether or not Hhcy impairs microvascular concomitant to macrovascular reactivity in humans has not been studied so far. The aim of this study, therefore, was to simultaneously determine macrovascular endothelial function (EF) in the brachial artery and microvascular EF in the skin microcirculation in healthy subjects before and during acute Hhcy.
Subjects and Methods Subjects Macro- and microvascular function before and during acute Hhcy was studied in 11 healthy subjects aged 25e40 years (5 females, 6 males). Study participants were free of overt cardiovascular disease, free of cardiovascular risk factors, had normal renal function and normal glucose tolerance. None of the subjects took any cardiovascular medications, vitamins or antioxidant supplements. The study protocol complied with the Declaration of Helsinki on ethical principles for medical research involving human subjects as revised in 2000 (20). Informed consent was obtained from all participants.
Experimental Protocol All measurements were performed in a temperaturecontrolled room (22e24 C) with the subjects in supine position and acclimatized at least 30 min before the experiments. During each study day, EF was assessed in the fasting state and 4 to 6 h after an oral methionine load (0.1 g/kg body weight) to induce acute Hhcy. Methionine powder was dissolved in apple juice. After performing the baseline studies and before performing the oral methionine load, subjects were allowed to have a light breakfast. Microvascular function tests were performed before measuring macrovascular function. All measurements were repeated on three (macrovascular) or two (microvascular) independent occasions with 6-week intervals each.
Macrovascular Endothelial Function To study endothelium-dependent vasoreactivity in the macrocirculation, the diameter of the brachial artery was measured using a high-resolution vascular ultrasound device equipped with a 7.5 kHz linear array transducer (Accuson 128XP/10; Siemens Medical Solutions, Mountain View, CA) both before and during reactive hyperemia, as described previously (21,22). Reactive hyperemia was induced by a blood pressure cuff positioned on the forearm distal to the brachial artery and inflated to suprasystolic levels for 5 min. The flow-mediated dilation (FMD) during reactive hyperemia, observed 45 60 sec after sudden deflation of the cuff and calculated using the formula [(maximum vessel diameter e baseline vessel diameter)/baseline vessel diameter], is a well-established parameter for endotheliumdependent vasodilation and represents an increased release of endothelium-derived vasodilatory NO. To control for any effects of the experimental procedure or the oral methionine challenge on endothelium-independent vasodilation, measurements of the diameter of the brachial artery were repeated 4 min after application of sublingual nitroglycerin, which directly acts on vascular smooth muscle cells. Examinations were done by the same trained examiner, stored on videotape, and analyzed offline. At least nine readings were performed for each measurement point, and averaged. Reproducibility of the measurements was assessed on three occasions. The within-subject coefficient of variation (CV), calculated using the formula standard deviation/mean (22), was 4.4%. Microvascular EF Microvascular EF was studied by measuring skin perfusion at the volar side of the forearm using a laser Doppler instrument (Periflux 5001; Perimed AB, Ja¨rfa¨lla, Sweden) equipped with a solid-state diode laser probe (780 nm). Flux was recorded on one arm at baseline and after application of increasing doses of acetylcholine, and on the contralateral arm at baseline and after application of sodium nitroprusside. Both drugs were applied by iontophoresis using a micropharmacology delivery system (Perilont; Perimed AB) as described previously (23). Briefly, laser Doppler is a non-invasive method to assess microvascular skin perfusion. A laser beam penetrates the skin to a depth of 1e1.5 mm and a fraction of the light is backscattered by moving blood objects undergoing a frequency shift according to the Doppler principle. Thereby a signal, proportional to tissue perfusion, is generated and expressed in arbitrary perfusion units. A drug delivery electrode was placed in the head of the laser probe, and the probe temperature was set to 32 C. The drug delivery electrode was filled with 140 mL acetylcholine (ACH) 1% (Sigma Chemicals, St. Louis, MO) or sodium nitroprusside (SNP) 1% (Sigma Chemicals). Both chemicals were dissolved in deionized and double-distilled water and kept on ice during the day.
Homocysteine-induced Endothelial Dysfunction
Fresh SNP solution was prepared every day. ACH solution was prepared once and stored in aliquots at e20 C until use. After recording of baseline perfusion for 3 min, an electric field (0.1 mA for 10 sec) was applied (anodal current for ACH, cathodal current for SNP, respectively) and the laser Doppler response was recorded for 3 min. Thereafter, electric current was increased to 0.2 mA for 10 sec and 0.2 mA for 20 sec, whereas the response was recorded for 3 min each. The electric current stimulation cascade was well tolerated by the subjects. Endothelium-dependent, ACH-induced vasodilation was expressed as the ratio of laser Doppler flow recorded during maximum stimulation (0.2 mA for 20 sec) divided by baseline flow. Endotheliumindependent, SNP-induced vasodilation was expressed accordingly. To control for the effects of pure vehicle (deionized and double-distilled water) on vasodilation in the laser Doppler experiment, the pure vehicle was iontophoresed by anodal current once in each volunteer. Cathodal current was applied on three different volunteers in control experiments. Negligible changes were induced by the current stimulation cascade used in the study (data not shown). Readings were stored on a personal computer and analyzed offline. The within-subject CV was calculated as described above. CV for the measurements was 30.3%. Blood Chemistry Blood samples were drawn before and 6e7 h after administration of methionine (after the micro- and macrovascular function studies had been performed) on each occasion. Total plasma Hcy levels were measured using an isocratic HPLC method after liberation of thiol compounds from plasma proteins and reduction of oxidized thiols with tri-n-butylphosphine followed by derivatization with the thiol-specific fluorogenic marker, 7-fluoro-benzo-2-oxa1,3-diazole-sulfonate, as described previously (24). Lipid profiles and basic clinical chemistry parameters [including full blood counts, glucose levels, levels of glycosylated hemoglobin (HbA1c), and liver and kidney function tests] were measured by standard clinical chemistry methods.
413
analyses were performed using the STATVIEW software package (Abacus Concepts, Berkeley, CA).
