Urinary excretion of nitrosodimethylamine and nitrosoproline in humans: interindividual and intraindividual differences and the effect of administered ascorbic acid and alpha-tocopherol.

Gas chromatography-high resolution mass spectrometry methods were developed for quantifying nitrosodimethylamine (NDMA) and nitrosoproline (NPRO) in human urine. The limits of quantitation of these methods, which utilize stable isotope analogues of NDMA and NPRO as internal standards, were 5 pg per ml for NDMA and 0.14 ng per ml for NPRO. The assays were used to quantify NDMA and NPRO in urine samples collected 4 times a wk for 5 wk from 24 healthy volunteers. The mean urinary excretion of NDMA during this time was found to be 38.2 ng per day, and the mean urinary excretion of NPRO was found to be 3.26 micrograms per day. Treatment of the volunteers with 600 mg of ascorbic acid and 100 IU of alpha-tocopherol 4 times a day for the final 3 wk of the study did not influence the urinary excretion of NDMA or NPRO. Considerable person-to-person and day-to-day variations were observed for the urinary excretion of both nitrosamines, but the urinary excretion of NDMA was not correlated with the urinary excretion of NPRO. Person-to-person and day-to-day differences in the urinary excretion were greater for NPRO than for NDMA. The mean urinary excretion of NDMA by the 24 subjects was as much as 5-fold higher on some days than on other days, but this was not observed for NPRO. Day-to-day differences in the mean urinary excretion of NDMA were correlated with the concentrations of nitrogen dioxide in the air.

Antiscorbutic activity of ascorbic acid phosphate in the rhesus monkey and the guinea pig.

Rhesus monkeys fed an ascorbic acid-free, purified liquid diet, developed scurvy in 70 to 105 days as evidenced by loss of weight, anemia, bleeding gums, inflamed palate, diarrhea, and inability to stand. Oral administration of either 10 mg/kg body weight of ascorbic acid or an equimolar amount of the magnesium salt of 1-ascorbic acid phosphate cured all symptoms of scurvy. Similarly, oral administration of 1-ascorbic acid phosphate cured all symptoms of scurvy in the guinea pig and resulted in liver ascorbate levels equal to those of animals feed ascorbic acid. It is concluded that ascorbic acid phosphate is a readily available source of ascorbic acid activity in vivo.

Lack of antiscorbutic activity of ascorbate 2-sulfate in the rhesus monkey.

Oral administration of 10 mg per kilogram of body weight of ascorbic acid (AA) completely prevented development of scurvy in juvenile rhesus monkey (Mucaca mulata) fed an AA-free liquid diet. The same dose cured scurvy when injected intramuscularly. An equimolar dose of ascorbic acid 2-sulfate (AA-2-S) did not prevent or cure scurvy. Neither AA nor AA-2-S altered serum cholesterol. AA but not AA-2-S reduced serum triglyceride. A case of scurvy in an AA-2-S treated monkey is described in detail.

Studies on the mutagenic activity of ascorbic acid in vitro and in vivo.

In vitro data are presented to show that ascorbic acid does not have intrinsic mutagenicity towards strain TA100 of S. typhimurium if deionized water is used to prepare the incubation medium. The addition of Cu2+ ions to the bacterial medium that contains ascorbic acid, or the use of tap water and ascorbic acid alone, causes a mutagenic and cytotoxic response that is blocked by EDTA. Additional in vitro data demonstrate that hydrogen peroxide is mutagenic to S. typhimurium strain TA100 and it is suggested that ascorbic acid may be mutagenic and cytotoxic through the generation of hydrogen peroxide. In vivo studies using a sensitive intrahepatic host-mediated mutagenicity assay indicate that ascorbic acid is not genotoxic in guinea pigs even when the dietary intake of vitamin C is above the level required for tissue saturation (5000 mg/kg body weight/day).

Studies on the antimutagenic activity of ascorbic acid in vitro and in vivo.

The possibility that ascorbic acid, as a nucleophile, may inhibit mutagenicity induced by electrophilic metabolites of N-nitroso compounds was examined. In vitro data are presented to show that ascorbic acid does not decrease the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in a modified Ames bacterial mutagenicity system if deionized water is used to prepare the incubation medium. However, ascorbic acid prevents the mutagenicity of MNNG in vitro if added to bacteria in a medium prepared with either sterile tap water or deionized water and Cu2+ ions and that this antimutagenic response is blocked by EDTA. Additional in vitro experiments suggest that when ascorbic acid and Cu2+ ions are mixed in aqueous solution, H2O2 and free radicals derived from H2O2 are formed and these compounds may deactivate N-nitroso compounds. In vivo data are presented to show that ascorbic acid supplementation to guinea pigs (2000 mg/kg body weight/day) has no effect on the mutagenicity of N-nitrosodimethylamine, MNNG, N-methylnitrosourea and streptozotocin using the intrahepatic host-mediated bacterial mutagenicity assay. Additional in vivo studies demonstrate that simultaneous oral administration of ascorbic acid prevents the mutagenicity that follows the intragastric nitrosation of aminopyrine by nitrite while dietary pre-treatment with ascorbic acid does not. These findings suggest that ascorbic acid can block the intragastric formation of mutagenic N-nitroso compounds but that ascorbic acid has no effect on mutagenicity of N-nitroso compounds once they are formed.

