Dicarboxylic aminoaciduria: An inborn error of amino acid conservation

422

September 1977 TheJournalofPEDIATRICS

Dicarboxylic aminoaciduria: An inborn error of amino acid conservation A 38-month-oM apparently healthy male has been followed for three years because of a massive glutamic and aspartic aminoaciduria detected shortly after birth in a neonatal screening program. Amino acid clearance studies revealed the presence o f renal wastage of dicarboxylic amino acids. Intestinal transport and in vitro oxidation of dicarboxylic amino acids were found to be intact. Clinical and metabolic data obtained on a previously described patient and the present case suggest that some patients with dicarboxylic aminoaciduria might have a selective renal conservation defect without clinical abnormalities, whereas others might demonstrate an additional defect in intestinal transport associated with fasting hypoglycemia.

Serge B. Melanqon,* L o u i s D a l l a i r e , Bernard Lemieux, Pierre Robitaille, and M i c h e l P o t i e r , M o n t r e a l , P . Q . , C a n a d a

THE DICARBOXYLIC AMINO ACIDS, aspartic a n d glutamic, are f o u n d in very small a m o u n t s in n o r m a l h u m a n urine even i n very y o u n g children. To our knowledge increased u r i n a r y excretion o f b o t h dicarboxylic a m i n o acids w i t h o u t generalized a m i n o a c i d u r i a has b e e n reported only once in a two-year-old athyreotic girl with oligophrenia, growth retardation, h y p e r p r o l i n e m i a a n d fasting hypoglycemia, a n d ketoacidosis.' W e report the results o f clinical a n d m e t a b o l i c observations in a 38m o n t h - o l d male infant, suggesting the occurence of dicarboxylic a m i n o a c i d u r i a as a n i n b o r n error o f a m i n o acid conservation w i t h o u t associated neurologic a n d developm e n t a l abnormalities.

CASE REPORT The patient (B.D.: 10-15-1973) was referred to us at the age o f two months for investigation of an abnormal glutamic aciduria

From the Departments of Pediatrics, University of Montreal and Sherbrooke, and the Medical Genetics Laboratory, Pediatric Research Center, Ste-Justine Hospital. Supported by grants from the Medical Research Council of Canada (MA-4767) and the Ministry of Social Affairs of the Province of Quebec. Reprint address: Section of Medical Genetics, Ste-Justine Hospital 3175 Cbte Ste-Catherine, Montreal, Quebec, H3T 1C5. *Recipient of a Scholarshipfrom the Medical Research Council of Canada. Vol. 91, No. 3, pp. 422-427

observed through the Quebec Network of Medical Genetics newborn detection program. 2 He was the second child of unrelated French-Canadian parents. A sister, age seven years, and both parents were in good health. Monthly physical examinations from two to 12 months of age remained essentially normal. At the age of one year, the child was admitted to Ste-Justine Hospital for detailed investigation. On physical examination, the height (72 cm), weight (10 kg), and head circumference (48 cm) were within normal limits. Psychomotor development agreed with expected standards for his age. Observed values for the following laboratory determinations were within normal range: fasting blood glucose, urea nitrogen, ammonia, creatinine, uric acid, calcium, phosphorus, alkaline phosphatase, serum protein, electrolytes,

Abbreviations used FIGLU: formiminoglutamic acid CSF: cerebrospinal fluid

lactate, pyruvate, lactic, and glutamic dehydrogenases, glutamicoxalacetic and glutamic-pyruvic transaminases. Urine obtained by catheterization showed no bacterial growth. There was no proteinuria, phosphaturia, or glycosuria a n d renal acidification of urine after an oral dose of sodium bicarbonate remained effective. Bone age was consistent with chronologic age; there was no evidence of rickets. A D-xylose absorption test and an electroencephalogram were interpreted as normal. Between 13 and 38 months of age, the child experienced three acute episodes of bronchial asthma, four brief bouts of diarrhea, and, on two occasions, atopic dermatitis over the legs and forearms. All of these minor illnesses responded well to therapy; the overall

