D-2-hydroxyglutaric aciduria: Case report and biochemical studies

J. Inher. Metab. Dis. 3 (1980) 11-15

D-2-Hydroxyglutaric Aciduria: Case Report and Biochemical Studies R. A. CHALMERS, A. M. LAWSON, R. W. E. WATTS and A. S. TAVILL

Divisions of lnherited Metabolic Diseases, CIh~ical Chemistry and Clinical hTvestigation, MRC Clinical Research Centre, WatJord Road, Harrow HA1 3UJ J. P. KAMERLING

Bio-organic Chemistry Department, University of Utrecht, The Netherlands E. HEy and D. OGILWE

The Hospital for Sick Children, Great Ormond Street, London A patient with protein-losing gastroenteropathy and egg allergy has been shown to have a previously unrecognized organic aciduria, D-2-hydroxyglutaric aciduria. The observations made are consistent with an inherited metabolic disorder in the catabolism of 5-aminolaevulinate possibly due to deficient activity of a specific D-2-hydroxyglutarate dehydrogenase. A patient with protein-losing gastroenteropathy and egg allergy was found to excrete greatly increased amounts of 2-hydroxyglutaric acid in his urine (Chalmers and Lawson, 1976). Clinical and biochemical studies during 6 years have shown this previously unrecorded organic aciduria to be maintained and recent work has identified the 2-hydroxyglutaric acid as the D (or R) enantiomer. The observations made are consistent with the presence of an inherited metabolic disorder in the catabolism of hydroxylysinr and particularly of 5-aminolaevulinic acid. This paper presents the case report of the patient and the results of studies on urinary organic acids.

biopsy, barium enema, barium meal and a modified Lundh test were all normal. Slit lamp examination for a Kayser-Fleischer ring was negative and stools on several occasions were free from parasites, ova and pathogenic bacteria. Further investigations at 8.5 years showed that serum B~z and folate, thyroid function and liver function were normal. 13rinary albumin was negative and lactose tolerance was normal. Stools were again free from parasites but showed Charcot-Leyden crystals. A skin test showed a strongly positive ~eaction to milk and eggs and a weakly positive reaction to fish, and, following ingestion of approximately 6g of egg, he immediately vomited and developed abdominal pain. There was an urticarial rash within 2-3 minutes and a mild wheeze, puffy eyes and an itch within half an hour. There was little or no reaction to milk. The [14C]glycocholate breath test was normal, indicating no blind loop or bacterial overgrowth. Plasma and urinary amino acids were normal. An albumin turnover study was carried out using intravenously administered [~31I]albumin. Plasma volume was 1226 ml, equivalent to 60.5 ml/kg body weight (normal 41.1 + 5.6). Plasma albumin concentration was 20 g/l. The intravenous albumin pool was 24.5 g, equivalent to 1.20 g/kg (normal 1.73 g + 0.02). The fractional catabolic rate of albumin was 44% of the intravenous pool per day (normal 7-13%) with a half-life of 2 days (normal 17-21 days). This gave an absolute catabolic rate of 10.8g/day, equivalent to 5 3 3 m g k g - ~ d a y -a (normal 188 + 14 mg k g - ~d a y - t), Intestinal plasma protein loss was measured by means of a StCr plasma clearance study. The plasma clearance of StCr into the stool was equivalent to the loss of 800ml/day. The normal clearance in the adult is usually less than 50 ml/day (equivalent to l - 2 g albumin/day). This suggests that the vast majority of the greatly increased albumin catabolic rate in this child could be accounted

PATIENT: CASE R E P O R T The patient was a Kenyan Asian boy, the first child of first cousin parents, normal at birth but presenting with anaemia and a history of recurrent antibiotic-resistant chest infections from 3 years of age. Significant findings on admission at 4.5 years included a haemoglobin of 5.9g per 100ml, eosinophils of 1520 per mm 3, serum iron of 27 lag per 100 ml (normal 80-120), and total iron binding capacity (TIBC) of 139 lag per 100 ml. Serum iron and TIBC rose to 320 lag per 100 ml and 408 ~tg per 100 ml respectively 3 hours after an oral iron load and iron deficiency anaemia was diagnosed. Serum albumin at this time was 2.6 g per 100 ml. He was discharged on oral iron. He was referred again at 7 years with recurrent chest infections and lethargy, with a haemoglobin of 4 g per 100 ml, eosinophils 2000 per mm 3, serum iron 7 lag per 100 ml, TIBC 219 lag per 100 ml, and serum albumin of 2.5 g per 100 ml. There was an increased loss of albumin in the stools. Serum copper and caeruloplasmin were reduced at 25lag per 100ml (normal 80-170) and 4lag per 100ml (normal >20) respectively. Bone marrow was diagnostic of iron deficiency anaemia, jejunal

