Determination of free and protein-bound N-nitrosoproline in nitrite-cured meat products

Levels of nitrosoproline (NPRO) in the urine of subjects on a Western-style free-choice diet typically ranged from 1 to 2 ng/ml. Higher levels of NPRO in the urine were associated with the consumption of nitrite-cured meat products. The elevation in NPRO excretion after the consumption of cured meats was not diminished by the concurrent consumption of the nitrosation inhibitor ascorbic acid, indicating the presence of preformed NPRO or some NPRO precursor in the meat. Cured meat samples were analysed for NPRO using methanolic extraction, followed by gas chromatography and nitrosamine quantification by means of a thermal energy analyzer. Levels of NPRO detected in meats were less than those indicated by the amount of NPRO excreted when the same meat was eaten. Digestion of meat samples by proteolytic enzymes sharply increased the amount of NPRO detectable by chemical analysis. Nitrite-cured meat products contained 39-3900 ng total NPRO/g product. Of this amount of NPRO, 3-93% was protein-associated and only detectable after proteolytic digestion. Protein-associated NPRO may represent nitrosated N-terminal proline in proteins or peptides. Such NPRO would not be detectable as free NPRO on direct chemical analysis, but would be released by proteolysis in the digestive tract when the meat is eaten.