Carnitine therapy and metabolism in the disorders of propionyl-CoA metabolism studied using 1H-NMR spectroscopy

Clinica Chimica Acta, 204 (1991) 263-278 0 Elsevier Science Publishers B.V. All rights reserved 0009-8981/91/%03.50

263

CCA 05184

Carnitine therapy and metabolism in the disorders of propionyl-CoA metabolism studied using ‘H-NMR spectroscopy S.E.C. Davies1y2**,R.A. Iles’, T.E. Stacey29**, C. de Sousa2 and R.A. Chalmers29f ‘Medical Unit (Cellular Mechanisms Research Group), The London Hospital Medical College, Whitechapel, London, 2Section of Perinatal and Child Health, MRC Clinical Research Centre. Harrow (VK)

(Received 30 April 1991; revision received 7 October 1991; accepted 9 October 1991) Key words: Camitine; Inherited metabolic disorder; Organic aciduria; Propionic acidaemia;

Methylmalonic aciduria; NMR spectroscopy

Summary ‘H-NMR spectroscopy has been used to study metabolic perturbations in patients with disorders of propionyl-CoA metabolism during the administration of oral and intravenous L-carnitine. The administration of L-carnitine either in the form of a challenge or as a therapeutic measure resulted in an increased excretion of propionylcarnitine, consistent with the removal of accumulated intramitochondrial propionyl-CoA esters. Additionally, during the therapeutic administration of Lcarnitine excretion of acetylcarnitine occurred, coincident with an improvement in clinical condition and confirming the intracellular propionyl-CoA depletion. An additional benefit from the formation of acylcarnitines may be an accompanying intracellular alkalinisation.

Correspondence to: R.A. Iles, Medical Unit, Cellular Mechanisms Research Group, The London Hospital Medical College, Whitechapel, London El lBB, UK. *Current Address: Department of Biochemistry and Molecular Biology, University of Manchester, Stopford Building, Oxford Road, Manchester Ml3 9PT, UK. **Current Address: Department of Child Health, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK.

264

Introduction Many of the inherited disorders of amino-acid metabolism are characterised by defects in the further metabolism and an acyl CoA intermediate which in consequence accumulates in the mitochondria with a variety of adverse effects on intermediary metabolism [l]. It has previously been shown [2] that children with such disorders excrete increased amounts of acylcarnitine compounds derived from these acyl-CoA intermediates; for example propionyl-carnitine in methylmalonic aciduria and propionic acidaemia [2-51, isovalerylcarnitine in isovaleric acidaemia [2, 61 and 3-methylglutarylcamitine and 3-hydroxyisovalerylcamitine in 3-hydroxy-3-methylglutaric aciduria [2]. Administration of oral r.-camitine was accompanied by increases in both plasma and urinary acylcamitine concentrations [2-71. This suggested that such patients have insufficient L-camitine for their increased metabolic needs and that they might benefit from L-carnitine therapy. Both oral and intravenous camitine therapies have been tried, with some success, both in the acute phase and as an important form of maintenance therapy [2,4,6, 81. In this paper we present the results of ‘H-NMR studies of urinary metabolites excreted by patients with propionic acidaemia and methylmalonic aciduria before and after oral or intravenous L-camitine. The studies described involve both carnitine challenges, given when the patients were relatively stable clinically, and therapeutic administration during episodes of metabolic decompensation. Preliminary reports of some of these studies have been made [9]. Methods Samples of urine were frozen at - 20°C until NMR analysis was carried out. Portions of urine (0.5 ml) were placed in a 5-mm NMR tube to which 50 ~1 of D20 (Goss Scientific Instruments, Ingatestone, Essex, UK) and 20 ~1 of 500 mmol/l3-trimethylsilyl-2,2,3,3,-tetradeuteropropionate (TSPG; B.D.H Ltd. Poole, Dorset, U.K.) were added to lock the magnetic field and to act as a chemical shift reference standard, respectively. The samples were analysed at room temperature in either a Bruker AM250 or WH400 spectrometer (Bruker Spectrospin Ltd., Coventry, U.K.) operating at a field frequency of 250 MHz and 400 MHz, respectively, A single pulse sequence was used, pulse angle 45”, with a pulse recycle time of 2 s. The water signal was suppressed by selective irradiation during the relaxation delay. Patients All of the patients studied had been diagnosed on the basis of the urinary excretion of abnormal organic acids and, where possible, enzymology on cultured skin fibroblasts. Studies on patients were approved by Harrow District Ethical Committee.

