Plasma Concentration of Asymmetric Dimethylarginine, an Endogenous Inhibitor of Nitric Oxide Synthase, Is Elevated in Monkeys With Hyperhomocyst(e)inemia or Hypercholesterolemia

Animals and Experimental Protocol

Sixteen adult cynomolgus monkeys (Macaca fascicularis) were studied in 2 separate groups. Details of the study protocols have been published previously.^{1 13} In group 1, 7 monkeys were assigned to receive, in a randomized crossover design, either a control diet (Purina Monkey Chow, Ralston-Purina) or a hyperhomocyst(e)inemic diet enriched in methionine (1.0 g/100 g), relatively depleted of folic acid (0.15 g/100 g), and free of choline, for 4 weeks, followed by the other diet for 4 weeks. In group 2, 9 monkeys were assigned to receive an atherogenic diet for 17 months, followed by an atherogenic diet supplemented with B vitamins (5 mg folic acid, 400 μg vitamin B₁₂, and 20 mg vitamin B₆ daily) for 6 months. The atherogenic diet contained 43% of total calories as fat, 0.7% as cholesterol, and small amounts of B vitamins (≈1.0 μg vitamin B₁₂, 0.75 mg vitamin B₆, and <25 μg folic acid daily).

After the first experimental period of both studies (4 weeks in group 1 and 17 months in group 2), the animals were sedated with ketamine hydrochloride (25 mg/kg IM) and anesthetized with sodium pentobarbital (30 mg/kg IV). A tracheotomy was performed, and the animals were intubated and ventilated with room air and supplemental oxygen. Venous blood was drawn for the measurement of plasma total cholesterol, homocyst(e)ine, l-arginine, and dimethylarginine concentrations. A nonobstructive multiple–side-hole catheter equipped with a Doppler transducer was inserted into the right femoral artery and positioned in the distal aorta, and the right femoral vein was cannulated for administration of supplemental anesthesia (15 mg/kg IV pentobarbital as needed). Heart rate, respiration, and blood pressure were monitored continuously. Changes in blood flow to the legs were measured in response to intra-arterial infusion of collagen (150 mg/min for 10 minutes) or intra-arterial injection of acetylcholine (3×10⁻⁸, 10⁻⁷, and 3×10⁻⁷ mol/L) by quantitative angiography and Doppler measurement of hindlimb flow velocity. Collagen activates platelet aggregation in vivo,¹⁶ which causes greater reduction of peripheral blood flow when endothelium-mediated inhibition of platelet activation is impaired than when endothelial function is normal.¹⁶ Cineangiograms of the distal descending aorta and the left iliac artery were obtained as described previously.¹ Quantification of arterial lumen diameter was performed by the use of computerized arterial lumen edge detection software as described previously.¹⁶ Velocity of blood flow to the leg was measured with a Doppler transducer at the time of angiography. By measuring the velocity of flow (by Doppler) and aortic mean diameter (by angiography), blood flow to the leg was calculated.

At the end of the procedure, 1 common carotid artery was surgically exposed and ligated proximally and distally, and the isolated segment of artery was removed and placed in oxygenated Krebs’ solution. Removal of 1 carotid artery did not produce stroke or other adverse effects in any monkey and did not alter arterial blood pressure. After the second experimental period (8 weeks in group 1 and 23 months in group 2), animals were anesthetized again, another venous blood sample was drawn, and measurement of the vasomotor response to collagen infusion in vivo was repeated. The remaining carotid artery was removed and placed in oxygenated Krebs’ solution. The animals were killed by administration of sodium pentobarbital (200 mg/kg IV) and exsanguinated. The study protocol was approved by the University of Iowa Animal Care and Use Committee.

