Pediatr. Res. 15: 115-119 (1981) amino acid lysinuric protein intolerance

Orotic Aciduria in Lysinuric Protein Intolerance: Dependence on the Urea Cycle Intermediates

JUKKA RAJANTIE'"' Children's Hospital, University of Helsinki. Helsinki. Finland

Summary feeding and an IV alanine load. As the hypothesis proved to be true, I proceeded to studying with this new sensitive indicator: (1) Urinary excretion of was measured in controls and the comparative value of oral supplements of citrulline, arginine, in subjects homozygous and heterozygous for lysinuric protein and ornithine in normalization of removal, (2) the intolerance under various conditions of amino nitrogen intake. In plasma ornithine level necessary for this normalization, using all situations, the controls excreted less than 28 pg/kg/hr. Only repeated oral ammonia loads with IV infusion of ornithine at one of the two heterozygotes studied differed from tbe controls. stepwise increasing rate, and (3) the heterozygous state for LPI. The orotic aciduria of homozygotes was normal in fasting but increased on a self-chosen low-protein diet (log mean, 80; range, 8 to 588 pg/kg/hr in 24-hr urine), after cow's milk protein, 0.5 g/ MATERIALS AND METHODS kg (76% 251 to 1747 pg/kg/hr in 4 to Chr urine), oral ammonium lactate, 2.5 mmoles/kg (95; 15 to 1127 pg/kg/hr in 1.5-hr urine), SUBJECTS and IV alanine, 6.6 mmoles/kg (51% 47 to 1831 pg/kg/hr in Chr urine). Twenty-four homozygotes for LPI (one infant, 14 prepubertal Giving ornithine or citrulline IV with the infusion of alanine children, one adolescent, eight young adults; 17 females) partici- prevented the increase in orotic acid excretion. Given orally, pated in this study. Fifteen of them have been described earlier citrulline was more efficient than ornithine or arginine. To prevent (26). The others similarly had the clinical picture typical of LPI, the hyperammonemic and orotic aciduric responses with ornithine, the diaminoaciduria and the hyperammonernic response to an IV its plasma concentrations needed to be higher than normal. alanine load (26). Orotic aciduria is a reliable indicator of the function of the urea Twenty-four-hr urine specimens were collected from 10 homo- cycle in lysinuric protein intolerance and facilitates monitoring of zygotes during a self-chosen, low-protein diet without amino acid the treatment of this disease. supplementation, 27 healthy obligate heterozygotes (parents of the homozygotes; 14 females), and 90 controls (healthy young adults and hospitalized children of all ages, with normal intake of urea Speculation cycle intermediates, not receiving cytostatic treatment and without The parallel changes in urinary orotic acid and blood ammonia evidence of urea cycle dysfunction or disorders of or concentrations in lysinuric protein intolerance suggest a close link purine ; 3 1 females) on a free or hospital diet. between the urea cycle and the pyrimidine pathway, probably via Twenty homozygotes, two heterozygotes, and nine controls carbamyl phosphate and asparate. Follow-up of orotic aciduria is (healthy adults or children first suspected of having impaired urea a simple and presumably accurate method for monitoring the home production, but then found to show normal elevations of serum treatment of disorders of the urea cycle. urea concentrations without after the IV ala- nine load) received one or more IV or oral loads each (see "Experiments"). Patients with enzyme defects of the urea cycle show increased All subjects and/or their guardians had given informed consent urinary excretion of orotic acid, an intermediate in the to these studies. of (22). Orotic aciduria after a protein load reveals hyterozygosity for ornithine transcarbamylase (EC 2.1.3.3) defi- EXPERIMENTS ciency (6, 8). In experimental animals, excretion rises after am- monium loads (12, 28) and on diets deficient in arginine (14, 15). The following loads were given after a 10-hr fast and after a Lysinuric protein intolerance (LPI) is an inherited defect of period of at least 24 hr without amino acid supplementation (19). diamino acid transport in the intestine (20). kidney, and liver (26). Only water was allowed during the tests. In the affected patients, concentrations of the diamino acid inter- L-Alanine, 6.6 mmoles/kg, was given as a constant 90-min mediates of the urea cycle, ornithine and arginine, are low in infusion of 5% (w/v) solution (9). On another day, the infusion plasma and presumably also in liver cells, which are the site of the was repeated with L-citrulline or L-ornithine-HC1, I. 1 mmoles/kg. cycle. Therefore, urea formation is impaired, and amino nitrogen Urine was collected for 6-hr periods, once before and for 1 to 3 loads lead to hyperammonemia. Oral ornithine and arginine