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Figure. Vascular Responses to L-Arginine
Image description not available.
Responses are expressed as absolute changes (10-minute values − baseline values) in mean blood pressure, platelet aggregation, and blood viscosity following each load. Asterisks indicate significant differences (P<.05) compared with baseline and with each of the other loads.
Table 1. Homocysteine, Lipid, Glucose, Blood Pressure, Coagulation, and Adhesion Molecule Parameters in 20 Healthy Subjects Before and After 3 Interventions*
Image description not available.
Table 2. Blood Pressure, Platelet Aggregation to Adenosine Diphosphate, and Blood Viscosity Parameters in 20 Healthy Subjects Before and After 3 Interventions*
Image description not available.
Table 3. Vascular Responses to L-Arginine in 20 Healthy Subjects Before and After 3 Interventions*
Image description not available.
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Nygard O, Nrdrehaug JE, Resfum H, Ueland PM, Farstad M, Vollset SE. Plasma homocysteine levels and mortality in patients with coronary artery disease.  N Engl J Med.1997;337:230-236.
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Anderson TJ, Uehata A, Gerhard MD.  et al.  Close relation of endothelial function in the human coronary and peripheral circulation.  J Am Coll Cardiol.1995;26:1235-1241.
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Clinical Investigation
June 9, 1999

Impairment of Endothelial Functions by Acute Hyperhomocysteinemia and Reversal by Antioxidant Vitamins

Author Affiliations

Author Affiliations: Departments of Geriatrics and Metabolic Diseases (Drs Nappo, De Rosa, Marfella, and Giugliano) and Immunology (Dr Farzati), Institute of General Pathology and Oncology (Dr De Lucia), and Institute of Biochemistry of Macromolecules (Drs Ingrosso and Perna), Second University of Naples, Naples, Italy.

JAMA. 1999;281(22):2113-2118. doi:10.1001/jama.281.22.2113
Context

Context Increased levels of homocysteine are associated with risk of cardiovascular disease. Homocysteine may cause this risk by impairing endothelial cell function.

Objective To evaluate the effect of acute hyperhomocysteinemia with and without antioxidant vitamin pretreatment on cardiovascular risk factors and endothelial functions.

Design and Setting Observer-blinded, randomized crossover study conducted at a university hospital in Italy.

Subjects Twenty healthy hospital staff volunteers (10 men, 10 women) aged 25 to 45 years.

Interventions Subjects were given each of 3 loads in random order at 1-week intervals: oral methionine, 100 mg/kg in fruit juice; the same methionine load immediately following ingestion of antioxidant vitamin E, 800 IU, and ascorbic acid, 1000 mg; and methionine-free fruit juice (placebo). Ten of the 20 subjects also ingested a placebo load with vitamins.

Main Outcome Measures Lipid, coagulation, glucose, and circulating adhesion molecule parameters, blood pressure, and endothelial functions as assessed by hemodynamic and rheologic responses to L-arginine, evaluated at baseline and 4 hours following ingestion of the loads.

Results The oral methionine load increased mean (SD) plasma homocysteine level from 10.5 (3.8) µmol/L at baseline to 27.1 (6.7) µmol/L at 4 hours (P<.001). A similar increase was observed with the same load plus vitamins (10.0 [4.0] to 22.7 [7.8] µmol/L; P<.001) but no significant increase was observed with placebo (10.1 [3.7] to 10.4 [3.2] µmol/L; P=.75). Coagulation and circulating adhesion molecule levels significantly increased after methionine ingestion alone (P<.05) but not after placebo or methionine ingestion with vitamins. While the mean (SD) blood pressure (−7.0% [2.7%]; P<.001), platelet aggregation response to adenosine diphosphate (−11.4% [4.5%]; P=.009) and blood viscosity (−3.0% [1.2%]; P=.04) declined in these parameters 10 minutes after an L-arginine load (3 g) following placebo, the increase after methionine alone (−2.3% [1.5%], 4.0% [3.0%], and 1.5% [1.0%], respectively; P<.05), did not occur following methionine load with vitamin pretreatment (−6.3% [2.5%], −7.9% [3.5%], and −1.5% [1.0%], respectively; P=.24).

Conclusion Our data suggest that mild to moderate elevations of plasma homocysteine levels in healthy subjects activate coagulation, modify the adhesive properties of endothelium, and impair the vascular responses to L-arginine. Pretreatment with antioxidant vitamin E and ascorbic acid blocks the effects of hyperhomocysteinemia, suggesting an oxidative mechanism.

