A GC/MS method for the quantitation of N-nitrosoproline and N-acetyl-S-allylcysteine in human urine: Application to a study of the effects of garlic consumption on nitrosation

• Published in: Analytical biochemistry 2009-07, Vol.394 (2), p.243-248
• Main Authors: Cope, Keary, Seifried, Harold, Seifried, Rebecca, Milner, John, Kris-Etherton, Penny, Harrison, Earl H.
• Format: Article
• Language: English
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2009.07.035

Biomarkers in urine can provide useful information about the bioactivation of chemical carcinogens and can be used to investigate the chemoprotective properties of dietary nutrients. N-nitrosoproline (NPRO) excretion has been used as an index for endogenous nitrosation. In vitro and animal studies have reported that compounds in garlic may suppress nitrosation and inhibit carcinogenesis. We present a new method for extraction and sensitive detection of both NPRO and N-acetyl-S-allylcysteine from urine. The latter is a major metabolite of S-allyl cysteine which is abundant in garlic. Urine was acidified and the organic acids extracted by reversed phase extraction (RP-SPE) and use of a polymeric weak anion exchange (WAX-SPE) resin. NPRO was quantified by isotope dilution gas chromatography-mass spectrometry using 13 C 5 NPRO and N-nitrosopipecolic acid (NPIC) as internal standards. This method was used to analyze urine samples from a study that was designed to test whether garlic supplementation inhibits NPRO synthesis. Using this method, 2.4 to 46 ng of NPRO per mL urine was detected. The method is straightforward, reliable and can be performed with readily available GC/MS instruments. N-acetyl-S-allylcysteine was quantified in the same fraction and detectable at levels of 4.1 to 176.4 ng per mL of urine. The results suggest that 3 to 5 grams of garlic supplements inhibited NPRO synthesis to an extent similar to a 0.5 g dose of ascorbic acid or a commercial supplement of aged garlic extract. Urinary NPRO concentration was inversely associated with the N-acetyl-S-allylcysteine concentration. It is possible that allyl sulfur compounds found in garlic may inhibit nitrosation in humans. .