Cell Culture

The spontaneously transformed human umbilical vein endothelial cell line ECV304 (American Type Culture Collection)¹⁷ was cultured in medium 199 (GIBCO-BRL) containing 10% FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin (GIBCO-BRL). This cell line retains many of the characteristics of primary endothelial cells, including synthesis of angiotensin-converting enzyme, NO, and prostacyclin, and the expression of endothelial adhesion molecules, including intercellular adhesion molecule-1 and lymphocyte function–associated antigen-3. ECV304 cells were maintained in medium 199 until they reached confluence. The cells were washed, and fresh medium was added. Cells were then maintained in culture for an additional 24 hours in the absence or presence of methionine (1×10⁻⁴ to 4×10⁻⁴ mol/L), dl-homocysteine (10⁻³ mol/L), dl-homocysteine plus S-adenosylhomocysteine (10⁻⁴ mol/L), or dl-homocysteine plus B vitamins (10⁻³ mol/L pyridoxine, 10⁻⁴ mol/L cobalamine, and 10⁻⁴ mol/L folic acid) before the cells and media were harvested for measurement of ADMA and SDMA levels.

Biochemical Analyses

Concentrations of l-arginine and dimethylarginines in plasma and in cell supernatants were determined by high-performance liquid chromatography (HPLC) with precolumn derivatization with o-phthalaldehyde by a modification of a previously described method.⁷ Samples were spiked with 10 μmol/L homoarginine as an internal standard and extracted on CBA solid-phase extraction cartridges (Varian). The eluates were dried under nitrogen, and residues were dissolved in bidistilled water for HPLC analysis. HPLC was carried out on a liquid chromatography system (Gynkotek) consisting of 2 HPLC pumps with a gradient controller (model M 480 HDG), a spectral fluorescence detector RF 1002, and an automatic injector (model GINA 160). Samples and standards were incubated for exactly 30 seconds with the o-phthalaldehyde reagent (5.4 mg/mL o-phthalaldehyde in borate buffer, pH 8.5, containing 0.4% 2-mercaptoethanol) before automatic injection into the HPLC system. Chromatographic separation was performed on a C₆H₅ column (Macherey and Nagel) with the fluorescence monitor set at excitation and emission wavelengths of 340 and 455 nm, respectively. Samples were eluted from the column isocratically with 0.96% citric acid/methanol (2:1 [vol/vol], pH 6.8) at a flow rate of 1 mL/min. The coefficients of variation of this method were 5.2% for within-assay determination and 5.5% for between-assay determination; the detection limit of the assay was 0.1 μmol/L.

Fasting plasma homocyst(e)ine concentrations were measured by HPLC and electrochemical detection, according to the method of Smolin and Schneider,¹⁸ as previously described.^{1 19}

Plasma total cholesterol levels were determined by using methods established by the Lipid Research Centers and standardized by the Centers of Disease Control as described previously.²⁰

Endothelium-Dependent Vascular Function Ex Vivo

After removal of loose connective tissue, the common carotid artery was cut into multiple rings, each 5 mm wide. Carotid artery rings were suspended in organ chambers containing oxygenated Krebs’ buffer maintained at 37°C and connected to force transducers to measure changes in isometric tension. Rings were precontracted to a tension of 1.0 g by stepwise addition of prostaglandin F_2α (1 to 3 μmol/L), and relaxation concentration response curves were generated by cumulative addition of acetylcholine or sodium nitroprusside (each 1 nmol/L to 10 μmol/L).

Calculations and Statistical Analyses

Data are given as mean±SEM. Statistical significance was tested by ANOVA, followed by the Fisher protected least significant difference test. Linear regression curves and correlation coefficients were calculated according to the least squares method. Multiple regression analysis was performed for endothelium-dependent vasodilation ex vivo, with cholesterol, ADMA, and homocyst(e)ine concentrations used as independent variables. A linear ADMA-homocyst(e)ine interaction term was also included as an independent variable to assess the potential interaction between both molecules on endothelial function. Statistical significance was accepted at P<0.05.

