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

Fd Chem. Toxic. Vol. 27, No. I, pp. 31-34, 1989 Printed in Great Britain.All rights reserved

0278-6915/89 $3.00+ 0.00 Copyright © 1989PergamonPress pie

FORMATION OF ALIPHATIC AMINE PRECURSORS OF N-NITROSODIMETHYLAMINE AFTER ORAL A D M I N I S T R A T I O N OF C H O L I N E A N D C H O L I N E A N A L O G U E S IN T H E RAT S. H. ZEISEL,S. GETTNER and M. YOUSSEF Nutrient Metabolism Laboratory, Room M I002, Departments of Pathology and Pediatrics, Boston University School of Medicine, 85 East Newton Street, Boston, MA 02118, USA (Receit'ed 2 June 1988; ret~isions received 29 August 1988)

Abstraet--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, p-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, 120 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 trimethylamin¢and dimethylamine formation. How then can we explain the persistence of trimethylamine and dimethylamine excretion observed in choline-deficient rats? We suggest that endogenous (nonbacterial) synthesis of trimethylamine and dimethylamine occurs within some tissue of the rat.

INTRODUCTION The aliphatic amines trimethylamine (TMA) and dimethylamine (DMA) are important precursors of N-nitrosodimethylamine (Johnson, 1977; Lijinsky et al., 1972; Zeisel et al., 1988), which is a potent carcinogen in a wide variety of animal species (Bartsch and Montesano, 1984). Choline, a component of the normal human diet (Zeisel, 1988), is metabolized by bacteria within the intestine to form TMA and DMA (Asatoor and Simenhoff, 1965; Johnson, 1977; Zeisel et al., 1983). Strains of bacteria from the genera Clostridia, Vibrio, Serratia, Pseudomonas and Proteus can catalyse choline N-dealkylation (Colby and Zatman, 1973; Johnson, 1977; Sandhu and Chase, 1986; Seim et al., 1982). For example, Proteus mirablis cleaves choline to form TMA and acetaldehyde. Choline cleavage is tightly coupled to the dismutation of acetaldehyde to ethanol and acetate (Sandhu and Chase, 1986). TMA, once DEA = diethylamine; DMA = dimethylamine; MMA = monomethylamine; NDMA = N-nitrosodimethylamine; TMA = trimethylamine; TMA-O = trimethylamine oxide.

Abbreviations:

formed, can be demethylated by bacteria to form DMA (Asatoor and Simenhoff, 1965; Colby and Zatman, 1973; Large, 1971). Most of the DMA formed within the intestine is absorbed and then excreted in the urine (Asatoor and Simenhoff, 1965; Ishiwata et al., 1984; Simenhoff, 1975; Zeisel et al., 1985). DMA is the major short-chain aliphatic amine in human and rat urine (Asatoor and Simenhoff, 1965). TMA formed within the intestine is also absorbed, and then much of it is oxidized within liver to form TMA N-oxide (TMA-O; A1 Waiz et aL, 1987b,c). Urinary excretion is the major mechanism for elimination of TMA and TMA-O from the body (AI Waiz et aL, 1987b,c). For these reasons, excretion of DMA, TMA and TMA-O in urine provides an excellent cumulative measure of the formation of these amines within the whole animal. Rats on a diet free of choline, DMA and TMA for more than 4 wk continue to excrete some TMA and DMA (Zeisel et aL, 1985). We have suggested that such data support the hypothesis that these methylamines can be synthesized by mammalian tissues; however, it is possible that intestinal bacteria are capable of converting other components of the 31

S. H. ZELSEL et al.

32

diet into TMA and DMA. Betaine and carnitine are commonly found in the diet, and share important structural features with choline; they could be precursors of TMA and DMA. With a better understanding of which structural features of the choline molecule are important for recognition by bacterial C - - N cleaving enzymes in vivo, we might be able to predict more accurately which components of the normal diet could be metabolized to form methylamines. We measured monomethylamine (MMA), DMA, TMA and TMA-O excretion in rat urine after administering compounds that share structural features with choline. MATERIALS AND METHODS

