J. Inher. Metab. Dis. 7 Suppl. 1 (1984) 74-78

Pyruvate Carboxylase Deficiency K. BARTLETT, H. K. GHNEIM, J.-H. STIRK, G. DALE and K. G. M. M. ALBERTI Department of Clinical Biochemistry and Metabolic Medicine, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7R U, UK The ca~ses. of congenital ,lactic acidaemia are outlined. Isolated pyruvate carboxylase deficiency is reviewed detatl wIth a repo,rt of a recent case and a discussion of the biochemical consequences. Other causes of defective pyruvate carboxylation are described, particularly the combined carboxylase defects.

10

intermediate formation of biotinyl adenylate. The reaction is catalysed by holocarboxylase synthetase, thought to be a single enzyme although it has not been purified to homogeneity. The reaction catalysed by PC can be expressed as two partial reactions: firstly the MgATP-dependent carboxylation of the enzyme-bound biotin, and secondly the binding of pyruvate with transfer ofCO z to yield oxaloacetate. The native enzyme is a tetramer of identical subunits, each with a molecular weight of 12.5 kd. There is one biotin and one acetyl CoA binding site per monomer. The enzyme is activated by K +, Mg Z + and acetyl CoA and inhibited by MgADP and some acyl-CoA esters. These and other properties of PC have been reviewed in detail elsewhere (Utter et al., 1975). Liver and kidney show the highest activities of PC, although the enzyme can be detected in most tissues. Available evidence suggests that the gut uptake of biotin required for PC and the other biotin-dependent carboxylases is facilitated (Berger et al., 1972). The biotin is carried in the circulation to tissues where it enters cells by a carrier mediated process (Ghneim and Bartlett, unpublished findings). Biotinylation of apoPC, apopropionyl CoA carboxylase and apo-3methylcrotonyl CoA carboxylase occurs in the mitochondrion. The turnover of the biotin-dependent enzymes yields biocytin, the lysyl derivative of biotin, rather than free biotin. There exists a specific hydrolase, biotinidase (EC 3.5.1.12), to regenerate free biotin. Biotinidase is present in plasma at about the same specific activity as in liver on a gram wet weight basis, although its function in plasma is unknown. The functional relationship of endogenous recycled biotin and biotin derived from the diet also remains uncertain.

INTRODUCTION Lactic acidaemia is a common finding and it is important to establish that it is not the consequence of either muscular exercise or stress. A screaming but perfectly normal infant can generate extremely high blood lactate concentrations (Kollee et aI., 1977). Similarly we have observed that a fitting child produced a marked lactic acidaemia of 16mmol/l. Thus it is not surprising that lactic acidaemia is found in some children with neurological damage in whom there is no other evidence of biochemical disorder. Lactic acidaemia may be a perfectly normal response to a number of situations and does not necessarily indicate either a primary defect of pyruvate metabolism or a secondary effect of some other organic acidaemia. However, deficiencies of pyruvate carboxylase, pyruvate dehydrogenase, phosphoenolpyruvate carboxykinase, glucose-6-phosphatase and terminal electron transport are known to result in a lactic acidaemia. The remainder of this discussion will be limited to pyruvate carboxylase (PC; Pyruvate:CO z ligase (ADP) EC 6.4.1.1) and its disorders. The known disorders of PC are summarized in Table 1. PYRUVATE CARBOXYLASE STRUCTURE AND FUNCTION The place of PC in intermediary metabolism is well known. PC has a dual role; it is essential for gluconeogenesis from lactate and pyruvate in which it is probably rate-determining, but it also has an anaplerotic function. PC is a biotin-dependent carboxylase. Biotin is attached to the apocarboxylase via the £-amino group of a lysine residue in a two step process with the

ISOLATED PC DEFICIENCY-A CASE REPORT The patient (M.K.) was born full term to unrelated parents after an uneventful pregnancy and was the first child. At 36 h he was found to be hypothermic, hypotonic and refused to feed. He was transfered to the special care baby unit where he was found to be acidaemic (pH 6.92, pCO z 3.0 kPa), his urine contained ketones and there was an anion gap of 31 mmoljl. Urinary organic acid screening showed a raised lactate (10.4 mmoljmmol creatinine) and 3-hydroxybutyrate (0.56 mmoljmmol creatinine). The urinary amino acids were also abnormal. Citrulline, arginine and lysine were

