Formation of aliphatic amine precursors of N-nitrosodimethylamine after oral administration of choline and choline analogues in the rat.

Trimethylamine and dimethylamine are important precursors of N-nitrosodimethylamine, which is a potent carcinogen in a wide variety of animal species. Choline, a component of the normal human diet, is metabolized by bacteria within the intestine to form trimethylamine and dimethylamine. However, animals on a choline-free diet continue to excrete some trimethylamine and dimethylamine, suggesting that other dietary precursors of these methylamines might exist. To determine whether C-N bond cleavage by the intestinal bacteria is specific to the choline molecule, we measured monomethylamine, dimethylamine, trimethylamine and trimethylamine oxide excretion in rat urine after the administration of compounds that shared structural features with choline. Water, choline, dimethylaminoethanol, diethylaminoethanol, phosphocholine, betaine, carnitine, beta-methylcholine or dimethylaminoethyl chloride were administered by orogastric intubation, and the urine was collected for 24 hr. Administration of choline (15 mmol/kg body weight) resulted in increased urinary excretion of dimethylamine, trimethylamine and trimethylamine oxide (increases of approximately twofold, 500-fold and 50-fold, respectively). Of the administered choline, 12% was converted to trimethylamine or trimethylamine oxide and excreted in the urine within 24 hr. Phosphocholine administration resulted in similar increases in dimethylamine, trimethylamine and trimethylamine oxide excretion by rats. Modification of the ethyl-backbone or quaternary amine end of the choline molecule resulted in marked suppression of methylamine formation. Though administration of some analogues of choline (methylcholine, betaine and carnitine) resulted in the formation of small amounts of trimethylamine or trimethylamine oxide, and the administration of others (dimethylaminoethanol and dimethylaminoethyl chloride) resulted in the formation of some dimethylamine, the amounts formed were minimal compared with the amounts of trimethylamine and trimethylamine oxide formed after choline administration. Thus, of the many components of foods, only choline and its esters are likely to be significant substrates for trimethylamine and dimethylamine formation. How then can we explain the persistence of trimethylamine and dimethylamine excretion observed in choline-deficient rats? We suggest that endogenous (non-bacterial) synthesis of trimethylamine and dimethylamine occurs within some tissue of the rat.

Endogenous formation of dimethylamine.

An understanding of the biosynthesis and metabolism of dimethylamine (DMA) is important because it is a precursor of dimethylnitrosamine (nitroso-DMA). DMA is the major short-chain aliphatic amine in human and rat urine. DMA is formed from trimethylamine (TMA), which, in turn, is a breakdown product of dietary choline. Enzymes within gut bacteria catalyse both of these reactions; it is not known whether mammalian cells can form DMA. To determine the relative importance of dietary choline, bacteria and other mechanisms for the formation of DMA, we measured DMA excretion in the urine of rats fed on a diet devoid of choline, and in urine of rats with no bacterial colonization of the intestines. We also describe an improved gas-chromatographic method for the measurement of methylamines in biological fluids. In control rats there were significant amounts of DMA within several biological fluids [urine, 54.2 +/- 3.0 mumol/kg body wt. per 24 h (556.2 +/- 37.5 nmol/ml); blood, 18.8 +/- 1.9 nmol/ml; gastric juice, 33.5 +/- 10.5 nmol/ml; means +/- S.E.M.]. Animals eating a diet containing no choline excreted as much MMA and DMA as did choline-supplemented rats (25-35 mumol/kg per 24 h), and they excreted slightly less TMA (2 versus 2.5 mumol/kg per 24 h). Rats with no gut bacteria excreted the same amount of DMA in their urine as did the control animals (45-55 mumol/kg per 24 h). They excreted much less MMA (16.3 +/- 1.5 versus 40.3 +/- 2.6 mumol/kg per 24 h; mean +/- S.E.M.; P less than 0.01), TMA (0.7 +/- 0.2 versus 2.5 +/- 0.5 mumol/kg per 24 h; mean +/- S.E.M.; P less than 0.01) and piperidine (2.0 +/- 0.3 versus 6.3 +/- 0.6 mumol/kg per 24 h; mean +/- S.E.M.; P less than 0.01) in their urine. From our studies we conclude that DMA is present in significant amounts within gastric fluid, an environment that is ideal for nitrosamine formation (under acidic conditions, nitroso-DMA is chemically formed by the reaction of nitrite with DMA). Results also indicate that dietary choline was not the sole precursor for DMA formation and that gut bacteria are not essential for the formation of DMA. Hence in mammals there must be endogenous pathways that are capable of forming DMA; however, these endogenous mechanisms remain unidentified.

