Clinical Science and Molecular Medicine (1977) 53, 173-181.

Nitrogen metabolism in neonatal citrullinaemia

M . WALSER, M. BATSHAW, G. SHERWOOD, B. ROBINSON A N D S. BRUSILOW Department of Pharmacology and Experimental Therapeutics, Department of Pediatrics, and Department of Medicine, Johns Hopkins University School of Medicine, and John F. Kennedy Institute, Baltimore, Maryland, U.S.A., and Department of Pediatrics, Hospital for Sick Children, Toronto, Ontario, Canada

(Received 24 November 1976; accepted 5 April 1977)

Summary 1. The pathways of disposition of waste nitrogen were studied in a male infant with neonatal citrullinaemia during 3 months of normal growth on a protein-restricted diet supplemented by a mixture of amino acids and nitrogen-free analogues of amino acids. 2. The rate of excretion of urea averaged onesixteenth, and the rate of excretion of total urinary nitrogen one quarter, of that reported in normal infants retaining the same amount of nitrogen per kg. Thus the efficiency of retention of dietary nitrogen for growth in this infant was very high. Plasma urea varied from 0.35 to 1.30 mmolil. 3. The small amount of urea formed was apparently derived entirely from dietary arginine via arginase, as indicated by the observation that urea excretion and plasma urea were linear functions of arginine intake with intercepts of zero or less. These relationships imply that urea synthesis would cease at zero arginine intake. This hypothesis could not be verified because arginine intakes below 2.2 mmol day-' kg- led to hyperammonaemia. 4. The high intake of arginine was required chiefly to replenish ornithine skeletons lost as citrulline in the urine (0.7 mmol day-' kg-I). Smaller amounts of arginine were estimated to be required for protein synthesis and for Correspondence: Dr Mackenzie Walser, Department of Pharmacology, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, Maryland 21205, U.S.A.

creatine synthesis. The rate at which ornithine was utilized in reactions other than citrulline synthesis appeared to be excessively high. 5. Citrulline was the principal end-product of nitrogen metabolism, accounting for 51 % of urinary nitrogen. 6. The results suggest that normal growth can be obtained in the virtual absence of the ability to dispose of waste nitrogen as urea. Key words: arginine metabolism, hyperammonaemia, neonatal citrullinaemia, nitrogen metabolism, ornithine metabolism.

Introduction Citrullinaemia is a rare inherited disorder caused by an abnormality of argininosuccinate synthetase (EC 6.3.4.5), the third enzyme in the Krebs-Henseleit urea cycle. It has been repeatedly observed that some urea production persists in this disorder as well as in the other urea-cycle enzymopathies (Levin, 1971). The portion of this residual urea synthesisthat can be attributed to the activity of arginase on dietary arginine has not been determined, nor has the optimal arginine intake for these patients, in whom arginine synthesis via the urea cycle is severely limited. In defects of the last three urea-cycle enzymes. this problem is further complicated by substantial urinary losses of the accumulated intermediates, citrulline, argininosuccinic acid and arginine, all containing the ornithine skeleton, which is normally only used catalytically in the urea cycle. 173