Table 1 Causes of defective pyruvate carboxylase (1) Isolated pyruvate carboxylase deficiency (2) Combined carboxylase deficiency (a) Holocarboxylase synthetase deficiency (b) Biotinidase deficiency (c) Defective gut absorption (d) Defective renal reabsorption (e) Dietary biotin deficiency (3) Secondary to other organic acidaemias

74 Journal.of Inh.erited Metabolic Disease. ISSN 0141-8955. Copyright © SSIEM and MTP Press Limited, Queen Square, Lancaster

UK. Pnnted m the Netherlands.

'

75

Pyruvate Carboxylase Deficiency

elevated (107, 26 and 460 ~mol/~mol creatinine respectively), the other amino acids, alanine in particular, were normal. The plasma amino acids showed gross disturbances and are given in Table 2. There was normoglycaemia (8.3 mmoljl), hyperketonaemia (3hydroxybutyrate = 1.22 mmol/l) and a marked lactic acidaemia (10.8 mmoljl) with an abnormally high lactate/pyruvate ratio (25.6). Measurement ofleukocyte carboxylases showed a complete absence of pyruvate carboxylase, a result which was subsequently confirmed in cultured fibroblasts. The acidaemia was partially controlled with bicarbonate and therapy with biotin and aspartate attempted, neither of which altered his lactic acidaemia or clinical state. He remained hypotonic and unresponsive with increasing dyspnoea until his death at about 3 months of age. Autopsy was refused by the parents. The mother subsequently became pregnant, refused antenatal diagnosis and a second child was born who has proved not to have the disease. ISOLATED PC DEFICIENCY There have been about 20 cases of isolated PC deficiency described (De Vivo et aI., 1977; Robinson et al., 1980; Wolf and Feldman, 1982). In most the presentation is in early life with hypotonia, metabolic acidosis, developmental delay, and death within the first few years of life. Surviving patients are severely retarded. There have been suggestions that PC deficiency is the underlying defect in the subacute necrotizing encephalomyelopathy of Leigh (SNE) (Tada et aI., 1973). Indeed the first Table 2 Plasma amino acids in Patient M.K. with pyruvate carboxylase deficiency llffioljl

Amino acid Patient M.K. Aspartate Threonine Serine Asparagine Glutamate Glutamine Proline Glycine Citrulline Alanine 1/2 Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Tryptophan Ornithine Lysine Arginine

40 307 112 79 58 534

o

1637 392 1477 103 475 67 200 382 342 156 224 18 117 1292 229

Normal range 20-70 100-400 50-350 0-175 0--250 400--1600 50--450 200--600

0-40 100-800 0-100 50--400 0-80 0--150 20--180 30--135 40-110 45-150 0-50 25-225 105-315 10-70

Amino acids in bold type indicate concentrations > 5 SD from the mean

patient with hepatic PC deficiency reported by Hommes et al. (1968) was described as a case of SNE. In several