265

Patient 1 was a boy who was diagnosed as having propionic acidaemia at 5 days of age using GCMS. He responded poorly to therapeutic measures and died at the age of four and a half years. Patient 2 was a boy, who was diagnosed as having propionic acidaemia shortly after birth using GCMS. He remained relatively well at all times during these studies and was maintained on a low protein diet. He died at the age of 1 year. Patient 3 is a girl, now aged eight years, who was diagnosed as having methylmalonit aciduria at 8 days of age using GCMS and who has subsequently been shown to have an apomutase (EC 5.4.99.2) deficiency which was unresponsive to Bi2 therapy. She has been maintained on oral carnitine (50 mg kg-’ day-‘). Results A. L-carnitine challenges Propionic acidaemia. Spectra from patient 1, with propionic acidaemia are shown in Fig. 1. The spectral resonances have been assigned previously [lo, 1l] with the exception of dimethylamine. This peak had been previously assigned to sarcosine by Bales et al. [12] but these authors are now in agreement with our assignment [13]. The first sample (Fig. la) collected before an oral L-carnitine challenge (100 mg/kg body weight) shows resonances from some of the metabolites characteristic of this disorder. Thus, 3-hydroxypropionate, tiglylglycine and propionylglycine are present in high concentration (> creatinine). However, the 2S,3S and 2S,3R isomers of methylcitrate, characteristic of propionic acidaemia and methylmalonic aciduria, are not readily detected in these spectra. The largest resonance, at 3.27 ppm, is from the trimethyl group of betaine, which we have previously observed in other patients with propionic acidemia and methylmalonic aciduria [lO,l l] and in urine samples from healthy neonates [14,15]. In addition to the two creatinine resonances there are two from the methyl (3.05) and methylene (3.94) resonances of creatine. After oral administration of 100 mg/kg body weight L-carnitine (Fig. lb) a prominent resonance appears at 3.19 ppm from the trimethyl resonance of propionylcarnitine and a smaller resonance at 3.23 ppm from the trimethyl group of free carnitine. Fig. 2 shows the changes in creatine, betaine, propionylcamitine and ‘total propionate excretion’, i.e., the sum (3-hydroxypropionate + propionylglycine + propionylcarnitine + tiglylglycine) expressed as a ratio to creatinine concentration over a period of 113 h. Figure 2 shows that overall carnitine accentuates not only the excretion of propionyl-CoA associated metabolites but also that of creatine and betaine. This continued until 6-7 h after ingestion when a fall in the excretion of some metabolites occurred. Propionylcamitine excretion rose to reach a peak between 5-6 h after carnitine administration when it accounted for 19$ of the total propionate excretion. The total propionate excretion was still twice the pre-administration value after 8.5 h.

266

Q

OHP

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2.0

10

1.5

PPm

PC CC b

OlY I

PC

m

OHP Al

Pa

Ill

ona

40

3.5

30

’

2.5

20

15

(

1.0

rm

Fig. 1. Spectra from patient 1 with propionic acidaemia (a) Before camitine challenge and (b) five hours after the oral administration of 100 mg/kg L-carnitine. Spectrum (a) is of 237 data accumulations and (b) 414. The spectra were run at room temperature at 400 MHz. Bt, betaine; C, carnitine; Cr, creatine; Cm, creatinine; DMN, dimethylamine; Gly, glycine; OHB, 3-hydroxybutyrate; OHP, 3-hydroxypropionate; PC, propionylcamitine; Pg, propionylglycine; Tg, tiglylglycine.

267 61

6-

Fig. 2. Changes in urinary metabolites in patient 1 with propionic acidaemia during the administration of 100 mg/kg L-camitine. Conditions were as described in Fig. 1 and the text. The results are expressed as a molar fraction of the creatinine excretion. Changes in: (0), betaine; (A), creatine; (Cl), propionylcarnitine and (0) ‘total propionate’ excretion (3-hydroxypropionate + propionylglycine + tiglylglcyine + propionylcarnitine). Zero time is the start of the camitine infusion.

