Organic aciduria in neonatal multiple carboxylase deficiency

J. Inher. Metab. Dis. 5 (1982) 49-53

Organic Aciduria in Neonatal Multiple Carboxylase Deficiency L. SWEETMAN and W. L. NYHAN Department of Pediatrics, University of California, San Diego, La Jolla, California, USA N. A. SAKATI and A. OHLSSON

Department of Pediatrics, King Faisal Specialist Hospital, Riyadh, Saudi Arabia M. S. MANGE Dhahran Health Center, Dhahran, Saudi Arabia R. B. BOYCHUK and R. KAYE Kapeolani Children's Medical Center, Honolulu, Hawaii, USA A Samoan patient and a Saudi-Arabian patient were found to have abnormalities in the pattern of organic acid metabolites characteristic of 3-methylcrotonylglycinuria, propionic acidaemia and lactic acidosis. Both patients died early in life. The metabolic pattern is diagnostic of multiple carboxylase deficiency and an enzymatic diagnosis was made in a subsequent affected sibling of the first patient. Deficiency of the three carboxylases suggests a primary defect in the metabolism of biotin which is required for their activity. The importance of the recognition of the clinical picture is highlighted by the fact that this lethal disease is readily treated with biotin. These patients have prominent skin lesions which can serve as alerting signs for the diagnosis. 3-Methylcrotonylglycinuria (McKusick 21020) is a disorder of leucine metabolism in which the accumulation of 3-methylcrotonyl-CoA is followed by conversion to 3methylcrotonic acid, 3-methylcrotonylglycine and 3hydroxyisovaleric acid (Edjarn et al., 1970). 3Methylcrotonyl-CoA carboxylase (EC 6.4.1.4) is a biotin-containing enzyme, and three infants with increased excretions of the abnormal metabolites consistent with a deficiency of this enzyme responded clinically and biochemically to treatment with large amounts of biotin (Gompertz et al., 1971; Gompertz et al., 1973; Keeton and Moosa, 1976). It has more recently become apparent that in the first biotin-responsive patient from England (Gompertz et al., 1971) there is also a defect in isoleucine metabolism in which the accumulation of propionyl-CoA leads to the presence of tiglyglycine (Gompertz and Draffan, 1972) and 3-hydroxypropionic acid and methylcitric acid (Chalmers et al., 1974; Sweetman et al., 1977). This chemical evidence of combined deficiency of two biotin-containing enzymes, propionyl-CoA carboxylase (EC 6.4.1.3) and 3methylcrotonyl-CoA carboxylase has been confirmed by assay of the enzymes in fibroblasts (Sweetman et al., 1977). The activities of the two enzymes were markedly deficient when the cells were cultured in medium containing low concentrations of biotin but were normal when cultured in the presence of high concentrations of biotin (Bartlett and Gompertz, 1976; Bartlett and Gompertz, 1978; Weyler et al., 1977). The kinetic properties of both carboxylases were normal, suggesting

that the fundamental defect is in the metabolism of biotin, presumably in the enzyme holocarboxylase synthetase, which attaches biotin to the carboxylases (Weyler et al., 1977). Another infant reported as having lactic acidosis and 3-methylcrotonic aciduria (Roth et aI., 1976) has now been shown to have an identical enzymatic and genetic defect by complementation analysis of hybridized fibroblasts (Saunders et al., 1979). Furthermore, fibroblasts from both patients have been shown to have a biotin-dependent deficiency of another carboxylase, pyruvate carboxylase (EC 6.4.1.1.) which would account for the lactic acidaemia (Saunders et al., 1979). Another patient presenting at a later age with intermittent acidosis has been shown to have low activities of the three carboxylases in fibroblasts cultured with low concentrations of biotin and marked increases in the activities when grown in medium with high concentrations of biotin (Bartlett et al., 1980). Another infant with a similar pattern of metabolite excretions, seizures, hypotonia, and seborrhoeic dermatitis also responded to treatment with biotin (Lehnert et al., 1979). Three somewhat older children have been reported (Cowan et al., 1979; Charles et al., 1979; Munnich et al., 1980) with biotin-responsive alopecia, dermatitis, lactic acidosis and increased metabolites characteristic of multiple carboxylase deficiency. In contrast to the patients of Gompertz et al. (1971), Roth et al. (1976) and Bartlett et al. (1980), these patients and the one reported by Keeton and Moosa (1976) studied by Chalmers and Spellacy (1980) do not have deficiencies of the carboxylases in fibroblasts cultured with low concentrations of biotin. When examined, these patients have shown a biotin-responsive deficiency of the carboxylases in

Addressfor correspondence:Dr. LawrenceSweetman,Departmentof Pediatrics M-009, University of California, San Diego, La Jolla, California 92093, USA. 49

Journal of Inherited Metabolic Disease. ISSN 0141-8955. Copyright © 1982 MTP Press Limited, Cable Street, Lancaster, UK. Printed in the Netherlands.