periods after the start of the infusion. Blood ammonia was mea- supplements partly prevent the hyperammonemia (9, 21, 26). sured at 0, 120, 180, and 270 min. Without such treatment, the patients excrete increased amounts of As an oral protein load, cow's milk was given to provide 0.5 g orotic acid into the urine (3, 21). of protein per kg. The load was repeated together with 2 or 4 I undertook the present work to prove the hypothesis that orotic tablets of citrulline (0.414 g each), arginine-HCI (0.5 g), or orni- aciduria is more sensitive and practical than ammonemia as thine-HC1 (0.5 g) on different days. Urine was collected for three indicator of urea cycle failure in LPI. For this purpose, the urinary subsequent 2-hr periods. excretion of orotic acid was measured in controls and homozygotes Ammonium lactate, 2.5 mmoles/kg, was given orally as a 1.25 for LPI in basal state and in such standard amino nitrogen loading M solution in orange juice. Five percent mannitol with 20 mEq/ situations as have previously been characterized for hyperammo- liter of NaCl and 10 mEq/liter of KC1 was infused at 12 ml/kg/ nemic response in the homozygotes. These were a cow's milk hr IV to ensure diuresis. Blood ammonia was measured at 0, 45, 60, 75, and 90 min. Urine was collected for 90 rnin. To raise the RESULTS plasma ornithine level, infusion of the same solution was then continued with ornithine-HCI added in concentrations increased Most untreated homozygotes had clear orotic aciduria (Table 1). The amount excreted varied greatly, roughly reflecting the stepwise at 2.5-hr intervals to 1.5, 3.0,4.5, and 6.0 mM. Each step IV, protein content of the diet. In the heterozygotes orotic acid excre- was preceded by a priming dose of 5% ornithine-HCI equal to tion was within the range of the controls. 7Wo of the amount of ornithine to be given in the next 60 min. The ammonium lactate load was repeated 60 min after each step. Blood for amino acid analysis was drawn before and 60 rnin after IV ALANINE LOAD (TABLE 2) each ammonium load. The mean of the two ornithine concentra- During the fast before the load, orotic acid excretion was always tions was taken as a steady-state concentration. Two patients (Fig. minimal. The alanine infusion provoked massive orotic aciduria 2, patients I and 2) were given 5% glucose instead of mannitol to in all homozygotes, but in none of the controls. The controls had provide energy during the long experiment. Their results did not no rise in blood ammonia (9). One of the heterozygotes was differ from those for the other patients. nauseated and drowsy at the end of the alanine infusion, her blood ammonia having risen from 55 (0 min) to 128 pmoles/liter (120 ANALYTICAL min). Her orotic acid excretion was also clearly increased (1 13 pg/ Blood ammonia was measured with an ammonia-specific elec- kg/hr). The other heterozygote tested responded like the controls. trode (Orion Research Inc., Cambridge, MA) within 30 rnin of The orotic aciduria of the homozygotes was most marked during drawing the sample (18). The concentrations of amino acids were the first 6 hr after the load; in some of them it continued for 12 to determined with an automatic amino acid analyzer (Beckman 18 hr. Blood ammonia normalizes much earlier, usually within 4.5 Unichrom or Beckman 121 M with citrate and lithium buffers, hr (9). respectively). Urinary orotic acid was analyzed by a modified The amount of nitrogen excreted by the homozygotes as orotic colorimetric method (1, 6) and creatinine by a modified Jaffe acid within 12 hr after the load, calculated as a fraction of the reaction (2). The peak blood ammonia level appeared between 60 alanine nitrogen load, was on the average 0.7% and maximally and 75 rnin after the ammonium lactate load. The mean of the 60- 4.5%. and 75-min values less the basal value was taken as an estimate of The hyperammonemia induced by the alanine load in homo- the maximal increment caused by this load. zygotes is prevented by giving ornithine IV (24). After such an alanine plus ornithine load, orotic acid excretion was within the MATERIALS same range in the homozygotes as in the controls after the alanine load. Similarly, hyperammonemia and orotic aciduria could be Amino acids were obtained from Tanabe Seiyako Co. Ltd., prevented by giving citrulline IV. Osaka or Ajinomoto Co. Inc., Tokyo, Japan, and manufactured as tablets and 5% solutions by the Pharmaceutical Plant Leiras, ORAL PROTEIN LOAD (FIG. I) Turku, Finland. Commercial solutions of pure mannitol and glucose were used. Ammonium lactate was obtained as a 60% In the homozygotes, after the intake of cow's milk protein, 0.5 solution (w/v) from BDH Chemicals, Ltd., Poole, England. g/kg, no hyperammonemia was detected. However, their orotic