The first spectrum (Fig. 3a) from patient 2, aged one month, with propionic acidaemia in contrast to patient 1, showed only elevated glycine, with no obvious propionate metabolites detectable by ‘H-NMR spectroscopy. Administration of L-carnitine (single oral dose 100 mg/kg body weight) resulted in the excretion of acylcarnitine, together with free carnitine. The major acylcarnitine was propionylcarnitine (identified by the triplet at 1.10 ppm) with acetyl-carnitine excretion (a singlet at 2.15 ppm), at about one third that of propionyl-carnitine. Fig. 4 shows the excretion of carnitine, propionylcarnitine and acetyl-carnitine expressed as mol/mol creatinine. Creatine excretion decreased to undetectable levels. This patient was relatively well and did not excrete any propionate metabolites detectable by ‘H-NMR spectroscopy other than propionylcamitine during this challenge. B. Car&tine Therapy Propionic acidaemia. Patient 1 was followed over several four years, during the development of a period of metabolic required the use of intravenous (i.v.) L-camitine therapy patient was then studied during a period of recovery and

days, when aged nearly decompensation, which for its resolution. The subsequently monitored

268

G’Y

Ala Lac

4.0

3.8

3.6

3.4

3.2

3.0

2.8

2.6

2.4

2.2

2.0

1.8

1.6

1.4

1.2

1.0

PPm

Fig. 3. ‘H-NMR spectra from patient 2 with propionic acidaemia during the administration carnitine (100 mg/kg body weight). (a) Before camitine administration;

of oral

L-

periodically over the next few months. At the time of this study the patient was receiving oral L-carnitine therapy (100 mg/kg body weight/day) but in spite of this a period of metabolic crisis ensued presumably caused by a mild intercurrent infection. Abnormal metabolite levels were increasing several days before the patient became unwell. Figure 5a shows the excretion of creatine, betaine, carnitine and propionylcarnitine. Fig. 5b shows the excretion of 3_hydroxypropionate, propionylglytine and glycine during the same period. Propionylglycine and 3-hydroxypropionate excretion were both high and were followed by a steady increase in creatine excretion from less than 0.5 to almost 4 mol/mol creatinine. A similar increase is seen in glycine excretion. Betaine excretion did not fluctuate as much, varying between 0.6 and just over 1 mol/mol creatinine. However, the level is abnormally high for a child of 4 years old; in healthy children of a similar age betaine excretion is less than 0.02 mol/ mol creatinine [16]. After the administration of i.v. L-carnitine (100 mg/kg body weight) a gradual clinical improvement was seen together with a large decrease in propionylglycine and a fall in 3-hydroxypropionate excretion. Changes in glycine lev-

269

PC

4.0

3.8

3.6

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3.2

3.0

2.8

2.6

2.4

2.2

2.0

1.8

1.6

1.4

1.2

1.0

wm

Fig. 3. (b) after camitine administration. Both spectra are the sum of 128 data accumulations and were run at room temperature at 400 MHz. Additional assignments; AC, acetylcamitine; Ala, alanine; Cit, citrate; Lac, lactate; Succ, succinate.

els showed an inverse relationship with those of propionylglycine. Little further change in carnitine or propionylcarnitine was observed, probably because most of the carnitine load had already been excreted before the next sample (6 h after the infusion). Creatine excretion recovered more slowly after an initial fall. This was accompanied by an improvement in the patient’s clinical condition. Methylmalonic aciduria.

Patient 3 was on a regime of 100 mg/kg body weight/day oral L-carnitine from the age of 21 months. At the age of 25 months, L-carnitine administration was stopped for further assessment of her treatment. She developed a severe metabolic crisis despite re-introduction of oral L-carnitine at 100 mg/kg body weight/day and was given an additional oral dose of 400 mg/kg in 4 equally divided doses over 8 h. The results are shown in Fig. 6. Methylmalonate excretion ninety minutes after the third dose had fallen by over 50%. This coincided with a rise in propionylcamitine excretion and the appearance of an additional carnitine conju-

210

time(hours) Fig. 4. Changes in urinary metabolites in patient 2 with propionic acidaemia during the administration of oral L-carnitine (100 mg/kg body weight). (0), acetylcarnitine; ( n), carnitine; (0) propionylcarnitine. Zero time is the start of the carnitine infusion.

a

) --1c

time

(days)

Fig. 5. Urinary metabolites in patient 1 during iv. rxarnitine administration (100 mg/kg body weight). (a) Changes in: (0), betaine; (H), carnitine; (A), creatine; (Cl), propionylcamitine.

