September 1978
The Journal o/PED I A T RIC S
421
Treatment of complete ornithine transcarbamylase deficiency with nitrogen-free analogues of essential amino acids A male infant with complete hereditary deficiency oj hepatic ornithine transcarbamylase was fed a low-protein diet (1 gmtkgrday] supplemented with nitrogen-free analogues oj essential amino acids from the age of 2 days until his death at 5 months. Blood ammonia and plasma amino acid concentrations were maintained In the near normal range during most of his lifetime. Growth and development were entirely normal. Abrupt, unprecipitated hyperammonemia, which could not be reversed by Intensive treatment, led to his death. To our knowledge, this child lived longer than any previously reported Infant with O'I'C deficiency of this severity.
John W. McReynolds, M.D., Stephanos Mantagos, M.D., Saul Brusilow, M.D., and Leon E. Rosenberg, M.D.,* New Haven, Conn.,
and Baltimore, Md.
of hepatic ornithine transcarbamylase, the second enzyme in the Krebs-Henseleit urea cycle, is an X-linked disorder resulting in partial deficiency of enzymatic activity in heterozygous females and in virtually complete absence of activity in most hemizygous males.' The resultant block in detoxification of ammonia causes protein intolerance and periodic ammonia intoxication in females; in males the usual course is one of fulminant hyperammonernia and metabolic encephalopathy, which is lethal during the first weeks of life without treatment.' Past therapeutic attempts in such male infants have been unrewarding.': a Recently, nitrogen-free analogues of certain essential amino acids in conjunction with the remaining essential amino acids and arginine have been used in the treatment of inherited disorders of the urea cycle. Two patients with INHERITED DEFI C IENc'Y
From the Departments of Human Genetics and Pediatrics, Yale University School of Medicine, and the Department of Pediatrics, Johns Hopkins University School of Medicine. Supported in part by a research grant from the National Institutes of Health (AM 09527). "Reprint address: Yale University School of Medicine, Department of Human Genetics, 333 Cedar SI., New Haven, CT065IO.
0022-3476178/0393-0421$00.70/0
e 1978 The e. V.
Mosby Co.
the severe neonatal form of citrullinemia" s have lived to about 8 months of age, and a single male with OTC deficiency" has survived to 7 weeks on this therapy. The therapeutic rationale is that the nitrogen-free analogues can be transaminated to their corresponding amino acids utilizing endogenous ammonia via glutamine: In this way nitrogen restriction can be accomplished without protein malnutrition. We now report our experience with this therapy in a male infant with virtually complete absence of hepatic GTe. Abbreviations used OTC: ornithine transcarbamylase CPS I: carbamyl phosphate synthetase I CASE REPORT
Patient R. B. was the 3.05 kg product of an uncomplicated 36-week gestation to a 24-year-old gravida 3, para 2 mother, whose first pregnancy resulted in the birth of a 2.51 kg male infant; he did weU during the first two days of life, but on the third day became lethargic, hypothermic, and poorly responsive to stimulation. Recurrent seizures developed and he lapsed into coma. He became apneic and, despite the ventilatory support, died late in the third day. Evaluation for intracranial hemorrhage, sepsis, electrolyte imbalance, and hypoglycemia gave no explanation for his clinical course. Autopsy was noncontribu lo-
ry.
Vol. 93, No, 3, pp. 421-427
422
McReynolds et al.