Table 1. Twentyfour-hr orotic acid excretion in controls and in heterozygotes and homozygotes for LPI on a self-chosen diet mg/liter mg/kg/24 hr' mg/g of creatinine Controls 4, CL162 (90)" 0.12. 0.00-0.7 1 (68) 11.9-15 (5) Heterozygotes 5,l-15 (27) 0.1 1, 0.05-0.25 (23) 5, 2-15 (27) Homozygotes 43, 2-533 (10) 1.93, 0.20- 14.12 (6) 61, 1-888 (10) The volume was not known for all wllections. Hence, the number of determinations is smaller in this column. ' The values are means (on log scale) and ranges. " Numbers in parentheses, numbers of determinations.

Table 2. Orotic acid excretion in controls and in heterozygotes and homozygotes for LPI before and afrer and I V alanine infusion' and effect of citrulline and ornithine2 supplements Orotic acid excretion (pg/kg/hr)

Before the load After the load

-6-0 hr 0-6 hr 6-12 hr 12-18 hr Controls Alanine 4, 2-g5 (3)' 7, 1-28 (8) 6, 2-10 (3) 2. (1) Heterozygotes ~laninc -5 35, 11-1 13 (2) - - Homozygotes Alanine 4, 3-8 (5) 519.4771831 (11) 77, 10-2042 (7) 16. 5-108 (5) Alanine + citrulline - 8, 6-1 1 (2) 5. 3-9 (2) - Alanine + ornithine - lO,9-12 (2) - - 6.6 mmol/kg was given as a constant infusion of 5% (w/v) solution in 90 min, starting at time 0. ' 1.1 mmol/kg was added to the alanine solution. Values are means (on log scale) and ranges. ' Numbers in parentheses, numbers of determinations. "ata not available. OROTIC ACIDURIA IN LYSINURIC PROTEIN INTOLERANCE 117

LPI CONTROL Citrulline thus seems to be at least as effective as arginine and ornithine in improving protein tolerance in the homozygotes. The doses have to be sufficiently large. Appparently, 0.5 rnmoles of citrulline per g of ingested protein will prevent the orotic aciduria. The protection afforded by citrulline seems to be rapid, but lasts for only 4 hr. This may be important in the treatment of acute hyperammonemia.

ORAL AMMONIUM LACTATE LOAD Ammonium lactate load was first given to 15 homozygotes and eight controls. The mean blood ammonia levels were at 0, 45. 60, 75, and 90 min in the homoozygotes 86, 117, 141, 155, and 114 pM and in the controls 40, 47, and 58 pM (90-min value not measured), respectively. The 90-min urinary orotic acid excretion, m LPI measured in 10 homozygotes and in five controls, was (log mean; range) 109; 15 to 1127 pg/kg/hr, and 6; 1 to 20 pg/kg/hr, respec- tively. When the load was then repeated in five homozygotes at elevated plasma ornithine concentrations, the hyperammonemic and orotic aciduric responses decreased (Fig. 2). However, the responses normalized fully only at supranormal ornithine concen- trations. The hyperammonemia was first abolished, and the orotic aciduria only at still higher ornithine concentrations.

0-2 2-4 4-6 0-2 2-4 4-6 TIME (HOURS)