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Dicarboxylic aminoaciduria

423

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Fig. 1. Tracing of major peaks from an ion-exchange chromatogram of urine on patient ( increased excretion of both dicarboxylic amino acids. development of the child has been assessed as adequate by his attending pediatrician. METHODS Analytical procedures. Plasma, cerebrospinal fluid, and urine amino acids were measured on a Technicon and a Beckman 121 automatic amino acid analyzers using the sodium citrate elution buffers recommended for physiologic fluid procedures 9 F o r m i m i n o g l u t a m i c acid was determined by a semiquantitative procedure using onedimensional paper chromatography in isopropanol-formic acid-water (75:12.5: 12.5, v / v ) and exposing the sheet to ammonia vapors before dipping in ninh~cdrin reagent. Organic acids in physiologic fluids were determined as trimethylsilyl esters and ethers on a Packard model 7300 gas-chromatograph after solvent extraction with ether and ethyl acetate? Quantitative extraction of a-ketoacids was achieved after oximation.' Further identification of the derivatized organic compounds was made by massspectrometry on a LKB model 9000 GC-MS. Cultured skin fibroblasts oxidation studies were performed according to procedures described by Blass and associates,:' using sodium pyruvate, L-aspartic acid, and L-glutamic acid, all U -~4 C-labeled (AmershamSearle).

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Table I. Fasting plasma and cerebrospinal fluid amino acid concentrations in patient with dicarboxylic aminoaciduria

Amino acids (~M/I) Aspartic acid l Glutamic acid I Patient plasma*

2.6 (tr-5.0)

Control plasmat Patient CSF~ Normal CSFw

15.5 (tr-32.1 tr 0.9 _+ 0.5

48.4 (24.8-87.2) 114.1 (75.2-203.1) 7.5 7.0 _+ 4.9

Proline

212 (70-388) 222 (159-278) 0 0.6 -

*Mean and range of five determinations in patient after an overnight fast. tMean and range in 13 children 4 weeks to 4 years of age. ~:One determination only. w _+ SD in 18 patients 4 months to 42 years of age;

M e t a b o l i c s t u d i e s . Oral loads of L-glutamic acid (0.1 g m / k g as the free acid) and histidine ( H C L monohydrate 0.5 gm/kg) were performed after an overnight fast. Urine was collected on ice 4 hours before and 8 hours after the load for amino acid and organic acid determinations. Venous blood was drawn using the heparin lock technique '~ at 0, 1, 2, and 4 hours, respectively, following the

424

Melan~on et al.

The Journal of Pediatrics September 1977

Table II. Urinary excretion (mole/100 mole creatinine) and clearance ( m l / m i n / 1 . 7 3 m ~) of dicarboxylic amino acids

Amino acids Aspartic acid

Glutamic acid Clear aspartic

Excretion I Clearance (mean +_ SD) (mean + SD)

Subject

I

Present case Teijema's case* Controls~

17.3 _+ 4.3 (4) 19.0 _+ 3.0 (6) 0.27 _+ 0.21(11)

Clear glutamic

Clear inulin

Excretion (mean + SD)

Clearance (mean +_SD)

1.72 (1) 1.50 (l)t' -

172 _+ 43 (4) 294 _+ 33 (6) 2.2 _+ 1.5(11)

155.6 _+ 25.7 (5) 305 (2) 2.6 + 2.2 (9)

88.7 _+ 17.0(5) 150 (1) 2.6 _+ 2.1 (9)

Clear inulin 3.02(1) 2.80 (2)t

*A 23-month-old athyreotic girl; values obtained during a fast of less than 8 hours? ]'Clearance of creatinine. :~Children two to 14 years; number of observations in parenthesis?

Table Ill. Endogenous renal clearance of amino acids ( m l / m i n / 1 . 7 3 m ~)

I

Control children

Amino acid

Present case Mean +_ SD*

Aspartic acid Glutamic acid Half-cystine Tyrosine Phenylalanine Ornithine Lysine Histidine

88.7 _+ 17.0 155.6 _+ 25.7 3.5 _+ 0.6 4.7 _+ 1.5 3.7 _+ 1.7 4,6 _+ 1.4 2.6 _+ 2.7 25.5 _+ 14.1

Mean

Range'~

2.8 0.8

tr- 8.8 0.1- 2.4 1.0- 1.4 0.8- 3.3 0.3- 2.3 0.2- 0.8 0.3- 2.4 1.%21.8

2.0 1.2 0.5 1.1 9.2

*Mean and SD of four 30-minute periods after an overnight fast and one 24-hour urine clearance. tScriver and Davies': nine children, 3-12 years of age: short-term clearance after overnight fast.