Animals. Male Sprague-Dawley rats weighing 133 + 3.5 g (SEM) and obtained from Charles River Breeding Laboratories Inc., Wilmington, MA, USA, were housed in metabolic cages in a controlled environment (24'~C, 12-hr light/dark cycle). They were offered food (Ralston Purina rat chow No. 5001 obtained from Farmers Exchange, Framingham, MA, USA; the diet contained 0.05% MMA, no detectable DMA, 0.41% TMA and 0.2% choline). Water and food were made available ad lib. for I wk before the animals were used in the study. Chemicals. TMA-HCI, DMA-HCI, MMA-HCI, carnitine, betaine, 2-dimethylaminoethyl chloride, and 2-diethylaminoethanol were obtained from Aldrich Chemicals (Milwaukee, WI, USA); 2-dimethylaminoethanol, choline-HCl, TMA-O and phosphocholine were obtained from Sigma Chemicals (St Louis, MO, USA). fl-Methylcholine was synthesized using a modification of the method of Simon et al. (1975). 3-Dimethylamino-l-propanol (Aldrich Chemicals) was mixed with iodomethane (Aldrich Chemicals) in a 1:2 ratio. The mixture was kept in the dark at room temperature for 48 hr. The product was purified by recrystallization from acetone-methanol-ether (1:i:1, v/v). Purity was ascertained by thin-layer chromatography on silica gel plates (Si250-PA, J. T. Baker, Phillipsburg, N J, USA) which were developed with H 2 0 - n - b u t a n o l methanol-acetic acid (3 : 8: 2:1, v/v). All treatments were dissolved in water. The MMA, DMA and T M A content of all treatments was determined by gas chromatography using the methods described below. Protocol. Experimental treatments (water [solvent control], choline, dimethylaminoethanol, diethylaminoethanol, phosphocholine, betaine, carnitine or fl-methylcholine at a dose of 15mmol/kg body weight; dimethylaminoethyl chloride at a dose of 7 mmol/kg body weight) were administered by orogastric intubation, and urine was collected for 24 hr in vials containing hydrochloric acid (final concentration, 0.1 M). Animals were denied access to food during this period, but were allowed to drink a 10% (w/v) dextrose--water solution ad lib. The acidified urines were centrifuged at 500 g for 5 rain at 4°C in a refrigerated centrifuge (model DPR-6000, IEC, Needham, MA, USA). Urine volumes were recorded and aliquots were frozen at - 9 0 ° C until the time of analysis. Animals were killed at the end of the experiment.

Methylamine determination. MMA, DMA, TMA and diethylamine (DEA) were determined using a gas chromatograph with nitrogen-phosphorus detection after samples had been extracted into isopropanol from alkalinized urine (Zeisel et al., 1985). TMA-O was assayed after conversion to TMA (Cohen et al., 1958). Equal volumes of urine and reducing agent (titanium chloride-10% formalin-l~-HCl (1:2:1, v/v)), were placed in a sealed vial under nitrogen, and kept at room temperature for I hr. TMA was then extracted and assayed using the method described earlier. TMA-O concentrations were calculated as the differences in TMA content after and before reduction, Calculations. The increment in excretion of MMA, DMA, TMA, TMA-O or DEA was calculated by subtracting the values obtained in controls from those obtained in the treatment group. Data was analysed by Student's t-test, or one way analysis of variance and Dunnett's test (Bruning and Kintz, 1977).