271

25

lb

-10

-6

-6

-4

-2

0

2

4

time (days) Fig. 5. (b) Changes in (O), glycine; (A) 3-hydroxypropionate; (0), propionylglycine. Zero time is the start of the carnitine infusion.

gate, acetylcamitine. At around this time there was a marked clinical improvement. Methylmalonate excretion continued to decline to c 30 mol/mol creatinine 24 h after the final carnitine dose. Creatine fell at the end of the study. The same patient was studied at the age of 3 years before and after two intravenous (100 mg/kg body weight) doses of L-carnitine. Spectra are shown in Fig. 7. In Figure 7a, from a sample taken 2 days before the first carnitine infusion, methylmalonate resonances from the methyl (1.30 ppm) and methine (3.27) groups dominate the spectrum and creatine peaks are also prominent (0.4 mol/mol creatinine). The position of the methylmalonate resonances at 1.33 ppm (methyl) and 3.27 (methine) and wide separation of the methyl resonances for creatine (3.04 ppm) and creatinine (3.1 ppm) indicate a very low urine pH. One day before the first camitine dose creatine had risen to > 1.Omol/mol creatinine and methylmalonate excretion to 28 mol/mol creatinine. After the first carnitine infusion there was little change in methylmalonate excretion and creatine excretion continued to rise. Four hours after the second camitine dose (Fig. 7b) the situation was unchanged with creatine/creatinine at 1.5. However, the urine pH had increased considerably, as demonstrated by the right-hand shift of the methylmalonate and creatinine resonances. As propionylcamitine and acetylcamitine continued to rise there was a rapid fall in methylmalonate excretion by over 50% (Fig. 7c) and 40 h later it was < 8 mol/mol creatinine. Creatine fell at a slower rate (Fig. 7b and 7c) but had declined to less then 0.4 mol/mol creatinine 40 h later (not shown).

212 10

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time (hours)

’5

b

50

:B ia 30

20!

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I

I

25

-5 t

t

t"

t

time(hours)

l5

Fig. 6. Changes in urinary metabolites in a 21-month-old girl with methylmalonic aciduria during the administration of 400 mg/kg L-camitine in 4 x 100 mg/kg doses. Conditions were as described in the text. The results are expressed as a molar fraction of the creatinine excretion. (a) Changes in: (m), camitine; (O), propionylcamitine; (0) acetylcamitine and, (b) changes in: (A), methylmalonate and (O), total propionate metabolites (methylmalonate + propionylcamitine + 3-hydroxypropionate). Zero time is the start of the first 100 mg/kg camitine infusion.

213

,‘,.,.,.,.,.i.I.I,I’I

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3.4

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2.8

2.6

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2.2

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1.6

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Fig. 7. rH-NMR spectra from a girl aged 3 years with methylmalonic aciduria. (a) Before the first 100 mg/kg camitine dose (see text), (b) four hours after the administration of the second 100 mg/kg dose and (c) nine hours after the administration of the second 100 mg/kg dose. Spectrum (a) is of 256 data accumulations, (b) 288 and (c) 168. The spectra were run at room temperature at 400 MHz. Assignments are as for Fig. 2 with the following additions: Me, methylmalonate.

Discussion This study demonstrates that ‘H-NMR, although insensitive, offers a distinct advantage over other methods of measurement of acylcarnitine moieties, for example fast atom bombardment mass spectrometry, by offering the opportunity for rapid results on unextracted samples with simultaneous measurement of organic acids, amino-acids and other metabolites in addition to the strongly basic acylcarnitines. The administration of an oral L-carnitine challenge provoked a common response in the two patients with propionic acidaemia: an increased excretion of propionyland acetyl-carnitine conjugates which reached a peak after 610 h followed by a slow fall over the succeeding 12-h period. The specific acylcamitine identified was characteristic of propionic acidaemia, as has been found in earlier studies when either endogenous propionylcarnitine was identified [2, 73 or carnitine was given therapeutically [4]. The excretion of acylcarnitine esters following administration of L-camitine is consistent with removal of the corresponding accumulated intramitochondrial acyl CoA esters. The provision of an increased camitine supply creates an additional means of reducing the propionyl pool, by propionylcamitine formation, passage out of the mitochondrion and subsequent excretion. Camitine challenge in the first patient with propionic acidaemia (patient 1)

274

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3.6

3.6

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26 PPm

2.4

2.2

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2.0

16

16

1.4

1.2

1.0

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+AC

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40

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3.6

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Fig. 7 continued.