Fig. I. Orotic acid excretion after an oral load of protein, 0.5 g/kg, as cow's milk: (A) in homozygotes for LPI (@) and in controls (0);no amino acid supplement; (B) in homozygotes for LPI with a citrulline (CIT), arginine (ARG), or ornithine (ORN) supplement given with the protein, in the doses indicated in the panels. Two patients had orotic aciduria even after the higher citrulline supple- ment (asterisks).This response normalized in both patients, when the load was repeated with the same citrulline supplement given twice, at times 0 and 4 hr (a,outside the columns). Logarithmic scale on ordinate. PLASMA ORNlTHlNE (pmol/l) Fig. 2. The effect of plasma ornithine wncentration in homozygotes acid excretion increased, with maximum values 4 to 6 hr after the for LPI on the hyperammonemic and orotic aciduric response to ammo- load (up to 437 times over that during fasting). Gitrulline, given nium lactate load. Ornithine was infused in stepwise increasing wncentra- as an oral supplement at 0.12 mmoles/kg, only partially prevented tions, each increase being preceded by a priming dose. After an equilibra- this rise. After a double dose, their orotic acid excretion remained tion period, an ammonium lactate load was given orally during each normal for 4 hr but then increased during the next 2 hr in two of ornithine concentration (for details, see "Materials and Methods"). the five homozygotes. This late orotic aciduria was abolished by To allow comparison of the two responses, the patients have been repeating the citrulline dose at 4 hr. In all but one patient, the identified with numbers. Broken lines, upper limits of the responses in the higher oral doses of arginine and ornithine failed to prevent the controls. The normal rage of plasma ornithine wncentration (26 to 75 orotic aciduria. pmol/liter in our laboratory; n = 17) is shaded on the horizontal axis. DISCUSSION In experimental animals, oral administration of urea cycle intermediates will prevent the induced hyperammonemia; the All homozygotes for LPI had orotic aciduria after amino nitro- most effective intermediate is arginine (7). In patients with LPI, gen and ammonia loads. The amounts excreted showed a clear arginine, ornithine, and citrulline IV all prevented hyperammo- correlation with the degree of hyperammonemia (Fig. 2). The nemia and ornithine and citrulline orotic aciduria. For long-term orotic aciduria thus seems to reflect a decreased capacity of the oral supplementation, the most effective is citrulline, probably urea cycle to detoxify ammonia. The enzymes of the pyrimidine because in contrast to arginine and ornithine, it is normally pathway have not been studied in LPI, but a defect or inhibition absorbed from the intestine of the patients (20). To normalize the of them is unlikely to be the cause of the orotic aciduria. Orotic function of the urea cycle, a supranormal plasma concentration of aciduria is associated with the hyperammonemic state in all ornithine was needed in LPI. This may be due to impaired enzyme defects of the urea cycle but not in deficiency of carbamyl transport of ornithine into the hepatic cells (24, 25). In patients phosphate (CP) synthetase I (22). The hyperammonemia of LPI with hyperornithinemia-hyperammonemia-homocitrullinuria is also believed to result from hypofunction of the urea cycle due plasma ornithine is constantly elevated (5, 23). The basic defect in to the decreased availability of diamino acid intermediates of the this disease has been suggested to lie in the mitochondrial uptake cycle (26). of ornithine. In these patients, supranormal ornithine concentra- The orotic aciduria in hyperammonemia is most readily ex- tions do not prevent hyperammonemia. However, feeding of plained as follows (Fig. 3). When the mitochondrial synthesis of supplementary ornithine decreased hyperammonemia even in CP from ammonia is normal but its further metabolism is blocked, these patients (4). CP will accumulate in the mitochondria. Some of it will leak into In contrast to the homozygotes, the heterozygotes for LPI have the cytoplasm (16, 29). Cytoplasmic CP and aspartate are the normal capacity to ammonia removal, according to absence of an substrates of the orotic acid-pyrimidine pathway. Aspartate, which increase in blood ammonia after the standard IV alanine load transfers the second ammonia moiety in urea synthesis is presum- (17). So far, the only established biochemical feature to distinguish ably also present in increased amounts. Accumulation of both the heterozygotes from controls is a decreased efficiency of intes- Drecursors will cause increased vroduction of orotic acid. In certain tinal lysine absorption; their plasma lysine levels after an oral karginal situations, this shunt& of CP may prevent the increase lysine load were intermediate between the levels observed in in blood ammonia concentration, as is seen in patients with LPI controls and homozygotes (20). Their intestinal absorption of who sometimes had orotic aciduria without apparent hyperam- ornithine and arginine has not been adequately studied. My monemia. The mitochondrial synthesis of CP may be regulated present finding of normal daily urinary excretion of orotic acid by the mitochondrial concentration of arginine (13). Although the indicates that the heterozygotes have suficient availability of the hepatocellular arginine concentration is believed to be decreased urea cycle intermediates in the basal state. Only two of them were in LPI (26). the production of CP is evidently not inhibited. available for testing with the alanine load. One of the two had a clearly increased response in orotic acid excretion. This suggests NORMAL that some of the heterozygotes may have a limited reserve to ammonia removal. Orotic acid seems to be only slightly toxic. It may disturb by depleting the liver of 5-phosphoribosyl-I-pyro- phosphate (10, 27). This is a substrate needed for the synthesis of pyrimidines from orotic acid, and is probably also the rate-limiting factor in purine synthesis. A resulting imbalance of purine and pyrimidine production may be one of the causes of the impaired growth and mental retardation associated with hyperammonemia. Feeding rats on a diet with added orotic acid produces fatty liver (30). Changes in liver morphology are also a constant finding in LPI (26). However, protein malnutrition and lysine deficiency are more likely reasons for the changes than orotic acid. Orotic acid stones have been found in hereditary orotic aciduria but not in OROTlC AClD hyperammonemic patients (I I). Presumably, the amounts excreted 1 by them are too small to produce concrements. PVRlMlDlNES In LPI and probably in other hyperammonemic diseases, mea- surement of orotic aciduria offers a valuable parameter of control. It is more sensitive than measurements of blood ammonia in LPI diagnosing the disease and in revealing the effect of oral protein load. It may be that orotic acid production increases more readily than blood ammonia rises. On the other hand, orotic aciduria gives an integrated measure for a period of several hr or even days, in contrast to blood ammonia. Easy collection of urine and the possibility for mailing urine samples from home make orotic UREA ,.'I aciduria a suitable parameter for following home treatment of -.,, ; patients with hyperammonemia. I

