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Intermittent Branched Chain Ketonuria
Clin. Biochem. 4, 52-58 (1971)
I N T E R M I T T E N T B R A N C H E D CHAIN K E T O N U R I A (VARIANT OF MAPLE SYRUP URINE DISEASE) W. C. IRWIN, S. B. MARTEL AND N. GOLUBOFF Saskatoon City Hospital, Saskatoon, Canada (Received ~(arch 24, 1971)
SUMMARY 1. A patient (N.R.) with Intermittent Branched Chain Ketonuria (Intermittent Maple Syrup Urine Disease) is described. It is the first case of this very rare abnormality to be documented in Canada. 2. Laboratory diagnosis was initially made on the basis of a maple syrup-like odor to the urine, along with a strongly positive ketone test and a negative reducing substance test. The diagnosis was confirmed by elevated urinary and plasma levels of leucine, isoleucine, alloisoleucine and valine, and by the demonstration of reduced branched-chain oxidative decarboxylase activity in cultured fibroblasts. Urinary keto acids were also elevated. 3. Dietary treatment of the patient (N.R.) consisted of replacement of oral feedings by I.V. fluids for 36 hours, then oral protein free fluids for a further 36 hours after which a gelatin based formula was given. The gelatin based formula was gradually replaced by Enfalac and was supplemented with regular commercial fruit, meat, and vegetable purees. In retrospect the dietary treatment was probably more rigorous than necessary.
MAPLE SYRUP URINE DISEASE (MSUD) WAS INITIALLYDESCRIBEDas a syndrome by Menkes, Hurst and Craig in 1954 (1). The untreated case manifests as an early onset, fatal disorder, in which neurological symptoms are accompanied by excretion of urine having a sweet maple syrup-like odor. Dancis demonstrated that the basic defect was a reduction of branched-chain keto acid decarboxylase activity (2). As a result the branched-chain keto acids and their respective aminoacids (leucine, isoleucine, and valine), are elevated in the blood and in the urine. Morris et al. in 1961 described an infant who suffered from transient episodes of MSUD (8). The child was completely normal between attacks. This variant of MSUD has been termed Intermittent Branched Chain Ketonuria (6). The intermittent variant has been diagnosed in two other infants in the United States, one of these being a sibling of the first case (5, 6). In addition, three cases Correspondence: Dr. W. C. Irwin, Department of Pathology, Saskatoon City Hospital. Saskatoon, Saskatchewan, Canada.
INTERMITTENT BRANCHED CHAIN KETONURIA
53
have been diagnosed in Norway, two of these being siblings (7, 8). A description of the first case of Intermittent Branched Chain Ketonuria to be diagnosed in Canada follows: CASE REPORT
A 9½ month old white female (N.R.) was admitted to Saskatoon City Hospital with a diagnosis of left otitis media and debility of unknown etiology. The infant was the product of a normal pregnancy, except that the date of partum exceeded the expected date by approximately five weeks. Weight at birth was 7 lbs 1 oz. The infant was breast fed for the first five months, after which she received Enfalac (Mead Johnson). The transition to Enfalac was without incidence. Commercial infant purees of meat, vegetables and fruit were well tolerated. At approximately 9 months of age the milk source was changed from Enfalac to homogenized whole milk, which had been used previously in preparing the child's pablum. Neurologically, the child's development was relatively normal. He sat with assistance at 5 months and unassisted at 7½ months. The mother felt his locomotor development had been retarded by the restriction of a brace applied for kyllosis correction. She described him as being alert, independent, active and happy. Approximately two weeks prior to admission the child had suddenly become irritable and whined constantly. This persisted for two days during which smaller feedings were noted. However the food was consumed willingly and without emesis. The only other symptom noticed by the mother was a "very peculiar smelling urine". On the third day the child awoke in a lethargic state and remained drowsy for the remainder of