II

12

Chalmers, Lawson, Watts. Tavill, Kamerling, Hey and Ogih,ie L-2-hydroxyglutarate (Fluka AG, Buchs, Switzerland) were converted into the corresponding acids using Dowex 50 (H+). ( - ) - 2 - B u t a n o l i c HCI (1 mol/I HCI) was prepared by bubbling dry HCI gas through ( - ) - 2 butanol (Fluka AG) and was stored at - 1 8 ~ in a desiccator. The commercially available ( - ) - 2 - b u t a n o l contains about 670 of the ( +)-enantiomer (Gerwig et al., 1978). The free 2-hydroxyglutaric acids (5 mg) and the urinary organic acids, extracted from 8 ml of urine with ethylacetate (6mg of 2-hydroxyglutaric acid), were esterified by adding 0.5 ml ( - ) - 2 - b u t a n o l i c I mol/I HCI

for by the exudative loss of intact plasma into the gut as a feature of a severe protein-losing enteropathy produced by an allergic response to the dietary intake of eggs. The patient, now aged 13 years, has remained clinically unchanged, with asthma and allergic rhinitis being his major symptoms. His weight gain has been poor (now below the third percentile for height and weight), iron deficiency anaemia persists, becoming more severe when he discontinues oral iron, and he shows persistent hypoalbuminaemia and intermittent eosinophilia. He shows low IgG and IgM, and high lgE to all allergens tested. He is not retarded and attends normal school. There is no family history of food allergy or pulmonary disease except for asthma in his maternal grandfather and hay fever of recent onset in mother and father. He has a younger, healthy sister whose urinary organic acid profile is normal.

!213 697

MATERIALS AND METHODS

Urinary organic acids were determined using established procedures involving extraction on to DEAE Sephadex and analysis by gas-liquid chromatography of their trimethylsilyl (TMS) and TMS-ethoxime derivatives (Chalmers and Watts, 1972; Healy et al., 1973; Chalmers et al., 1977). Deproteinized blood plasma and whole blood were examined by similar methods. Organic acids were identified by gas chromatography and mass spectrometry using repetitive scanning and maximizing ion techniques, the latter giving 'purer' mass spectra and the impression of components eluting in smaller retention volumes, enabling the presence of certain peaks to be detected that are obscured in conventional chromatograms due to lack of resolution on packed columns (Chalmers et al., 1977). The absolute configuration of 2-hydroxyglutaric acid was determined by capillary gas-liquid chromatography after esterification with ( - ) - 2 - b u t a n o l and acylation with acetic anhydride. Zinc DL-2-hydroxyglutarate (Sigma Chemical Co., St Louis, Mo., USA) and sodium

I

l-~J~~

I I I

I I I I" I I I I I

Minutes

Figure 1 Gas-liquid chromatogram of urinary organic acids

from a patient with D-2-hydroxyglutaric aciduria. Separated as their trimethylsilyl (TMS) and TMS-ethoxime derivatives on 10~o OV 101 on Chromosorb W (HP), 80-100 mesh, by temperature programming frorn 110~ to 285~ at 4 ~ min- ~, with a 5-minute initial isothermal delay. Peak identifications are : (1) glycollic acid; (2) sulphate; (3) phosphate; (4) succinic acid: (5) 2-deoxytetronic acid; (6) 2-hydroxyglutaric acid (o); (7) 2-oxoglutaric acid; (8) 4-hydroxyphenylacetic acid; (9) citric acid; (10) undecandioic acid (internal standard); (11) urate: (12) n-tetracosane; (13) n-hexacosane (internal standard)

Table 1 Quantitative excretion of urinary organic acids in a patient with D-2-hydroxyglutaric aciduria

Glycollic Succinic D-2-Hydroxy- 2-Oxoghttaric glutaric mg per g creatinine at 8.6 years at 10.3 years normal controls mg per 24 hours (at 10.3 years) mmol per 24 hours (at 10.3 years)

230 110 > 70 8 O.l

90 5 2-75 10 0.1

Total Total volume creathlhw (ml) (rag) at 10.3 years 0900-1800 1800~)800

270 315

192 274

1200 1980 :1,20 799 5.4

60 250 2-51 65 0.4

D-2-Hydroxyghttaric acid Total (rag)

(mg/h)