The Journal oj Pediatrics September 1978
Table I. Hepatic urea cycle enzyme activities Enzyme activity (pM/hr /mg protein) Subject
Patient's sister (LB.) Patient (R.B.) Adult control range (autopsy and needle biopsy)
CPS J 1.33 2.53 1.35-3.74 (n = 10)
Arginase 4.48
0.00 20.1-72.1 (n = 12)
346 544 136-550 (n =7)
CPS I - Carbamyl phosphate synthetase I; OTC - ornithine transcarbarnylase,
Her second pregnancy ended in the birth of a 3.18 kg female infant, L. B., who thrived on standard infant formula until four months of age when cow milk was introduced. Intermittent vomiting supervened thereafter, Developmental milestones were normal until 10 months, when normal progression ceased. At 13 months she became increasingly irritable. anorectic, and ataxic over a two-week period of time. On admission to an other hospital, a deteriorating clinical Coursewith generalized seizures, progressivehepatomegaly , elevated SOOT, prolonged prothrombin ~d partial thromboplastin times, and blood ammonia of9oo Jlg/dl prompted transfer to Yale-New' Haven Hospital with a presumptive diagnosis of Reye syndrome. Examination revealed a comatose, flaccid, hyperventil ating child unresponsive to deep pain. Weight was 7.3 kg « 3%), length 70 C/Il (~%) , and head circumference 43 cm « 3%). The liver was palpable 7 embelow the right costal margin.. Based on the above clinical and 'laboratory information, plus the fact that the mother had voluntarily restricted her own protein intake for as long as she could remember, a presumptive diagnosis of O'I'C deficiency was made; this thesis was later supported by an abnormal ammonia tolerance test' on the mother. WIth supportive therapy, blood ammonia gradually fell to normal after six days, Thereafter she did well, tolerating a regimen of dietary protein restriction (1.0 gm/kg/day) and orallactulose with blood ammonia values in the range of 110 to 220 I'g/dl on most occasions. At the age of 20 months, percu taneous biopsy of the liver and assay for urea cycle enzymesrevealed O'I'C activity to be 10% of normal (Table I). At age 25 months, formal assessment showed a developmental age of 20 months. Current ly she is thriv ing on a protein intake of about 0.9 gm/kg/day at the age of 36 months . Liver biopsy had been planned for the mother but this was precluded when she became pregnant with Patient R, B. After appropriate counseling, amniocentesis was performed for sex determination. Although the fetus was male, she elected to continue the pregnancy si~ce there was a 50% chance that he would be unaffected. Following an uneventful labor and delivery, physical examination of patient R_B. was entirely normal. He was giver glucose feedings, and blood ammonia concentration was monitored. At 24 hours of age he was irritable, somewhat jittery, and mildly hypertonic. His blood ammonia concentration was 300.Jlg/ dl, at the upper limits of normal for his age,- He was given a small protein feeding and three hours later his ammonia level was 370
Jlg/dl. A tentative diagnosis of OTC deficiency was made and confirmed three weeks later when assay for hepatic O'TC showed undetectable activity (Table 1), After detailed discussion of the potential risks and benefits, consent for experimental therapy with nitrogen-free analogues of essential amino acids was obtained from the mother. The infant was started on a proteinrestricted formula containing these analogues on the second day of life.
MATERIALS AND METHODS Infant formula. The formula consisted of standard infant formula in a quantity sufficient to supply the desired amount of milk protein, and a synthetic, proteinfree preparation (Product 80056, Mead Johnson Laboratories), supplying calories, vitamins, and trace elements, diluted appropriately with water. To the daily allotment of this formula was added a supplement consisting of the keto analogues of leucine. isoleucine. and valine, the hydroxy analogues of phenylalanine and methionine, and the amino acids lysine, histidine, threonine, tryptophan, and arginine, each In a quantity exceeding the infant's daily requirements. This daily volume of formula was divided into aliquots suitable for individual feedings. Supplementary pyridoxine (5 mg/day) was administered to assure adequate cofactor for transamination. Blood studies, Blood ammonia was determined by the method of Seligson and Hirahara" on heparinized specimens transported to the laboratory on ice and analyzed immediately. The normal range for blood ammonia with this method is SO to 150 Jlg/dl in children and adults. During the first three days of life, blood ammonia values are higher and can reach 300 ILg/ dl in normal neonates.v" In premature infants, higher values are common and can persist for three to five weeks." Plasma amino acid concentrations were measured by column chromatography with a Beckman am ino acid analyzer (Model B), Plasma proteins were precipitated immediately after sample collection by mixing equal portions of plasma and 10% sulfosalicylic acid. Urine studies. Orotic acid in urine was measured by a modification of the colorimetric method described by Adachi et al," and reported per mg creatinine. Orotic acid in amniotic fluid was measured with the same assay following precipitation of proteins by uranyl acetate." Urinary ammonia was assayed on refrigerated samples by the Berthelot reaction, I< Urea nitrogen was determined by a modification of the technic described by Kaplan," creatinine by the Jaffe jechnic," and uric acid by a modification of the method of Caraway." Total urinary nitrogen was assayed by the Kjeldahl method." Hepatic urea cycle enzyme assays. OTC was measured by colorimetric determination of citrulline production as described by Nuzum and Snodgrass" with the following
Treatment of ornithine transcarbamylase deficiency
Volllme 93 3
423
Number
300 ~
..... ~
200
10 0
Cl
1.0
...