Results The oral methionine load resulted in an approximately 5.1fold increase in plasma Hcy levels (from 5.9 0.7 mmol/L to 30.0 3.0 mmol/L, p !0.005) (Figure 1). Acute Hhcy had no effect on heart rate or systolic and diastolic blood pressure values, which did not differ between baseline and post-methionine load examinations (Table 1). The oral methionine challenge slightly increased total triglyceride and decreased HDL cholesterol levels but had no effect on total or LDL cholesterol levels (Table 1). Standard biochemical parameters were within normal ranges in the subjects studied (data not shown). The effect of acute Hhyc on macrovascular EF was measured by following FMD of the brachial artery. FMD during acute Hhyc was significantly reduced from 8.1 0.5% to 3.6 0.6% ( p !0.005) compared to fasting examinations (Figure 2A). The oral methionine challenge had no effect on the resting diameter of the brachial artery (data not shown). Endothelium-independent vasodilation after application of sublingual nitroglycerin was not affected by the oral methioine challenge (Figure 2B). This indicates that acute Hhcy impairs endothelium-dependent vasodilation, presumably due to a decrease in bioavailable NO, but does not affect endothelium-independent vasodilation in large conductance arteries, like the brachial artery. EF in microvascular circulation was assessed by laser Doppler skin perfusion measurements in reaction to ACH or SNP. At baseline measurements, iontopheresis of acetylcholine, as a stimulator of endothelial-dependent vasodilation, led to a maximum 9.2 1.0 fold increase in laser Doppler flow. During acute Hhcy maximum increase in
Statistical Analysis Replicate vascular function data obtained during the three (macrovascular function) and two (microvascular function) examinations, respectively, were averaged for each individual, and expressed as median SEM for the whole group. Replicate plasma Hcy measurements of an individual during each of the three examinations and before and after the oral methionine were averaged for each individual and expressed accordingly for the whole group. Differences before and after the oral methionine challenge were compared using Wilcoxon Matched Pairs Signed Rank Test (25); p values !0.05 were considered significant. Statistical
Figure 1. Plasma homocysteine concentrations before and 6e7 h after an oral methionine challenge. Triplicate measurements of each individual were performed during the three study time points, and averaged. Values indicate the median SEM of all subjects (n 5 11, *p !0.005 vs. before).
Abahji et al./ Archives of Medical Research 38 (2007) 411e416
414
Table 1. Blood pressure, heart rate, and lipid levels before and after an oral methionine challenge Before
After
p value
Systolic blood pressure (mmHg) 116.7 2.7 114.4 1.8 0.534 Diastolic blood pressure (mmHg) 75.0 1.8 75.0 1.9 0.173 Heart rate (per minute) 65.3 2.9 65.0 2.6 0.575 Total cholesterol (mg/dL) 169.5 4.9 169.0 5.1 0.623 Total triglyceride (mg/dL) 63.0 9.4 117 17.8 !0.01 LDL cholesterol (mg/dL) 103.5 5.6 100.0 4.6 0.139 HDL cholesterol (mg/dL) 48 2.8 42.5 2.9 !0.01 Triplicate measurements of each individual were performed during the three study time points, and averaged. Values indicate the median SEM of all subjects (n 5 11).
laser-Doppler flow in response to ACH compared to baseline flow was significantly reduced to 7.8 1.3 fold ( p !0.01) (Figure 3A). In contrast, acute Hhcy had no effect on SNPinduced increase in skin perfusion, an endotheliumindependent vasodilator (Figure 3B). We further studied whether or not there is any correlation between the magnitude of ED that had been induced by the oral methionine challenge in the two vascular territories studied. Pearson regression coefficient was calculated between FMD or SNP-induced vasodilation in the brachial artery on the one hand, and ACH- or
Figure 3. Ratio of endothelium-dependent acetylcholine-stimulated vs. baseline laser Doppler flow (A) and the ratio of endothelium-independent sodium nitroprusside-stimulated vs. baseline laser Doppler flow (B) in the forearm skin before and after an oral methionine challenge. Duplicate measurements of each individual were performed during the second and third study time points, and averaged. Values indicate the median SEM of all subjects (n 5 11, *p !0.05 vs. before).
SNP-induced increases in skin perfusion on the other hand, both before and after the oral methionine load. No significant correlations were found (data not shown).
Discussion
Figure 2. Endothelium-dependent flow-mediated vasodilation (A) and endothelium-independent nitroglycerin-induced vasodilation (B) before and hours after an oral methionine challenge. Triplicate measurements of each individual were performed during the three study time points, and averaged. Values indicate the median SEM of all subjects (n 5 11, *p !0.005 vs. before).
Our study confirms previous data showing that acute Hhcy induced by an oral methionine challenge induces ED in large conduction vessels, like the brachial artery (Figure 2) (8e12). These functional alterations of large vessel function in Hhcy may contribute to the increased risk for coronary artery disease, cerebrovascular disease and peripheral arterial occlusive disease in hyperhomocysteinemic subjects (26). Mechanistically, this effect is thought to be due to interference of Hhcy with vascular NO signaling. Incubation of cultured endothelial cells with Hcy results in a reduction of NO released from endothelial cells after stimulation with acetylcholine or bradykinin and in decreased levels of cyclic guanosine monophosphate in vascular smooth muscle cells coincubated with Hcy-incubated endothelial cells (27). Hcy increases vascular oxidant stress and superoxide anion output resulting in chemical inactivation of NO (2), and/or increased levels of the endogenous
Homocysteine-induced Endothelial Dysfunction
NO synthase inhibitor asymmetric dimethylarginine (28). Both mechanisms lead to a decrease in bioavailable NO. The novel finding of our study is that acute Hhcy simultaneously impaired endothelium-dependent ACH-induced vasodilation in skin microvessels (Figure 3). This observation may be linked to recent clinical findings that elevated Hcy levels are associated with microvascular complications. For example, Hhcy has been associated with subcortical vascular encephalopathy, a small vessel disease leading to dementia (15), and with microvascular complications in diabetics leading to nephropathy and retinopathy (16). Microangiopathy is also described as part of the hereditary disorder of homocystinuria (17). We did not study any mechanisms underlying Hcy’s effects on microvascular function. EDHF, and not NO, is thought to be the major determinant of vascular tone in microvessels and thereby seems to be a critical determinant of organ perfusion (18). In rats with chronic Hhcy, an impairment of the renal blood flow response to intrarenal acetylcholine has been demonstrated even during systemic NO synthase and cyclooxygenase inhibition. This suggests a decreased action of vasodilator EDHF in this model of Hhcy (19). The observed effects of the oral methionine challenge on macro- and microvascular endothelial functions are not due to changes in hemodynamic parameters as blood pressure and heart rate remained unaffected (Table 1). Furthermore, it seems very unlikely that the effects are due to the slight increase in triglyceride and the slight decrease in HDL cholesterol levels after the oral methionine challenge. Although postprandial hyperlipidemia is known to induce macrovascular ED in hyperlipidemic subjects, mild