glutamic acid load. Inulin clearance and short-term amino acid clearance were calculated over six 30-minute periods with venous blood being taken at 15 minutes and urine being collected from the bladder through a catheter. The effect of a sustained low- and high-protein intake on blood a m m o n i a and urinary amino acid levels was studied over a three-week period during which the child was offered a daily protein load of 2, 4, and 6 g m / k g body weight for one week. Venous blood for a m m o n i a measurements was obtained 2 hours after breakfast on the seventh day of the test. RESULTS The massive urinary excretion of glutamic acid and to a lesser degree of aspartic acid is illustrated in Fig. 1. With the exception of the dicarboxylic amino acids, all other urinary amino acids were within normal range and have remained so for the past two years.

Levels of plasma and cerebrospinal fluid amino acids were recorded as normal after a 5- to 8-hour fast. Glutamic and aspartic acid concentrations became markedly reduced, however, in plasma samples studied after an overnight fast (Table I) while proline levels remained unchanged. The p l a s m a : C S F ratio for glutamic acid was calculated once at 6.45 (normal, 8),7; aspartic acid concentration was too low for accurate quantitation and proline was undetectable. Results of the urinary excretion and renal clearance of the dicarboxylic amino acids in our patient as compared with Teijema's case are shown in Table II. Our patient's total diurnal loss was about 60 times control values for aspartic acid and close to 80 times control values for glutamic acid. The clearance of cystine, tyrosine, phenylalanine, ornithine, lysine, and histidine were, respectively, above mean control values while other amino acids remained within normal range (Table III). The plasma amino acid response to oral L-glutamic acid resulted in a comparable increase in glutamic acid both in patient and control (Fig. 2). Attempts to detect an elevation of a-ketoglutaric acid in plasma remained unsuccessful and no measurable peak of either a-ketoglutaric or pyroglutamic acid could be demonstrated in urine by GC-MS methods. Results of the oral histidine loading test disclosed a minimal increase in the urinary excretion of FIGLU. The correlation between blood a m m o n i a levels and dicarboxylic amino acid output while receiving high- and low-protein diets is shown in Table IV. Blood a m m o n i a concentration increased with increasing protein intake. There was, however, no evidence o f h y p e r a m m o n e m i a of the order expected with a possible glutamine synthesis or urea cycle defect. Adjunction of pyridoxine to the child's diet did not change the dicarboxylic aminoaciduria. Skin fibroblast studies showed normal oxidation of

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Dicarboxylic aminoaciduria

pyruvic, aspartic, and glutamic acid in our patient's cells.

425

ASPARTIC ACID 400

DISCUSSION The existence of a common renal transport mechanism for dicarboxylic amino acids has been known for 25 years) Yet unlike the other four major amino acid transport groups (#-amino acids; dibasic amino acids; imino acids and glycine; and neutral amino acids) no disorder had been identified in man which implicated this system prior to 1974.1 The initial report referred to dicarboxylic aminoaciduria as a transport defect with metabolic implications. The present case of dicarboxyiic aminoaciduria has come to our attention through a newborn screening program and shares in common with the previously reported case 1 a unique metabolic abnormality characterized by massive renal wastage of glutamic and aspartic acid. None of the symptoms and metabolic abnormalities observed in the initial patient have been found in our patient. These included oligophrenia, periodic rapid loss of weight with ponderal and statural growth retardation, a tendency toward fasting hypoglycemia and ketoacid0sis, hyperprolinemia, and evidence for an intestinal glutamic acid transport defect. One possible explanation for some of these discrepancies might be the apparent normal intestinal absorption of glutamate in our patient as opposed to the previous one. Preservation of an adequate intestinal transport system for the dicarboxylic amino acids in our patient is evidenced by the elevation of plasma glutamic acid after oral loading and the increase in dicarboxylic aminoaciduria while receiving a high-protein intake (Table IV). In addition, our patient could fast for periods up to 16 hours without hypoglycemia or hyperprolinemia while maintaining plasma concentrations of glutamic acid down to levels comparable with Teijema and associates I patient. These observations and the presence of comparable urinary dicarboxylic amino acid losses in each patient suggest that renal wastage cannot by itself be responsible for the hypoglycemia and the related clinical abnormalities. Normal or low plasma levels of the dicarboxylic amino acids do not suggest an overflow phenomenon as seen in many cases of hyperaminoacidemia. The observed gross aminoaciduria in the present and in the previously reported case must be of renal origin. Renal overproduction might be a conceivable explanation in view of the excretion rate in excess of the glomerular filtration rate. This phenomenon is not new, having been observed during dicarboxylic amino acid infusion experiments in dog#' and in the case of cystinuriaY' The former authors ~ postulated a direct renal synthesis of glutamic and