Conversion of dietary choline to trimethylamine and dimethylamine in rats: dose-response relationship.

Trimethylamine (TMA) and dimethylamine (DMA) are normal components of human urine and are precursors of dimethylnitrosamine, a potent carcinogen. In part, DMA and TMA are products of the metabolism of dietary choline by intestinal bacteria. Most TMA formed in the intestinal tract is later oxidized and excreted as trimethylamine oxide (TMAO). Humans treated with large doses of choline smell "fishy" (the odor of TMA). Humans ingest choline as part of foods, and yet rarely smell fishy, suggesting that TMA formation must depend upon the dose of choline ingested. We found that, in adult rats, at low doses of choline (1.5 mmol/kg body wt) only 9 mumol choline (6% of the dose) reached the part of the intestine which is colonized by bacteria (the cecum and colon). After administration of 15 mmol choline/kg body wt, 237 mumol (16% of the dose) reached the cecum and colon. At both doses, 64-65% of the administered choline was absorbed from the intestine by 3 h after the dose. We found that orally administered choline slightly increased TMA and TMAO excretion at doses of choline smaller than 7 mmol/kg body wt, but that there was a disproportionately large increase in TMA excretion per 24 h when larger doses were administered (from 11 mumol TMA and 100 mumol TMAO per kg body wt in controls to 226 mumol TMA and 3617 mumol TMAO per kg body wt in rats treated with 15 mmol choline/kg body wt).(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro production of N-nitrosodimethylamine and other amines by proteus species.

N-nitrosodimethylamine, a potent carcinogen, was produced by three strains each of Proteus mirabilis, P. morganii, and P. rettgeri, but not by three strains of P. vulgaris grown under the same conditions. Many of the alkaline-extractable volatile metabolites elaborated by these organisms are the same, but there are some qualitative and quantitative differences among species. Representative gas-liquid chromatographic profiles of the four species are presented, and the significance of the differences is discussed. Primary emphasis, however, is given to the importance of the production of N-nitrosodimethylamine by these microorganisms and the conditions under which it is produced.

The influence of creatinine, lecithin and choline feeding on aliphatic amine production and excretion in the rat.

The excretion of aliphatic amines, methylamine, dimethylamine and trimethylamine in the urine and faeces of rats fed on a control diet and diets supplemented with creatinine, lecithin or choline were measured over a 14 d feeding period. The rats were then killed and concentrations of amines in small and large intestinal contents measured. Adding creatinine to the diet resulted in a significant increase of methylamine excretion in the faeces and urine. The amount of methylamine found in all parts of the intestine increased, especially in the caecum. Adding lecithin to the diet resulted in an increase in the methylamine excretion only, and no change in the concentrations of amines found in the intestine, except for trimethylamine which was significantly increased in the caecum and colon. Adding choline to the diet resulted in a significant increase in excretion of trimethylamine and, to a lesser extent, methylamine. The levels of amines found in the gut increased, dimethylamine being increased in the small bowel, and methylamine and trimethylamine in the caecum.

Volatile compounds produced in sterile fish muscle (Sebastes melanops) by Pseudomonas perolens.

Volatile compounds produced by Pseudomonas perolens ATCC 10757 in sterile fish muscle (Sebastes melanops) were identified by combined gas-liquid chromatography and mass spectrometry. Compounds positively identified included methyl mercaptan, dimethyl disulfide, dimethyl trisulfide, 3-methyl-1-butanol, butanone, and 2-methoxy-3-isopropylpyrazine. Compounds tentatively identified included 1-penten-3-ol and 2-methoxy-3-sec-butylpyrazine. The substituted pyrazine derivative 2-methoxy-3-isopropylpyrazine was primarily responsible for the musty, potato-like odor produced by P. perolens.

Microbial formation of nitrosamines in vitro.

Mortierella parvispora and an unidentified bacterium converted trimethylamine to dimethylamine, and the bacterium (but not the fungus) formed dimethylnitrosamine in the presence of nitrite. Dimethylnitrosamine also appeared in cell suspensions of Escherichia coli and Streptococcus epidermidis and in hyphal mats of Aspergillus oryzae incubated with dimethylamine and nitrate. Suspensions of a number of microorganisms produced N-nitrosodiphenylamine from diphenylamine and nitrite at pH 7.5, and soluble enzymes catalyzing the N-nitrosation of diphenylamine were obtained from two of these organisms. In the presence of these enzymes, several dialkylamines were converted to the corresponding N-nitroso compounds.