Monitoring N-nitrosamino acids excreted in the urine and feces of rats as an index for endogenous nitrosation

Abstract

A simple and sensitive method for the quantitative estimation of endogenous N-nitrosation in rats has been developed. This approach is based on the findings that N-nitrosamino acids (e.g., nitrosoproline (NPRO), nitrosohydroxyproline (NHPRO) and nitrososarcosine (NSAR)) when administered orally to rats, are excreted unchanged almost quantitatively (88-96% of the dose) in the urine and feces. After sequential administration of a nitrosatable amino acid and sodium nitrite the nitrosamino acid excreted in the urine and feces was analyzed. The amount of NPRO excreted in the urine of rats was proportional to the dose of proline and to the square of the nitrite dose. Co-administration of ascorbic acid and alpha-tocopherol together with the precursors decreased the urinary NPRO whereas thiocyanate increased the yield. After feeding an amino acid precursor and nitrite, the yield of nitrosamino acids formed in vivo and excreted in the urine increased in the order: NPRO less than NSAR less than NHPRO. The same order was seen when the nitrosation rates of the amino acids in vitro were compared. Thus N-nitrosation in vivo in rats occurs via a similar mechanism as observed in vitro. Monitoring of N-nitrosamino acids excreted in the urine and feces thus appears to provide a valuable index for endogenous N-nitrosation.