RESULTS Some of the treatments (dimethylaminoethanol, phosphocholine, carnitine, fl-methylcholine, dimethylaminoethyl chloride) contained trace amounts of methylamines which could not be removed completely by recrystallization. The amounts of MMA, D M A or TMA excreted in urine that might have been derived from such contamination was modest (1-5%) when compared with the amount of these amines detected in urine. For this reason we did not correct our data for this source of methylamines. Control rats excreted M M A (11.5 +0.5 [SEM] pmol/kg body weight/24 hr), DMA (28.7 + 2.6 #mol/ kg body weight/24hr), TMA ( I . 3 + 0 . 3 p m o l / k g body weight/24 hr) and TMA-O (24.9 + 2.8/~mol/kg body weight/24hr). They did not excrete DEA. Rats treated with choline excreted 1.6 times as much M M A (18.4 + 4.5 #mol/kg body weight/24 hr), twice as much DMA ( 6 5 . 1 + 7 . 8 # m o l / k g body weight/24hr), almost 500 times as much TMA (641 + 87 pmol/kg body weight/24 he), and almost 50 times as much TMA-O (1172 + 126 gmol/kg body weight/24hr) as did controls. Twelve percent of the administered choline was excreted as TMA or TMA-O within 24hr (Fig. 1). Rats treated with phosphocholine excreted amounts of these amines that were similar to those excreted in the cholinetreated group (Fig. 1). Animals treated with dimethylaminoethyl chloride excreted more of the dose as DMA and M M A than did choline-treated rats (DMA excretion was increased more than fivefold to 167 + 32 gmol/kg body weight/24hr; significantly different from control by paired t-test, P < 0.01. Rats treated with all of the other compounds excreted much smaller total amounts of MMA, DMA, T M A and TMA-O than did the cholinetreated group (Fig. i). Several treatments did increase D M A or TMA excretion compared with the control values, but the magnitudes of these changes were very small when compared with that of the change after choline treatment. Betaine increased T M A excretion more than sixfold (to 8.5 + 1.1 [SEM] pmol/kg body weight/24 hr; significantly different from the control

Formation of amine precursors of NDMA

33

,R= R, ,R, R,-- C--C-- N - - R s H R~

H

R~ Ra

R6 R4

R_~ R6.

TMA

modkdNand H

H

CH3

CH:I

OH

H

H

CIHs

C:H$

OPOsN:

H

H

CH3

CH3

CH3

~/~O1~

OH

¢H3

H

CH3

CH3

CH3

~i~lllW

OH

O

H

CH3

CH3

CH3

rf,=lledOH~ t=MOI4=~e C ~ CH2COOH OH

H

CH3

CH3

CH3

H

CH3

CH3

maddb¢OHmdi Nn l

Dlmetllytm l ~

Cl

H

DMA

MMA

•

OH

m d ~ OHe'~ II~)~0dt011m

TMA-O

t

•

t

t

)

t

2

4

6

12

0 0.5 1

0

0.2

0.4

Percent of Dose Excreted as Product/24 hr

Fig. I. Formation of methylamines from analogues of choline administered to five rats per treatment by orogastric intubation (as single doses of 15 mmol/kg body weight, except for dimethylaminoethyl chloride which was at a dose of 7 mmol/kg body weight). Urine was collected for 24 hr. Urinary trimethylamine (TMA), trimethylamine oxide (TMA-O), dimethylamine (DMA), monomethylamine (MMA) and diethylamine were measured using gas chromatography. Data are mean differences in amine excretion (treatmentcontrol) and are expressed as percentages of the administered dose + SEM. Values marked with asterisks differ significantly (one way analysis of variance and Dunnett's test) from those for choline (*P < 0.01).

value by paired t-test; P < 0.01) carnitine increased T M A excretion more than eightfold (to 11.4 ± 3.7 /~mol/kg body weight/24hr; significantly different from the control value by paired t-test; P < 0.01), dimethylarninoethanol increased TMA excretion more than I l-fold (to 14.8 ± 2.6 ~mol/kg body weight/ 24 hr; significantly different from the control value by paired t-test; P < 0.01) and increased D M A excretion almost twofold (to 53.4+ 12.4/~mol/kg body weight/24 hr; significantly different from the control value by paired t-test; P < 0.05), and methylcholine increased T M A excretion almost 12-fold (to 15.5 + 3.2 (SEM)/~mol/kg body weight/24 hr; significantly different from the control value by paired t-test; P <0.01). Carnitine and methylcholine increased TMA-O excretion by more than fourfold (Fig. 1). Diethylaminoethanol was the only treatment which resulted in the excretion of diethylamine (Fig. 1).