the day. The parents at this time noted a newly erupted tooth. The infant was seen by her pediatrician who did not detect any specific abnormalities. The following day the infant was once again alert and active. Approximately five days prior to admission the child again became irritable. The episode repeated itself in an identical manner except that the lethargy noted on the third day persisted the following day as well. The attending physician examined the child in the Saskatoon City Hospital Emergency department and subsequently admitted her to hospital that day for further observation. The child's weight on admission was 17 lbs 1 oz. Initial laboratory urinalysis results were as follows: specific gravity 1.025, pH 5.5, trace of protein, negative for reducing substances, large quantity of ketones (by Ames Labstix), and a positive test for occult blood. Blood hemoglobin concentration was 13.2 g/100 ml and the white cell count was 12,400 cells/cu mm, 460-/o neutrophils, 51°'/o lymphocytes, and 30-/0 monocytes. Blood sugar on the second day of admission was 71 mg/100 ml. Spinal fluid obtained on the second day of admission and again on the fourth day was clear and had normal protein and glucose levels. The infant was given Ampicillin drops, 125 mg q 6 h starting the second day. Despite treatment the infant's clinical condition did not improve and by the third day of admission was taking fluids only by force. The nurses had noted a "sweet" odor to the urine from admission on. However,
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INTERMITTENT BRANCHED CHAIN KETONURIA
55
it was not until the fifth d a y t h a t the odor was characterized as similar to t h a t of maple syrup. A t e n t a t i v e diagnosis of I n t e r m i t t e n t Branched Chain K e t o n u r i a was made. All oral feedings were then discontinued and T r a v e r t 10%, Electrolyte #2 (Baxter) I.V. was started at a rate of 30 ml per hour. T h e following d a y elevated urinary levels of branched-chain amino acids were d e m o n s t r a t e d b y paper chromatography, confirming the diagnosis. A s u m m a r y of plasma aminoacid concentrations along with other pertinent d a t a is given in Table 1. After 24 hrs of intravenous fluids the infant's condition had improved. She appeared to recognize her mother, which she had not done the two days previous. However, there was still marked ptosis of the eyelids, ataxia, and occasionally opisthotonos. Intravenous fluids were discontinued after 36 hrs. B y this time the infant, although still ataxic had improved considerably. She would drink with encouragement and was given Pedialyte (Ross L a b o r a t o r i e s - - D i v i s i o n of Abbott) orally. Twelve hours later she was able to sit alone, was more alert, played with toys, and was drinking well. Seventy-two hours after the s t a r t of the protein-free fluid therapy, she was given a gelatin based formula. T h e basic formula used is given below. Protein Gelatin 30 g (i.e. 26 g protein) Fat Corn Oil 28 g Carbohydrate Dextri-maltose 61 g Minerals Calcium gluconate 4.0 g Copper 0.5 mg Dipotassium hydrogen phosphate 1.0 g Iodine 75 ug Iron 18 mg Magnesium 3 mg Manganese 0.5 mg Molybdenum 0.1 mg Monopotassium dihydrogen phosphate 1.0 g Sodium chloride 1.3 g Vitamins A 5000 IU D 400 I U B~ 3.0 ug Thiamine 2.0 mg Riboflavin 2.0 mg Niacinamide 10.0 mg Pyridoxine 1.0 mg Ascorbic acid 40.0 mg Water 900 ml T h e r e was some variation in this basic formula. During the first two days of feeding, only 20 g of gelatin was added rather than the 30 g used thereafter. Also after five days the corn oil was replaced b y an equivalent a m o u n t of Lipomul (Up john). T h e gelatin formula was gradually replaced b y Enfalac as given in T a b l e 1. Also fruit juices and fruit, vegetable and meat purees were added to the diet as given in T a b l e 1. After six days of intravenous and oral dietary t r e a t m e n t the infant's condition was almost normal, a slight irritability being the only s y m p t o m at this time. She remained in the hospital another two weeks for further dietary adjustment.
S6
IRWIN et al.