During the 1990s, there have been many reports associating elevated plasma homocysteine levels with arteriosclerotic cardiovascular disease.17 Although not all new studies are consistent regarding the risk of homocysteine,8 a relative risk of 1.4 for the difference between homocysteine levels higher than 15 µmol/L compared with levels less than 10 µmol/L after adjustment for other cardiovascular risk factors seems to be the best estimate.9

Homocysteine is a sulfur-containing amino acid that is an intermediary product in methionine metabolism. Methionine taken orally is converted to homocysteine by demethylation, and the effect of an oral load can be used as a diagnostic test to identify individuals with enzyme defects or poor vitamin status who show an exaggerated rise in homocysteine levels.10,11 Several mechanisms are likely to be involved in the induction of vascular damage by homocysteine, including endothelial cell desquamation,12 oxidation of low-density lipoprotein,13 increased monocyte adhesion to the vessel wall,14 and impaired vascular response to the endothelium-dependent relaxing factor nitric oxide.15 Impaired flow-mediated vasodilation has been demonstrated in healthy humans after acute increases in plasma homocysteine concentrations following ingestion of a methionine load.16,17 Although flow-mediated vasodilation is largely dependent on the release of nitric oxide,18 it focuses only on the vasomotor response of the endothelium, which, owing to its strategic position, plays an important role in regulation of the atherogenetic process, including monocyte adhesion, platelet aggregation, coagulation, and blood rheology.1921

In this study, we investigate whether a mild to moderate increase in plasma homocysteine concentration following an oral methionine load in healthy subjects alters their cardiovascular risk profile and impairs endothelial functions assessed by hemodynamic and rheologic responses to L-arginine, the natural precursor of nitric oxide.19 We also investigate whether acutely induced endothelial dysfunction is influenced by preadministration of antioxidant vitamin supplements.

METHODS

The study population consisted of 20 healthy, physically active subjects aged 25 to 45 years (10 men and 10 women; mean [SD] age, 28.4 [6.1] years) recruited among hospital staff. No subject had a history of hypertension, diabetes mellitus, hyperlipidemia, or tobacco abuse or a family history of premature cardiovascular disease. Mean (SD) body mass index (weight in kilograms divided by the square of height in meters) was 24 (2) kg/m2. All subjects were clinically healthy and following ad libitum diets; no subject was taking medications or vitamin supplements. Subjects had normal red blood cell folate (16.3 [5.6] nmol/L) and plasma vitamin B12 (369.6 [69] pmol/L) concentrations. All subjects volunteered (without being paid) for the study and gave written informed consent. The protocol was approved by the institutional committee of ethical practice of the Second University of Naples, Italy.

Studies were begun at 8 AM, after a 12-hour overnight fast. After fasting blood sampling for determination of serum lipid and glucose levels, coagulation parameters, and soluble cell adhesion molecule count, endothelial functions were assessed. Following this assessment, all subjects were submitted in varying order to receive (1) an oral methionine load (100 mg/kg in fruit juice); (2) the same methionine load immediately following oral ingestion of antioxidant vitamin E (800 IU) and ascorbic acid (1000 mg); and (3) methionine-free fruit juice (placebo). Ten of the 20 subjects (5 men and 5 women) also ingested a methionine-free load after the vitamin intake. Administration of loads was separated by 1-week intervals. A person who was not involved in trial management randomly assigned the patients using random numbers derived from published tables. Blinding to the loads was ensured by administering them in a room separated from the vascular laboratory.

Endothelial functions in the form of hemodynamic and rheologic responses to L-arginine were assessed in the fasting state and again at 4 hours following ingestion of each load, as previously described.22 In brief, the subjects were placed in a supine comfortable position with a room temperature between 20°C and 24°C; following cannulation of a large antecubital vein with an intravenous line kept open with a 0.9% saline drip, the subjects' blood pressure was automatically recorded with a noninvasive technique (Finapres Ohmeda 2300, Englewood, Calif). Blood pressure was also recorded with a random zero sphygmomanometer. After a 10-minute equilibration period, an intravenous bolus of 3 g of L-arginine (10 mL of a ready 30% solution of L-arginine monochloride) was injected within 60 seconds. Blood pressure, platelet aggregation response to adenosine diphosphate, and blood viscosity were measured before the L-arginine injection and every 5 minutes thereafter up to 15 minutes. Subjects then received oral methionine, oral methionine with vitamins, or placebo, and endothelial functions were assessed again 4 hours later. Subjects were allowed to walk or sit, as they wished, during the interval between the first and second assessments. Parameters were analyzed by independent investigators blinded to the subject's identity, methionine load status, and temporal sequence.