Effects of Hyperhomocyst(e)inemic Diet on Plasma Homocysteine, Total Cholesterol, l-Arginine, and ADMA Levels

The hyperhomocyst(e)inemic diet increased plasma homocyst(e)ine levels 2.7-fold compared with the control diet (P<0.05, Table⇓). Plasma total cholesterol concentrations were not significantly different between these groups (Table⇓).

Plasma ADMA levels were elevated 3-fold in animals fed the hyperhomocyst(e)inemic diet (P<0.05, Figure 1A⇓). Plasma SDMA levels did not differ significantly between the 2 groups of animals (Table⇑). Plasma concentrations of l-arginine were elevated during the hyperhomocyst(e)inemic diet (P<0.05, Table⇑). The l-arginine/ADMA ratio was significantly lower in the group fed the hyperhomocyst(e)inemic diet (P<0.05, Figure 1B⇓).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

A, Plasma concentrations of the endogenous NO synthase inhibitor ADMA. B, Plasma l-arginine/ADMA ratios. Monkeys were fed hyperhomocyst(e)inemic (HyperHC) or control diets or an atherogenic (AS) diet with or without supplemental B vitamins (Vit). Data are mean±SEM of 7 to 9 animals in each group. *P<0.05 vs control.

Effects of Atherogenic Diet and B Vitamin Supplementation on Plasma Homocyst(e)ine, Total Cholesterol, l-Arginine, and ADMA Levels

The atherogenic diet increased plasma total cholesterol and homocyst(e)ine concentrations (Table⇑).¹³ B vitamin supplementation decreased homocyst(e)ine to control levels but did not significantly affect total cholesterol levels (Table⇑).

ADMA levels were elevated in monkeys fed the atherogenic diet and remained unchanged after B vitamins were added to the diet (Figure 1A⇑). Plasma concentrations of l-arginine and SDMA were not significantly changed by the atherogenic diet with or without supplemental vitamins (Table⇑). The l-arginine/ADMA ratio was significantly decreased in these 2 groups compared with monkeys fed the control diet; vitamin supplementation did not affect this ratio (Figure 1B⇑).

Regression Analysis of Plasma ADMA and Homocyst(e)ine Concentrations

There was a significant correlation between plasma ADMA concentration and plasma homocyst(e)ine concentration in monkeys fed the hyperhomocyst(e)inemic or control diet (R=0.83, P=0.01; n=14; Figure 2A⇓). When all animals were included in the analysis, the correlation was also significant (R=0.40, P=0.02; n=32; Figure 2B⇓).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

A, Correlation between plasma homocyst(e)ine and ADMA concentrations in monkeys fed control or HyperHC diets. B, Correlation between plasma homocyst(e)ine and ADMA concentrations in monkeys fed 4 different dietary regimens.

Regression Analysis of Plasma ADMA Concentration and Endothelium-Dependent Vasodilator Function Ex Vivo

Isolated carotid arteries from monkeys showed a concentration-dependent relaxation response to acetylcholine, and maximum acetylcholine-induced relaxation was significantly impaired in carotid arteries from hyperhomocyst(e)inemic animals compared with control animals.¹ Maximum relaxation was 80.7±7.7% in monkeys fed the control diet, 48.7±12.2% in monkeys fed the hyperhomocyst(e)inemic diet (P<0.05 versus the control diet), and 53.3±9.4% in monkeys fed the atherogenic diet without supplemental B vitamins.^{1 13} Supplementation of the atherogenic diet with B vitamins did not improve maximum relaxation to acetylcholine (54.6±9.4%), although sensitivity to the lowest concentrations of acetylcholine was modestly increased.¹³

In simple regression analysis, a strong inverse correlation was found between ADMA plasma concentration and maximum acetylcholine-induced vasodilation of carotid artery ex vivo for monkeys fed control or hyperhomocyst(e)inemic diets (R=0.65, P=0.01; n=14). A significant inverse correlation was also observed when data from all animals were included in the analysis (R=0.50, P=0.004; n=32; Figure 3A⇓).

