Hypercholesterolemia impairs basal nitric oxide synthase turnover rate: a study investigating the conversion of L-[guanidino-(15)N(2)]-arginine to (15)N-labeled nitrate by gas chromatography--mass spectrometry.

Endothelial function is impaired in hypercholesterolemia and atherosclerosis, which is probably due to reduced biological activity of endothelium-derived nitric oxide (NO). NO is synthesized in functionally intact endothelium by oxidation of the terminal guanidino nitrogen atom(s) of the amino acid precursor, L-arginine. We applied stable isotope dilution techniques and gas chromatographic-mass spectrometric approaches to investigate metabolism of L-[guanidino-(15)N(2)]-arginine to (15)N-labeled nitrate in hypercholesterolemic rabbits and controls. After 4 weeks on control or 1% cholesterol-enriched diet, rabbits received 267 +/- 6 micromol of L-[guanidino-(15)N(2)]-arginine/kg of body weight via gastric cannulation. (15)N-isotope content of L-arginine in plasma and in platelet lysates increased 2h later in both groups, and almost returned to baseline until 24h. (15)N-isotope content of plasma nitrite and nitrate also increased in both groups at 2h, and had almost returned to natural content 24h later. (15)N-isotope content of urinary nitrate was significantly increased in control animals in urines collected from 0 to 12, 12 to 24, and had returned to baseline in the urine sample collected from 24 to 48 h. In the cholesterol group only a slight, insignificant elevation of (15)N-isotope content was observed for urinary nitrate. The extent of conversion of L-[guanidino-(15)N(2)]-arginine to (15)N-labeled nitrate was strongly and inversely correlated to plasma concentration of the endogenous NO synthase inhibitor, asymmetric dimethylarginine (ADMA), which was elevated in cholesterol-fed rabbits (R=0.77; p < 0.05). Our data show that baseline NO synthase turnover rate is reduced in rabbits during early hypercholesterolemia. Our study gives evidence that the mechanism of the impaired conversion of L-[guanidino-(15)N(2)]-arginine to (15)N-labeled nitrate most likely involves inhibition of NO synthase by ADMA, which is present in elevated concentrations in hypercholesterolemia.