Consumption of Nitrate-Rich Beetroot Juice with or without Vitamin C Supplementation Increases the Excretion of Urinary Nitrate, Nitrite, and N-nitroso Compounds in Humans.

Consumption of nitrate-rich beetroot juice (BRJ) by athletes induces a number of beneficial physiological health effects, which are linked to the formation of nitric oxide (NO) from nitrate. However, following a secondary pathway, NO may also lead to the formation of N-nitroso compounds (NOCs), which are known to be carcinogenic in 39 animal species. The extent of the formation of NOCs is modulated by various other dietary factors, such as vitamin C. The present study investigates the endogenous formation of NOCs after BRJ intake and the impact of vitamin C on urinary NOC excretion. In a randomized, controlled trial, 29 healthy recreationally active volunteers ingested BRJ with or without additional vitamin C supplements for one week. A significant increase of urinary apparent total N-nitroso Compounds (ATNC) was found after one dose (5 to 47 nmol/mmol: p < 0.0001) and a further increase was found after seven consecutive doses of BRJ (104 nmol/mmol: p < 0.0001). Vitamin C supplementation inhibited ATNC increase after one dose (16 compared to 72 nmol/mmol, p < 0.01), but not after seven daily doses. This is the first study that shows that BRJ supplementation leads to an increase in formation of potentially carcinogenic NOCs. In order to protect athlete's health, it is therefore important to be cautious with chronic use of BRJ to enhance sports performances.

Endogenous formation of nitrosamines and oxidative DNA-damaging agents in tobacco users.

One-third of all cancers worldwide can be attributed to various tobacco habits. Both in tobacco smoke and smokeless tobacco, carcinogenic N-nitroso compounds (NOC) are implicated as DNA-damaging agents in cancers of the aerodigestive tract and the pancreas. The exposure from nitrosamines in certain types of tobacco use such as "toombak" in Sudan could be as high as a few milligrams per day. Using the N-nitrosoproline test, it has been shown that smoking contributes to endogenous nitrosation and likely increases NOC formation in vivo. Smokeless tobacco, most widely used in the form of chewing of betel quid (BQ) with tobacco, was shown to particularly enhance endogenous nitrosation in the oral cavity, a site where chewing habits are causally associated with cancer. Poor oral hygiene was found to contribute to the formation of nitrosamines in the oral cavity. The evidence so far accumulated demonstrates that tobacco habits increase endogenous NOC formation, thus adding to the burden of exposure by preformed carcinogenic NOC in tobacco products. In snuff dippers, the unexpected higher level of HPB released from hemoglobin, an exposure marker for carcinogenic tobacco-specific nitrosamines, has been attributed to the endogenous formation of these carcinogens. Recent studies have demonstrated that besides carcinogenic tobacco-specific nitrosamines, reactive oxygen species derived from BQ ingredients could also play a role in the etiology of oral cancer in chewers. Although the use of chemopreventive agents may block nitrosation reactions in vivo in tobacco users, cessation of tobacco habits is the only safe way for an efficient reduction of cancer risk, in view of the high exposure to other (preformed) tobacco-related carcinogens.

Studies on endogenous formation of N-nitroso compounds in the guinea pig supplemented with proline or thioproline and sodium nitrate.

The endogenous formation of N-nitrosoproline (NPRO) and N-nitrosothioproline (NTPRO, N-nitrosothiazolidine-4-carboxylic acid) was studied by monitoring their excretion in the urine of guinea pigs given oral doses of 10 mg proline or thioproline after supplementation with 34 mg (0.4 mmol) sodium nitrate. In order to estimate the conversion of nitrate to nitrite, the animals were also supplemented with 3.5 mg (0.05 mmol) sodium nitrite instead of sodium nitrate. In animals fed commercial diets, the excretion of NPRO and NTPRO under supplementation with sodium nitrate was 2.0 micrograms and 28.7 micrograms/animal/day, respectively, whereas the excretion under supplementation with sodium nitrite was 0.7 micrograms and 13.3 micrograms/animal/day, respectively. The higher excretion of NTPRO than NPRO in each case shows that thioproline is more effective for nitrite trapping than proline. The animals supplemented with nitrate excreted more than twice the amounts of NPRO or NTPRO than those supplemented with nitrite. It is assumed, therefore, that more than 0.1 mmol nitrate is reduced to nitrite and takes part in the endogenous nitrosation of the guinea pig. When various concentrations of L-ascorbic acid (AsA), known to inhibit the formation of N-nitroso compounds, were also administered orally to animals immediately after supplementation with sodium nitrate, the NPRO excretion decreased with increasing AsA concentration. These data indicate that the guinea pig, which is unable to synthesize AsA as well as humans, may be an appropriate animal model for evaluation of the endogenous nitrosation ability of humans ingesting nitrate.

Marked nitrosation by stimulation with lipopolysaccharide in ascorbic acid-deficient rats.

Marked formation of N-nitrosothioproline (N-nitrosothiazolidine-4-carboxylic acid) by stimulation with Escherichia coli lipopolysaccharide (LPS) was demonstrated in ascorbic acid-deficient mutant rats (osteogenic disorder syndrome rats; ODS rats) unable to synthesize ascorbic acid. The amounts of urinary nitrate and N-nitrosothioproline excretion after thioproline administration was measured in ODS rats with and without ascorbic acid supplement before and after the injection of LPS. LPS caused marked increase of urinary nitrate excretion in both groups. Urinary N-nitrosothioproline excretion increased 6-fold after LPS injection in ODS rats not supplied with ascorbic acid, but supplement with ascorbic acid markedly decreased the excretion of N-nitrosothioproline.