ARG REFERENCES AND NOTES ',, ...-._, dachi. T.. Tanimura. A,. and Asahina. M.: A colorimetric determination of ,. ,. ASA orotic acid. J. Vitaminol.. 9: 217 (1963). FUMARATE 2. Bartels. H.. Bohmer. M.. and Heierli. C.: Serum Kreatininbestimmung ohne OROTIC AClD Enteiweissen. Clin. Chim. Acta. 37: 193 (1972). 3. Carson. N. A. J.. and Redmond, 0. A. B.: Lysinuric protein intolerance. Ann. 1 Clin. Biochem.. 14: 135 (1977). PVRlMlDlNES 4. Fell. V.. Poll~tt.R. J.. Sampson. G. A,. and Wright. T.: Ornithinemia. hyperam- monemia, and homocit~lllnuria.A disease associated with mental retardation & and possibly caused by defective mitochondrial transport. Am. J. Dis. Child.. Fig. 3. The suggested mechanism of increased orotic acid production 127: 752 (1974). in LPI. -, increased concentration/flux; - - - -, decreased reaction. 5. Gatfield. P: D.. Taller. E.. Wolfe. D. M.. and Haust. M. D.: Hyperornithinemia. OROTIC ACIDURIA IN LYSINURIC PROTEIN INTOLERANCE