Platelet aggregation was determined according to the method of Born.23 Platelet-rich plasma was obtained by centrifuging each sample at 200g for 10 minutes, and platelet-poor plasma was prepared by centrifuging the remaining volume of blood at 2000g for 20 minutes. The aggregometer was adjusted before each test, and aggregation was induced using a final concentration of 1.25 µmol/L of adenosine diphosphate. Blood viscosity at high rates of shear (225/sec-1) was assessed with a digital viscosimeter 0.8° cone, using aliquots of blood anticoagulated with 0.77 mol/L of EDTA (ratio of blood to EDTA, 1:20). The coefficient of variation was 2% for blood viscosity and 4.5% for platelet aggregation. The overall reproducibility of the L-arginine test in the same subject (the 3 baseline L-arginine tests) was 0.75% (SD of the difference) and the coefficient of variation was 2.9%. All hemodynamic and rheologic parameters after L-arginine administration were confirmed as having returned to baseline by 15 to 20 minutes.

Assays for serum total and high-density lipoprotein cholesterol, triglyceride, and glucose levels were performed in the hospital's chemistry laboratory. Total plasma homocysteine level was measured by high-performance liquid chromatography.24 Serum concentrations of soluble intracellular adhesion molecule 1 and soluble vascular cell adhesion molecule 1 were determined using commercially available immunosorbent kits (R & D Systems, Minneapolis, Minn). Coagulative parameters were also measured using immunosorbent kits (fibrinopeptide A, plasminogen activator inhibitor 1, and tissue plasminogen activator: Boehringer Mannheim–Roche Diagnostics, Mannheim, Germany; prothrombin fragments 1 and 2 and D-dimers: Behringwerke, Malburg, Germany).

Data are presented as group mean (SD). Sample size was determined on the basis of 2 preliminary experiments with placebo, 2 with a methionine load, and 2 with a methionine load and vitamins. These experiments allowed us to estimate the SD and the difference between the means. For a desired P value of .05 and 80% power to detect an actual difference, a sample size of 10 per group was considered satisfactory.25 Analysis of variance for repeated measures followed by 2-tailed paired t test was used to test for differences in the response to different loads and to L-arginine. The effect of order was tested by analysis of variance. Linear regression analysis was used as appropriate. P<.05 was considered significant.

RESULTS

Total homocysteine, lipid, coagulative, glucose, blood pressure, and soluble cell adhesion molecule parameters before and after the placebo, methionine, and methionine with vitamin loads are shown in Table 1. Preload plasma total homocysteine levels were similar on each of the 3 study days and there was no evidence of an order effect. Administration of the methionine load increased the mean (SD) total homocysteine level from 10.5 (3.8) to 27.1 (6.7) µmol/L at 4 hours (P<.001). A similar increase was seen 4 hours following the oral methionine load with vitamins (P<.001), whereas no increase in plasma total homocysteine levels occurred following placebo ingestion (P=.75). No significant changes in lipoprotein, glucose, and blood pressure levels were observed after the methionine load. All coagulation parameters showed significant increments following ingestion of methionine (P<.05), but not placebo (Table 1). When vitamin supplementation accompanied the methionine load, there was a significant reduction of the rise of the coagulation parameters, with values not significantly different from those seen after the placebo load. Plasma levels of soluble adhesion molecules rose after the methionine load; no significant increase was observed following ingestion of placebo or methionine load with vitamins (Table 1).

Baseline and postload mean blood pressure, platelet aggregation, and blood viscosity values are shown in Table 2. No load caused significant change in any of the parameters after 4 hours.

Postload vascular responses to L-arginine are shown in Table 3 and Figure 1. Following the placebo load, L-arginine produced a significant decrease in blood pressure, platelet aggregation, and blood viscosity levels. Mean (SD) percentages of decrease at 10 minutes were −7.0% (2.7%), −11.4% (4.5%), and −3.0% (1.2%), respectively (P<.05 for all). These responses were not significantly different from those obtained in the baseline L-arginine test in the 3 studies. Following the methionine load, the vascular responses to L-arginine were significantly attenuated compared with placebo (−2.3% [1.5%] for blood pressure decrease; P=.009) or presented a paradoxical increase (4.0% [3.0%] and 1.5% [1.0%] for platelet aggregation and blood viscosity, respectively; P<.05). The methionine load with ingestion of vitamins produced changes in the vascular responses to L-arginine similar to those seen after ingestion of placebo (−6.3% [2.5%], −7.9% [3.5%], and −1.5% [1.0%], respectively, for blood pressure, platelet aggregation, and blood viscosity). Thus, the reduction of endothelial functions in terms of impairment of vascular responses to L-arginine following the methionine load differed significantly from the responses following the other 2 loads.