Inhibitory effect of alpha-tocopherol on the formation of nitrosomorpholine in mice treated with morpholine and exposed to nitrogen dioxide.

The possibility that alpha-tocopherol (vitamin E) inhibits the formation of nitrosomorpholine (NMOR) in vivo was investigated in mice orally pretreated with alpha-tocopherol (2.5-100 mg/kg body wt) once daily for 6 days. Twenty-four hours later, the animals were injected i.p. with 2 mg of morpholine (MOR) per animal followed by exposure to 47 p.p.m. of NO2 for 2 h. Under these conditions, low oral doses of alpha-tocopherol (2.5-5 mg/kg body wt) significantly decreased NMOR formation in vivo. As total body alpha-tocopherol levels increased, in vivo NMOR formation decreased, and a maximal 50-70% inhibition of NMOR formation occurred at alpha-tocopherol levels of 5 micrograms/g body wt. Additional results showed that NMOR was rapidly eliminated in mice, so that studies which measure the levels of NMOR found in animals treated with MOR and then exposed to NO2 may underestimate the amount of NMOR that is actually formed. Finally, oral pretreatment of up to 100 mg of alpha-tocopherol/kg body wt had no effect on NMOR elimination.

Formation of N-nitrosomorpholine in mice treated with morpholine and exposed to nitrogen dioxide.

The possibility of N-nitrosomorpholine formation was investigated in mice treated with morpholine and then exposed to 45 p.p.m. nitrogen dioxide in an inhalation chamber for 2 h. Following this treatment, the mice were frozen and pulverized in liquid nitrogen and concentrated extracts from the powders of these animals were analyzed for N-nitrosomorpholine using a thermal energy analyzer interfaced to a gas chromatograph. The data indicate that nitrogen dioxide exposure causes the nitrosation of morpholine in vivo. Additional data show that significant levels of artifactually formed N-nitrosomorpholine are found in control animals that are treated with morpholine after exposure to nitrogen dioxide for 2 h unless a combination of L-ascorbic acid and d,1-alpha-tocopherol are used to inhibit nitrosation during the homogenization, extraction, and analysis of the samples. The need for both a lipid phase nitrosation blocker (d,1-alpha-tocopherol) and an aqueous phase nitrosation blocker (L-ascorbic acid) indicates that the nitrosation of morpholine occurs in both a lipid and an aqueous phase in vitro and therefore may occur in both a lipid and an aqueous environment in vivo. The data from this study also demonstrate the importance of adding suitable inhibitors of nitrosation, such as L-ascorbic acid and d,1-alpha-tocopherol to the extraction solution to prevent possible artifactual formation of N-nitrosomorpholine during the extraction and analysis of the samples.

Caffeic and ferulic acid as blockers of nitrosamine formation.

Caffeic acid and ferulic acid, which are naturally occurring phenols present in a wide variety of plants, were examined for their ability to react with nitrite in vitro and to inhibit nitrosamine formation in vivo. Their activities were compared with other phenols (butylated hydroxyanisole and Trolox) and with a non-phenolic polyhydroxylated compound, glycerol guaiacolate. In simulated gastric fluid, caffeic acid and ferulic acid reacted rapidly and completely with an equimolar quantity of sodium nitrite. In rats receiving aminopyrine and nitrite, caffeic acid and ferulic acid blocked the elevation of serum N-nitrosodimethylamine (NDMA) levels and the serum glutamic pyruvic transaminase levels associated with hepatotoxicity. Neither phenol had any effect on serum levels of NDMA in rats treated with NDMA. In both the in vitro (reaction with nitrite) and in vivo (inhibition of hepatotoxicity) systems, caffeic acid was more effective than ferulic acid. Butylated hydroxyanisole and Trolox were partially effective, and glycerol guaiacolate was inactive. The results of this study suggest that dietary caffeic acid and ferulic acid may play a role in the body's defense against carcinogenesis by inhibiting the formation of N-nitroso compounds.