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The identification of a patient with neonatal citrullinaemia has made possible the study of nitrogen metabolism in this disorder during management with protein restriction and supplements of nitrogen-free analogues of essential amino acids. These studies aimed to characterize nitrogen metabolism in a subject growing normally, but virtually lacking any ability to synthesize urea. Clinical detaiIs A male infant weighing 2620 g at term developed lethargy, vomiting and focal seizures at 4 days of age while ingesting a proprietary infant food. At 5 days of age the child became comatose. The liver was enlarged. Plasma ammonium measured by an enzymatic method was 670 pmol/l (normal c 150 ,amol/l). An amino acid chromatogram revealed an increase in several amino acids; glutamine and citrulline were off scale. Continuous peritoneal dialysis from the fifth to the eighth days of life lowered the plasma ammonium to 300 pmol/l, with marked clinical improvement. On the ninth day of life, the child was given an intravenous infusion containing 36 mmol(5-3 g) of the a-keto analogues of five essential amino acids [valine 6.5 mmol (0.9 g), leucine 8-7 mmol (1.2 g), isoleucine 5-3 mmol (0.8 g), methionine 10.5 mmol (1.2 g) and phenylalanine 6.4 mmol (1.2 g)] and the following amino acids: L-histidine 1.3 mmol (0.2 g), L-threonine 1-7mmol (0.2 g), L-tryptophan 0.5 mmol (0.1 g), L-lysine/HCl 1.6 mmol (0.3 g) and L-arginine/HCl 9.5 mmol (2.0 g)]. The effects of this infusion on plasma amino acid concentrations are discussed below. Plasma ammonium rose transiently during the infusion to 563 pmolll but fell 12 h later to 332 pmoI/1, the child's clinical condition remaining stable. An oral mixture of the nitrogen-free analogues and essential amino acids started 36 h later. The composition was initially as follows (mmol day- kg- l) : sodium a-keto-isocaproate 2 3 (0.38 g), sodium a-keto-8-methylvalerate 1.8 (0.28 g), sodium a-keto-isovalerate 2.0 (0.28 g), sodium L-phenyl-lactate 1.0 (0.2 g), sodium m-a-hydroxy-y-methylthiobutyrate1.2 (0.2 g), ~-Iysine/HC10-4(0.8 g), L-histidine 0.3 (0-5g), L-threonine 0.5 (0.6 g), L-tryptophan 0.16 (0.33 g) and L-arginine 2.3 (0.4 g). He became alert and responsive, and seizure activity ceased during the following week. He accepted a diet of

a proprietary infant food (Enfamil, MeadJohnson Laboratories) and a carbohydrate supplement (Polycose, Ross Laboratories), initially providing 0-5 g of protein day-' kg-l. Multivitamins, including folk acid and supplemental pyridoxine, were given daily. Plasma ammonium fell to normal, although plasma citrulline remained elevated. The composition of the diet and the dietary supplement was altered several times in an attempt to maintain normal plasma concentrations of ammonium and essential amino acids. The child continued to thrive until 7 months of age, having gained 7.4 kg from birth, when he developed an acute hyperammonaemic crisis precipitated by an ear infection and died despite continuous peritoneal dialysis. Argininosuccinate synthetase was assayed in liver tissue obtained post mortem and stored frozen, by the method of Raiha & Suihkonen (1968), citrulline of high specific activity being used. Five assays gave a mean of 0.29+_0.18 (SEM) pmol h-' g-', compared with 92 & 9 pmol h- g- in four unaffected infants. Theoretical considerations An abridged diagram of the urea cycle as it pertains to those intermediates containing the Protein

A

intake

Urine

-4w

Urine

FIG. 1. An abridged diagram of the Krebs-Henseleit urea cycle. Under steady-state conditions, the rate of production of each of the substrates (Arg, arginine; Cit, citrulline; Om, ornithine) shown, in mmol/day, is equal to its rate of metabolism and/or excretion; italic letters represent these rates. The broken line represents the metabolic block in argininosuccinic acid synthesis in this patient.

Nitrogen metabolism in neonatal citrullinaemia

ornithine skeleton is shown in Fig. 1. The reaction rates shown symbolically pertain to semisteady-state conditions during growth, The broken line represents the metabolic block in citrullinaemia, through which residual enzyme activity may permit arginine synthesis at rate y. Citrulline is lost into the urine at rate x . Citrulline is synthesized from ornithine and carbamoyl phosphate at rate u. Thus under steady-state conditions: u =x+y

(1)