and acute hypertriglyceridemia do not lead to ED in normolipidemic individuals (29). All subjects studied were normolipidemic. In addition, previous studies have shown that postprandial hyperlipidemia to or above the extent seen in our study does not induce ED in forearm resistance vessels (30). We could not demonstrate any significant correlation between the magnitude of impairment of ED in the two vascular territories studied. This might be due to different mechanisms leading to decreased vasodilation in the two vascular beds examined. Furthermore, our study showed that Hhcy had no significant effect on smooth muscle (endothelium-independent) vasodilator responses to exogenous nitrate, e.g., nitroglycerin and SNP, in both vascular territories. Overall, the observations point to the vascular endothelium as the primary target of Hhcy in different vascular territories. In contrast to our data, a previous study in healthy male volunteers could not demonstrate any effect of acute Hhcy on skin microvascular responses (31). The reasons for these differing results are unclear. They may be related to different ages of subjects studied, to gender differences, or to
415
different methodology. Both studies examined young and healthy subjects; therefore, age is unlikely to cause the different results. Davis et al. (31) studied only males, whereas we studied both female and male subjects without identifying any gender differences. Therefore, the differences might not be explained by gender. Davis’s study neither simultaneously measured macrovascular endothelial function, nor was any systemic endothelial cell activation seen, as von Willebrand factor levels remained unchanged after methionine loading. We could simultaneously demonstrate macro- and microvascular ED. Nevertheless, our findings have to be confirmed before drawing firm conclusions on the effect of Hcy on microvascular EF. Our study has several limitations. First, the sample size of this pilot study is rather small. The effect of the sample size, however, was partly compensated as we repeated the measurements during two or three independent occasions with consistent results. Second, analyses of macro- and microvascular EF were performed offline from data stored on videotapes or on computer, but were not done blinded. This might introduce some bias. Third, we did not introduce a placebo arm into the study protocol to control for any potential effects of the daytime on FMD or microvascular function. Room temperature and skin temperature of the subjects were well-controlled and constant during the day. A recent study that systematically evaluated the effect of daytime on non-invasive measurements of vascular function could not find any influence on FMD measurements as long as exogenous conditions were controlled (32). In addition, in a subset of subjects we studied effects of the daytime on skin perfusion responses to ACH without a methionine challenge and could not find any significant effect (data not shown). Fourth, assessment of microvascular endothelial function using a laser Doppler device inherently exhibits a substantial variability due to spatial and temporal variations of laser Doppler flux. This is indicated by within-subject CVs of |30% in our, as well as in other, studies (23). Accuracy of our data indicating that acute Hhcy impairs microvascular EF, however, is supported by the fact that we repeated the measurements on two independent occasions in each individual with consistent results. In summary, this pilot study showed that acute Hhcy not only impairs macrovascular but also microvascular EF. The latter observation may at least partly explain the clinical observation that Hhcy is associated with small vessel complications. If this finding can be reproduced in larger studies and by independent investigators, it may have impact on our pathophysiological understanding of microvascular complications in certain patient groups where Hhcy interacts with other cardiovascular risk factors on small vessel complications, like patients with diabetes or chronic kidney disease. Further studies are warranted to explore the potential mechanisms by which Hcy may interfere with non-NO pathways in endothelial cells that regulate vascular tone and function in the microcirculation.
416
Abahji et al./ Archives of Medical Research 38 (2007) 411e416
Acknowledgments The work was supported by research grants from Wakunaga of America, Co. Ltd., Mission Viejo, CA and from the FriedrichBaur Foundation, Munich, Germany. The authors would like to thank all study participants for their cooperation, Ilona Fialla and Pirkko Koelle for excellent technical assistance, and Gabriele Ho¨lscher, Institute for Medical Information Technology, Biometry and Epidemiology at the University of Munich, Germany, for biostatistical consulting.
References 1. Weiss N, Keller C, Hoffmann U, Loscalzo J. Endothelial dysfunction and atherothrombosis in mild hyperhomocysteinemia. Vasc Med 2002; 7:227e239. 2. Weiss N. Mechanisms of increased vascular oxidant stress in hyperhomocysteinemia and its impact on endothelial function. Curr Drug Metab 2005;6:27e36. 3. Guerra R Jr, Brotherton AF, Goodwin PJ, Clark CR, Armstrong ML, Harrison DG. Mechanisms of abnormal endothelium-dependent vascular relaxation in atherosclerosis: implications for altered autocrine and paracrine functions of EDRF. Blood Vessels 1989;26: 300e314. 4. Luscher TF, Richard V, Tschudi M, Yang ZH, Boulanger C. Endothelial control of vascular tone in large and small coronary arteries. J Am Coll Cardiol 1990;15:519e527. 5. Woo KS, Chook P, Lolin YI, Cheung AS, Chan LT, Sun YY, et al. Hyperhomocyst(e)inemia is a risk factor for arterial endothelial dysfunction in humans. Circulation 1997;96:2542e2544. 6. Tawakol A, Omland T, Gerhard M, Wu JT, Creager MA. Hyperhomocyst(e)inemia is associated with impaired endothelium-dependent vasodilation in humans. Circulation 1997;95:1119e1121. 7. Holven KB, Holm T, Aukrust P, Christensen B, Kjekshus J, Andreassen AK, et al. Effect of folic acid treatment on endotheliumdependent vasodilation and nitric oxide-derived end products in hyperhomocysteinemic subjects. Am J Med 2001;110:536e542. 8. Hanratty CG, McAuley DF, McGurk C, Young IS, Johnston GD. Homocysteine and endothelial vascular function. Lancet 1998;351: 1288e1289. 9. Chambers JC, McGregor A, Jean-Marie J, Obeid OA, Kooner JS. Demonstration of rapid onset vascular endothelial dysfunction after hyperhomocysteinemia: an effect reversible with vitamin C therapy. Circulation 1999;99:1156e1160. 10. Kanani PM, Sinkey CA, Browning RL, Allaman M, Knapp HR, Haynes WG. Role of oxidant stress in endothelial dysfunction produced by experimental hyperhomocyst(e)inemia in humans. Circulation 1999;100:1161e1168. 11. Lambert J, van den Berg M, Steyn M, Rauwerda JA, Donker AJ, Stehouwer CD. Familial hyperhomocysteinaemia and endotheliumdependent vasodilatation and arterial distensibility of large arteries. Cardiovasc Res 1999;42:743e751. 12. Chao CL, Kuo TL, Lee YT. Effects of methionine-induced hyperhomocysteinemia on endothelium-dependent vasodilation and oxidative status in healthy adults. Circulation 2000;101:485e490. 13. Bellamy MF, McDowell IF, Ramsey MW, Brownlee M, Bones C, Newcombe RG, et al. Hyperhomocysteinemia after an oral methionine load acutely impairs endothelial function in healthy adults. Circulation 1998;98:1848e1852. 14. Chambers JC, Ueland PM, Wright M, Dore CJ, Refsum H, Kooner JS. Investigation of relationship between reduced, oxidized, and proteinbound homocysteine and vascular endothelial function in healthy human subjects. Circ Res 2001;89:187e192.
