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Proton NMR Spectroscopic Analysis of Multiple Acyl-Coa Dehydrogenase Deficiencyðcapacity of the Choline Oxidation Pathway for Methylation in Vivo

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Proton NMR Spectroscopic Analysis of Multiple Acyl-Coa Dehydrogenase Deficiencyðcapacity of the Choline Oxidation Pathway for Methylation in Vivo Biochimica et Biophysica Acta 1406Ž. 1998 274±282 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Proton NMR spectroscopic analysis of multiple acyl-CoA dehydrogenase deficiencyÐcapacity of the choline oxidation pathway for methylation in vivo Shamus P. Burns a, Heather C. Holmes a, Ronald A. Chalmers c, Andrew Johnson b, Richard A. Iles a,) a Medical Unit, Cellular and Molecular Mechanisms Research Group, St. Bartholomew's and The Royal London School of Medicine and Dentistry, Whitechapel, London E1 1BB, UK b Biochemistry Unit, Institute of Child Health, London WC1N 1EH, UK c Paediatric Metabolism Unit, Department of Child Health, St. George's Hospital Medical School, London SW17 ORE, UK Received 20 January 1998; accepted 16 February 1998 Abstract 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 characteris- tic 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, dimethylglycineŽ. 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. q 1998 Elsevier Science B.V. All rights reserved. Keywords: NMR spectroscopy; Betaine; Choline; Organic acidurias; Multiple acyl-CoA dehydrogenase deficiency; Homocysteine 1. Introduction intermediateswx 1±3 . It is caused by a defect either in the electron transfer flavoproteinŽ. ETF or electron The inherited disorder multiple acyl-CoA dehydro- transfer flavoprotein ubiquinone oxidoreductase genase deficiencyŽ. MADD has been characterised by Ž.ETFQO which are needed for the transfer of elec- the accumulation of several short chain fatty acyl-CoA trons via FAD from these acyl-CoA compounds to the electron transport chainŽ. ETCwx 4,5 . The acyl- CoA dehydrogenases affected include both those in- ) Corresponding author. Medical UnitŽ Cellular Mechanisms volved with fatty acid oxidation and branched-chain Research Group. , St. Bartholomew's and The Royal London amino-acid catabolism. School of Medicine and Dentistry, Alexandra Wing, The Royal London Hospital, Whitechapel, London E1 1BB, UK. Tel.: q44- Two neonatal onset forms of MADD have been 0171-377-7293; fax: q 44-0171-377-7636; E-mail: recognised. The first is associated with severe hypo- [email protected] glycaemia and metabolic acidosis and congenital ab- 0925-4439r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S0925-4439Ž. 98 00015-5 S.P. Burns et al.rBiochimica et Biophysica Acta 1406() 1998 274±282 275 Fig. 1. Sites of inhibition of choline oxidation in multiple acyl-CoA dehydrogenase deficiency. Abbreviations: BADH, betaine aldehyde dehydrogenase; BHMT, betaine-homocysteine methyl transferase; CDH, choline dehydrogenase; DMGDH, dimethylglycine dehydroge- nase; GNM, glycine-N-methylase; SDH, sarcosine dehydrogenase; FMHT, 5-methyltetrahydrofolate-homocysteine methyltransferase; 5-CH34 ±H folate, 5-methyltetrahydrofolate. normalities and presents during the first 24±48 h of hepatic choline oxidase activity indicated that the life. Infants in the second category do not have activity of the human enzyme, in adults, is very low congenital abnormalities, but also develop hypogly- compared to several other mammalian species being caemia and metabolic acidosis within the first few only one sixtieth that of the rat, which had the highest days of lifewx 5 . A third, less severely affected cate- activitywx 12 of the species studied. gory, has a later, variable onset and agewx 5 . We have used proton nuclear magnetic resonance Gas chromatographic and mass spectrometric Ž1H-NMR. spectroscopy to characterise a number of Ž.GC±MS analysis of urine from affected patients has disorders of organic acid and amino-acid metabolism, detected large increases in several short- and and thereby provide a means for their rapid diagnosis, medium-chain dicarboxylic acids and mono- and di- by exploiting the unique ability of the technique to carboxylate glycine conjugateswx 6 . In this respect, the select C±HŽ.n containing metabolites irrespective of GC±MS urinary profile shows some similarities to chargewx 13±15 . The technique is particularly applica- that found in medium-chain acyl-CoA dehydrogenase ble to the detection of intermediates in the choline deficiencywx 7 but differs appreciably with the addi- oxidation pathway, e.g., betaine, dimethylglycine and tional detection of variable amounts of ethylmalonate, sarcosineŽ. Fig. 1 . We have applied the technique to glutarate, isovalerylglycine, iso- and n-butyrylgly- investigate the abnormal metabolites in urine from cine, 