DISCUSSION

We found that orally administered choline was metabolized to form TMA, TMA-O, D M A and M M A (Fig. 1). This is consistent with previous reports (Asatoor et aL, 1967; Asatoor and Simenhoff, 1965; de la Huerga and Popper, 1951; Lowis, Eastwood and Brydon, 1985; Zeisel et al., 1983). It is not likely that absolutely all of the methylamines formed from precursors within the intestine were excreted within 24 hr. Therefore, our data represent underestimates of the methylamines formed from any given precursor. Phosphocholine was also metabo-

lized to form TMA, TMA-O, D M A and M M A (Fig. I), suggesting that substitution at the hydroxyl end of the choline molecule does not make the compound unusable as a substrate for N-deaikylation by gut bacteria. However, phosphocholine is cleaved to choline by alkaline and acidic phosphatases which may be present within the intestinal lumen. It is possible that phosphocholine was converted to choline before it was metabolized to form methylamines. Modification of the ethyl-backbone or quaternary amine end of choline resulted in almost complete suppression of metabolism to methylamines (Fig. I). The dimethyl- or diethyl-analogues of choline were not well accepted as substrates. Methylation of the /~-carbon of choline also decreased its acceptability as a substrate. Betaine, a naturally occurring metabolite of choline, was not a good substrate for methylamine formation when compared with choline, and therefore it is not likely that dietary betaine accounts for the urinary TMA or D M A that is observed when choline is missing from the diet. Carnitine, also a common constituent of the diet, was not a good substrate for T M A or D M A formation when compared with choline (Fig. I). Seim et aL (1982) have identified a strain of acinetobacter which is capable of cleaving the C - - N bond of carnitine to form TMA. Our data suggest that this bacterial pathway for the metabolism of carnitine is not very important in the rat intestine. We conclude that the bacterial enzymes catalysing the N-dealkylation of choline are relatively specific for choline as a substrate. It is possible that choline esters, such as phosphocholine, may be direct substrates,

S. H. ZEISELet al.

34

but we cannot rule out conversion to choline before their use as substrates for methylamine synthesis. Our data suggest that quaternary amines which differ from choline in that they are substituted on the backbone, or have backbones which vary in length or are substituted on the nitrogen, are not used by gut bacteria to make appreciable amounts o f T M A or DMA. Patients with trimethylaminuria lack the ability to oxidize trimethylamine, due to an inborn error of metabolism, and they smell like rotten fish because of the odour of T M A (AI Waiz et al., 1987a; Brewster and Schedewie, 1983; Humbert et al., 1970; Lee et al., 1976; Shelley and Shelley, 1984). Removal of choline from their diets decreased T M A excretion and odour, but did not return it to normal levels (Brewster and Schedewie, 1983; Lee et al., 1976; Shelley and Shelley, 1984). Our data suggest that it is unlikely that removal of other components of their diets will be of further benefit. In our earlier experiments, we showed that rats fed a choline-free diet continued to excrete T M A and D M A (Zeisel et al., 1985). This diet did not contain choline esters, therefore these compounds could not have been a source of T M A or D M A . It is unlikely that another dietary component, such as betaine or carnitine, could have been present in sufficient quantities to have been a significant source of T M A and D M A . Germ-free rats, and rats treated with neomycin to sterilize their intestines, continue to excrete D M A and T M A (Asatoor et al., 1967; Zeisel et al., 1985). These observations suggest that bacterial degradation of dietary components is not the only source o f D M A and T M A . The persistence of methylamine excretion in choline-deficient, or germfree rats was probably due to synthesis of T M A and D M A within some tissue of the rat. Acknowledgements--This work was supported by a grant

from the National Institutes of Health (No. CA-26731). Some of the data were presented in preliminary form as an abstract at the Federation of American Societies for Experimental Biology meeting in May, 1988.

REFERENCES

AI Waiz M., Ayesh R., Mitchell S. C., Idle J. and Smith R. (1987a). Trimethylaminuria (fish-odour syndrome): an inborn error of metabolism. Lancet il, 634. A1 Waiz M., Mitchell S. C., Idle J. and Smith R. (1987b). The relative importance of N-oxidation and Ndemethylation in the metabolism of trimethylamine in man. Toxicology 43, 117. A1 Waiz M., Mitchell S. C., Idle J. and Smith R. (1987c). The metabolism of ~4C labelled trimethylamine and its N-oxide in man. Xenobiotica 17, 551. Asatoor A. M., Chamberlain M. J., Emmerson B. T., Johnson J. R., Levi A. J. and Milne M. D. (1967). Metabolic effects of oral neomycin. Clin. Sci. 33, 111. Asatoor A. M. and Simenhoff M. L. (1965). The origin of urinary dimethylamine. Biochem. biophys. Acta !11,384.