15. Bertsch T, Mielke O, Holy S, Zimmer W, Casarin W, Aufenanger J, et al. Homocysteine in cerebrovascular disease: an independent risk factor for subcortical vascular encephalopathy. Clin Chem Lab Med 2001;39:721e724. 16. Looker HC, Fagot-Campagna A, Gunter EW, Pfeiffer CM, Narayan KM, Knowler WC, et al. Homocysteine as a risk factor for nephropathy and retinopathy in Type 2 diabetes. Diabetologia 2003; 46:766e772. 17. McCully KS. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am J Pathol 1969;56:111e128. 18. Triggle CR, Ding H, Anderson TJ, Pannirselvam M. The endothelium in health and disease: a discussion of the contribution of non-nitric oxide endothelium-derived vasoactive mediators to vascular homeostasis in normal vessels and in type II diabetes. Mol Cell Biochem 2004;263: 21e27. 19. De Vriese AS, Blom HJ, Heil SG, Mortier S, Kluijtmans LA, Van de Voorde J, et al. Endothelium-derived hyperpolarizing factor-mediated renal vasodilatory response is impaired during acute and chronic hyperhomocysteinemia. Circulation 2004;109:2331e2336. 20. World Medical Association Declaration of Helsiniki. Ethical Principles for Medical Research Involving Human Subjects. http://www. wma.net/e/policy/pdf/GGTSPU-11623-65270-DAT/17c.pdf. Accessed July 17, 2006. 21. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, et al. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 1992;340: 1111e1115. 22. De Roos NM, Bots ML, Schouten EG, Katan MB. Within-subject variability of flow-mediated vasodilation of the brachial artery in healthy men and women: implications for experimental studies. Ultrasound Med Biol 2003;29:401e406. 23. Farkas K, Kolossvary E, Jarai Z, Nemcsik J, Farsang C. Non-invasive assessment of microvascular endothelial function by laser Doppler flowmetry in patients with essential hypertension. Atherosclerosis 2004;173:97e102. 24. Feussner A, Rolinski B, Weiss N, Deufel T, Wolfram G, Roscher AA. Determination of total homocysteine in human plasma by isocratic high-performance liquid chromatography. Eur J Clin Chem Clin Biochem 1997;35:687e691. 25. Wilcoxon F. Individual comparisons by ranking methods. Biometrics 1945;1:80e83. 26. Bautista LE, Arenas IA, Penuela A, Martinez LX. Total plasma homocysteine level and risk of cardiovascular disease: a meta-analysis of prospective cohort studies. J Clin Epidemiol 2002;55:882e887. 27. Weiss N, Zhang YY, Heydrick S, Bierl C, Loscalzo J. Overexpression of cellular glutathione peroxidase rescues homocyst(e)ine-induced endothelial dysfunction. Proc Natl Acad Sci USA 2001;98:12503e 12508. 28. Dayal S, Lentz SR. ADMA and hyperhomocysteinemia. Vasc Med 2005;10(Suppl 1):S27eS33. 29. Giannattasio C, Zoppo A, Gentile G, Failla M, Capra A, Maggi FM, et al. Acute effect of high-fat meal on endothelial function in moderately dyslipidemic subjects. Arterioscler Thromb Vasc Biol 2005;25: 406e410. 30. Gudmundsson GS, Sinkey CA, Chenard CA, Stumbo PJ, Haynes WG. Resistance vessel endothelial function in healthy humans during transient postprandial hypertriglyceridemia. Am J Cardiol 2000;85: 381e385. 31. Davis KR, Pearson H, Moat S, Bonham JR, Donnelly R. Acute hyperhomocysteinaemia affects pulse pressure but not microvascular vasodilator function. Br J Clin Pharmacol 2001;52:327e332. 32. ter Avest E, Holewijn S, Stalenhoef AF, de Graaf J. Variation in non-invasive measurements of vascular function in healthy volunteers during daytime. Clin Sci (Lond) 2005;108:425e431.
ORIGINAL ARTICLE
Acute Hyperhomocysteinemia Induces Microvascular and Macrovascular Endothelial Dysfunction Thomas N. Abahji,a Lars Nill,a Nagatoshi Ide,b Christiane Keller,b Ulrich Hoffmann,a and Norbert Weissa,b a
Department of Vascular Medicine, bSection General Internal Medicine and Metabolic Diseases, Medical PolicliniceCity Campus, University of Munich Medical Center, Munich, Germany Received for publication August 2, 2006; accepted January 4, 2007 (ARCMED-D-06-00327).
Background. Hyperhomocysteinemia (Hhcy) has been shown to induce endothelial dysfunction due to a decrease in bioavailable nitric oxide (NO) by increased vascular oxidant stress. This can be detected as an impairment of endothelium-dependent vasodilation in conductance arteries, like brachial or coronary arteries. The effect of Hhcy on endothelial function (EF) in small resistance vessels that critically determine organ perfusion, however, has not been studied systematically in humans. Therefore, we simultaneously determined macro- and microvascular EF in 11 healthy subjects before and during acute Hhcy induced by an oral methionine challenge. Methods. Macrovascular EF was determined by measuring endothelium-dependent flowmediated vasodilation of the brachial artery by vascular ultrasound and microvascular EF by measuring skin perfusion during iontophoresis of acetylcholine using laser Doppler fluxmetry. Results. Oral methionine significantly increased homocysteine levels by about 5.1-fold. Acute Hhcy leads to a significant decrease in flow-mediated vasodilation of the brachial artery from 8.1 0.5% to 3.6 0.6% and to a significant decrease in the ratio of acetylcholine-stimulated vs. baseline laser Doppler flow in the forearm skin (from 9.2 1.0- to 7.8 1.3-fold). Conclusions. Acute Hhcy impairs macro- as well as microvascular (EF) in humans. Ó 2007 IMSS. Published by Elsevier Inc. Key Words: Homocysteine, Endothelial dysfunction, Nitric oxide, Endothelium-derived hyperpolarizing factor, Microcirculation.
