The choline content of human breast milk doubles 6-7 days after birth and, unlike that of many formula feeds, appears to be sufficient to account for betaine excretion in healthy full-term neonates. However, for premature babies who usually receive much lower quantities of milk, yet have a higher demand for choline, the intake may be inadequate.

We have previously shown that primary trimethylaminuria, or fish-odour syndrome, is caused by an inherited defect in the flavin-containing monooxygenase 3 (FMO3) catalysed N-oxidation of the dietary-derived malodorous amine, trimethylamine (TMA). We now report a novel causative mutation for the disorder identified in a young girl diagnosed by proton nuclear magnetic resonance (NMR) spectroscopy of her urine. Sequence analysis of genomic DNA amplified from the patient revealed that she was homozygous for a T to C missense mutation in exon 3 of the FMO3 gene. The mutation changes an ATG triplet, encoding methionine, at codon 82 to an ACG triplet, encoding threonine. A polymerase chain reaction/restriction enzyme-based assay was devised to genotype individuals for the FMO3Thr82 allele. Wild-type and mutant FMO3, heterologously expressed in a baculovirus-insect cell system, were assayed by ultraviolet spectrophotometry and NMR spectroscopy for their ability to catalyse the N-oxidation of TMA. The latter technique has the advantage of enabling the simultaneous, direct and semi-continuous measurement of both of the products, TMA N-oxide and NADP, and of one of the reactants, NADPH. Results obtained from both techniques demonstrate that the Met82Thr mutation abolishes the catalytic activity of the enzyme and thus represents the genetic basis of the disorder in this individual. The combination of NMR spectroscopy with gene sequence and expression technology provides a powerful means of determining genotype-phenotype relationships in trimethylaminuria.

A variable fraction of ingested choline and trimethylamine-Noxide (TMAO) is converted to trimethylamine (TMA) by bacterial fermentation in the human colon. Eggs and liver are particularly rich dietary sources of choline and TMAO is found in marine fish. TMA is absorbed 6om the gut and is then usually converted to TMAO by (liver) flavin monooxygenases (FMOs) before excretion. Some TMA may also be converted to dimethylamine (DMA) in the liver which is also excreted.In the inherited disorder trimethylaminuria [I] it is thought that an FMO is defective [2]: TMA is excreted if choline or TMAO containing foods are ingested.We have investigated a 4 year old female patient with suspected trimethylaminuria. A number of timed urine samples were collected whilst the patient was taking in a normal diet and following a fish meal and an oral choline load. The urine samples were analysed by 'H nuclear magnetic resonance spectroscopy (nmr). To 0.5 ml aliquots of urine was added 50 of 2H20 containing 20 mM 3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionate (TSPd4) for field locking and as an internal chemical shift reference respectively. The samples were tun at room temperature in a Jeol GSX5OO or Bruker AM400 spectrometer using a single pulse sequence (30" pulse angle, 2.73 s acquisition time and a 5 s recycling time). Urinary metabolites were quantitated by measuring peak heights relative to the TSP& signal at 0.00 ppm or the creatinine signals at 3.05 and 4.08 ppm ifthere was no timed volume available.With a normal diet, avoiding fish and sources of excess choline this patient excreted an average of 5.1 pmol/hr TMA, 6.4 pmol/br TMAO and 6.4 p m o b DMA over a 25 hour period. This was 0.05, 0.06 and 0.06 moVmol creatinine respectively. Following a meal of fish all these metabolites were excreted at increased amounts, 4.6, 2.1 and 0.2 moVmol creatinine respectively. After a choline bitartrate dose of 7.5 g (30 mmol) there was an extremely large increase in excretion of TMA and a smaller increase in excretion of TMAO and DMA (Fig. 1). During the 7 hour period after the dose the excretion of TMA was 1300 pmovhr, TMAO 43 pmol/hr and DMA 16 pmol/hr (1 I, 0.4 and 0.5 moVmol creatinine respectively). The excretion of these three metabolites represents 33 % of the choline dose. As excretion of all three was still elevated by at least 12-20 hours after the dose it is likely that more than halfofthe choline had been metabolised to TMA.Normal healthy volunteers after a fish meal excreted mainly TMAO but very little TMA. The ratio W T M A O was 0.004 or less. The average ratio for this patient on a normal diet was 0.8. After a fish meal this ratio rose to 2.2. The choline load caused this ratio to increase to 30 during the 7 hour period immediately after the dose. Choline therefore provided a larger increase in TMA/TMAO than TMAO from the fish meal and a lower rate of TMAO excretion in spite of giving a greater total TMA load. Since choline does not give rise to TMAO directly it is likely that a substantial amount of TMAO from the fish i...