Urinary N-nitrosamino acids as indices of endogenous formation of N-nitroso compounds.

Exposure to their precursors (e.g., amines, nitrate/nitrite, NOx) can lead to formation in the human body of N-nitroso compounds (NOC), a class of potent animal carcinogens, which are also suspected of being carcinogenic in man. A non-invasive method, the 'N-nitrosoproline (NPRO) test', for estimating endogenous nitrosation in man was developed in our laboratory. This test, which monitors 24-hr-excretion of urinary N-nitrosamino acids, is now applied in clinical and field studies, with the aim of measuring nitrosamine exposure and of identifying dietary, life-style, and host factors, or disease states, that affect nitrosation in man. Results from such studies are used to identify populations/individuals at high risk for cancers of the stomach, oesophagus, and oral cavity possibly caused by endogenous nitrosamines, and to indicate preventive measures by which the body burden of endogenous nitroso carcinogens can be lowered efficiently.

N-nitrosoproline in urine from patients and healthy volunteers after administration of large amounts of nitrate.

Urines from 12 healthy volunteers, sampled from 0-24 h after the volunteers had been administered 9.5 g sodium nitrate intravenously or up to 10.5 g ammonium nitrate orally, were analysed for N-nitrosoproline (NPRO). The mean NPRO content in urines voided just before administration of nitrate was 4.1 micrograms/L. Mean NPRO levels remained low until 6 h after nitrate intake. Mean NPRO contents in urines voided 6-8 h, 8-10 h, 10-15 h and 15-24 h after nitrate administration were 10, 40, 74 and 53 micrograms/L. respectively. The highest NPRO content found was 320 micrograms/L. These findings demonstrate that intake of one large dose of nitrate leads to enhanced nitrosation in the body, which lasts at least one day. Surprisingly, in urine from 21 patients who had been ingesting 2.5-9 g ammonium nitrate daily for several months to prevent the redevelopment of calcium phosphate renal stones, only slightly enhanced NPRO levels were found: less than 1-32 micrograms/L; mean value, 6.2 micrograms/L. In most of the urines, from the healthy volunteers as well as from the patients, N-nitrosothiazolidine-4-carboxylic acid was also detected. Results from the volunteers indicated that urinary excretion of this compound also increases several hours after intake of one large dose of nitrate.

Occurrence in human urine of new sulphur-containing N-nitrosamino acids N-nitrosothiazolidine 4-carboxylic acid and its 2-methyl derivative, and their formation.

To quantitate endogenous nitrosation reactions in man, the quantity of N-nitrosoproline (NPRO) excreted in the urine after ingestion of proline and/or nitrate was estimated. When this monitoring method (NPRO test) was applied in clinical and field studies, several hitherto unidentified N-nitroso compounds were frequently detected. These were recently identified as sulphur-containing N-nitrosamino acids, N-nitrosothiazolidine 4-carboxylic acid (NTCA), and trans- and cis-isomers of N-nitroso-2-methylthiazolidine 4-carboxylic acid (NMTCA). NTCA and NMTCA were readily formed in vitro following nitrosation at acidic pH of the respective precursor, thiazolidine 4-carboxylic acid (TCA) or of 2-methylthiazolidine 4-carboxylic acid (MTCA). As the latter compounds can be formed by reaction of L-cysteine with formaldehyde or acetaldehyde, respectively, NTCA and NMTCA were also formed by reacting L-cysteine with the respective aldehyde and with nitrite at optimal pH (2.5 for NTCA and 4.5 for NMTCA). Up to 95% of NTCA and NMTCA given orally to fasted rats was recovered as such in urine and faeces within 2 days. Administration of TCA or MTCA, together with nitrite increased the urinary excretion of NTCA and NMTCA, as did co-administration of L-cysteine, nitrite, and the respective aldehyde. NTCA and NMTCA were also detected in the 24-h urine of human volunteers, and smokers tended to excrete higher levels than nonsmokers. Daily excretion levels varied, however, and a diet supplemented with ascorbic acid significantly decreased the total amount of nitrosamino acids. NTCA and NMTCA may occur in human urine as a result of (i) intake of preformed N-nitroso compounds; (ii) intake of thiazolidine 4-carboxylic acid or its 2-methyl derivative and subsequent nitrosation in vivo; (iii) endogenous two-step synthesis by the reaction of L-cysteine with the respective aldehyde and a nitrosating agent. Thus, measurement of NTCA and NMTCA together with NPRO in urine may provide an index for the exposure of human subjects to nitrosamines or their precursors, i.e., nitrosating agents, certain aldehydes, or aldehyde-generating compounds. Our data demonstrate unequivocally that N-nitroso compounds are formed in the human body, as suggested previously by Druckrey. Their relevance to human cancer at specific sites should now be investigated.

Failure of ascrobic acid to inhibit the metabolic N-oxidation of the bladder carcingen 4-biphenylamine.

The effect of L-ascorbic acid on the in vivo metabolic N-oxidation of the bladder carcinogen 4-biphenylamine has been investigated in the dog. Gas chromatographic analysis of the urines of dogs receiving concomitant oral doses of L-ascorbic acid and 4-biphyenylamine showed no significant difference in the N-oxidized metabolites when compared to dogs receiving arylamine alone. The results indicate that pretreatment with large doses of L-ascorbic acid has no effect on the metabolic N-oxidation of 4-biphenylamine or on the urinary concentration of these metabolites. The failure of L-ascrobic acid to lower the urinary concentration of these presumed proximate urinary carcinogens casts doubt on its efficacy in bladder tumor prophylaxis.