Introduction The pathobiological mechanisms that lead to the atherogenic propensity associated with elevated homocysteine (Hcy) levels suggest that a key target is the vascular endothelium leading to endothelial dysfunction (ED) and structural endothelial injury (1,2). An impairment of the endotheliumdependent regulation of vascular tone in large conductance vessels like the coronary arteries or the brachial artery is an integral component of ED and is indicative of a reduction in the bioavailability of the endothelium-derived signaling
Address reprint requests to: Norbert Weiss, Medical PolicliniceCity Campus, University of Munich Medical Center, Pettenkoferstrasse 8a, D-80336 Munich, Germany; E-mail: [email protected]
molecule nitric oxide (NO) (3,4). This can be detected in chronic hyperhomocysteinemic subjects free of overt cardiovascular disease (5e7) as well as in healthy subjects during acute hyperhomocysteinemia (Hhcy) induced by an oral methionine challenge (8e12). After an oral methionine challenge, the time course of the impairment of endothelium-dependent vasodilation strongly correlates with the time-course of the increase of total homocysteine (Hcy) (13) and especially in the plasma levels of free reduced Hcy (14). This suggests that reduced Hcy may be the form of Hcy that impairs NO bioavailability. In addition to Hcy’s effect on large artery function, recent studies have associated Hhcy with small vessel complications, e.g., with subcortical vascular encephalopathy, a small vessel disease leading to dementia (15), and with
0188-4409/07 $esee front matter. Copyright Ó 2007 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2007.01.004
412
Abahji et al./ Archives of Medical Research 38 (2007) 411e416
microvascular complications in diabetics leading to nephropathy and retinopathy (16). Furthermore, microangiopathy is described as part of the hereditary disorder of homocystinuria (17). Several lines of evidence have indicated that endothelium-derived hyperpolarizing factor (EDHF), and not NO, is a major determinant of vascular tone in microvessels and thereby seems to be a critical determinant of organ perfusion (18). In rats with chronic hyperhomocysteinemia (Hhcy) subsequent to intravenous methionine supplementation, EDHF-mediated vasodilation was impaired, as evaluated as the renal blood flow response to intrarenal acetylcholine during systemic NO synthase and cyclooxygenase inhibition (19). Whether or not Hhcy impairs microvascular concomitant to macrovascular reactivity in humans has not been studied so far. The aim of this study, therefore, was to simultaneously determine macrovascular endothelial function (EF) in the brachial artery and microvascular EF in the skin microcirculation in healthy subjects before and during acute Hhcy.
Subjects and Methods Subjects Macro- and microvascular function before and during acute Hhcy was studied in 11 healthy subjects aged 25e40 years (5 females, 6 males). Study participants were free of overt cardiovascular disease, free of cardiovascular risk factors, had normal renal function and normal glucose tolerance. None of the subjects took any cardiovascular medications, vitamins or antioxidant supplements. The study protocol complied with the Declaration of Helsinki on ethical principles for medical research involving human subjects as revised in 2000 (20). Informed consent was obtained from all participants.
Experimental Protocol All measurements were performed in a temperaturecontrolled room (22e24 C) with the subjects in supine position and acclimatized at least 30 min before the experiments. During each study day, EF was assessed in the fasting state and 4 to 6 h after an oral methionine load (0.1 g/kg body weight) to induce acute Hhcy. Methionine powder was dissolved in apple juice. After performing the baseline studies and before performing the oral methionine load, subjects were allowed to have a light breakfast. Microvascular function tests were performed before measuring macrovascular function. All measurements were repeated on three (macrovascular) or two (microvascular) independent occasions with 6-week intervals each.
Macrovascular Endothelial Function To study endothelium-dependent vasoreactivity in the macrocirculation, the diameter of the brachial artery was measured using a high-resolution vascular ultrasound device equipped with a 7.5 kHz linear array transducer (Accuson 128XP/10; Siemens Medical Solutions, Mountain View, CA) both before and during reactive hyperemia, as described previously (21,22). Reactive hyperemia was induced by a blood pressure cuff positioned on the forearm distal to the brachial artery and inflated to suprasystolic levels for 5 min. The flow-mediated dilation (FMD) during reactive hyperemia, observed 45 60 sec after sudden deflation of the cuff and calculated using the formula [(maximum vessel diameter e baseline vessel diameter)/baseline vessel diameter], is a well-established parameter for endotheliumdependent vasodilation and represents an increased release of endothelium-derived vasodilatory NO. To control for any effects of the experimental procedure or the oral methionine challenge on endothelium-independent vasodilation, measurements of the diameter of the brachial artery were repeated 4 min after application of sublingual nitroglycerin, which directly acts on vascular smooth muscle cells. Examinations were done by the same trained examiner, stored on videotape, and analyzed offline. At least nine readings were performed for each measurement point, and averaged. Reproducibility of the measurements was assessed on three occasions. The within-subject coefficient of variation (CV), calculated using the formula standard deviation/mean (22), was 4.4%. Microvascular EF Microvascular EF was studied by measuring skin perfusion at the volar side of the forearm using a laser Doppler instrument (Periflux 5001; Perimed AB, Ja¨rfa¨lla, Sweden) equipped with a solid-state diode laser probe (780 nm). Flux was recorded on one arm at baseline and after application of increasing doses of acetylcholine, and on the contralateral arm at baseline and after application of sodium nitroprusside. Both drugs were applied by iontophoresis using a micropharmacology delivery system (Perilont; Perimed AB) as described previously (23). Briefly, laser Doppler is a non-invasive method to assess microvascular skin perfusion. A laser beam penetrates the skin to a depth of 1e1.5 mm and a fraction of the light is backscattered by moving blood objects undergoing a frequency shift according to the Doppler principle. Thereby a signal, proportional to tissue perfusion, is generated and expressed in arbitrary perfusion units. A drug delivery electrode was placed in the head of the laser probe, and the probe temperature was set to 32 C. The drug delivery electrode was filled with 140 mL acetylcholine (ACH) 1% (Sigma Chemicals, St. Louis, MO) or sodium nitroprusside (SNP) 1% (Sigma Chemicals). Both chemicals were dissolved in deionized and double-distilled water and kept on ice during the day.