294 475

32.7 33.9

D-2-Hydroxyglutaric A ciduria : Case Report and Biochemical Studies

and heating for 2 hours at 100~ The solvent was evaporated under reduced pressure and the residue acetylated in pyridine-acetic anhydride (1:1 v/v, I ml) for 30 minutes at 100~ After evaporation in the presence of absolute ethanol, the residue was dissolved in chloroform. The O-acetylated 2-hydroxyglutaric acid di-(-)-2-butyl esters were analysed by capillary gasliquid chromatography on SP 1000 as non-chiral stationary phase at 160~ as described elsewhere (Kamerling et al., 1977). RESULTS Figure 1 illustrates the gas chromatogram of urinary organic acids from the patient. The TMS-2-hydroxyglutaric acid peak is grossly increased and there are

13

slight increases in 2-oxoglutaric, succinic and glycollic acid excretion. Excretion of 4-hydroxyphenylacetic acid and other urinary organic acids are normal. Quantitative levels of excretion are given in Table 1.2-Hydroxyglutaric acid was not detectable in the patient's blood. Figure 2 compares the capillary gas chromatograms of authentic DL- and L-2-hydroxyglutaric acid (O-acetyl, (-)-2-butyl esters) with that of the urinary 2-hydroxyglutaric acid. The absolute configuration of the acid extracted from the patient's urine is D (or R). The L-enantiomer gas chromatographic peak shows a shoulder with a higher retention time corresponding to the position of the D-enantiomer owing to the presence of a small amount of (+)-enantiomer in the (--)-2butanol sample. A similar small peak, corresponding to the position of the L-enantiomer was observed in the extract of the patient's urine. The esterification procedure also gave rise to lactone formation with the production of an additional gas chromatographic peak.

A

|octone

O

lacton~~ LD

D

toctone

Figure 2 Capillary gas-liquid chromatograms of O-acetylated di-(-)-2-butyl esters of (A) authentic L-2-hydroxyglutaric acid, (B) authentic DL-2-hydroxyglutaric acid, and (C) of the urinary acid, separated on SPI000 at 160~

DISCUSSION The patient has a protein-losing gastroenteropathy secondary to egg allergy. Allergic gastroenteropathy is rare, the clinical manifestation usually resulting from a type I, anaphylactic reagin-dependent hypersensitivity to a particular protein (in this case an egg protein, possibly ovomucoid or ovalbumin), probably by direct exposure of the intestinal surface to the antigen (Golbert, 1972; Freier, 1973). While allergic gastroenteropathy is frequently of familial occurrence, there is no evidence in the present case to link the disorder to the abnormal organic aciduria. It has not proved possible to undertake stress tests of his allergy while collecting blood and urine specimens for organic acid analysis. 2-Hydroxyglutaric acid is a normal constituent of human urine at low concentrations ( < 20 mg/g creatinine) (Lawson et al., 1975) and is increased in some patients with abnormal 2-oxoglutaric aciduria, for example pyruvate decarboxylase deficiency (Chalmers et al., 1977) and pyruvate carboxylase deficiency (Van Biervliet et al., 1977), but not in others with gross 2-oxoglutaric aciduria, for example glucose-6-phosphatase deficiency (Chalmers et al., 1978). The absolute configuration of this acid is not known. The occurrence of a grossly increased excretion of 2-hydroxyglutaric acid is extremely rare and has not been observed by the present authors in urine from more than 3500 normal healthy individuals including newborns and infants, mentally-subnormal patients, or acutely ill newborns, infants and children. It has not been observed by other workers either, although Duran et al. (1979) have recently reported increased excretion of L-2-hydroxyglutaric acid in a retarded dystrophic patient. The significance of this finding in relation to the present case is unclear, although it is of interest that the patient of Duran et al. also had severe anaemia. The origin of L-2-hydroxyglutarate is unknown. It has been proposed that it is produced by the anaerobic dismutation of 2-oxoglutarate in animal tissues (WeilMalherbe, 1937), but this has not been confirmed by later workers (Lindahl et al., 1967). It may be the

