Am J Hum Genet 29:272-279, 1977
Maple Syrup Urine Disease: Branched-Chain Keto Acid Decarboxylation in Fibroblasts as Measured with Amino Acids and Keto Acids JOSEPH DANCIS,1 JOEL HUTZLER,1 AND RODY P. Cox2.3
Maple syrup urine disease (MSUD) is an autosomal recessive disease in which the primary defect is that of oxidative decarboxylation of the branched-chain keto acids (BCKA). In the original demonstration of the metabolic defect, leukocytes were incubated with radioactive branched-chain amino acids (BCAA) labeled in the carboxyl carbon. There was a sharp reduction in the evolution of radioactive CO2 by leukocytes from patients with MSUD [1]. Appropriate studies eliminated a defect in transamination, the first step in the degradative pathway of the BCAA. The essentials of the original technique have been retained in this laboratory in all subsequent studies of MSUD, except for one in which the BCKA were used as substrate [2]. Extensive experience has demonstrated a correlation between results using BCAA as substrate and the clinical history, indicating that the assay provided a reasonably accurate measure of the patient's ability to degrade dietary BCAA [3, 4]. Several laboratories have preferred to use radioactive BCKA as substrate, thereby avoiding the interposed step of transamination [5-8]. Recently, Wendel et al. [6] have reported an extensive study of the kinetics of decarboxylation of radioactive BCKA by intact skin fibroblasts using a microassay developed in their laboratory. They reported that at low substrate concentrations (5 x 10-4 M) the metabolic defect in patients with MSUD was readily demonstrable. At high substrate concentrations (15 -160 x 10-4 M), significant decarboxylation was exhibited by fibroblasts from patients with MSUD, including those with the classical disease (with one exception), and there was extensive overlap in results between controls and mutants. In the present study we have reinvestigated the important and unexpected observations of Wendel et al. [6]. Subjects that had been carefully classified as classical or variant types of MSUD according to criteria previously established in this laboratory were selected for study. Skin fibroblasts were assayed concurrently with BCAA Received September 16, 1976; revised January 20, 1977. This work was supported by grants HD 04526 and AM 14528 from the National Institutes of Health and by the Samuel A. Berger Foundation. 1 Department of Pediatrics, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10006. 2 Department of Medicine and Pharmacology, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10016. 3 Division of Human Genetics, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10016. © 1977 by the American Society of Human Genetics. All rights reserved.
272
MAPLE SYRUP URINE DISEASE
273
or BCKA. The results with leucine confirmed our previous experience, demonstrating its usefulness in establishing the clinical diagnosis. In contrast, the studies with a-ketoisocaproic acid at substrate concentrations of 1 mM and above were similar to the observations of Wendel et al. [6], showing limited correlation with the clinical diagnosis. MATERIALS AND METHODS
Radioactive Substrates L-leucine- 1-14C was purchased from New England Nuclear (Cambridge, Mass.). Purity was checked by ascending chromatography in n-butanol:acetic acid:water, 60:15:25 (v/v/v) and phenol:water, 80:20 (w/v). L-leucine was added to the radioactive amino acid to make a final solution of 20 mM in 0.1 M NaCl, 0.5 Ac/Amole. The pH was adjusted to 3 with HCl. Sodium a-ketoisocaproate- 1- 14C was prepared from L-leucine- 1- 14C by oxidation with L-amino acid oxidase [9]. The keto acid was checked for purity by ascending chromatography in n-butanol:acetic acid:water, 60:15:25, in n-butanol:water:concentrated ammonia, 14:2: 1, and by high voltage electrophoresis in 1 M acetic acid. The keto acid was diluted to a 200 mM solution, 0.05 ucL/imole using sodium-a-ketoisocaproate prepared synthetically [10]. The pH was approximately 3 without further adjustment. Substrates were stored at -20'C in a 50 ml Erlenmeyer flask with a well containing 0.1 ml 1 N NaOH. The flasks were thawed 30 min prior to use and agitated on a Dubnoff water bath at 20°C to trap volatile radioactive materials. The pH of the solution was checked frequently and maintained at 3. Tissue Culture Biopsies of skin were grown in monolayer culture in Waymouth's medium containing 10% fetal calf serum and antibiotics (penicillin 50 U, streptomycin 50 ,ug, and kanamycin 30 ,ug per ml). For many subjects, the fibroblasts had been maintained in liquid nitrogen for several months to several years. These were thawed, grown to confluence and harvested 4 to 10 days after the last medium change.