• Download figure
• Open in new tab
• Download powerpoint

Figure 3.

A, Correlation between plasma ADMA concentration and endothelium-dependent relaxation of isolated carotid artery segments in response to 10 μmol/L acetylcholine (ACh) ex vivo from monkeys on 4 different dietary regimens. B, Correlation between plasma ADMA levels and blood flow response to intra-arterial infusion of collagen in vivo in monkeys on 4 different dietary regimens.

Acetylcholine-induced relaxation of the carotid artery ex vivo also correlated inversely with the plasma homocyst(e)ine concentration (R=0.42, P=0.02; n=32). Multiple regression analysis indicated that plasma ADMA concentration was the only independent predictor of endothelium-dependent vasodilation ex vivo (R=0.553, P=0.02), whereas plasma homocyst(e)ine or total cholesterol levels were not independent predictors of endothelium-dependent relaxation. Inclusion of an ADMA-homocyst(e)ine interaction term did not increase the correlation coefficient (R=0.554, P=0.03), indicating the absence of an additive effect of ADMA and homocyst(e)ine on endothelial dysfunction of the carotid artery (P=0.82 for interaction).

Regression Analysis of Plasma ADMA and Blood Flow Response to Collagen In Vivo

Intra-arterial infusion of collagen decreased hindlimb blood flow by 42±9% in monkeys fed the hyperhomocyst(e)inemic diet and by 14±11% in monkeys fed the control diet (P<0.01).¹ In monkeys fed the atherogenic diet, reduction in hindlimb blood flow in response to collagen was 30±3% before and 38±5% after supplementation with B vitamins (P=NS).¹³

There was a significant inverse correlation between plasma ADMA concentration and the change in hindlimb blood flow in response to collagen for all animals (R=0.49, P<0.01; Figure 3B⇑) and for monkeys fed the hyperhomocyst(e)inemic or control diet (R=0.56, P<0.05). Plasma ADMA concentration did not correlate with a change in limb blood flow in response to acetylcholine (R=0.14, P=NS).

Effects of Methionine and Homocysteine on ADMA Release by Endothelial Cells

Under control conditions (medium free of methionine and homocysteine), ECV304 human endothelial cells produced 13.7±1.3 pmol of ADMA per microgram of protein over 24 hours and 10.3±3.6 pmol of SDMA per microgram of protein over 24 hours. ADMA formation was concentration-dependently increased by increasing the concentrations of methionine (P<0.05 versus control for 200 and 400 μmol/L, Figure 4⇓). Incubation with 1.0 mmol/L homocysteine also significantly elevated ADMA production (27.8±2.9 pmol/μg protein over 24 hours, P<0.05 versus control). SDMA formation was not significantly affected under the same conditions. The increased production rate of ADMA in the presence of homocysteine was completely reversed in the presence of the methylation inhibitor S-adenosylhomocysteine (13.6±1.8 pmol/μg protein over 24 hours, P<0.05 versus homocysteine; P=NS versus control), but it was not affected by the addition of B vitamins (34.2±4.4 pmol/μg protein over 24 hours, P=NS versus homocysteine).

• Download figure
• Open in new tab
• Download powerpoint

Figure 4.

Formation of ADMA by cultured ECV304 human endothelial cells. Confluent endothelial cells were incubated in methionine-free and homocysteine-free medium (control), in the presence of increasing concentrations of exogenous methionine (1×10⁻⁴ to 4×10⁻⁴ mol/L), and in the presence of homocysteine (10⁻³ mol/L), homocysteine plus S-adenosylhomocysteine (S-Adeno-hcys, 10⁻⁴ mol/L), or homocysteine plus B vitamins (10⁻³ mol/L pyridoxine, 10⁻⁴ mol/L cobalamine, and 10⁻⁴ mol/L folic acid). Data are mean±SEM *P<0.05 vs control.