14

Chahners, Lawson, Watts, Tavill, Kamerling, He), and Ogih,ie

product of L-malate dehydrogenase (L-malate:NAD oxidoreductase, EC 1.1.1.37) action on 2-oxoglutarate (Davies and Kun, 1957). Jerzykowski et al. (1973) have shown that D-2-hydroxyglutarate occurs in mammalian systems as an intermediate between ~,~5-dioxovalerate and 2-oxoglutarate in the succinate-glycine cycle originally proposed by Shemin et al. (1955) (Nemeth et al., 1957; Tubbs and Greville, 1961) (Figure 3). It has also been proposed as an intermediate in the metabolism of 5-hydroxy-L-lysine (Lindahl et al., 1967), but this has not been confirmed by other workers (Polan et al., 1967; Hammerstedt et al., 1968) and tiffs hydroxyamino acid is believed to be metabolized primarily via phosphohydroxylysine and 2-aminoadipic acid and thence to glutaryl CoA and acetyl CoA via the normal pathway of L-lysine degradation in mammals (Hiles et al., (1972). It is of interest, however, that D-2-hydroxyglutarate may be a major obligate intermediate in the metabolism of 2-amino-L-adipate by Pseudomonas putida (Kopchick and Hartline, 1979), this bacterium also exhibiting the alternative pathway via glutaryl CoA as in mammals and in P. fluorescens (Numa et al., 1964), when the pathway is induced by glutarate. These observations suggest that o-2-hydroxyglutarate may arise by other m e c h a n i s m s than the s u c c i n a t e - g l y c i n e cycle in mammalian systems.

mitochondria. Thus it is most probable that a specific o-2-hydroxyglutarate dehydrogenase also occurs in mammalian mitochondria. The constant excretion by the present patient of large amounts of o-2-hydroxyglutarate despite treatment of his iron-deficiency anaemia and chest infections (antibiotics), and the absence of evidence for bacterial colonization of the gut (normal [a4C]glycocholate breath test and 4-hydroxyphenylacetic acid excretion), suggests that he has an inborn metabolic disorder possibly, in the absence of other metabolic products at increased levels, of a specific D-2-hydroxyglutarate dehydrogenase. With an unremarkable family history, a normal healthy younger sibling and first cousin parents, an autosomal recessive mode of inheritance is possible. The enzyme defect may be relatively benign and any clinical consequences are probably masked in the present case by those due to his gastroenteropathy. We have had interesting discussions with Dr M. Duran and Prof. Dr S. K. Wadman on the metabolic origins and fate of 2-hydroxyglutaric acid. MS received 1.1.80 A cceptedfor publication 22.1.80

Porphynns, Haern.

1

5,aminolaevulinale . ~ C H : N H : 9CO ' (CH=)=" COOH

2-amino-3-oxoadipate HOCO" CHNH= * CO(CH=}= 9COOH

S-2 - hydloxyglularyl- GSH SG" CO" CHOH 9(CH=)=" COOH

I Glycine

oo. I l~

Succinyl CoA ~r

References

2(4)-o~oglularate semialdehyde (y.6-dioxovalelate) OCH, CO. (CHz) ~ 9COOH

2-oxoglularate

CO" (CH=)= 9COOH I

PnOPOSEODEFECT "

~'

L-2-hydroxyglutarate

?

COOH

G

Glyoxylate I

=* Gi,/collate

Figure 3 Metabolic pathway involved in D-2-hydroxyglutarate metabolism with site of proposed defect D-2-Hydroxyglutarate, produced from 5-aminolaevulinic acid via the intermediate y,~5-dioxovalerate (2(4)oxoglutarate semialdehyde) (Figure 3), is oxidized by mammalian tissues to 2-oxoglutarate, and thence into the tricarboxylic acid cycle, by mitochondrial D-2hydroxyglutarate dehydrogenase (EC 1.1.99.6) (Tubbs and Greville, 1961). This mitochondrial enzyme resembles those found in yeast, Escherichia coil and other bacteria, the enzyme in E. coli being associated with cytochrome-containing particles. A specific o-2-hydroxyglutarate oxidoreductase of P. putida that catalyses the oxidation of D-2-hydroxyglutarate to 2-oxoglutarate has been reported by Reitz and Rodwell (1969) who described it as an inducible membrane-bound enzyme of the electron transport particle of the bacterium that resembled, in enzymic properties and chemical composition, the electron transport particles of beef heart