Homocysteine-induced Endothelial Dysfunction
Fresh SNP solution was prepared every day. ACH solution was prepared once and stored in aliquots at e20 C until use. After recording of baseline perfusion for 3 min, an electric field (0.1 mA for 10 sec) was applied (anodal current for ACH, cathodal current for SNP, respectively) and the laser Doppler response was recorded for 3 min. Thereafter, electric current was increased to 0.2 mA for 10 sec and 0.2 mA for 20 sec, whereas the response was recorded for 3 min each. The electric current stimulation cascade was well tolerated by the subjects. Endothelium-dependent, ACH-induced vasodilation was expressed as the ratio of laser Doppler flow recorded during maximum stimulation (0.2 mA for 20 sec) divided by baseline flow. Endotheliumindependent, SNP-induced vasodilation was expressed accordingly. To control for the effects of pure vehicle (deionized and double-distilled water) on vasodilation in the laser Doppler experiment, the pure vehicle was iontophoresed by anodal current once in each volunteer. Cathodal current was applied on three different volunteers in control experiments. Negligible changes were induced by the current stimulation cascade used in the study (data not shown). Readings were stored on a personal computer and analyzed offline. The within-subject CV was calculated as described above. CV for the measurements was 30.3%. Blood Chemistry Blood samples were drawn before and 6e7 h after administration of methionine (after the micro- and macrovascular function studies had been performed) on each occasion. Total plasma Hcy levels were measured using an isocratic HPLC method after liberation of thiol compounds from plasma proteins and reduction of oxidized thiols with tri-n-butylphosphine followed by derivatization with the thiol-specific fluorogenic marker, 7-fluoro-benzo-2-oxa1,3-diazole-sulfonate, as described previously (24). Lipid profiles and basic clinical chemistry parameters [including full blood counts, glucose levels, levels of glycosylated hemoglobin (HbA1c), and liver and kidney function tests] were measured by standard clinical chemistry methods.
413
analyses were performed using the STATVIEW software package (Abacus Concepts, Berkeley, CA).
Results The oral methionine load resulted in an approximately 5.1fold increase in plasma Hcy levels (from 5.9 0.7 mmol/L to 30.0 3.0 mmol/L, p !0.005) (Figure 1). Acute Hhcy had no effect on heart rate or systolic and diastolic blood pressure values, which did not differ between baseline and post-methionine load examinations (Table 1). The oral methionine challenge slightly increased total triglyceride and decreased HDL cholesterol levels but had no effect on total or LDL cholesterol levels (Table 1). Standard biochemical parameters were within normal ranges in the subjects studied (data not shown). The effect of acute Hhyc on macrovascular EF was measured by following FMD of the brachial artery. FMD during acute Hhyc was significantly reduced from 8.1 0.5% to 3.6 0.6% ( p !0.005) compared to fasting examinations (Figure 2A). The oral methionine challenge had no effect on the resting diameter of the brachial artery (data not shown). Endothelium-independent vasodilation after application of sublingual nitroglycerin was not affected by the oral methioine challenge (Figure 2B). This indicates that acute Hhcy impairs endothelium-dependent vasodilation, presumably due to a decrease in bioavailable NO, but does not affect endothelium-independent vasodilation in large conductance arteries, like the brachial artery. EF in microvascular circulation was assessed by laser Doppler skin perfusion measurements in reaction to ACH or SNP. At baseline measurements, iontopheresis of acetylcholine, as a stimulator of endothelial-dependent vasodilation, led to a maximum 9.2 1.0 fold increase in laser Doppler flow. During acute Hhcy maximum increase in
Statistical Analysis Replicate vascular function data obtained during the three (macrovascular function) and two (microvascular function) examinations, respectively, were averaged for each individual, and expressed as median SEM for the whole group. Replicate plasma Hcy measurements of an individual during each of the three examinations and before and after the oral methionine were averaged for each individual and expressed accordingly for the whole group. Differences before and after the oral methionine challenge were compared using Wilcoxon Matched Pairs Signed Rank Test (25); p values !0.05 were considered significant. Statistical
Figure 1. Plasma homocysteine concentrations before and 6e7 h after an oral methionine challenge. Triplicate measurements of each individual were performed during the three study time points, and averaged. Values indicate the median SEM of all subjects (n 5 11, *p !0.005 vs. before).
Abahji et al./ Archives of Medical Research 38 (2007) 411e416
414
Table 1. Blood pressure, heart rate, and lipid levels before and after an oral methionine challenge Before
After
p value
Systolic blood pressure (mmHg) 116.7 2.7 114.4 1.8 0.534 Diastolic blood pressure (mmHg) 75.0 1.8 75.0 1.9 0.173 Heart rate (per minute) 65.3 2.9 65.0 2.6 0.575 Total cholesterol (mg/dL) 169.5 4.9 169.0 5.1 0.623 Total triglyceride (mg/dL) 63.0 9.4 117 17.8 !0.01 LDL cholesterol (mg/dL) 103.5 5.6 100.0 4.6 0.139 HDL cholesterol (mg/dL) 48 2.8 42.5 2.9 !0.01 Triplicate measurements of each individual were performed during the three study time points, and averaged. Values indicate the median SEM of all subjects (n 5 11).
laser-Doppler flow in response to ACH compared to baseline flow was significantly reduced to 7.8 1.3 fold ( p !0.01) (Figure 3A). In contrast, acute Hhcy had no effect on SNPinduced increase in skin perfusion, an endotheliumindependent vasodilator (Figure 3B). We further studied whether or not there is any correlation between the magnitude of ED that had been induced by the oral methionine challenge in the two vascular territories studied. Pearson regression coefficient was calculated between FMD or SNP-induced vasodilation in the brachial artery on the one hand, and ACH- or
Figure 3. Ratio of endothelium-dependent acetylcholine-stimulated vs. baseline laser Doppler flow (A) and the ratio of endothelium-independent sodium nitroprusside-stimulated vs. baseline laser Doppler flow (B) in the forearm skin before and after an oral methionine challenge. Duplicate measurements of each individual were performed during the second and third study time points, and averaged. Values indicate the median SEM of all subjects (n 5 11, *p !0.05 vs. before).
SNP-induced increases in skin perfusion on the other hand, both before and after the oral methionine load. No significant correlations were found (data not shown).
Discussion
Figure 2. Endothelium-dependent flow-mediated vasodilation (A) and endothelium-independent nitroglycerin-induced vasodilation (B) before and hours after an oral methionine challenge. Triplicate measurements of each individual were performed during the three study time points, and averaged. Values indicate the median SEM of all subjects (n 5 11, *p !0.005 vs. before).
