P97	NO synthase activity in healthy and hypercholesterolemic humans: application of stable isotope infusion technique.
E.Schwedhelm, R.Maas, L.Schneider, R.Benndorf, M.Kastner, A.Steenpaß, R.H.Böger
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Hamburg, DE.

Background: Endogenous nitric oxide (NO) is generated by NO synthase (NOS) from L-arginine. NO is rapidly degraded to nitrite and subsequently to nitrate, the latter being excreted into the urine. Theoretically, NOS activity is best assessed in vivo by measurement of urinary nitrate. In reality, majority of urinary nitrate originates from dietary source. This problem can be overcome by stable isotope infusion of non-radioactive 15N-labeled L-arginine which is converted to 15N-labeled NO and excreted as 15N-labeled nitrate. 15N-labeled nitrate can be discriminated from the naturally abundant 14N isotope by gas chromatography-mass spectrometry (GC-MS). Methods: NOS activity was measured in 12 healthy and 24 hypercholesterolemic subjects (mean ±SD LDL-C: 124 ±21 vs. 216 ±43 mg/dL) by short-term infusion of 500 mg 15N-labeled L-arginine. In addition, NOS activity was determined in patients before and after 6 weeks of 40 mg atorvastatin (n=12) or placebo (n=12) treatment. Approval by the local ethics committee and written informed consent were obtained. Enrichment of 15N-labeled nitrate was determined with GC-MS after derivatization with pentafluorobenzyl bromide as m/z-ratio of 63 to 62. Results: NOS activity was impaired in all hypercholesterolemic patients. Urinary excretion of 15N nitrate was 6.8±2.4 vs. 9.6±5.0 µmol/24h (p<0.05 vs. controls). LDL-C and NOS activity were inversely correlated (Spearmans rho = -0.44, p<0.01). Atorvastatin significantly reduced LDL-C by 55% (p<0.001 vs. placebo). NOS activity was not significantly improved in the active treatment group. Urinary excretion of 15N nitrate was 6.5±2.8 µmol/24h before and 7.2±4.0 µmol/24h after treatment (p=0.42). Conclusion: NOS activity determined by stable isotope technique in vivo is impaired in hypercholesterolemia. Short term treatment with 40 mg atorvastatin significantly reduces LDL-C by 55% in hypercholesterolemic patients but fails to improve NOS activity in vivo.

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