Chalmers, R. A. and Watts, R. W. E. The quantitative extraction and gas-liquid chromatographic determination of organic acids in urine. Analyst 97 (1972) 958-967 Chalmers, R. A. and Lawson, A. M. 2-Hydroxyglutaric aciduria: gas chromatographic and mass spectrometric studies of organic acids in a patient. Proc. 2rid European Congress on Clinical Chemistry, Prague (Abstract) (I 976) Chalmers, R. A., Lawson, A. M. and Borud, O. Gas chromatographic and mass spectrometric studies on urinary organic acids in a patient with congenital lactic acidosis due to pyruvate decarboxylase deficiency. Cfin. Chhn. Acta 77 (1977) 117-124 Chahners, R. A., Ryman, B. E. and Watts, R. W. E. Studies on a patient with hi vivo evidence of glycogenosis type I and normal enzyme activities in vitro. Acta Paediatr. Stand. 67 (1978) 201-207 Davies, D. D. and Kun, E. Isolation and properties of malic dehydrogenase from ox-heart mitochondria. Biochem. J. 66 (1957) 307-316 Duran, M., Kamerling, J. P., Bakker, H. D., van Gennip, A. H. and Wadman, S. K. L-2-Hydroxyglutaricaciduria : an inborn error ofmetabolism ? J. lnher. Metab. Dis. 3 (1980)in press Freier, S. Paediatric gastrointestinal allergy. Clin. Allergy 3 (1973) 597~518 Gerwig, G. J., Kamerling, J. P. and Vliegenthart, J. F. G. Determination of the D and L configuration of neutral monosaccharides by high resolution capillary GLC. Carbohydrate Res. 62 (1978) 349-357 Golbert, T. M. Food allergy. In Patterson, R. (ed.) Allergic Diseases: Diagnosis and Management. J. B. Lippincott Co., Toronto, 1972, p. 355 Hammerstedt, R. H., Swan, P. B. and Henderson, L. M. Degradation of 5-hydroxylysine in the rat and in the perfused liver. Arch. Bioehem. Biophys. 128 (1968) 243-251

o-2-Hydroxyglu taric A eiduria : Case Report and Biochemical Studies

Healy, M. J. R., Chalmers, R. A. and Watts, R. W. E. Reduction of data from the automated gas-liquid chromatographic analysis of complex extracts from human biological fluids using a digital electronic integrator and an off-line computer program. J. Chromatogr. 87 (1973) 365-377 Hiles, R. A., Willett, C. J. and Henderson, L. M. Hydroxylysine metabolism in rats, mice and ch'ickens. J. Nutr. 102 (1972) 195-204 Jerzykowski, T., Winter, R. and Maturzewski, W. ,/,6-Dioxovalerate as a substrate for the glyoxalase enzyme system. Biochem. J. 135 (1973) 713-719 Kamerling, J. P., Gerwig, G. J., u J. F. G., Duran, M., Ketting, D. and Wadman, S. K, Determination of the configurations of lactic and glyceric acids from lmman serum and urine by capillary gas-liquid chromatography. J. Chromatogr. 143 (1977) 117-121 Kopchick, J. J. and Hartline, R. A. ct-Hydroxyglutarate as an intermediate in the catabolism of c~-aminoadipate by Pseudomonas putida. J. Biol. Chem. 254 (1979) 3259-3263 Lawson, A. M., Chalmers, R. A. and Watts, R. W. E. Urinary organic acids in man. I. Normal patterns. Clin. Chem. 22 (1976) 1283-1287 Lindahl, G., Lindstedt, G. and Lindstedt, S. Metabolism of 2-amino-5-hydroxyadipic acid in the rat. Arch. Biochem. Biophys. 119 (1967) 347-352

15

Nemeth, A. M., Russell, C. S. and Shemin, D. The succinateglycine cycle. II. Metabolism of 8-aminoiaevulinic acid. J. Biol. Chem. 229 (1957) 415M22 Numa, S., Ishimura, Y., Nakazawa, T., Okazaki, T. and Hayaishi, O. Enzymic studies on the metabolism~ of glutarate in Pseudomonas. J. Biol. Chem. 239 (1964) 391~-3926 .Polan, C. E., Smith, W. G., Ng, C. Y., Hammerstedt, R. H. and Henderson, L. M. Metabolism of hydroxylysine by rats. J. Nutr. 91 (1967) 143-150 Reitz, M. S. and Rodwell, V. W. u-Hydroxyglutarate oxidoreductase of Pseudomonas putida. J. BacterioL 100 (1969) 708-714 Shemin, D., Russell, C. J. and Abramsky, T. The succinateglycine cycle. II. The mechanism of pyrrole synthesis. J. Biol. Chem,. 215 (1955) 613~i26 Tubbs, P. K. and Greville, G. D. The oxidation of D-cthydroxyacids in animal tissues. Biochem. J. 81 (1961) 104-114 Van Biervliet, J. P. G. M., Bruinvis, L., van der Heiden, C., Ketting, D., Wadman, S. K., Willemse, J. L. and Monnens, L. A. H. Report of a patient with severe, chronic lactic. acidaemia and pyruvate carboxylase deficiency. Devel. Med. Child. Neurol. 19 (1977) 392--401 WeiI-Malherbe, H. The oxidation of/(-)