50

Sweetman, Nyhan, Saka~i, Ohtsson, Mange, Boychuk and Kaye

teukocytes in vivo. We have thought that these patients may have abnormalities in the absorption or transport of biotin in vivo. It is the purpose of this report to describe two patients with the neonatal pattern of disease in whom there were concentrations of metabolites characteristic of multiple carboxylase deficiency° Occurrence in two very different ethnic groups suggests that the gene is widely distributed. Although each of these infants died of the disease before the samples collected could be sent for analysis, their documentation may call attention to the possibility of the diagnosis in other infants who may be successfully treated. A sibling of one of the patients has now been diagnosed and has responded dramatically to treatment with biotin.

CASE REPORTS Case 1 M.Z., a Saudi-Arabian male infant, was referred for admission at 3 months of age with diagnoses of convulsive disorder, degenerative brain disease and seborrhoeic dermatitis. He had been born after an uneventful pregnancy and delivery to parents who were young, healthy, and first cousins. Birth weight was 3 kg and length 51 crn (head circumference 35crn). Of five siblings, a sister had died at 3-4 months of age with convulsions, which began at 2 months, poor feeding, vomiting and failure to thrive, a clinical picture reminiscent of that of the patient. A living sister had blindness thought to be due to congenital optic atrophy. The patient was irritable from birth. He was breast fed for the first 2 months of life, but he did not gain weight adequately during this period and had repeated vomiting. At 2 months, he developed generalized tonic convulsions. Despite therapy with phenobarbital, the number and duration of the seizures increased over the 5 days before admission. A generalized scaly eruption began a week before admission. Physical examination revealed an irritable infant with a generalized, erythernatous scaly eruption (Figure 1). There was crusting, cracking, and some secondary pustular infection over the scalp. The skin lesions prevented him from completely closing the eyes. The weight was 4050 g, the length 53 crn and the head circumference 37 cm. He was hypertonic, had clenched fists, and the appearance of early spastic contractures of the arms and legs. Deep tendon reflexes were increased. Plantar responses were extensor. He did not fix on an object, or follow a light. He was not seen to smile but according to the mother had done so in the past. His haemoglobin concentration was 9.1 g/dl, haematocrit 28 %, MCV 79, MCH 26.5 and MCHC 33.3. The leukocyte count was 10 500 per mrn a and the differential count normal. A platelet count was 410 000 per mm 3. A large amount of ketones were present in the urine. The sodium concentration was 163mmol/1, potassium 4.4, chloride 124 and bicarbonate 11 mmol/1. The concentration of uric acid was 12.6 mg/dl. The BUN was 28 and the creatinine concentration 1.0mg/dl; calcium concentration was l l.0mg/dl, phosphorus concentration

Figure 1 Case 1 at 3 months of age. The scaly eruption was erythematous and present throughout the body. He had almost complete alopecia of the scalp, except for a small amount of occipital hair, but there were sparse eyebrows and eyelashes 4.4 mg/dl and the sugar concentration 81 mg/dL SGOT activity was 30IU, SGPT activity 21 IU, and the bilirubin concentration 0.6mg/dl. Qualitative analysis of the urine for amino acids was unrernarkable. Cultures of the nose, throat, and scalp revealed Staphylococcus aureus and Pseudomonas aeruginosa, and culture of the urine revealed more than 105 colonies of Ps. aeruginosa. Culture of the blood was negative. Despite vigorous parenteral fluid therapy the acidosis worsened. The following day the concentration of bicarbonate was 6mrnol/1 and the pH 7.19. He developed Kussrnaul breathing, and he died on the third day after admission. Case 2 Baby girl F. developed hypothermia, jaundice and vomiting at 24 hours of life; she developed apnoeic episodes and complete unresponsiveness and died at 3 days of age. The patient was born after an uneventful 43 weeks pregnancy, labour and delivery in a 28-year-old Samoan. There was no parental consanguinity. The birth weight was 2892 g, and the Apgar score 9 at 1 and 5 min. The infant was alert and active. She was moderately hirsute, slightly hypotonic and had peeling of skin of the soles. The length was 49.5 cm and the head