Our study confirms previous data showing that acute Hhcy induced by an oral methionine challenge induces ED in large conduction vessels, like the brachial artery (Figure 2) (8e12). These functional alterations of large vessel function in Hhcy may contribute to the increased risk for coronary artery disease, cerebrovascular disease and peripheral arterial occlusive disease in hyperhomocysteinemic subjects (26). Mechanistically, this effect is thought to be due to interference of Hhcy with vascular NO signaling. Incubation of cultured endothelial cells with Hcy results in a reduction of NO released from endothelial cells after stimulation with acetylcholine or bradykinin and in decreased levels of cyclic guanosine monophosphate in vascular smooth muscle cells coincubated with Hcy-incubated endothelial cells (27). Hcy increases vascular oxidant stress and superoxide anion output resulting in chemical inactivation of NO (2), and/or increased levels of the endogenous
Homocysteine-induced Endothelial Dysfunction
NO synthase inhibitor asymmetric dimethylarginine (28). Both mechanisms lead to a decrease in bioavailable NO. The novel finding of our study is that acute Hhcy simultaneously impaired endothelium-dependent ACH-induced vasodilation in skin microvessels (Figure 3). This observation may be linked to recent clinical findings that elevated Hcy levels are associated with microvascular complications. For example, Hhcy has been associated with subcortical vascular encephalopathy, a small vessel disease leading to dementia (15), and with microvascular complications in diabetics leading to nephropathy and retinopathy (16). Microangiopathy is also described as part of the hereditary disorder of homocystinuria (17). We did not study any mechanisms underlying Hcy’s effects on microvascular function. EDHF, and not NO, is thought to be the major determinant of vascular tone in microvessels and thereby seems to be a critical determinant of organ perfusion (18). In rats with chronic Hhcy, an impairment of the renal blood flow response to intrarenal acetylcholine has been demonstrated even during systemic NO synthase and cyclooxygenase inhibition. This suggests a decreased action of vasodilator EDHF in this model of Hhcy (19). The observed effects of the oral methionine challenge on macro- and microvascular endothelial functions are not due to changes in hemodynamic parameters as blood pressure and heart rate remained unaffected (Table 1). Furthermore, it seems very unlikely that the effects are due to the slight increase in triglyceride and the slight decrease in HDL cholesterol levels after the oral methionine challenge. Although postprandial hyperlipidemia is known to induce macrovascular ED in hyperlipidemic subjects, mild and acute hypertriglyceridemia do not lead to ED in normolipidemic individuals (29). All subjects studied were normolipidemic. In addition, previous studies have shown that postprandial hyperlipidemia to or above the extent seen in our study does not induce ED in forearm resistance vessels (30). We could not demonstrate any significant correlation between the magnitude of impairment of ED in the two vascular territories studied. This might be due to different mechanisms leading to decreased vasodilation in the two vascular beds examined. Furthermore, our study showed that Hhcy had no significant effect on smooth muscle (endothelium-independent) vasodilator responses to exogenous nitrate, e.g., nitroglycerin and SNP, in both vascular territories. Overall, the observations point to the vascular endothelium as the primary target of Hhcy in different vascular territories. In contrast to our data, a previous study in healthy male volunteers could not demonstrate any effect of acute Hhcy on skin microvascular responses (31). The reasons for these differing results are unclear. They may be related to different ages of subjects studied, to gender differences, or to
415
different methodology. Both studies examined young and healthy subjects; therefore, age is unlikely to cause the different results. Davis et al. (31) studied only males, whereas we studied both female and male subjects without identifying any gender differences. Therefore, the differences might not be explained by gender. Davis’s study neither simultaneously measured macrovascular endothelial function, nor was any systemic endothelial cell activation seen, as von Willebrand factor levels remained unchanged after methionine loading. We could simultaneously demonstrate macro- and microvascular ED. Nevertheless, our findings have to be confirmed before drawing firm conclusions on the effect of Hcy on microvascular EF. Our study has several limitations. First, the sample size of this pilot study is rather small. The effect of the sample size, however, was partly compensated as we repeated the measurements during two or three independent occasions with consistent results. Second, analyses of macro- and microvascular EF were performed offline from data stored on videotapes or on computer, but were not done blinded. This might introduce some bias. Third, we did not introduce a placebo arm into the study protocol to control for any potential effects of the daytime on FMD or microvascular function. Room temperature and skin temperature of the subjects were well-controlled and constant during the day. A recent study that systematically evaluated the effect of daytime on non-invasive measurements of vascular function could not find any influence on FMD measurements as long as exogenous conditions were controlled (32). In addition, in a subset of subjects we studied effects of the daytime on skin perfusion responses to ACH without a methionine challenge and could not find any significant effect (data not shown). Fourth, assessment of microvascular endothelial function using a laser Doppler device inherently exhibits a substantial variability due to spatial and temporal variations of laser Doppler flux. This is indicated by within-subject CVs of |30% in our, as well as in other, studies (23). Accuracy of our data indicating that acute Hhcy impairs microvascular EF, however, is supported by the fact that we repeated the measurements on two independent occasions in each individual with consistent results. In summary, this pilot study showed that acute Hhcy not only impairs macrovascular but also microvascular EF. The latter observation may at least partly explain the clinical observation that Hhcy is associated with small vessel complications. If this finding can be reproduced in larger studies and by independent investigators, it may have impact on our pathophysiological understanding of microvascular complications in certain patient groups where Hhcy interacts with other cardiovascular risk factors on small vessel complications, like patients with diabetes or chronic kidney disease. Further studies are warranted to explore the potential mechanisms by which Hcy may interfere with non-NO pathways in endothelial cells that regulate vascular tone and function in the microcirculation.
416
Abahji et al./ Archives of Medical Research 38 (2007) 411e416
Acknowledgments The work was supported by research grants from Wakunaga of America, Co. Ltd., Mission Viejo, CA and from the FriedrichBaur Foundation, Munich, Germany. The authors would like to thank all study participants for their cooperation, Ilona Fialla and Pirkko Koelle for excellent technical assistance, and Gabriele Ho¨lscher, Institute for Medical Information Technology, Biometry and Epidemiology at the University of Munich, Germany, for biostatistical consulting.