Choline is a conditionally essential nutrient required by the human body for synthesis of the neurotransmitter acetylcholine and the phospholipids phosphatidylcholine (PtdCho), lysophosphatidylcholine, sphingomyelin (SM) and plasmalogen. It also undergoes irreversible oxidation to form betaine, a major source of methyl groups. Betaine is excreted by human infants in large quantities (up to 1 mol/mol creatinine) during the first year reaching a maximum at 2-3 months of age. Since the betaine content of breast milk is very low and choline is the only known endogenous source of betaine, the supply of dietary choline to neonates may be critical particularly for premature babies whose intake may be restricted in the early days of life [ 11. Some premature babies receive expressed breast milk and we have measured the choline content, distribution and temporal variation to determine whether its supply is sufficient. Choline is present in breast milk as free choline (Cho), phosphorylcholine (PC), glycerophosphocholine (GPC), PtdCho and SM [2]. Previous studies have not considered the contributions of GPC and PC [3].Milk samples were obtained from the Neonatal Intensive Care Unit of the Royal London Hospital. Samples were expressed by mothers and then frozen at -20°C until such time as the baby required the milk. At that time samples were defrosted and an aliquot taken for preparation for 'H nmr spectroscopy.The water soluble metabolites (Cho, PC and GPC) were measured in perchloric acid extracts of the milk, neutralised with KOH. Samples were run at pH values of 2 and 7 to distinguish the three metabolites from each other and from other metabolites with similar chemical shift values such as carnitme. Following lyophilisation the samples were dissolved in 0.6 ml 'H20 with 50 p1 lOmM fiunarate and 20 p1 20 mM 3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionate (TSPQ) for field locking, quantification and chemical shift referencing respectively. The samples were run at room temperature in a Jeol GSXSOO or Bruker AM400 spectrometer using a single pulse sequence (30" pulse angle, 2.73 s acquisition time and a 5 s recycling time).The choline containing phospholipids were measured in chloroform extracts. After separation the lower chloroform layer was evaporated under nitrogen, redissolved in 0.6 ml C2HCI3:C2H3O2H (2: 1) containing tetramethykhe (TMS; 0.03% v h in C2HCl3) and 0.3 mM 1,3,5-trichlorobee as the chemical shift reference and quantification standard respectively. The samples were run as descriied above. Figure 1 shows the concentration of choline metabolites in the milk expressed by one mother. This is qualitatively similar to the pattern observed for milk fiom 4 other mothers who have expressed milk over a similar time period and whose babies were born at between 28 and 37 weeks gestation. Until about one week after birth the total choline content is relatively low and is mostly present as Cho, PtdCho and SM. After that the total choline concentration rises quite markedly with the greatest increase being shown by GPC ...

Proton NMR spectra of urine from subjects with multiple acyl-CoA dehydrogenase deficiency, caused by defects in either the electron transport flavoprotein or electron transport flavoprotein ubiquinone oxidoreductase, provide a characteristic and possibly diagnostic metabolite profile. The detection of dimethylglycine and sarcosine, intermediates in the oxidative degradation of choline, should discriminate between multiple acyl-CoA dehydrogenase deficiency and related disorders involving fatty acid oxidation. The excretion rates of betaine, dimethylglycine (and sarcosine) in these subjects give an estimate of the minimum rates of both choline oxidation and methyl group release from betaine and reveal that the latter is comparable with the calculated total body methyl requirement in the human infant even when choline intake is very low. Our results provide a new insight into the rates of in vivo methylation in early human development.