O.~
o
0
:>
The Journal o/PED I A T RIC S
421
Treatment of complete ornithine transcarbamylase deficiency with nitrogen-free analogues of essential amino acids A male infant with complete hereditary deficiency oj hepatic ornithine transcarbamylase was fed a low-protein diet (1 gmtkgrday] supplemented with nitrogen-free analogues oj essential amino acids from the age of 2 days until his death at 5 months. Blood ammonia and plasma amino acid concentrations were maintained In the near normal range during most of his lifetime. Growth and development were entirely normal. Abrupt, unprecipitated hyperammonemia, which could not be reversed by Intensive treatment, led to his death. To our knowledge, this child lived longer than any previously reported Infant with O'I'C deficiency of this severity.
John W. McReynolds, M.D., Stephanos Mantagos, M.D., Saul Brusilow, M.D., and Leon E. Rosenberg, M.D.,* New Haven, Conn.,
and Baltimore, Md.
of hepatic ornithine transcarbamylase, the second enzyme in the Krebs-Henseleit urea cycle, is an X-linked disorder resulting in partial deficiency of enzymatic activity in heterozygous females and in virtually complete absence of activity in most hemizygous males.' The resultant block in detoxification of ammonia causes protein intolerance and periodic ammonia intoxication in females; in males the usual course is one of fulminant hyperammonernia and metabolic encephalopathy, which is lethal during the first weeks of life without treatment.' Past therapeutic attempts in such male infants have been unrewarding.': a Recently, nitrogen-free analogues of certain essential amino acids in conjunction with the remaining essential amino acids and arginine have been used in the treatment of inherited disorders of the urea cycle. Two patients with INHERITED DEFI C IENc'Y
From the Departments of Human Genetics and Pediatrics, Yale University School of Medicine, and the Department of Pediatrics, Johns Hopkins University School of Medicine. Supported in part by a research grant from the National Institutes of Health (AM 09527). "Reprint address: Yale University School of Medicine, Department of Human Genetics, 333 Cedar SI., New Haven, CT065IO.
0022-3476178/0393-0421$00.70/0
e 1978 The e. V.
Mosby Co.
the severe neonatal form of citrullinemia" s have lived to about 8 months of age, and a single male with OTC deficiency" has survived to 7 weeks on this therapy. The therapeutic rationale is that the nitrogen-free analogues can be transaminated to their corresponding amino acids utilizing endogenous ammonia via glutamine: In this way nitrogen restriction can be accomplished without protein malnutrition. We now report our experience with this therapy in a male infant with virtually complete absence of hepatic GTe. Abbreviations used OTC: ornithine transcarbamylase CPS I: carbamyl phosphate synthetase I CASE REPORT
Patient R. B. was the 3.05 kg product of an uncomplicated 36-week gestation to a 24-year-old gravida 3, para 2 mother, whose first pregnancy resulted in the birth of a 2.51 kg male infant; he did weU during the first two days of life, but on the third day became lethargic, hypothermic, and poorly responsive to stimulation. Recurrent seizures developed and he lapsed into coma. He became apneic and, despite the ventilatory support, died late in the third day. Evaluation for intracranial hemorrhage, sepsis, electrolyte imbalance, and hypoglycemia gave no explanation for his clinical course. Autopsy was noncontribu lo-
ry.
Vol. 93, No, 3, pp. 421-427
422
McReynolds et al.