There was no significant correlation between changes in vascular responses to L-arginine and baseline, postload, or increase in plasma homocysteine levels (r=−0.04, P=.79). Three subjects reported mild nausea after oral methionine that disappeared after the first hour. Vascular responses to L-arginine in the 10 subjects who ingested vitamins with placebo did not differ from the results when the same subjects were given placebo alone.

COMMENT

We found that an oral methionine load, which produces an increase in plasma homocysteine, acutely affects the level of cardiovascular risk in healthy subjects. To our knowledge, this is the first demonstration that an acute increase in plasma homocysteine level is associated with activation of coagulation, as indicated by increased levels of fibrinopeptide A and prothrombin fragments 1 and 2, and modification of the adhesive properties of the endothelium. Soluble forms of cellular adhesion molecules found in plasma are considered an index of endothelial activation and even a molecular marker of early atherosclerosis.26

Another important finding of this study is that acute hyperhomocysteinemia impairs hemodynamic and rheologic responses to L-arginine, the natural precursor of nitric oxide. Although other studies have demonstrated impairment of flow-mediated brachial artery dilatation in healthy subjects receiving a methionine load,16,17 the L-arginine test is able to assess more than a specific endothelial function. In fact, we found an attenuated blood pressure decline after L-arginine injection and a paradoxical (positive) response of both platelet aggregation and blood viscosity in condition of hyperhomocysteinemia. Pretreatment with antioxidant vitamin supplements normalized both the level of cardiovascular risk and the impairment of endothelial functions following acute hyperhomocysteinemia, but did not increase these functions in normal conditions. Taken together, these findings suggest that acute hyperhomocysteinemia deteriorates the cardiovascular risk profile and impairs endothelial functions through oxidative stress, which is blocked by pretreatment with antioxidant vitamins.

Endothelial dysfunction appears to be an important early process in atherogenesis.27 All major cardiovascular risk factors are associated with impaired endothelial function, which precedes the appearance of atherosclerosis.2830 Tests that assess endothelial function may therefore help detect early vascular abnormalities in patients with cardiovascular risk factors but without evident clinical disease. L-Arginine is the natural precursor of nitric oxide. Systemic intravenous infusion of L-arginine in healthy humans lowers mean blood pressure levels and peripheral arteriolar resistance, and inhibits platelet aggregation and blood viscosity.31,32 We have previously shown that the vascular responses following an intravenous bolus of 3 g of L-arginine represent a likely consequence of a rise of endothelial nitric oxide production because they can be largely blocked by NG-monomethyl-L-arginine, the nonmetabolized L-arginine analog that antagonizes the synthesis of nitric oxide from L-arginine in a competitive manner,33 and are not reproduced by similar doses of D-arginine.22 Moreover, the similarity between the vascular effects of L-arginine and those currently attributed to nitric oxide is intriguing.

Endothelial dysfunction, when present, tends to be generalized; a close relationship has been demonstrated between endothelial function in human peripheral and coronary circulation.34 In addition to the globalization of endothelial dysfunction, however, it is also important to focus on the multiplicity of biological effects that result from endothelial dysfunction: vasoconstriction, increased platelet aggregation, monocyte adhesion to the endothelium, and activation of coagulation, all of which promote atherosclerosis. Homocysteine may promote atherogenesis by various mechanisms.1215,35,36 In vitro studies show that exposure of endothelial cells to homocysteine results in oxidative effects, including generation of superoxide anion radicals and hydrogen peroxide,37 which leads to inactivation of nitric oxide and endothelial damage. The various mechanisms proposed to explain the toxic effects of homocysteine, including endothelial dysfunction, lipoprotein oxidation, increased monocyte adhesion, and smooth muscle cell proliferation, are to a large extent mediated by nitric oxide availability.38 Moreover, our results show that acute hyperhomocysteinemia impairs L-arginine–induced vascular responses, which are largely nitric oxide–mediated. Finally, antioxidant vitamins C and E are effective scavengers of superoxide and other oxygen-reactive species39 and prevent endothelial dysfunction caused by hyperhomocysteinemia. It therefore seems possible that deactivation of nitric oxide through an oxidative stress is the predominant mechanism through which acute hyperhomocysteinemia exerts vascular damage.