References 1. Weiss N, Keller C, Hoffmann U, Loscalzo J. Endothelial dysfunction and atherothrombosis in mild hyperhomocysteinemia. Vasc Med 2002; 7:227e239. 2. Weiss N. Mechanisms of increased vascular oxidant stress in hyperhomocysteinemia and its impact on endothelial function. Curr Drug Metab 2005;6:27e36. 3. Guerra R Jr, Brotherton AF, Goodwin PJ, Clark CR, Armstrong ML, Harrison DG. Mechanisms of abnormal endothelium-dependent vascular relaxation in atherosclerosis: implications for altered autocrine and paracrine functions of EDRF. Blood Vessels 1989;26: 300e314. 4. Luscher TF, Richard V, Tschudi M, Yang ZH, Boulanger C. Endothelial control of vascular tone in large and small coronary arteries. J Am Coll Cardiol 1990;15:519e527. 5. Woo KS, Chook P, Lolin YI, Cheung AS, Chan LT, Sun YY, et al. Hyperhomocyst(e)inemia is a risk factor for arterial endothelial dysfunction in humans. Circulation 1997;96:2542e2544. 6. Tawakol A, Omland T, Gerhard M, Wu JT, Creager MA. Hyperhomocyst(e)inemia is associated with impaired endothelium-dependent vasodilation in humans. Circulation 1997;95:1119e1121. 7. Holven KB, Holm T, Aukrust P, Christensen B, Kjekshus J, Andreassen AK, et al. Effect of folic acid treatment on endotheliumdependent vasodilation and nitric oxide-derived end products in hyperhomocysteinemic subjects. Am J Med 2001;110:536e542. 8. Hanratty CG, McAuley DF, McGurk C, Young IS, Johnston GD. Homocysteine and endothelial vascular function. Lancet 1998;351: 1288e1289. 9. Chambers JC, McGregor A, Jean-Marie J, Obeid OA, Kooner JS. Demonstration of rapid onset vascular endothelial dysfunction after hyperhomocysteinemia: an effect reversible with vitamin C therapy. Circulation 1999;99:1156e1160. 10. Kanani PM, Sinkey CA, Browning RL, Allaman M, Knapp HR, Haynes WG. Role of oxidant stress in endothelial dysfunction produced by experimental hyperhomocyst(e)inemia in humans. Circulation 1999;100:1161e1168. 11. Lambert J, van den Berg M, Steyn M, Rauwerda JA, Donker AJ, Stehouwer CD. Familial hyperhomocysteinaemia and endotheliumdependent vasodilatation and arterial distensibility of large arteries. Cardiovasc Res 1999;42:743e751. 12. Chao CL, Kuo TL, Lee YT. Effects of methionine-induced hyperhomocysteinemia on endothelium-dependent vasodilation and oxidative status in healthy adults. Circulation 2000;101:485e490. 13. Bellamy MF, McDowell IF, Ramsey MW, Brownlee M, Bones C, Newcombe RG, et al. Hyperhomocysteinemia after an oral methionine load acutely impairs endothelial function in healthy adults. Circulation 1998;98:1848e1852. 14. Chambers JC, Ueland PM, Wright M, Dore CJ, Refsum H, Kooner JS. Investigation of relationship between reduced, oxidized, and proteinbound homocysteine and vascular endothelial function in healthy human subjects. Circ Res 2001;89:187e192.
15. Bertsch T, Mielke O, Holy S, Zimmer W, Casarin W, Aufenanger J, et al. Homocysteine in cerebrovascular disease: an independent risk factor for subcortical vascular encephalopathy. Clin Chem Lab Med 2001;39:721e724. 16. Looker HC, Fagot-Campagna A, Gunter EW, Pfeiffer CM, Narayan KM, Knowler WC, et al. Homocysteine as a risk factor for nephropathy and retinopathy in Type 2 diabetes. Diabetologia 2003; 46:766e772. 17. McCully KS. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am J Pathol 1969;56:111e128. 18. Triggle CR, Ding H, Anderson TJ, Pannirselvam M. The endothelium in health and disease: a discussion of the contribution of non-nitric oxide endothelium-derived vasoactive mediators to vascular homeostasis in normal vessels and in type II diabetes. Mol Cell Biochem 2004;263: 21e27. 19. De Vriese AS, Blom HJ, Heil SG, Mortier S, Kluijtmans LA, Van de Voorde J, et al. Endothelium-derived hyperpolarizing factor-mediated renal vasodilatory response is impaired during acute and chronic hyperhomocysteinemia. Circulation 2004;109:2331e2336. 20. World Medical Association Declaration of Helsiniki. Ethical Principles for Medical Research Involving Human Subjects. http://www. wma.net/e/policy/pdf/GGTSPU-11623-65270-DAT/17c.pdf. Accessed July 17, 2006. 21. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, et al. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 1992;340: 1111e1115. 22. De Roos NM, Bots ML, Schouten EG, Katan MB. Within-subject variability of flow-mediated vasodilation of the brachial artery in healthy men and women: implications for experimental studies. Ultrasound Med Biol 2003;29:401e406. 23. Farkas K, Kolossvary E, Jarai Z, Nemcsik J, Farsang C. Non-invasive assessment of microvascular endothelial function by laser Doppler flowmetry in patients with essential hypertension. Atherosclerosis 2004;173:97e102. 24. Feussner A, Rolinski B, Weiss N, Deufel T, Wolfram G, Roscher AA. Determination of total homocysteine in human plasma by isocratic high-performance liquid chromatography. Eur J Clin Chem Clin Biochem 1997;35:687e691. 25. Wilcoxon F. Individual comparisons by ranking methods. Biometrics 1945;1:80e83. 26. Bautista LE, Arenas IA, Penuela A, Martinez LX. Total plasma homocysteine level and risk of cardiovascular disease: a meta-analysis of prospective cohort studies. J Clin Epidemiol 2002;55:882e887. 27. Weiss N, Zhang YY, Heydrick S, Bierl C, Loscalzo J. Overexpression of cellular glutathione peroxidase rescues homocyst(e)ine-induced endothelial dysfunction. Proc Natl Acad Sci USA 2001;98:12503e 12508. 28. Dayal S, Lentz SR. ADMA and hyperhomocysteinemia. Vasc Med 2005;10(Suppl 1):S27eS33. 29. Giannattasio C, Zoppo A, Gentile G, Failla M, Capra A, Maggi FM, et al. Acute effect of high-fat meal on endothelial function in moderately dyslipidemic subjects. Arterioscler Thromb Vasc Biol 2005;25: 406e410. 30. Gudmundsson GS, Sinkey CA, Chenard CA, Stumbo PJ, Haynes WG. Resistance vessel endothelial function in healthy humans during transient postprandial hypertriglyceridemia. Am J Cardiol 2000;85: 381e385. 31. Davis KR, Pearson H, Moat S, Bonham JR, Donnelly R. Acute hyperhomocysteinaemia affects pulse pressure but not microvascular vasodilator function. Br J Clin Pharmacol 2001;52:327e332. 32. ter Avest E, Holewijn S, Stalenhoef AF, de Graaf J. Variation in non-invasive measurements of vascular function in healthy volunteers during daytime. Clin Sci (Lond) 2005;108:425e431.