recent studies of autopsy-proven SNE, however, PC has been shown to be entirely normal (Hansen et al., 1982; Atkin et aI., 1979). Previous reports ofthe association of PC with SNE were ascribed to poor technique-the estimation of PC in liver biopsy being notoriously unreliable (Murphy et aI., 1981). It seems therefore that SNE and PC deficiency are separate entities, and that ifa deficiency in cultured cells or leukocytes cannot be shown then a diagnosis of PC deficiency is excluded. Isolated PC deficiency is inherited as an autosomal recessive condition (Atkin, 1979) and prenatal diagnosis has been reported (Marsac et al., 1981, 1982). Even if the reports of hepatic PC deficiency are discounted there still remains heterogeneity with respect to age at presentation, severity and metabolic consequences of PC deficiency. Somatic cell hybridization has, however, failed to demonstrate complementation (Feldman and Wolf, 1980) although biochemical heterogeneity has been shown (Hansen et aI., 1983; Robinson et aI., 1983; Robinson and Sherwood, 1984). BIOCHEMICAL CONSEQUENCES OF ISOLATED PC DEFICIENCY The citrullinaemia was the most striking of the amino acid changes observed in our patient. This has been observed previously in four patients reported by Coude et al. (1981), and is probably a result of aspartate shortage due to oxaloacetate depletion. This interpretation is consistent with the low level of TCA intermediates in liver (De Vivo et aI., 1977). Attenuation of the urea cycle due to substrate depletion is probably the mechanism of the hyperammonaemia sometimes seen. Although PC is essential for gluconeogenesis from lactate and pyruvate, hypoglycaemia was never observed in our patient and seems to be an inconsistent finding. Hyperketonaemia is however usually observed. It has been suggested that this is due simply to overflow of acetyl CoA from pyruvate due to oxaloacetate depletion. However, this is unlikely since pyruvate dehydrogenase is inactivated by high acetyl CoA/CoA ratios (Randle et al., 1979). Since the insulin/glucagon ratio is low in PC deficiency (De Vivo et aI., 1977), there will be increased lipolysis with increased delivery offatty acids to the liver. Furthermore since liver citrate islow, cytosolic acetyl CoA and hence malonyl CoA will be low, allowing unrestricted entry of fatty acids into the mitochondrial compartment and hence increased ~­ oxidation and ketogenesis (McGarry et aI., 1977). A feature of some patients with PC deficiency is a raised lactate/pyruvate ratio indicating that the cytosol is more reduced than normal. The reverse is true ofthe 3hydroxybutyrate/acetoacetate couple probably because ofimpaired activity ofredox shuttles due to oxaloacetate shortage. TREATMENT OF ISOLATED PC DEFICIENCY There appears to be no effective treatment for isolated PC deficiency. High fat diets exacerbate the hyperketonaemia, whereas high carbohydrate diets cause

Bartlett et al.

76 increased lactic acidaemia (De Vivo et aI., 1977). Aspartate treatment has been reported in a child with mild lactic acidaemia (1.9 mmol/l), low hepatic PC activity but normal leukocyte and fibroblast enzyme (Baal et at., 1981). Thiamine has also been reported to produce an amelioration of the lactic acidaemia (Brunette et al., 1972). Thiamine as its pyrophosphate is an inhibitor of PDH kinase and thus high thiamine may result in increased activity of PDH and therefore diversion of pyruvate to acetyl CoA (Randle, 1982). Biotin, when it has been tried, has never produced any clinical or biochemical response. PC DEFICIENCY IN COMBINED CARBOXYLASE DEFICIENCY Combined or multiple carboxylase deficiency (CCD) is a heterogenous group of disorders which have in common the excretion of a constellation of abnormal metabolites characteristic ofdefective activity ofall three mitochondrial carboxylases, defective activity of these enzymes in leukocytes and a marked and dramatic clinical and biochemical biotin-responsiveness (Leonard et at., 1981; Wolf and Feldman, 1982). Two variants have been distinguished on the basis of age at presentation-i.e. early- and late-onset CCD (Sweetman, 1981). In most instances the late-onset form is also characterized by the stigmata of dietary biotin deficiency, i.e. alopecia and skin rash (Sweetman et aI., 1981), in addition to the metabolic changes seen in the early-onset form of the disease. In early-onset CCD we have shown that there is defective activity of all three mitochondrial carboxylases in cultured cells, which can be reversed by the addition oflarge amounts of biotin to the culture medium (Bartlett et aI., 1980). In these individuals there is defective activity of the enzyme which attaches biotin to the apocarboxylases, holocarboxylase synthetase (Burri et al., 1981; Ghneim and Bartlett, 1982). However, in late-onset CCD no carboxylase defects can be demonstrated in cultured fibroblasts no matter what the concentration ofbiotin in the medium (Figure 1) although there is a marked

40

Normal

Biotinidase deficient

~30

~

~o

.

of

""'20

:I:

C, ~ V>

-aE'" c:

10

1234567

1234567

BIOTIN CONCENTRATION IN MEDIUM Inmoles/I)