Bartsch H. and Montesano R. (1984). Relevance of nitrosamines to human cancer. Carcinogenesis 5, 1381. Brewster M. A. and Schedewie H. (1983). Trimethylaminuria. Ann. clin. lab. Sci. 13, 20. Bruning J. L. and Kintz B. L. (1977). Computational Handbook o f Statistics. Scott, Foresman & Company, Glenview, IL. Cohen J., Krupp M. and Chidsey C. (1958). Renal conversion of trimethylamine oxide by the spiny dogfish Squalus Acanthus. Am. d. Physiol. 194, 229. Colby J. and Zatman L. J. (1973). Trimethylamine metabolism in obligate and facultative methyltrophs. Biochem. or. 132, 101. de la Huerga J. and Popper H. (1951). Urinary excretion of choline metabolites following choline administration in normals and patients with hepatobiliary disease, or. clin. Invest. 30, 463.

Humbert J. R., Hammond K. B., Hathaway W. E.. Marcoux J. G. and O'Brien J. G. (1970). Trimethylaminuria: the fish odour syndrome. Lancet ii, 770. lshiwata H., Iwata R. and Tanimura A. (1984). Intestinal distribution, absorption and secretion of dimethylamine and its biliary and urinary excretion in rats. Fd Chem. Toxic. 22, 649. Johnson K. A. (1977). The production of secondary amines by the human gut bacteria and its possible relevance to carcinogenesis. Med. Lab. Sci. 34, 131. Large P. (1971). Non-oxidative demethylation of trimethylamine N-oxide by Pseudomonas aminovoras. FEBS Lett. 18, 297. Lee C. W. G., Yu J. S., Turner B. B. and Murray K. E. (1976). Trimethylaminuria: fishy odours in children. New Engl. or. Med. 295, 937. Lijinsky W., Keefer L., Conrad E. and Van de Bogart R. (1972). Nitrosation of tertiary amines and some biologic implications. J. ham. Cancer Inst. 49, 1239. Lowis S., Eastwood M. A. and Brydon W. G. (1985). The influence of creatinine, lecithin and choline feeding on aliphatic amine production and excretion in the rat. Br. J. Nutr. 54, 43. Sandhu S. and Chase T. (1986). Aerobic degradation of choline by proteus mirablis: enzymatic requirements and pathway. Can. J. Microbiol. 32, 743-50. Seim H., Loster H., Claus R., Kleber H. and Strack E. (1982). Splitting of the C - - N bond in carnitine by an enzyme (trimethylamine forming) from membranes of Acinetobacter calcoaceticus. F E M S Microbiol. Lett. 15, 165. Shelley E. D. and Shelley W. B. (1984). The fish odor syndrome, trimethylaminuria. J. Am. Med. Ass. 251, 253. Simenhoff M. L. (1975). Metabolism and toxicity of aliphatic amines. Kidney Int. 7, 314. Simon J. R., Mittag T. and Kuhar M. (1975). Inhibition of synaptosomal uptake of choline by various choline analogs. Biochem. Pharmac. 24, 1139. Zeisel S. H. (1988). Choline. In Modern Nutrition in Health and Disease. Edited by M. Shils and V. Young. pp. 440--452. Lea & Febiger, Philadelphia. Zeisel S. H., daCosta K. and Fox J. G. (1985). Endogenous formation of dimethylamine. Biochem. J. 232, 403. Zeisel S. H., daCosta K. and LaMont J. T. (1988). Mono-, di- and trimethylamine in human gastric fluid: potential substrates for nitrosodimethylamine formation. Carcinogenesis 9, 179. Zeisel S. H., Wishnok J. S. and Blusztajn J. K. (1983). Formation of methylamines from ingested choline and lecithin. J. Pharmac. exp. Ther. 225, 320.