2.2

16

14

1.2

10

215

increased the excretion of several other metabolites suggesting possibly that renal function was affected (improved). However, patient 1 was rarely in a stable condition compared to the other subjects, as revealed by the excretion of abnormal metabolites prior to the challenge, and it is therefore possible that a partial therapeutic effect occurred. In comparison, patient 2, (who also had propionic acidaemia), did not show any such increase in metabolite excretion when he was given a carnitine challenge while metabolically stable. Nevertheless, after L-carnitine challenges both of these patients subsequently excreted propionylylcamitine, indicating the accumulation of intramitochondrial propionyl-CoA even in the absence of detectable pre-challenge propionylcarnitine excretion. Presumably such excretion was occurring at a much lower level, limited by the endogenous camitine supply, which was below NMR detection level. Patient 3 (who had methylmalonic aciduria) received relatively high (therapeutic) doses of carnitine on the two occasions when she had periods of severe metabolic decompensation. Propionylcamitine excretion was correspondingly high, reaching levels of 10 (Fig. 6) and 16 (Fig. 7c) mol/mol creatinine, accompanied by an increase in acetylcamitine. It is therefore possible that intramitochondrial propionyl groups were fully depleted allowing formation and excretion of acetylcamitine (Figs. 6, 7), presumably derived from intramitochondrial acetyl CoA. The excretion of acetylcarnitine coincided with a considerable improvement in clinical condition, consistent also with the depletion of the intramitochondrial propionyl CoA pool during the period concerned in both instances. It is therefore possible in retrospect that the dose given to patient 1 could have been increased as no acetylcamitine was detected in the urine after carnitine therapy. Throughout this period creatine excretion fell. A similar fall in creatine excretion was noted in the other patients studied which suggests there may be some correlation between clinical improvement and creatine excretion [16]. We have also demonstrated the converse situation in a patient with methylmalonic aciduria when creatine excretion rose with deteriorating clinical status and fell on recovery, when camitine was not involved [ll]. A more detailed discussion of the relationship between creatine excretion and clinical condition has recently been presented [17] and these results both complement and extend our previous findings. The accumulation of propionyl CoA causes perturbations in acetyl CoA metabolism, for example, by sequestering CoA [7, 181 and inhibiting citrate synthase [5]. Resolution of these perturbations resulted in L-camitine combining with available acetyl-CoA to form sutlicient excess acetylcarnitine to permit excretion. The appearance of acetylcarnitine in the urine in this instance could therefore indicate the successful resolution of a metabolite crisis. When L-camitine is given to healthy control subjects [16, 191 most of the carnitine appears unchanged in the urine but a small fraction, up to 30% of the excreted camitine, is converted to acetylcarnitine. The observations are consistent with carnitine acetyltransferase showing a greater affinity towards propionyl-CoA [18]. The large amounts of propionylcamitine relative to acetylcamitine in patients with propionic acidaemia and methylmalonic aciduria pro-

216

fraction, up to 30% of the excreted carnitine, is converted to acetylcarnitine. The observations are consistent with carnitine acetyltransferase showing a greater affinity towards propionyl-CoA [18]. The large amounts of propionylcarnitine relative to acetylcarnitine in patients with propionic acidaemia and methylmalonic aciduria provide further evidence for a very large propionyl CoA pool. In the patient with methylmalonic aciduria who excreted acetylcarnitine its appearance coincided with a large reduction in methylmalonate excretion (Fig. 6). The present results in patients with methylmalonic aciduria and propionic acidaemia clearly provide a scientific basis for the metabolic and clinical effects of L-carnitine. An additional benefit from the use of carnitine may derive from the removal of one proton per mol of acylcarnitine formed. This is illustrated (for propionylcarnitine) below together with the formation of propionylglycine which, by contrast, is a neutral reaction: NH3 +-CHrCOO-

+ CH3-CH2-COO -G= CH3-CH2-CONH-CH2-COO(Propionylglycine) (CH&+N-CHrCHOH-CH+OO+ CHrCH&OO+ H+ = (CH&+N-CH2- CH-CH2-COO- + Hz0

+ Hz0

0

CH3-CH2- C =0 Propionylcarnitine In terms of whole-body acid-base balance the contribution will be small but the localised effect in cells or indeed mitochondria where the formation of propionylcarnitine occurs may be significant. Thus we have found that the synthesis of methylcitrate, which may disrupt the citric acid cycle [20], from propionyl-CoA and oxaloacetate is increased by acidosis in vitro [21]. Alkalinisation may therefore reverse this increase and aid the resolution of the acute metabolic perturbation. Acknowledgements We are grateful to both the University of London Intercollegiate Research Service at Queen Mary College and the MRC Biomedical NMR Centre for NMR facilities. We are indebted to Sigma-Tau s.p.a., Rome, Italy for generous supplies of L-carnitine for these studies. S.E.C. Davies acknowledges a MRC studentship.