On discharge, the mother was given tables of protein content of foods the infant would receive. The only dietary restrictions imposed were a maximum limit on protein intake of 2.0 g/lb/day, with Enfalac being used as the main milk source. Whole milk was allowed only for pablum in the morning. The infant has continued to do well since discharge. DISCUSSION
Laboratory Diagnosis: The maple-syrup odor of the urine is practically pathognomonic of MSUD. Some caution should be exercised in respect to judgment of odors in urine in that a proper specimen should be collected. It is to be noted that the nursing staff had observed a "sweet" odor to the urine but had not characterized it as being similar to that of maple syrup. In addition the mother had noticed the urine to have a "peculiar odor" during the first attack and also prior to admission to the hospital. Later on observing the odor of a urine specimen collected in a disposable infant urine collection bag she stated that it was different than that which she had observed on the diapers. The odor may have been less intense at earlier stages of the attack, making it more difficult to characterize. It is also possible however that the odor was masked by remanents of compounds used for soaking, washing, and/or softening of the diapers. Positive urine ketone body tests with negative tests for glucose and reducing substances is further evidence for MSUD. However a negative test for ketone in the urine does not eliminate a diagnosis of MSUD. Scriver (9) recently described a variant of MSUD in which the urinary excretion of branched-chain keto acids did not seem to be raised. However there were excessive amounts of the equivalent hydroxy acids identified in the urine by gas chromatography. Reagents used for ketone body detection include ferric chloride and 2-4 dinitrophenylhydrazine. The keto acids may also be detected by commercial stick tests such as Ames Ketostix and Labstix (alkaline nitroprusside reagent). The colour produced on addition of ferric chloride solution to the urine of patients with MSUD has varied considerably. Fenichel (10) states the characteristic color produced is navy blue or black, Henry (11) grey with green tinge, and Lonsdale (6) brown-green. We obtained a brown color with a slight reddish tinge using 10% W / V ferric chloride in 2 N HCI. Labstix (Ames) test for ketone indicated large quantities present in the urine specimens prior to intravenous fluid treatment. After 18 hrs of IV protein-free fluid therapy both Labstix test for ketone bodies and the ferric chloride test were negative. At this time the urine still had a maple syrup odor. Phenistix (Ames) is a buffered ferric chloride stick test with magnesium ions added to minimize interference by phosphates. It is less sensitive to keto acids than the ferric chloride solutions and therefore has been reported negative in a number of instances (11, 1~). The Phenistix test on urine from patient N.R. was negative. The tests just described enable one to make an initial diagnosis so that treatment may begin without delay. Further verification is necessary in the form of
INTERMITTENT BRANCHED CHAIN KETONURIA
57
quantitative or semiquantitative demonstration of excess leucine, isoleucine and valine in both plasma and urine, demonstration of alloisoleucine in plasma and urine, and demonstration of excess branched-chain keto acids in plasma and urine. Excellent reviews of suitable procedures for assay of the above compounds have been written by Dancis and Levitz (13) and Hsia and Inouye (15). Plasma levels of leucine, isoleucine and valine for patient N.R. are given in Table 1. The analysis were done on a Technicon amino-acid auto-analyzer. Alloisoleucine was also clearly demonstrated in the first plasma specimen analyzed. Large quantities of leucine, isoleucine, and valine in urine were demonstrated by paper chromatography. No attempt was made to demonstrate excess branched-chain keto acids in patients N.R.'s plasma. However total keto acid in urine as measured by the 2-4 dinitrophenylhydrazine reaction is given in Table 1. Final confirmation of a diagnosis of MSUD is made by demonstration of reduced branched-chain oxidative decarboxylase activity of white cells or fibroblasts (~, lg). The decarboxylase activity of cultured skin fibroblasts of N.R. against each of the three branched-chain keto acids was approximately 10% of normal. Dietary Treatment: Initial intravenous, protein-free-fluid treatment of the patient resulted in a rapid decline of plasma leucine, isoleucine, and valine levels to normal values. Urine keto acids dropped even more rapidly (Table 1). In retrospect our dietary regimen was probably more rigorous than necessary for treatment of the intermittent phenotype. When the plasma amino-acid levels had returned to normal we could probably have started the infant on Enfalac and omitted the gelatin formula. It is thought that meat purees could also have been introduced at an earlier stage. Scriver el al. recently described a variant of MSUD which responded to relatively large doses of thiamine (9). Steen-Johnsen et al. also claim some success in the treatment of an intermittent phenotype during acute attacks with "Vitamin B" therapy (8). Since the dietary treatment of our case has been relatively simple, augmentation of the diet with co-factors of branched-chain decarboxylase has not been tried. ACKNOWLEDGMENTS
The authors wish to thank Dr. Joseph Dancis, New York Medical University Center, New York, for the oxidative decarboxylase enzyme studies. They are also grateful to Drs. A. Hill and W. A. Zaleski, Alvin Buckwold Mental Retardation unit, University of Saskatchewan, Saskatoon, for the amino acid analyses. REFERENCES
I. MENKES, J. H., HURST, P. L., & CRAIG, J.M. A new syndrome: progressive familial infantile cerebral dysfunction associated with an unusual urinary substance. Pediatrics 14, 462--466 (1954). ;~. DANCIS, J., HUTZIER, J., & LEVITZ, M. Diagnosis of maple syrup urine disease
58
8. 4. 5. 6. 7. 8. 9. 10. 11.