hyperammonemia. and homocitrullinuria associated with decreased carbamyl 20. Rajantie, J., Simell. O., and Perheentupa, J.: Intestinal absorption in lysinuric phosphate synthetase I activity. Pediatr. Res., 9: 488 (1975). protein intolerance: impaired for diamino acids, normal for citrulline. Gut. 21: oldstein. A. S.. Hoogenraad, N. J., Johnson. J. D.. Fukanaga, K.. Swiermwski. 519 (1980). E., Cann, H. M., and Sunshine. P.: Metabolic and genetic studies of a family 21. Russell, A,. Statter. M., and Ben-Zvi, A.: Ornithine administration as a therapeu- with ornithine transcarbamylase deficiency. Pediatr. Res.. 8: 5 (1974). tic tool in dibasic aminoaciduric protein intolerance. Hum. Her., 27: 206 7. Greenstein. J. P., Winitz. M., Gullino. P.. Bimbaum. S. M.. and Otey, M. C.: ( 1977). Studies on the metabolism of amino acids and related compounds in vivo. 111. 22. ~hih,V: E.: Urea cycle disorders and other congenital hyperammonemic syn- Prevention of ammonia toxicity by arginine and related compounds. Arch. dromes. In: J. B. Stanbury. J. B. Wyngaarden. D. S. Fredrickson: the Metabolic Biochem. Biophys., 64: 342 (1956). Basis of Inherited Disease. Ed. 4. Chap. 18, pp. 362-386 (McGraw-Hill Book Hokanson. J. T., O'Brien. W. E.. Idemoto. J.. and Schafer, I. A,: Carrier detection Co., New York, 1978). in ornithine transcarbamylase deficiency. J. Pediatr.. 93: 75 (1978). 23. Shih, V. E.. Efron. M. L.. and Moxr. H. W.: Hypcrornithinemia. hyperammo- 9. Kekomaki. M.. Visakorpi, J. K., Perheentupa, J.. and Saxin, L.: Familial protein nemia and homocitrullinuria. A new disorder of amino acid metabolism intolerance with deficient transport of basic amino acids. An analysis of 10 associated with myoclonic seizures and mental retardation. Am. J. Dis. Child., patients. Acta Paediatr. Scand., 56: 617 (1967). 11 7: 83 (1969). 10. Kelley. W. N.. Greene. M. L.. Fox, I. H., Rosenbloom. F. M.. Levy, R. I., and 24. Simell. 0.:Diamino acid transport into granulocytes and liver slices of patients Seegmiller. J. E.: Effects of orotic acid on purine and lipoprotein metabolism with lysinuric protein intolerance. Pediatr. Res.. 9: 504 (1975). in man. Metabolism. 19: 1025 (1970). 25. Simell. 0..and Perheentupa, I.: Defective metabolic clearance of plasma arginine I I. Kelley. W. N., and Smith. L. H.: Hereditary orotic aciduria. In: J. B. Stanbury, and ornithine in lysinuric protein intolerance. Metabolism, 23: 691 (1974). J. B. Wyngaarden, D. S. Fredrickson: The Metabolic Basisof Inherited Disease. 26. Simell, 0.. Perheentupa. J.. Rapola. J.. Visakorpi. J. K.. and Eskelin. L-E.: Ed. 4. Chap. 46, pp. 1047-1071 (McGraw-Hill Book Co., New York, 1978). Lysinuric protein intolerance. Am. J. Med.. 59: 229 (1975). 12. Kesner, L.: The effect of ammonia administration on orotic acid excretion in 27. Skaper. S. D., O'Brien. W. E.. and Schafer. I. A,: The influence of ammonia on purine and pyrimidine biosynthesis in rat liver and brain in vitro. Biochem. J., 172: 457 (1978). 28. Statter. M.. Russell. A,. Abzug-Horowitz S.. and Pinson, A,: Abnormal orotic acid metabolism associated with acute hyperammonaemia in the rat. Biochem. Med., 9: 1 (1974). 29. Tremblay. G. C., Crandall, D. E.. Knott. C. E., and Alfant. M.: Orotic acid biosynthesis in rat liver: studies on the source of carbamoylpbosphate. Arch. 16. Natale. P. J., and Tremblay. G. C.: On the availability of intramitochondrial Biochem. Biophys., 178: 264 (1977). carbamylphosphate for the extramitochondrial biosynthesis of pyrimidines. 30. Von Euler, L. H.. Rubin. R. J., and Handschumacher, R. E.: Fatty liver induced Biochem. Biophys. Res. Commun., 37: 512 (1969). by orotic acid. 11. Changes in nucleotide metabolism. J. Biol. Chem., 238: 2464 17. Norio, R.. Perheentupa. J.. Kekomaki, M.. and Visakorpi. J. K.: Lysinuric protein (1963). intolerance. an autosomal recessive disease. A genetic study of 10 Finnish 3 1. The author is indebted to Miss Majatta Viikari for skillful technical assistance. families. Clin. Genet., 2: 214 (1971). 32. Requests for reprints should be addressed to: Dr. J. Rajantie. Children's Hospital, 18. Proelss. H. F., and Wright, 8. W.: Rapid determination of ammonia in a University of Helsinki. SF-00290 Helsinki 29, Finland. perchloric acid supernate from blood. by use of an ammonia-specific electrode. 33. This research was supported by the Foundation for Pediatric Research, Finland. Clin. Chem.. 19: 1162 (1973). the Sigrid JusClius Foundation. Helsinki. Finland. and by the Association of 19. Rajantie, J.. Simell, 0.. and Perheentupa. J.: Lysinuric protein intolerance (LPI): the Finnish Life Insurance Companies. a two-year trial of dietary supplementation therapy with citmlline and lysine. 34. Received for publication November 7. 1979. Pediatr. Res. (in press). 35. Accepted for publication May 8. 1980

Copyright @ 1981 International Pediatric Research Foundation. Inc. Printed in U.S. A. 003 1-3998/8 1/ 1502-0 115$02.00/0