Arginine could be derived in part from residual enzyme activity at rate y, but results chiefly from arginine ingested at rate w f z . Arginine is metabolized to ornithine at rate z, and either incorporated into protein at rate w or released by proteolysis at rate -w, and also hydrolysed to urea and ornithine via arginase at rate u. In addition, arginine can be converted into ornithine via glycine transamidinase at rate t , thereby producing guanidinoacetic acid, which may be used for creatine synthesis. It follows that in the steady state: y+z = u+t (2) Ornithine, derived from arginine via these two pathways, may be utilized for citrulline synthesis or may be diverted to other pathways via ornithine transaminase or ornithine decarboxylase. Ornithine may be synthesized from glutamate or proline via ornithine transaminase. The net flux of ornithine out of (or into) the cycle is defined as a, and is taken to be positive for net flux out of the cycle. Thus:

u+t = a t u (3) Thus net diversion of ornithine from the cycle, a, must equal the difference between ornithine skeletons entering as arginine, z, and those lost as urinary citrulline, x (assuming that urinary excretion of arginine and ornithine is negligible). Complete urinary amino acid data were not obtained in this patient. However, we have analysed urine from another patient with neonatal cirtullinaemia and found no increase in arginine or ornithine; only citrulline, glycine and alanine were present in abnormal amounts. Hence : a =z-x (4) In normal subjects, urinary citrulline is negligible and therefore a = z. Normal sub-

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jects fed with an argininefree diet synthesize ornithine de nouo at rate - a and thereby synthesize extra arginine, which is diverted from the cycle at an equal rate, -z, and used for protein synthesis at rate w. Urea hydrolysis by intestinal bacteria, which amounts to 20% of urea production in normal adults (Walser &, Bodenlos, 1959), could make measured urinary urea somewhat less than u. The resulting ammonium would not alter the above equations, and in any event would be a trivially small quantity in our patient [about 0.1mmol day-' kg-l (see below)]. Methods Informed consent was obtained from the parents after full explanation of the purpose, nature, and risks of all procedures. These studies were approved by the Joint Committee on Clinical Investigation of the Johns Hopkins Medical Institutions. Nitrogen-free analogues of essential amino acids were administered under authorization from the Food and Drug Administration (IND 8472). Informed consent was obtained from the parents before therapy was begun. Metabolic study

Seventy-four morning blood samples and 40 24 h urine samples were obtained between age 19 days and 109 days. 24 h urine collections, with 5 ml of HCl (1 mol/l) as a preservative, were made by external catheterization, with sufficient interruptions to prevent skin maceration. Capillary blood was obtained each week day. Venous blood was obtained for measurement of plasma amino acids twice weekly. Laboratory methods Ammonium in urine and capillary plasma was measuredwith cation-exchangeresinas described by Batshaw, Brusilow & Walser (1975). Normal plasma ammonium by this method is 20-33 pmol/l in infants. Capillary plasma urea concentrations were measured by the urease method of Chaney & Marbach (1962). Urinary urea was similarly determined after absorption of preformed ammonium with Permutit (Fisher Scientific Co.). After hydrolysis of urine and capillary plasma samples with urease, citrulline

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was assayed by the method of Archibald (1944) as modified by Ceriotti & Spandrio (1973). Venous plasma amino acids were measured on sulphosalicylic acid filtrates by automated ionexchange chromatography. Total urine nitrogen was measured by a Coleman Nitrogen Analyzer (Coleman Instruments Division, Perkin-Elmer Corp., Maywood, Ill., U.S.A.). Urinary orotate was measured by the method of Goldstein, Hoogenrad, Johnson, Fukanaga, Swierczewski, Cann & Sunshine (1974). Monosubstituted guanidines were measured by the Sakaguchi reaction (Van Pilsum, 1959). Measured creatinine excretion was used to detect incomplete urine collections; days were omitted on which creatinine excretion was less than 60% of the amount predicted from the linear relationship observed between all meas ured values for creatinine excretion in mg/day (Cr) and age in days (d) throughout the study, namely Cr = 0.29d+8.9; r = 0.64, P

Nitrogen metabolism in neonatal citrullinaemia.

Clinical Science and Molecular Medicine (1977) 53, 173-181. Nitrogen metabolism in neonatal citrullinaemia M . WALSER, M. BATSHAW, G. SHERWOOD, B. R...
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