I~. 18.
14. I6.
IRWIN et al. (branched chain ketoaciduria) by in vitro study of peripheral leukocyte. Pediatrics 32,234--238 (1963). MORRIS, M. D., LEWIS, B. D., DOOLAN, P. D. & HARPER, H . A . Clinical and biochemical observations on an apparently nonfatal variant of branched-chain ketoaciduria (maple syrup urine disease). Pediatrics 28, 918--923 (1961). DANCIS, J., HUTZLER, J. & ROKKONES, T. Intermittent branched-chain ketonuria. New Eng. J. Med. 276, 84-89 (1967). MORRIS, M. D., FISHER, D. A. & FISHER, R. Late-onset branched-chain ketoaciduria (Maple syrup urine disease). J. Lancet 86, pp. 149-152 (1966). LONSDALE, D., MERCER, R. D., & FAULKNER, W . R . Maple syrup urine disease. Am. J. Dis. Child, 106, 258-266 (1963). KIIL, R. & ROKKONES, T. Late manifesting variant of branched-chaln ketoaciduria (maple syrup urine disease). Acta Paediat., 53, 356--364 (1964). STEEN-JOHNSEN, J., VELLAN, E. J. & GJESSING, L . R . Maple syrup urine disease variant--amino acid pattern and problems of treatment during acute attacks. Acta Paediat. Scand., Supp. 206, 71-73 (1970). SCRIVER, C. R., CLOW, C. L., MACKENZIE, S. & DELVIN, E. Thiamine-responslve maple-syrup-urine disease. The Lancet I, 310-312 (1971). FENICHEL, G. M. Maple syrup urine disease. The Pediatric Clinics of North America, 17, 326-355 (1970). HENRY, R . J . Clinical Chemistry--Principles and techniques, Hoeber Medical Division. Harper and Row, New York, 1964, p. 337. LONSDALE, D. & BARBER, D . H . Maple-syrup-urine disease. New Eng. J. Med., 271, 1338-1341 (1964). DANCIS, J. & LEVITZ, M. Maple syrup urine disease (Branched chain ketonuria). In The Metabolic Basis of Inherited Disease, Stanbury, J. R., Wyngaarden, J. B. & Frederickson, D. S., eds. McGraw-Hill, New York, 1966, pp. 353-365. HSIA, D. Y. & INOUYE, T. Inborn errors of metabolism, Part 2 Laboratory Methods, Year Book Medical Publishers Inc., Chicago, 1966, pp. 54-69 and pp. 79-83. DANCIS, J., HUTZLER, J. & COX, R.P. Enzyme defect in skin fibroblasts in intermittent branched-chain ketonuria and in maple syrup urine disease. Biochem. Med., 2,407-411 (1969).
I N T E R M I T T E N T B R A N C H E D CHAIN K E T O N U R I A (VARIANT OF MAPLE SYRUP URINE DISEASE) W. C. IRWIN, S. B. MARTEL AND N. GOLUBOFF Saskatoon City Hospital, Saskatoon, Canada (Received ~(arch 24, 1971)
SUMMARY 1. A patient (N.R.) with Intermittent Branched Chain Ketonuria (Intermittent Maple Syrup Urine Disease) is described. It is the first case of this very rare abnormality to be documented in Canada. 2. Laboratory diagnosis was initially made on the basis of a maple syrup-like odor to the urine, along with a strongly positive ketone test and a negative reducing substance test. The diagnosis was confirmed by elevated urinary and plasma levels of leucine, isoleucine, alloisoleucine and valine, and by the demonstration of reduced branched-chain oxidative decarboxylase activity in cultured fibroblasts. Urinary keto acids were also elevated. 3. Dietary treatment of the patient (N.R.) consisted of replacement of oral feedings by I.V. fluids for 36 hours, then oral protein free fluids for a further 36 hours after which a gelatin based formula was given. The gelatin based formula was gradually replaced by Enfalac and was supplemented with regular commercial fruit, meat, and vegetable purees. In retrospect the dietary treatment was probably more rigorous than necessary.