Figure 1 The specific activities ofpropionyl CoA carboxylase (e--e), 3-methylcrotonyl CoA carboxylase (0--0), pyruvate carboxylase (D-~D), acetyl CoA carboxylase (. - - . ) and citrate synthase (L::-.--L:J in biotin-deficient fibroblasts from control and biotinidase-deficient subjects cultured in the presence of increasing concentrations of biotin

deficiency of all four enzymes in leucokytes which is reversed by biotin treatment (Table 3). This paradox has been recently resolved by the elegant studies ofWolfand his co-workers (Wolf et al., 1983a, b), who demonstrated that some patients with late-onset CCD have defective activity of biotinidase. These individuals become functionally biotin-deficient because they are unable either to recycle endogenous biotin derived from the turnover of the biotin-dependent enzymes, or to liberate dietary protein-bound biotin. There have been reports of defective gut transport and renal reabsorption (Munnich et al., 1981; Baumgartner et al., 1982), although the interpretation of the former has been questioned (Thoene and Wolf, 1983). We have confirmed biotinidase deficiency in a total of six cases, including the patient shown in Table 3 and Figure 1, of late-onset CCD using both the original colorimetric

Table 3 Leukocyte carboxylase activity in a patient with biotinidase deficiency before and after biotin treatment nmolmg- t h- t Enzyme

Propionyl CoA carboxylase 3-Methylcrotonyl CoA carboxylase Pyruvate carboxylase Acetyl CoA carboxylase Citrate synthase* • Units: nmolmg- 1 min- 1

Pre-biotin

2 months post-biotin

12 months post-biotin

Normal range

0.04

1.7

6.2

1.6-3.7

0.004

0.9

4.5

0.2-1.3

0

0.2

0.6

0.1-0.5

0

0.04

0.33

0.06-0.2

7.3

5.9

10.4

5.5-17.3

Pyruvate Carboxylase Deficiency

assay of Knappe et al. (1963) and a new sensItIve f1uorimetric assay we have developed (Wastell, Dale and Bartlett, 1984). We have recently drawn attention to the auditory and visual defects in these children, a previously unrecognized problem ofbiotinidase deficiency treated with high doses of biotin (Taitz et al., 1983). At present it is not dear if this is a consequence ofearly metabolic insult, biotin treatment or the accumulation of biocytin. The early reports of low to normal biotin in these children must now be discounted because existing biotin assays do not distinguish biotin and biocytin. PC DEFICIENCY SECONDARY TO OrnER ORGANIC ACIDAEMIAS Lactic acidaemia is frequently observed in patients in whom there is clearly no primary defect in pyruvate metabolism and is secondary to some other organic acidaemia. It is possible that this is due to inhibition of PC by the accumulation of abnormally high concentrations of acyl-CoA esters within the mitochondrial matrix. Evidence for this mechanism has been recently reported (Stirk et al., 1983). Pyruvate carboxylation was measured in intact rat liver mitochondria and was inhibited by some acids. This inhibition could be partially relieved by glycine, presumably because formation of the corresponding acylglycine lowered the matrix concentration of abnormal acyl-CoA. Similar effects on PDH have been reported (Gregersen, 1981), which would of course also contribute to impaired pyruvate disposal.

