DOSING TIME WITH ASCORBIC-ACID AND NITRATE, GUM AND TOBACCO CHEWING, FASTING, AND OTHER FACTORS AFFECTING N-NITROSOPROLINE FORMATION IN HEALTHY-SUBJECTS TAKING PROLINE WITH A STANDARD MEAL

The N-nitrosoproline (NPRO) test measures the potential for intragastr ic formation of carcinogenic nitrosamines in humans. Nitrate and L-pro line are administered to volunteers. Noncarcinogenic NPRO is produced by an acid-catalyzed reaction of proline (a model for ingested amines) with nitrate-derived nitrite in the stomach. It is then absorbed and excreted in the urine, which is analyzed for NPRO. We studied the effe ct of certain dietary and other factors on the levels of urinary NPRO. For (generally) 5 days, healthy adult subjects (mostly men) followed a diet low in preformed NPRO, nitrate, proline, and (on days 4 and 5) ascorbic acid. The tests were conducted on days 4 and 5. In the standa rd test, the subjects took 400 mg nitrate at II a.m., and at noon they ate a standard 700-calorie meal containing 500 mg proline. (In previo us tests, proline was given 1 h after or between meals.) Urines were c ollected for 24 h, and samples were analyzed for NPRO by published met hods. This standard test yielded 26 +/- 2 (mean +/- SE) nmol NPRO comp ared with 5 a 1 mmol NPRO when proline alone was taken. In variations of the standard test, NPRO yield was not significantly affected by the subjects' gender, the time at which the standard meal was eaten, the size of the meal, or the drinking of extra water after the meal. Doses of 100 and 200 mg nitrate had lesser effects on NPRO yield than did t he dose of 400 mg nitrate. Nitrate (400 mg) produced the most NPRO whe n it was given 1 h before the meal. Pasting increased NPRO yield by 3- 4 times compared to giving proline with a meal. One g of ASC given 5 o r 2 h before, with, or 1 or 2 h after the meal with proline inhibited NPRO formation by mean values of 0, 71, 71, 67, and 19%, respectively. Chewing gum or tobacco for 2-3 h after the test meal did not increase NPRO formation or salivary nitrate levels, but salivary nitrite level s were reduced, especially when gum was chewed. When nitrate was not t aken, chewing tobacco appeared to increase salivary nitrite and nitrat e levels. The weak carcinogen N-nitrososarcosine (NSAR) was also detec ted in some tests, and the standard group showed 21 +/- 3 nmol NSAR. A high NSAR result (44 +/- 7 nmol) for women undergoing the standard te st should be reexamined. We discuss applying these results to the cond uct of future NPRO tests, as well as their implications for reducing t he potential production of carcinogenic nitrosamines in the stomach.