2-methylbutyrylglycine and methylsuccinate. three patients who were diagnosed with MADD in Elevation of sarcosine in both urine and plasma order to investigate the flux through the choline has also been reportedwx 2 . Two dehydrogenases which oxidation pathway andŽ. simultaneously determine mediate reactions of choline catabolism, dimethyl- whether the NMR spectrum itself was diagnostic for glycine dehydrogenase and sarcosine dehydrogenase the disorder. A preliminary report was made on a contain covalently bound FAD and also require ETF sample from one of these patientswx 16 . and ETFQO for electron transfer to the ETCwx 8,9 . Conversion of betaine to dimethylglycine is poten- tially an important reaction in the transfer of methyl 2. Materials and methods groups to form methionineŽ. Fig. 1 , the precursor of S-adenosyl methionine which is the ultimate methyl 2.1. Patients donor for creatine formation and many other impor- tant biological methylations. Choline oxidase activity Patient 1 is a boy who presented at the age of 8 is highest in liver and kidneywx 10 and betaine homo- months with fits and hepatomegaly. At the time of cysteine methyl transferase activity is thought to be samplingŽ. 2 weeks later he was still unwell, receiv- confined to these organs in Manwx 11 . However, the ing glucose and saline only and therefore, negligible quantitative significance of the choline oxidation dietary choline. He was unresponsive to riboflavin pathway in vivo is uncertain. A comparative study of but subsequently, he recovered rapidly and has been 276 S.P. Burns et al.rBiochimica et Biophysica Acta 1406() 1998 274±282 maintained subsequently on a normal diet with atten- congenital abnormalities. All diagnoses were made tion given to regular feeding to avoid hypoglycaemia initially by GC±MS and confirmed when possible by and consequent stimulation of fatty acid and protein the detection of CO2 from oxidation of fatty acids of catabolism. He is now 8 years old and is well having various chain lengths and leucine using cultured skin developed normally. Patient 2 was a girl who pre- fibroblasts. sented at the age of 24 h with severe compensated metabolic acidosis after an uneventful birth. She was 2.2. Samples given D,L-carnitine orally from day 2Ž 3=100 mg kgy1 dayy1. , stabilised with intravenous glucose and Random urine samples were analysed which had sodium bicarbonate, then maintained on a diet with a either been taken for diagnosis or therapeutic moni- reduced fat intake and discharged at 7 weeks. At the toring. time of samplingŽ. 4 weeks , her condition was rela- tively stable. She was still receiving oral carnitine 2.3. NMR spectroscopy and a fat free diet containing protein and carbo- hydrate. Her rate of growth was 4.5 gŽ kg body For 1H-NMR spectroscopy urine samples were weight.y1 dayy1 Ž mean for healthy babies approx. 7 prepared as follows: 0.5 ml of urine was transferred g. The choline intake of Patient 2 was also negligible to a 5-mm outer diameter NMR tubeŽ Goss Instru- at the time of study. She was re-admitted to hospital ments, Ingatestone, Essex, UK. and 50 ml of 20-mM at 4 months and died at the age of 4.5 months. Patient sodiumÐ2,2,3,3Ž. trimethylsilyl tetradeutero-pro- 2 3 is a girl aged 11.5 years with a mild form of the pionateŽ. B.D.H., Poole, Dorset, UK in H2 OŽ. Goss disorder and has been receiving a protein reduced was added to act as a chemical shift reference, quan- diet with regular meals. None of the patients had tification standard and a spectrometer lock. Normal Fig. 2. Proton NMR spectrumŽ. 500 MHz of a sample of urine from a 3-month-old healthy girl. The spectrum is of 128 transientsŽ 11 min. Ac: acetate; Ala: alanine; Bt: betaine; Cit: citrate; Cr: creatine; Crn: creatinine; Dma: dimethylamine; Dmg: dimethylglycine; Gly: glycine; Lac: lactate. For more details of assignments, see text. S.P. Burns et al.rBiochimica et Biophysica Acta 1406() 1998 274±282 277 one dimensionalŽ. 1D spectra were collected for each Peak assignments were made as previously de- urine sample. Spectra were recorded either on a Jeol scribedwx 13±15 . In summary, where available, au- GSX 500 MHz or a Bruker AM600 MHz spectrome- thentic standards were run under the same conditions ter with a 30±458 excitation pulse, an acquisition as the urine samples. Some standards were also run time of 1.7 s, a sweep width of 12 ppm, and a after varying the pH over a range which included the recycling time of 5 s. SolventŽ. H2 O suppression was urine pH values. Hahn spin-echo spectra aided spec- performed using pre-saturation of the water reso- tral interpretation by inverting even-numbered multi- nance with a 2-s irradiation pulse of 0.2 W prior to plets. In addition, a spectrum using two dimensional excitation and acquisition.
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