References Atkin, B. M. Carrier detection of pyruvate carboxylase in fibroblasts and lymphocytes. Pediatr. Res. 13 (1979) 1101-1104 Atkin, B. M., Buist, N. M. R., Utter, M. F., Leiter, A. B. and Banker, B. Q. Pyruvate carboxylase deficiency and lactic acidosis in a retarded child without Leigh's disease. Pediatr. Res. 13 (1979) 109-116 Baal, M. G., Gabreels, F. J. M., Renier, W.O., Hommes, F. A., Gijshers, Th. H. 1., Lamers, K. J. B. and Kok, J. C N. A patient with pyruvate carboxylase deficiency in the liver: treatment with aspartatic acid and thiamine. Dev. M ed. Child. Neural. 23 (1981) 521-530 Bartlett, K., Ng, H. and Leonard, J. V. A combined defect of three mitochondrial carboxylases presenting as biotinresponsive 3-methylcrotonylglycinuria and 3-hydroxyisovaleric aciduria. CUn. Chim. Acta 100 (1980) 183-185 Baumgartner, R., Suormala, T., Wick, H., Geisert, J. and Lehnert, W. Infantile multiple carboxylase deficiency: Evidence for normal absorption but renal loss of biotin. Helv. Paediatr. Acta 37 (1982) 499-502 Berger, E., Long, E. and Semenza, G. The sodium activiation of biotin absorption in hamster small intestine in vitro. Biochem. Biophys. Acta 255 (1972) 873-887 Brunette, M. G., Delvin, E., Hazel, B. and Scriver, C R. Thiamine-responsive lactic acidosis in a patient with deficient low-K m pyruvate carboxylase in liver. Paediatrics 50 (1972) 702-711 Burri, B. J., Sweetman. L. and Nyhan, W. L. Mutant holocarboxylase synthetase: evidence for the enzyme defect in early infantile biotin-responsive multiple carboxylase deficiency. J. Clin. Invest. 68 (1981) 1491-1495

77 Coude, F. X., Ogier, H., Marsac, C, Munnich, A., Charpentier, C and Saudubray, J.-M. Secondary citrullinaemia with hyperammonaemia in four neonatal cases of pyruvate carboxylase deficiency. Paediatrics 68 (1981) 914 De Vivo, D. C, Haymond, M. W., Leckie, M. P., Bussman, Y. L., McDougal, D. B. and Pagliara, A. S. The clinical and biochemical implications of pyruvate carboxylase deficiency. J. Clin. Endocrinol. Metab. 45 (1977) 1281-1296 Feldman, G. L. and Wolf, B. Evidence for two complementation groups in pyruvate carboxylase-deficient human fibroblast cell lines. Biochem. Genet. 18 (1980) 617-624 Ghneim, H. K. and Bartlett, K. Mechanism of biotinresponsive combined carboxylase deficiency. Lancet 1 (1982) 1187-1188 Gregersen, N. The specific inhibition of the pyruvate dehydrogenase complex from pig kidney by propionyl-CoA and isovaleryl-CoA. Biochem. Med. 26 (1981) 20-27 Hansen, T. L., Christensen, E. and Brandt, N. J. Studies of pyruvate carboxylase, pyruvate decarboxylase and lipoamide dehydrogenase in subacute necrotizing encephalomyelopathy. Acta Paediatr. Scand. 71 (1982) 263-267 Hansen, T. L., Christensen, E., Willems, J. L. and Trijbels, J. M. F. A mutation of pyruvate carboxylase in fibroblasts from a patient with severe, chronic lactic acidaemia. CUn. Chim. Acta 131 (1983) 39-44 Hommes, F. A., Polman, H. A. and Reerink, 1. D. Leigh's encephalopathy: an inborn error of gluconeogenesis. Arch. Dis. Child. 43 (1968) 423-426 Knappe, J., Brummer, W. and Biederbick, K. Reinung und Eigenschaften der biotinidase aus schweinenieren und Lactobacillus Casei. Biochem. Z. 338 (1963) 599-613 Kollee, L. A. A., Willems, J. L., De Kort, A. F. M., Monnens, L. A. H. and Trijbels, 1. M. F. Blood sampling technique for lactate and pyruvate estimation in children. Ann. Clin. Biochem. 14 (1977) 285-287 Leonard, J. V., Seakins, J. W. T., Bartlett, K., Hyde, J., Wilson, 1. and Clayton, B. Inherited defects of 3-methylcrotonylCoA carboxylation. Arch. Dis. Child. 56 (1981) 53-59 McGarry, J. D., Mannaerts, G. P. and Foster, D. W. A possible role for malonyl-CoA in the regulation of hepatic fatty acid oxidation and ketogenesis. J. Clin. Invest. 60 (1977) 265-270 Marsac, C, Augereau, Ch., Boue, 1. and Vidailhet, M. Antenatal diagnosis of pyruvate carboxylase deficiency. Lancet 1 (1981) 675 Marsac, C, Augereau, Ch., Feldman, G., Wolf, B., Hansen, T. L. and Berger, R. Prenatal diagnosis ofpyruvate carboxylase deficiency. CUn. Chim. Acta 119 (1982) 121-127 Munnich, A., Saudubray, 1.-M., Carre, G., Coude, F. X., Ogier, H., Charpentier, C and Frezal, J. Defective biotin absorption in multiple carboxylase deficiency. Lancet 2 (1981) 263 Murphy, J. V., Isohashi, F., Weinberg, M. B. and Utter, M. F. Pyruvate carboxylase deficiency: An alleged biochemical cause of Leigh's disease. Pediatrics 68 (1981) 401-404 Randle, P. J. Congentiallactic acidoses. In Metabolic Acidoses (Ciba Foundation Symposium No 87), Pitman Press, Bath, 1982, p. 354 Randle, P. J., Sugden, P. H., Kerbey, A. L., Radcliffe, P. M. and Hutson, N. 1. Regulation of pyruvate oxidation and the conservation of glucose. Biochem. Soc. Symp. 43 (1979) 47-67 Robinson, B. H., Taylor, 1. and Sherwood, W. G. The genetic heterogeneity of lactic acidosis: occurrence of recognisable inborn errors of metabolism in a paediatric population with lactic acidosis. Paediatr. Res. 14 (1980) 956-962 Robinson, B. H., Oei, J., Saunders, M. and Gravel. R. [3H]Biotin-labelled proteins in cultured human skin

