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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.

The activity of phosphatidylethanolamine N-methyltransferase (PeMT), an enzymic system that catalyses the synthesis of phosphatidylcholine (PtdCho) via sequential methylation of phosphatidylethanolamine (PtdEtn) using S-adenosylmethionine (AdoMet) as a methyl donor, was examined in brain homogenates from rats of various ages. The data thus obtained were consistent with the existence of two distinct enzyme activities within this enzyme system, i.e. one catalysing the methylation of PtdEtn [to form phosphatidyl-N-monomethylethanolamine (PtdMeEtn)], and the other catalysing the methylations of PtdMeEtn and phosphatidyl-NN-dimethylethanolamine (PtdMe2Etn) (to form PtdMe2Etn and PtdCho, respectively). PeMT (PtdEtn-methylating) activity per g of brain was 4-fold higher in neonatal than in adult brains. The enzyme activity in adult brains exhibited Michaelis-Menten kinetics for AdoMet, and its affinity for AdoMet was high (apparent Km 1.6 microM). In neonatal brain the relationships between AdoMet concentrations and PtdMeEtn formation were more complex: a sigmoidal component (with a Hill coefficient of 2.7), requiring 90 microM-AdoMet for half-saturation predominated over the high-affinity component (similar to that of the adult brain). PeMT (PtdMe2Etn-methylating) activity per g of brain increased 2-fold between the 5th and the 20th postnatal days and remained constant thereafter; it was higher than that of PeMT (PtdEtn-methylating) activity at all ages studied, and its affinity for AdoMet was low (apparent Km 99 microM). No sexual dimorphism in brain PeMT activity was observed at any age. We conclude that PeMT (PtdEtn-methylating) catalyses the rate-limiting step in PtdCho synthesis in rat brain, and that PtdCho formation via this pathway may be greatest during the neonatal period.

Choline and C1 metabolism pathways intersect at the formation of methionine from homocysteine. Hepatic S-adenosylmethionine (AdoMet) concentrations are decreased in animals ingesting diets deficient in choline, and it has been suggested that this occurs because the availability of methionine limits AdoMet synthesis. If the above hypothesis is correct, changes in hepatic AdoMet concentrations should relate in some consistent manner to changes in hepatic methionine concentrations. Rats were fed on a choline-deficient or control diet for 1-42 days. Hepatic choline concentrations in control animals were 105 nmol/g, and decreased to 50% of control after the first 7 days on the choline-deficient diet. Hepatic methionine concentrations decreased by less than 20%, with most of this decrease occurring between days 3 and 7 of choline deficiency. Hepatic AdoMet concentrations decreased by 25% during the first week, and continued to decrease (in total, by over 60%) during each subsequent week during which animals consumed a choline-deficient diet. Hepatic S-adenosylhomocysteine (AdoHcy) concentrations increased by 50% when animals consumed a choline-deficient diet. AdoHcy is formed when AdoMet is utilized as a methyl donor. In summary, choline deficiency can deplete hepatic stores of AdoMet under dietary conditions that only minimally decrease the availability of methionine within liver. Thus decreased availability of methionine may not have been the only mechanism whereby choline deficiency lowers hepatic AdoMet concentrations. We suggest that increased utilization of AdoMet might also have occurred.

The neonatal mammal requires especially large amounts of choline to sustain growth. Much of this choline is derived from the newborn's only source of food, milk. The concentration of choline in rat milk [182 +/- 24 microM (S.E.M.)] was much higher than that in maternal serum (11.6 +/- 0.9 microM), suggesting that a mechanism capable of concentrating choline into milk must exist. We characterized choline uptake by mammary epithelial cells (the site of milk production) of the lactating rat. We observed two uptake processes, one saturable and obeying Michaelis-Menten kinetics, and the other non-saturable and linear. At physiological blood choline concentrations, the saturable component of choline uptake predominated. The saturable component had Kapp. = 35 +/- 16 microM, and Vmax. = 1.24 +/- 0.19 nmol/h per mg of protein. Saturable uptake of choline was inhibited by hemicholinium-3. Ca2+ was required for uptake, but Mg2+ was not. Replacement Na+ with K+, Li+ or sucrose inhibited transport. Ouabain did not inhibit choline uptake. Choline concentration in epithelial cells was 67.7 +/- 1.9 nmol/g wet wt. at the start of incubation at 37 degrees C and rose to 80.9 +/- 6.5 nmol/g wet wt. over 30 min. Much of the choline accumulated by the mammary gland (in the presence of endogenous concentrations of choline) remained in the form of choline (50 +/- 1.2%), phosphatidylcholine (12 +/- 2.3%), lysophosphatidylcholine (0.1 +/- 0.03%), betaine (7 +/- 0.3% and phosphocholine (6 +/- 0.5%). In addition, we isolated 25 +/- 1.2% of choline-derived radiolabel in an unidentified compound.