Study Limitations

The present study does not exclude the possibility of a direct effect of methionine or an alteration in methylation reactions independent of methionine-homocysteine interconversion. The methionine loading test, specifically designed to challenge the degradation pathway of homocysteine through transsulfuration, obtains information in a setting not altogether assimilable to biological conditions, ie, a protein meal. In addition, the metabolic adjustments associated with methionine load cannot be explained merely by the activation of transsulfuration.40 Nevertheless, both fasting and postload homocysteine concentrations are associated with cardiovascular disease.6 Moreover, this test can uncover 39% of subjects with homocysteine-related cardiovascular disease risk who have normal baseline homocysteine levels.41 Despite its intrinsic limitations, the methionine test was used to create a condition of acute moderate hyperhomocysteinemia capable of eliciting a response at the endothelial level.17 The plasma homocysteine levels obtained after methionine loading were 2- to 3-fold higher than fasting levels seen in the general population.

It is possible that the observed reversal of homocysteine-induced endothelial dysfunction by antioxidant vitamins is by a nonspecific mechanism, not involving a reduction of oxidative stress. This interpretation, however, is in contrast with the absence of an effect of vitamins on endothelial functions in the absence of a methionine load. Supplementation with B-group vitamins (a combination of folic acid, pyridoxine hydrochloride, and cyanocobalamin) in the attempt to reduce postload hyperhomocysteinemia was not addressed, and an attempt was not made to differentiate the effects of vitamin C and vitamin E. Finally, this study was based on surrogate end points; the general principle of multi–risk factor prevention of a multifactorial disease, such as atherosclerosis, may require more than a combination of 2 vitamins.

Clinical Implications

This study shows that mild to moderate elevations of plasma homocysteine level in healthy humans impairs many aspects of endothelial function, including vasodilatation, platelet function, coagulation, and cell adhesion. A likely mechanism for this multifaceted injury is the deactivation of nitric oxide by oxidative stress, which is corrected by supplementation of antioxidant vitamins. Although the expected rise in postprandial homocysteine level is 1 to 2 µmol/L, in theory too small to induce changes in endothelial function, a difference of 0.6 µmol/L was observed between patients with myocardial infarction and control subjects in the US Physicians' Health Study.42 Mild to moderate elevations in plasma homocysteine level may also occur in subjects homozygous for the thermolabile variant of the enzyme methylene tetrahydrofolate reductase (10%-13% of the white population), particularly in the presence of suboptimal vitamin status.43 The identification of people with raised basal homocysteine levels or those with impaired methionine tolerance may provide a better mechanistic approach to target the intervention for reducing high homocysteine levels (for example, with B-group vitamins) or the biological effects of hyperhomocysteinemia (for example, with antioxidant vitamins).

References
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Malinow MR. Homocyst(e)inemia: a common and easily reversible risk factor for occlusive atherosclerosis.  Circulation.1990;81:2004-2006.
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Clarke R, Daly L, Robinson K.  et al.  Hyperhomocysteinemia: an independent risk factor for vascular disease.  N Engl J Med.1991;324:1149-1155.
3.
Selhub J, Jacques PF, Bostom AG.  et al.  Association between plasma homocysteine concentrations and extracranial carotid artery stenosis.  N Engl J Med.1995;332:286-291.
4.
Robinson K, Mayer EL, Miller D.  et al.  Hyperhomocysteinemia and low pyridoxal phosphate: common and independent reversible risk factor for coronary artery disease.  Circulation.1995;92:2825-2830.
5.
Boushey CJ, Beresford SA, Omenn GC, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: probable benefits of increasing folic acid intakes.  JAMA.1995;274:1049-1057.
6.
Graham IM, Daly LE, Resfum HM.  et al.  Plasma homocysteine as a risk factor for vascular disease: the European Concerted Action Project.  JAMA.1997;277:1775-1781.
7.
Nygard O, Nrdrehaug JE, Resfum H, Ueland PM, Farstad M, Vollset SE. Plasma homocysteine levels and mortality in patients with coronary artery disease.  N Engl J Med.1997;337:230-236.
8.
Evans RW, Shaten BJ, Hempel JD, Cutler JA, Kuller LH. Homocyst(e)ine and risk of cardiovascular disease in the Multiple Risk Factor Intervention Trial.  Arterioscler Thromb Vasc Biol.1997;17:1947-1953.
9.
Omenn GS, Beresford SAA, Motulsky AG. Preventing coronary heart disease: B vitamins and homocysteine.  Circulation.1998;97:421-424.
10.
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