78 fibroblasts from patients with pyruvate carboxylase deficiency. J. Bio/. Chem. 258 (1983) 6660-6664 Robinson, B. H. and Sherwood, W. G. Lactic acidaemia. J. Inher. Melab. Dis. 7, Supp!. 1 (1984) 69-73 Stirk, J.-H., Bartlett, K. and Sherratt, H. S. A. The effects of some short-chain fatty acids on pyruvate carboxylase activity in intact isolated rat liver mitochondria. Biochem. Soc. Trans. 11 (1983) 286-287 Sweetman, L. Two forms of biotin-responsive multiple carboxylase deficiencies. J.lnher. Metab. Dis. 4 (1981) 53-54 Sweetman, L., Suhr, L., Baker, H., Petersen, R. M. and Nyhan, W. L. Clinical and metabolic disorders in a boy with dietary deficiency of biotin. Pediatrics 68 (1981) 553-558 Tada, K., Sugita, K., Fujikawi, K., Kesai, T., Takada, G. and Omura, K. Hyperalaninaemia with pyruvicaemia in a patient suggestive of Leigh's encephalomyelopathy. J. Pediatr. 109 (1973) 13-18 Taitz, L. S., Green, A., Strachan, I., Bartlett, K. and Bennet, M. Biotinidase deficiency and the eye and ear. Lancet 2 (1983) 918

Bartlett et al. Thoene, 1. and Wolf, B. Biotinidase deficiency in juvenile multiple carboxylase deficiency. Lancet 2 (1983) 398 Utter, M. F., Barden, R. E. and Taylor, B. L. Pyruvate carboxylase: an evaluation of the relationships between structure and mechanisms and between structure and catalytic activity. Adv. Enzymol. 42 (1975) 1-72 Wastell, H., Dale, G. and Bartlett, K. A sensitive fluorometric rate assay for biotinidase using a new derivative of biotin, biotinyl-6-aminoquinoline. Anal. Biochem. (1984) in press Wolf, B. and Feldman, G. L. The biotin-dependent carboxylase deficiencies. Am. J. Hum. Genet. 34 (1982) 699-716 Wolf, B., Grier, R. E., Parker, W. D., Goodman, S. I. and Allen, R. J. Deficient biotinidase activity in late-onset multiple carboxylase deficiency. N. Eng/. J. Med. 308 (1983a) 161 Wolf, B., Grier, R. E., Allen, R. 1., Goodman, S. I. and Kien, C. L. Biotinidase deficiency: the enzymatic defect in late-onset multiple carboxylase deficiency. Clin. Chim. Acta 131 (1983b) 273-281