The normal mammal requires large amounts of choline for maintenance and growth of tissue mass. Since milk, the only food for neonates, has many-fold higher free choline concentration than does maternal plasma, it is possible that mammary gland can synthesize choline molecules. The only known mammalian pathway for the synthesis de novo of choline molecules is catalysed by phosphatidylethanolamine N-methyltransferase (PeMT), which synthesizes phosphatidylcholine (PtdCho) via sequential methylation of phosphatidylethanolamine (PtdEtn) using S-adenosylmethionine (AdoMet) as a methyl donor. We identified PeMT activity in rat mammary tissue, and differences in affinities for substrate, as well as in activities as a function of pH, suggest that at least two distinct enzyme activities are involved [i.e. one catalysing the methylation of PtdEtn to form phosphatidyl-N-methylethanolamine (PtdMeEtn) and the other catalysing the methylation of PtdMeEtn and phosphatidyl-NN-dimethylethanolamine (PtdMe2Etn) to form PtdMe2Etn and PtdCho, respectively]. The relationships between AdoMet concentrations and PtdCho formation from endogenous PtdEtn in rat mammary homogenate were complex: a sigmoidal component (with a Hill coefficient of 2.2), requiring 55 microM-AdoMet for half saturation (Vmax. = 9 pmol/h per mg of protein), and a high affinity component (Kapparent = 8.7 microM and Vmax. = 3.8 pmol/h per mg of protein) were identified. When exogenous PtdMe2Etn was added as substrate, PtdCho formation exhibited Michaelis-Menten kinetics for AdoMet, and its affinity for AdoMet was high (Kapparent = 9 microM, Vmax. = 85 pmol/h per mg of protein). In the presence of endogenous substrates, the rates of PeMT-catalysed PtdCho formation within homogenates of rat mammary tissue were similar in tissue from lactating and non-lactating animals. When exogenous PtdMe2Etn was added to homogenates of rat mammary tissue, tissue from lactating rats made twice as much PtdCho as did tissue from non-lactating rats. Isolated mammary epithelial cells also exhibited PeMT activity; the rate of formation of PtdCho was much greater in intact versus broken cells. We also identified PeMT activity in homogenates of mammary tissue from non-lactating humans. The rate of PtdCho formation was of similar magnitude to that seen in rat tissue. This evidence supports the hypothesis that some of the choline found in milk could have been synthesized de novo in the mammary gland.

1. Serum choline concentration in the newborn rat is extremely high and declines as the rat matures until adult values are attained at 20 days of age. 2. Rat milk is a rich source of choline, and rat pups denied access to milk had significantly lower serum choline concentrations than did fed littermates. We conclude that dietary intake of choline contributes to the maintenance of high serum choline concentrations in the neonatal rat. 3. In vivo, choline disappears with a half-life of 70 min. It is converted into betaine, phosphocholine and phosphatidylcholine. The rate of phosphocholine formation is identical in 3- and 10-day-old rats (3.3 mumol/h), whereas the rate of betaine formation is slower in younger animals (0.15 mumol/h at 3 days versus 0.69 mumol/h at 10 days). In vitro, choline oxidase activity [choline dehydrogenase (EC 1.1.99.1) and betaine aldehyde dehydrogenase (EC 1.2.1.8)] increased between birth and 40 days of age. The age-related acceleration in choline's conversion into betaine probably tends to diminish unesterified choline concentration in the rat.