MAPLE SYRUP URINE DISEASE (MSUD) WAS INITIALLYDESCRIBEDas a syndrome by Menkes, Hurst and Craig in 1954 (1). The untreated case manifests as an early onset, fatal disorder, in which neurological symptoms are accompanied by excretion of urine having a sweet maple syrup-like odor. Dancis demonstrated that the basic defect was a reduction of branched-chain keto acid decarboxylase activity (2). As a result the branched-chain keto acids and their respective aminoacids (leucine, isoleucine, and valine), are elevated in the blood and in the urine. Morris et al. in 1961 described an infant who suffered from transient episodes of MSUD (8). The child was completely normal between attacks. This variant of MSUD has been termed Intermittent Branched Chain Ketonuria (6). The intermittent variant has been diagnosed in two other infants in the United States, one of these being a sibling of the first case (5, 6). In addition, three cases Correspondence: Dr. W. C. Irwin, Department of Pathology, Saskatoon City Hospital. Saskatoon, Saskatchewan, Canada.
INTERMITTENT BRANCHED CHAIN KETONURIA
53
have been diagnosed in Norway, two of these being siblings (7, 8). A description of the first case of Intermittent Branched Chain Ketonuria to be diagnosed in Canada follows: CASE REPORT
A 9½ month old white female (N.R.) was admitted to Saskatoon City Hospital with a diagnosis of left otitis media and debility of unknown etiology. The infant was the product of a normal pregnancy, except that the date of partum exceeded the expected date by approximately five weeks. Weight at birth was 7 lbs 1 oz. The infant was breast fed for the first five months, after which she received Enfalac (Mead Johnson). The transition to Enfalac was without incidence. Commercial infant purees of meat, vegetables and fruit were well tolerated. At approximately 9 months of age the milk source was changed from Enfalac to homogenized whole milk, which had been used previously in preparing the child's pablum. Neurologically, the child's development was relatively normal. He sat with assistance at 5 months and unassisted at 7½ months. The mother felt his locomotor development had been retarded by the restriction of a brace applied for kyllosis correction. She described him as being alert, independent, active and happy. Approximately two weeks prior to admission the child had suddenly become irritable and whined constantly. This persisted for two days during which smaller feedings were noted. However the food was consumed willingly and without emesis. The only other symptom noticed by the mother was a "very peculiar smelling urine". On the third day the child awoke in a lethargic state and remained drowsy for the remainder of the day. The parents at this time noted a newly erupted tooth. The infant was seen by her pediatrician who did not detect any specific abnormalities. The following day the infant was once again alert and active. Approximately five days prior to admission the child again became irritable. The episode repeated itself in an identical manner except that the lethargy noted on the third day persisted the following day as well. The attending physician examined the child in the Saskatoon City Hospital Emergency department and subsequently admitted her to hospital that day for further observation. The child's weight on admission was 17 lbs 1 oz. Initial laboratory urinalysis results were as follows: specific gravity 1.025, pH 5.5, trace of protein, negative for reducing substances, large quantity of ketones (by Ames Labstix), and a positive test for occult blood. Blood hemoglobin concentration was 13.2 g/100 ml and the white cell count was 12,400 cells/cu mm, 460-/o neutrophils, 51°'/o lymphocytes, and 30-/0 monocytes. Blood sugar on the second day of admission was 71 mg/100 ml. Spinal fluid obtained on the second day of admission and again on the fourth day was clear and had normal protein and glucose levels. The infant was given Ampicillin drops, 125 mg q 6 h starting the second day. Despite treatment the infant's clinical condition did not improve and by the third day of admission was taking fluids only by force. The nurses had noted a "sweet" odor to the urine from admission on. However,
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INTERMITTENT BRANCHED CHAIN KETONURIA
55