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Protein intake (gm/kg/24 hr) 2 3.5* 4 6t Controls

Aspartic acid I Glutamic acid L (mole/lO0 mole creatinine) 23 15.8 23.6 43 0.27 0.21

124 106 216 2OO 2.2 1.55

Blood ammonia (l~g/dl) 43.05 72 95.7 114 30-120

*Normal diet plus pyridoxine (20 mg/day). ]'Diet ceased after 36 hours because of vomiting. SData from Teijema and associates' in children two to 14 years old.

aspartic acid or release of these amino acids from tissues to account for the "negative reabsorption" or "net secretion" observed when other amino acids such as alanine and histidine were infused. It may be of interest that the amino acids mentioned-histidine and a l a n i n e - m a y be readily converted to dicarboxylic amino acids and participate in transamination reactions but have never been

426

Melanqon et al.

elevated in our patient's plasma. Thus the more likely explanation for dicarboxylic amino acids wastage in our patient could be a defective etttux system at the plasma m e m b r a n e with exaggerated exodus of the relevant intracellular amino acids. Such an alteration could result from a defect in mitochondrial energy metabolism in which dicarboxylic amino acids play an important role. Bergeron and Vadeboncoeur have demonstrated direct transtubular backttux and reduced cellular accumulation of leucine following peritubular capillary injection of the amino acid in the maleic acid model of the Fanconi syndrome.'" 1,_, According to Bergeron and Vadeboncoeur's TM 15 studies, the aminoaciduria proceeds through two related energy dependable mechanisms. One is the loss of punctate contacts between the cells, the other concerns the carrier. At the present time, we have not been able to collect enough evidence of kidney dysfunction in our patient to suggest some indication for a kidney biopsy on Which to assess transport and intracellular concentration of dicarboxylic amino acids. Results of the oxidation studies performed on cultured fibroblasts tend to rule out the possibility of a metabolic defect involving dicarboxylic amino acids. There remains a possibility, however, that such a defect be presen! in the kidney alone. The increased renal clearance of amino acids from two other major groups, namely, cystine, lysine, ornithine (dibasic) and tyrosine, phenylalanine, histidine (neutral), is another indication that whatever the primary defect may be, it may affect other transport systems in addition to the dicarboxylic amino acid one. Formiminoglutamic aciduria ':~ and the kinky hair syndrome 14 are two other rare familial metabolic disorders in which an increased glutamic aciduria can be found. The glutamic aciduria in the former is an artifact owing to the "in vitro" degradation of F I G L U in glutamic acid, and should be easily identified through proper chromatographic techniques. '~ Menkes kinky hair syndrome has been associated with glutamic aciduria as a part of a generalized aminoaciduria, and elevated glutamic acid in the CSF. 16 The absence of neurologic abnormalities and a normal C S F level of glutamic acid in our patient should make it easy to distinguish between the two conditions. In conclusion, we suggest that dicarboxylic aminoaciduria be recognized as the "fifth" inherited disorder of group-specific amino acid transport in man. Available evidence indicates that more than one genotype can produce a metabolic alteration characterized by dicarboxylic aminoaciduria as recognized previously for other amino acid transport defects, namely, familial iminogly-