Organic Aciduria in Multiple Carboxylase Deficiency circumference 35 cm. She fed poorly at breast 3 times during the first day. At 24 hours she was found to have a temperature of 35 ° C. She was placed in a warmer and the temperature rose to 35.7 °C, but she appeared seriously ill. She was lethargic, icteric, and mottled. The respiratory rate was 40 per min., but she had an irregular respiratory pattern. The heart rate was 120 per min and the systolic blood pressure 38 mmHg. There were mild subcostal retractions, rales over the chest, and a systolic murmur. The liver wa~ palpable 1 cm below the right costal margin. The baby was thought to have sepsis, and treatment was initiated with ampicillin and kanamycin. CSF obtained was bloody but contained 70 mg/dl of glucose. The haemoglobin concentration was 14.7 g/dl and the leukocyte count 26 600 per mm 3 with 73 ~o polymorphonuclear forms, 2 0 ~ lymphocytes and 7 ~ monocytes. Platelets appeared adequate. The total concentration of bilirubin in the serum was 8.6mg/dl. Cultures of the blood and urine revealed no growth. A chest film was interpreted as normal. The infant became flaccid and developed spells of apnoea. At 2 days the blood ammonia concentration was 217 gg/dl. A screening test for urinary amino acids was negative. By the 3rd day of life the infant was completely unresponsive. The fontanelle was full. The liver was 3 crn below the costal margin. The mucous membranes were dry and the skin turgor poor. The arterial blood pH was 6.84; pCO2 27.9 and pOz 59.8mmHg. The base deficit was -30. An exchange transfusion lowered the blood ammonia concentration to 79 gg/dl, but there was no change in her level of consciousness. She developed bradycardia 6 h later, and despite intubation and efforts at resuscitation she died. Postmortem examination revealed pulmonary oedema and intra-alveolar haemorrhage. The brain weighed 478 g (expected weight 333 g) and the liver and kidneys were also heavier than normal. There was microscopic evidence of cerebral oedema, but no evidence of hypoxic degeneration of the hippocampus or type I Alzheimer cells. MATERIALS AND METHODS Non-volatile organic acids in blood and urine were analysed quantitatively by liquid partition chromatography (LPC) on sulphuric acid-hydrated silicic acid columns eluted with a gradient of 2-methyl-2-butanol of chloroform (Sweetman, 1972). The eluted acids were continuously titrated with sodium o-nitrophenol, the titration of indicator recorded spectrophotometrically by absorbance at 350 nm, and the areas under the curves were determined using an Autolab I integrator. Concentrations of acids were calculated from peak areas relative to peak area of standard acids analysed similarly. Retention times were expressed as Ry values relative to citric acid, which was standardized at 100.0. Fractions were collected and individual peaks were evaporated to dryness, 50gg n-triacontane added as internal standard, the trimethylsilyl (TMS) derivatives formed, and analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GCMS) on