it was not until the fifth d a y t h a t the odor was characterized as similar to t h a t of maple syrup. A t e n t a t i v e diagnosis of I n t e r m i t t e n t Branched Chain K e t o n u r i a was made. All oral feedings were then discontinued and T r a v e r t 10%, Electrolyte #2 (Baxter) I.V. was started at a rate of 30 ml per hour. T h e following d a y elevated urinary levels of branched-chain amino acids were d e m o n s t r a t e d b y paper chromatography, confirming the diagnosis. A s u m m a r y of plasma aminoacid concentrations along with other pertinent d a t a is given in Table 1. After 24 hrs of intravenous fluids the infant's condition had improved. She appeared to recognize her mother, which she had not done the two days previous. However, there was still marked ptosis of the eyelids, ataxia, and occasionally opisthotonos. Intravenous fluids were discontinued after 36 hrs. B y this time the infant, although still ataxic had improved considerably. She would drink with encouragement and was given Pedialyte (Ross L a b o r a t o r i e s - - D i v i s i o n of Abbott) orally. Twelve hours later she was able to sit alone, was more alert, played with toys, and was drinking well. Seventy-two hours after the s t a r t of the protein-free fluid therapy, she was given a gelatin based formula. T h e basic formula used is given below. Protein Gelatin 30 g (i.e. 26 g protein) Fat Corn Oil 28 g Carbohydrate Dextri-maltose 61 g Minerals Calcium gluconate 4.0 g Copper 0.5 mg Dipotassium hydrogen phosphate 1.0 g Iodine 75 ug Iron 18 mg Magnesium 3 mg Manganese 0.5 mg Molybdenum 0.1 mg Monopotassium dihydrogen phosphate 1.0 g Sodium chloride 1.3 g Vitamins A 5000 IU D 400 I U B~ 3.0 ug Thiamine 2.0 mg Riboflavin 2.0 mg Niacinamide 10.0 mg Pyridoxine 1.0 mg Ascorbic acid 40.0 mg Water 900 ml T h e r e was some variation in this basic formula. During the first two days of feeding, only 20 g of gelatin was added rather than the 30 g used thereafter. Also after five days the corn oil was replaced b y an equivalent a m o u n t of Lipomul (Up john). T h e gelatin formula was gradually replaced b y Enfalac as given in T a b l e 1. Also fruit juices and fruit, vegetable and meat purees were added to the diet as given in T a b l e 1. After six days of intravenous and oral dietary t r e a t m e n t the infant's condition was almost normal, a slight irritability being the only s y m p t o m at this time. She remained in the hospital another two weeks for further dietary adjustment.
S6
IRWIN et al.
On discharge, the mother was given tables of protein content of foods the infant would receive. The only dietary restrictions imposed were a maximum limit on protein intake of 2.0 g/lb/day, with Enfalac being used as the main milk source. Whole milk was allowed only for pablum in the morning. The infant has continued to do well since discharge. DISCUSSION
Laboratory Diagnosis: The maple-syrup odor of the urine is practically pathognomonic of MSUD. Some caution should be exercised in respect to judgment of odors in urine in that a proper specimen should be collected. It is to be noted that the nursing staff had observed a "sweet" odor to the urine but had not characterized it as being similar to that of maple syrup. In addition the mother had noticed the urine to have a "peculiar odor" during the first attack and also prior to admission to the hospital. Later on observing the odor of a urine specimen collected in a disposable infant urine collection bag she stated that it was different than that which she had observed on the diapers. The odor may have been less intense at earlier stages of the attack, making it more difficult to characterize. It is also possible however that the odor was masked by remanents of compounds used for soaking, washing, and/or softening of the diapers. Positive urine ketone body tests with negative tests for glucose and reducing substances is further evidence for MSUD. However a negative test for ketone in the urine does not eliminate a diagnosis of MSUD. Scriver (9) recently described a variant of MSUD in which the urinary excretion of branched-chain keto acids did not seem to be raised. However there were excessive amounts of the equivalent hydroxy acids identified in the urine by gas chromatography. Reagents used for ketone body detection