The Journal oj Pediatrics September 1978
Table I. Hepatic urea cycle enzyme activities Enzyme activity (pM/hr /mg protein) Subject
Patient's sister (LB.) Patient (R.B.) Adult control range (autopsy and needle biopsy)
CPS J 1.33 2.53 1.35-3.74 (n = 10)
Arginase 4.48
0.00 20.1-72.1 (n = 12)
346 544 136-550 (n =7)
CPS I - Carbamyl phosphate synthetase I; OTC - ornithine transcarbarnylase,
Her second pregnancy ended in the birth of a 3.18 kg female infant, L. B., who thrived on standard infant formula until four months of age when cow milk was introduced. Intermittent vomiting supervened thereafter, Developmental milestones were normal until 10 months, when normal progression ceased. At 13 months she became increasingly irritable. anorectic, and ataxic over a two-week period of time. On admission to an other hospital, a deteriorating clinical Coursewith generalized seizures, progressivehepatomegaly , elevated SOOT, prolonged prothrombin ~d partial thromboplastin times, and blood ammonia of9oo Jlg/dl prompted transfer to Yale-New' Haven Hospital with a presumptive diagnosis of Reye syndrome. Examination revealed a comatose, flaccid, hyperventil ating child unresponsive to deep pain. Weight was 7.3 kg « 3%), length 70 C/Il (~%) , and head circumference 43 cm « 3%). The liver was palpable 7 embelow the right costal margin.. Based on the above clinical and 'laboratory information, plus the fact that the mother had voluntarily restricted her own protein intake for as long as she could remember, a presumptive diagnosis of O'I'C deficiency was made; this thesis was later supported by an abnormal ammonia tolerance test' on the mother. WIth supportive therapy, blood ammonia gradually fell to normal after six days, Thereafter she did well, tolerating a regimen of dietary protein restriction (1.0 gm/kg/day) and orallactulose with blood ammonia values in the range of 110 to 220 I'g/dl on most occasions. At the age of 20 months, percu taneous biopsy of the liver and assay for urea cycle enzymesrevealed O'I'C activity to be 10% of normal (Table I). At age 25 months, formal assessment showed a developmental age of 20 months. Current ly she is thriv ing on a protein intake of about 0.9 gm/kg/day at the age of 36 months . Liver biopsy had been planned for the mother but this was precluded when she became pregnant with Patient R, B. After appropriate counseling, amniocentesis was performed for sex determination. Although the fetus was male, she elected to continue the pregnancy si~ce there was a 50% chance that he would be unaffected. Following an uneventful labor and delivery, physical examination of patient R_B. was entirely normal. He was giver glucose feedings, and blood ammonia concentration was monitored. At 24 hours of age he was irritable, somewhat jittery, and mildly hypertonic. His blood ammonia concentration was 300.Jlg/ dl, at the upper limits of normal for his age,- He was given a small protein feeding and three hours later his ammonia level was 370
Jlg/dl. A tentative diagnosis of OTC deficiency was made and confirmed three weeks later when assay for hepatic O'TC showed undetectable activity (Table 1), After detailed discussion of the potential risks and benefits, consent for experimental therapy with nitrogen-free analogues of essential amino acids was obtained from the mother. The infant was started on a proteinrestricted formula containing these analogues on the second day of life.
MATERIALS AND METHODS Infant formula. The formula consisted of standard infant formula in a quantity sufficient to supply the desired amount of milk protein, and a synthetic, proteinfree preparation (Product 80056, Mead Johnson Laboratories), supplying calories, vitamins, and trace elements, diluted appropriately with water. To the daily allotment of this formula was added a supplement consisting of the keto analogues of leucine. isoleucine. and valine, the hydroxy analogues of phenylalanine and methionine, and the amino acids lysine, histidine, threonine, tryptophan, and arginine, each In a quantity exceeding the infant's daily requirements. This daily volume of formula was divided into aliquots suitable for individual feedings. Supplementary pyridoxine (5 mg/day) was administered to assure adequate cofactor for transamination. Blood studies, Blood ammonia was determined by the method of Seligson and Hirahara" on heparinized specimens transported to the laboratory on ice and analyzed immediately. The normal range for blood ammonia with this method is SO to 150 Jlg/dl in children and adults. During the first three days of life, blood ammonia values are higher and can reach 300 ILg/ dl in normal neonates.v" In premature infants, higher values are common and can persist for three to five weeks." Plasma amino acid concentrations were measured by column chromatography with a Beckman am ino acid analyzer (Model B), Plasma proteins were precipitated immediately after sample collection by mixing equal portions of plasma and 10% sulfosalicylic acid. Urine studies. Orotic acid in urine was measured by a modification of the colorimetric method described by Adachi et al," and reported per mg creatinine. Orotic acid in amniotic fluid was measured with the same assay following precipitation of proteins by uranyl acetate." Urinary ammonia was assayed on refrigerated samples by the Berthelot reaction, I< Urea nitrogen was determined by a modification of the technic described by Kaplan," creatinine by the Jaffe jechnic," and uric acid by a modification of the method of Caraway." Total urinary nitrogen was assayed by the Kjeldahl method." Hepatic urea cycle enzyme assays. OTC was measured by colorimetric determination of citrulline production as described by Nuzum and Snodgrass" with the following
Treatment of ornithine transcarbamylase deficiency
Volllme 93 3
423
Number
300 ~
..... ~
200
10 0
Cl
1.0
...
O.~
o
0
:>