51 2mm x 180crn columns of 3 Y/oDexsil 300 on 100/120 mesh Supelcoport using a Hewlett-Packard 5830A gas chromatograph and an LKB 9000 gas chromatograph-mass spectrometer as previously described (Sweetman et al., 1978). The concentrations of the acids were calculated from peak areas using GC response factors relative to n-triacontane determined for authentic standards carried through identical procedures. Concentrations of amino acids were determined quantitatively using-ion-exchange chromatography (Spackman et al., 1958). RESULTS The pattern of excretion of the organic acids in case 2 is shown in Figure 2. The concentrations of the significant acids in the urine of both patients are given in Table 1. The most striking quantitative abnormality in case 2 was the massive excretion of lactic acid (Rf42.3). The value of 668 ~nol/mg creatinine was more than 700 times the upper limit of normal. This was accompanied by large excretions of 2-hydroxybutyric acid (Rf21.0) of 9.4~nol/mg creatinine and pyruvic acid (Rf 19.1) of 8.7 gin,l/rag creatinine. Hippuric acid (Rf 9.8) was also increased at 32.4 ~tmol/mg creatinine. An increased concentration of 3-hydroxybutyric acid (Rf 36.2) at 19.8 gmol/mg creatinine was indicative of ketosis. The presence of large amounts of 3-methylcrotonylglycine (Rf 12.9) and 3-hydroxyisovaleric acids (Rf 16.1) in the urine made the diagnosis of 3-methylcrotonylglycinuria. The presence of increased quantitities of the two diastereoisomers ofmethylcitricacid (Rf83.4, 86.0) and 3-hydroxypropionic acid (Rf61.6) made the diagnosis of propionic acidaemia. The lactic and pyruvic aciduria is consistent with a defect in pyruvate metabolism. The urine sample from case 1 was less well preserved either as a result of shipment or the patient's urinary tract infection, and thus some of the amounts were lower than in case 2. The presence of a highly increased concentration of 3hydroxyisovaleric acid, as well as the increased amounts of 3-hydroxypropionic acid and methylcitric acid, established the presence of the same disorder in both infants. 3-Methylcrotonylglycinewas not detected in the urine of case 1 but 3-methylcrotonic acid was 8.2 gmol/mg creatinine, thus bacterial glycine deacylase probably cleaved 3-methylcrotonylglycine to its components; similarly hippuric acid had also been cleaved to yield benzoic acid (Hansen et al., 1972). The organic acid profile obtained by LPC of the serum of case 2 (Figure 2) was similar to that of the urine. Lactic acid (Rf 43.0) was 20 mmol/1 and 3-hydroxyisovaleric acid (Rf 16.3) at 2.14 mmol/l was highly increased. The concentration of propionic acid in the serum was 0.11 mmol/1 (normal < 0.01). 3-Methylcrotonic acid and 3-methylcrotonylglycine were not detected. Analysis of the amino acids in case 2 revealed a mild generalized aminoaciduria including iminoaciduria, suggesting a renal tubular defect that could have been a consequence of overwhelming illness. Plasma amino acid concentrations were not unusual. The serum organic acid concentrations and urine excretions were normal in a subsequent unaffected sibling

Sweetman, Nyhan, Sakati, Ohlsson, Mange, Boychuk and Kaye

52

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Figure 2 Liquid partition chromatograms of serum (upper panel) and urine (lower panel) in case 2. The amount of acid is proportional to the indicator titration at 350 nm. The numbers above the peaks are Rf values calculated with citric acid equated to 100. The significantacids are: Rf 9.8, hippuric acid; Rf12.9, 3-methylcrotonylglycine;Rf 16.1 and 16.3, 3-hydroxyisovalericacid; Rf 19.1, pyruvic acid and 2-hydroxybutyric acid; Rf 36.2 and 36.8, 3-hydroxybutyricacid, Rf 42.3 and 43.0, lactic acid; Rf 61.6, 3-hydroxypropionate; Rf 83.4 and 86.0, methylcitric acid diastereoisomers of case 2. Quite recently another sibling of case 2 has been born. This male infant was found to have increased concentrations of the metabolites characteristic of multiple carboxylase deficiency and to have biotinresponsive deficiencies of the enzymes of 3methylcrotonyl-CoA, propionyl-CoA and pyruvate deficiencies of the enzymes of 3-methylcrotonyl-CoA, propionyl-CoA and pyruvate carboxylases in leukocytes in vivo and fibroblasts in vitro (Wolf et al., 1980). DISCUSSION Both patients (cases 1 and 2) had presentations that permit the recognition of the diagnosis or at least the suspicion of the presence of a disorder of organic acid metabolism. The first patien', had a classic alerting finding, the presence of the skin eruption. This was present in the initial patient (Gompertz et al., 1971) and in four other patients (Cowan et al., 1979; Charles et al., 1979; Lehnert et al., 1979; Munnich et al., 1980). It has been variously described as resembling ichthyosis, seborrhoeic dermatitis, and acrodermatitis enteropathica; it most resembles the eruption and alopecia we have seen in acquired biotin deficiency (Sweetman et al., 1979). In the patients with multiple carboxylase Table 1 Excretion of organic acids in the urine (as pmol/mg creatinine)

Organic acid

Case 1

Case 2 Normal

3-Methylcrotonylglycine 3-Hydroxyisovalericacid Lactic acid 3-Hydroxypropionic acid Methylcitric acid