include ferric chloride and 2-4 dinitrophenylhydrazine. The keto acids may also be detected by commercial stick tests such as Ames Ketostix and Labstix (alkaline nitroprusside reagent). The colour produced on addition of ferric chloride solution to the urine of patients with MSUD has varied considerably. Fenichel (10) states the characteristic color produced is navy blue or black, Henry (11) grey with green tinge, and Lonsdale (6) brown-green. We obtained a brown color with a slight reddish tinge using 10% W / V ferric chloride in 2 N HCI. Labstix (Ames) test for ketone indicated large quantities present in the urine specimens prior to intravenous fluid treatment. After 18 hrs of IV protein-free fluid therapy both Labstix test for ketone bodies and the ferric chloride test were negative. At this time the urine still had a maple syrup odor. Phenistix (Ames) is a buffered ferric chloride stick test with magnesium ions added to minimize interference by phosphates. It is less sensitive to keto acids than the ferric chloride solutions and therefore has been reported negative in a number of instances (11, 1~). The Phenistix test on urine from patient N.R. was negative. The tests just described enable one to make an initial diagnosis so that treatment may begin without delay. Further verification is necessary in the form of
INTERMITTENT BRANCHED CHAIN KETONURIA
57
quantitative or semiquantitative demonstration of excess leucine, isoleucine and valine in both plasma and urine, demonstration of alloisoleucine in plasma and urine, and demonstration of excess branched-chain keto acids in plasma and urine. Excellent reviews of suitable procedures for assay of the above compounds have been written by Dancis and Levitz (13) and Hsia and Inouye (15). Plasma levels of leucine, isoleucine and valine for patient N.R. are given in Table 1. The analysis were done on a Technicon amino-acid auto-analyzer. Alloisoleucine was also clearly demonstrated in the first plasma specimen analyzed. Large quantities of leucine, isoleucine, and valine in urine were demonstrated by paper chromatography. No attempt was made to demonstrate excess branched-chain keto acids in patients N.R.'s plasma. However total keto acid in urine as measured by the 2-4 dinitrophenylhydrazine reaction is given in Table 1. Final confirmation of a diagnosis of MSUD is made by demonstration of reduced branched-chain oxidative decarboxylase activity of white cells or fibroblasts (~, lg). The decarboxylase activity of cultured skin fibroblasts of N.R. against each of the three branched-chain keto acids was approximately 10% of normal. Dietary Treatment: Initial intravenous, protein-free-fluid treatment of the patient resulted in a rapid decline of plasma leucine, isoleucine, and valine levels to normal values. Urine keto acids dropped even more rapidly (Table 1). In retrospect our dietary regimen was probably more rigorous than necessary for treatment of the intermittent phenotype. When the plasma amino-acid levels had returned to normal we could probably have started the infant on Enfalac and omitted the gelatin formula. It is thought that meat purees could also have been introduced at an earlier stage. Scriver el al. recently described a variant of MSUD which responded to relatively large doses of thiamine (9). Steen-Johnsen et al. also claim some success in the treatment of an intermittent phenotype during acute attacks with "Vitamin B" therapy (8). Since the dietary treatment of our case has been relatively simple, augmentation of the diet with co-factors of branched-chain decarboxylase has not been tried. ACKNOWLEDGMENTS
The authors wish to thank Dr. Joseph Dancis, New York Medical University Center, New York, for the oxidative decarboxylase enzyme studies. They are also grateful to Drs. A. Hill and W. A. Zaleski, Alvin Buckwold Mental Retardation unit, University of Saskatchewan, Saskatoon, for the amino acid analyses. REFERENCES
I. MENKES, J. H., HURST, P. L., & CRAIG, J.M. A new syndrome: progressive familial infantile cerebral dysfunction associated with an unusual urinary substance. Pediatrics 14, 462--466 (1954). ;~. DANCIS, J., HUTZIER, J., & LEVITZ, M. Diagnosis of maple syrup urine disease
58
8. 4. 5. 6. 7. 8. 9. 10. 11.
I~. 18.
14. I6.
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