1 Striver CR, Beau&t AL, Sly WS and Valle D. Eds, The Metabolic Basis of Inherited Disease, 6 ed, New York, London, McGraw-Hill Book Co., 1989.

211 2 Chalmers RA, Roe CR, Stacey TE and Hoppel CL. Urinary excretion of L-camitine and acylcamitines in patients with disorders of organic acid metabolism: Evidence for secondary insufficiency of L-camitine. Pediatr Res 1984; 18: 1325-1338. 3 DiDonato S, Rimoldi M, Garavaglia B and Uziel G. Propionylcamitine excretion in propionic acidaemia and methylmalonic aciduria: a cause of camitine deficiency. Clin Chim Acta 1984; 139: 13-21. 4 Duran M, Ketting D, Beckeringh TE, Leupold D, Wadman SK. Direct identification of propionylcarnitine in propionic acidaemia: biochemical and clinical results of oral carnitine supplementation. J Inher Metab Dis 1986; 9: 202-207. 5 Roe CR, Hoppel CL, Stacey TE, Chalmers RA, Tracey BM, Millington DS. Metabolic response to carnitine in methylmalonic aciduria. Arch Dis Child 1983; 58: 91&920. 6 de Sousa C, Chalmers RA, Stacey TE, Tracey BM, Weaver CM, Bradley D. The response to L-camitine and glycine therapy in isovaleric acidaemia. Eur J Pediatr 1986; 144: 451-456. 1 Chalmers RA, Roe CR, Tracey BT, Stacey TE, Hoppel CL, Millington DS. Secondary camitine insufficiency in disorders of organic acid metabolism: modulation of acyl-CoA/CoA ratios by t-camitine in vivo. Biochem Sot Trans 1983; 11: 724725. 8 Bain MD, Jones M, Borriello SP, Reed PJ, Tracey BM, Chalmers RA, Stacey TE. Contribution of gut bacterial metabolism to human metabolic disease. Lancet 1988; i: 10781079. 9 Iles RA, Jago JR, Williams SR, Stacey TE, de Sousa C, Chalmers RA. Human camitine metabolism studied by ‘H-nuclear magnetic resonance spectroscopy. Biochem Sot Trans 1986; 14: 702-703. 10 Iles RA, Hind AJ, Chalmers RA. The use of proton nuclear magnetic resonance spectroscopy for the detection and study of organic acidurias. Clin Chem 1985; 31: 17951801. 11 Iles RA, Chalmers RA, Hind AJ. Methylmalonic aciduria and propionic acidaemia studied by proton nuclear magnetic resonance spectroscopy. Clin Chim Acta 1986; 173: 173-189. 12 Bales JR, Sadler PJ, Nicholson HJ, Timbre11 JA. Urinary excretion of acetaminophen and its metabolites as studied by proton NMR spectroscopy. Clin Chem 1984; 30: 1631-1636. 13 Woodham RH, Bell JD, Sadler PJ, Moore, PJ, Lee HA, Lee JA, Wilkie DR. NMR studies of human plasma and urine: chronic renal failure and amine metabolism. Abstracts, Ninth Annual Meeting Society Magnetic Resonance in Medicine, 1989; p. 380. 14 Davies SEC, Chalmers RA, Randall EW, Iles RA. Betaine metabolism in the human neonate and developing rat. Clin Chim Acta 1988; 178: 241-250. 15 Davies SEC, Chalmers RA, Woolf DA, Iles RA. Betaine metabolism in the human neonate. Biochem Sot Trans 1988; 16: 788-789. 16 Davies SEC. PH D. Thesis. University of London, 1989. 17 Davies SEC, Iles RA, Stacey TE, Chalmers RA. Creatine metabolism during metabolic perturbations in patients with organic acidurias. Clin Chim Acta 1990; 194: 203-218. 18 Bieber LL. Camitine. Annu Rev Biochem 1988; 57: 261-283. 19 Iles RA, Davies SEC, Chalmers RA, Rafter JEM. Metabolism of exogenously administered camitine in human volunteers. Abstracts 20th FEBS Meeting, Budapest, 1990, p. 52. 20 Cheema-Dhadi S, Leznoff CC, Halperin ML. Effect of 2-methylcitrate on citrate metabolism, implications for the management of patients with propionic acidemia and methylmalonic aciduria. Pediatr Res 1975; 9: 905-908. 21 Davies SEC, Chalmers RA, Iles RA. Intluences on the stereochemistry of citrate-synthase catalysed methylcitrate formation. Biochem Sot Trans 1987; 15: 841-842.