The Journal of Pediatrics September 1977

cinuria 17 and cystinuria. 18 On the analogy of this latter disorder, we suspect that homozygotes will be detected through screening programs with increased urinary dicarboxylic amino acids associated with impaired intestinal absorption (type I) and increased urinary excretion alone (type II). Whether patients should be told that they suffer from a nondeleterious mutation that necessitates no therapy remains to be confirmed as more cases emerge from newborn screening programs and metabolic studies are completed on their respective families. We are indebted to Dr. Damien Pomerleau who referred the patient; to Dr. Orval A. Mamer, Mass Spectrometry Unit, Royal Victoria Hospital, for determining the mass spectra of unknown organic compounds; to Danielle Lepage, Marie Debeau, Robert Gigu6re, Beno~t Grenier and Bernard Grignon for technical assistance and to Francine Lamothe who supervised the diets. Madeleine Gagnon, Diane Leblanc and Suzanne LallettiMorneau realized this manuscript. REFERENCES

1. Teijema HL, Van Gelderen HH, Giesberts MAH, and Laurent d e Angulo MSL: Dicarboxylic aminoaciduria: An inborn error of glutamate and aspartate transport with metabolic implications, in combination with a hyperprolinemia, Metabolism 23:115, 1974. 2. Haworth JC, Miller JR, and Scriver CR: Screening, counselling and treatment of hereditary metabolic disease; a survey of resources in Canada, Can Med Assoc J 16:1147, 1974. 3. Crawhall JC, Mamer O, Tjoa S, and Claveau JC: Urinary phenolic acids in tyrosinemia. Identification and quantitation by gas chromatography-mass spectrometry, Clin Chim Acta 34:47, 1971. 4. Lancaster G, Mamer OA, and Scriver CR: Branched-chain alpha-keto acids isolated as oxime derivatives: Relationship to the corresponding hydroxy acids and amino acids in maple syrup urine disease, Metabolism 23:257, 1974. 5. Blass JP, Avignan JA, and Uhlendorf BW: A defect in pyruvate decarboxylase in a child with an intermittent movement disorder, J Clin Invest 49:423, 1970. 6. Stern RC, Pittman S, Doershuk CF, and Matthews LW: Use of a "heparin lock" in the intermittent administration of intravenous drugs, Clin Pediatr 11:521, 1972. 7. Dickinson JC, and Hamilton PB: The free amino acids of human spinal fluid determined by ion exchange chromatography, J Neurochem 13:1179, 1966. 8. Scriver CR, and Davies E.: Endogenous renal clearance rates of free amino acids in prepuberal children, Pediatrics 32:592, 1965. 9. Kamin H, and Handler P: Effect of infusion of single amino acids upon excretion of other amino acids, Am J Physiol 164:654, 1951. 10. Crawhall JC, Scowen EF, Thompson CJ, and Watts RWE: The renal clearance of amino acids in cystinuria, J Clin Invest 41:1162, 1967. 11. Bergeron M, and Vadeboncoeur M: Microinjection of L-leucine into tubules and pericapillaries of the rats: II. The maleic acid model, Nephron 8:367, 1971.

Volume 91 Number 3

12. Bergeron M: Renal amino acid accumulation in maleatetreated rats, Rev Can Biol 30:267, 1971. 13. Arakawa T, Ohara K, Kudo Z, Tada K, Hayashi T, and Mizuno T: Hyperfolic-acidemia with formiminoglutamic aciduria following histidine loading, Tohoku J Exp Med 80:370, 1963. 14. Menkes JH, Alter M, Steigleder GK, Weakley DR, and Sung JH: A sex-linked recessive disorder with retardation of growth, peculiar hair, and focal cerebral and cerebellar degeneration, Pediatrics 29:764, 1962. 15. Perry TL, Applegarth DA, Evans ME, and Hansen S: Metabolic studies of a family with massive formiminoglutamic aciduria, Pediatr Res 9:117, 1975.

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16. Yoshida T, Tada K , Mizuno T, Wada Y, Akabane J, Ogasawara J, Minagawa A, Morikawa T, and Okamura T: A sex-linked disorder with mental and physical retardation characterized by cerebro-cortical atrophy and increase of glutamic acid in the cerebrospinal fluid, Tohoku J Exp Med 83:261, 1964. 17. Scriver CR, and Rosenberg LE: Nature and disorders of amino acid and glycine transport, in Amino acid metabolism and its disorders, pp 178-186. WB Saunders Company, Philadelphia, 1973. 18. Rosenberg LE, Downing SJ, Durand JL, and Segal S: Cystinuria: Biochemical evidence for three genetically distinct diseases, J Clin Invest 45:365, 1966.