Enzyme Assays Fibroblasts were collected with a rubber policeman and incubated in 0.35 ml of solution containing 0.25 ml Krebs-Ringer phosphate buffer, pH 7.1, 0.005 ml thiamine HCl, 100 mg/ml and radioactive substrate. When necessary, pH was readjusted to 7.1 with 1 N NaOH. Incubations were for 120 min at 35°C with '4C02 collected in a center well charged with 0.1 ml 1 N NaOH. At the end of the incubation, the NaOH was transferred to a scintillation cocktail to determine radioactivity. The cells were digested and protein determined by the method of Lowry et al. [11]. The procedure has been previously described in detail [12]. Heat Stability Cells were harvested into buffer and heated on a water bath for 10 min at indicated temperatures and then assayed for decarboxylase activity. Source of Fibroblasts Fibroblasts from six control subjects were selected to provide an equal sex distribution and an age range that would cover that represented by the patients with MSUD. Many of the patients with MSUD have been previously reported. Of these, TeHe, CaGu, and MeCa represent classical cases as previously defined; ElHa and ThAd are variants [4]. BeRo is the mother of NiRo and is also a variant [13]. GM612 was received from the Genetic Mutant Cell Repository, Camden, N.J., and described as a classical case. DaMa was referred by Dr. David L. Valle, Johns Hopkins University School of Medicine and AnKl, by Dr. A. M. 0. Veale, University of Auckland, New Zealand. The former is consistent with a classical case by clinical history and
274
DANCIS ET AL.
enzyme level, and the latter was classified by clinical history and this study as a variant with a low tolerance for the BCAA. FeHu was referred by Dr. Selma Snyderman of this institution. RESULTS
In previous reports from this laboratory using BCAA as substrate, decarboxylase activity was reported as the ratio of CO2 evolution to the incorporation of the radioactive amino acid into cell protein. The latter established a baseline of metabolic activity against which to measure decarboxylase activity. The results were reproducible and provided a fairly sharp definition of the magnitude of the metabolic defect. In the present study, the MSUD fibroblasts had passed through many additional generations after storage, and the rate of incorporation of radioactive amino acids into protein was lower and more erratic. For that reason, and for better comparison with the experiments with BCKA, the results are now expressed as nmoles CO2 evolved per mg cell protein. The results with leucine are presented in figure 1. Each point for the MSUD cases, classical and variant, represent the mean of two to four determinations. Variability Te So Ba St
1.6-
'.4-
.12 C31
Kr St
\La 0Wi
E .0
Do Ve
0 0.8D 0 0
0.14
0.2
~~ ,4
~
WI ~~~~~~~Sh
x
-
~ ~ ~~~..Th Ad
~ ~ ~
~~~~~
~El Ho n~ Fr Hu, Do Ma
0.1 0.5
1.5
4.5
1GM612
Leucine conc. (mM) FIG. 1.-Fibroblasts were incubated for 120 min with leucine-1-14C at different concentrations (1.5 ); variants (-- -); classical mM), and the liberated radioactive CO2 was measured. Controls ( MSUD (--- -). The five classical MSUD cases had values close to background and are represented by one line. The results with classical and variant MSUD represent mean of 2-4 determinations per point; controls are single determinations.
MAPLE SYRUP URINE DISEASE
275
about the means were relatively small. Radioactivity liberated by fibroblasts from classical MSUD never exceeded 0.1 nmole per mg. Maximal decarboxylase activity was approached or reached at levels of substrate concentration of 1.5 mM leucine. Consistent with previous reports is the ability to distinguish among the classical, variant, and control subjects [3, 4]. There was some minor irregularity in BCAA decarboxylase activity of fibroblasts from several variants when compared to the severity of their disease. For example, ElHa fibroblasts had less activity than previously reported which may reflect the higher passage (more advanced age) of her cells. Exact subclassification among variants is inherently difficult, requiring maximal control of the assay procedure, because of the small differences in decarboxylase activity among the subgroups. The results with a-ketoisocaproate are presented in figure 2. Variability about the means of replicate determinations for the classical and variant cases of MSUD was much broader than observed with leucine. The range for some points was as broad as 100%, though it was generally less than 50%. Maximal decarboxylase activity was approached or reached at 15 mM, 10 times the substrate concentration for leucine. Using this level to compare the decarboxylase activity among the various subjects, we find the MSUD fibroblasts functioning at a generally lower level than the controls. Seven of nine MSUD cases liberated 4 nmoles or less of radioactive CO2 per mg protein per 120 min as compared to 2 of seven controls. However, there were many striking inconsistencies between the results with a-ketoisocaproate and the clinical history of the classical cases of MSUD. Only DaMa remained at very low levels. All other classical MSUD cases had easily detectable decarboxylase activity, and two (CaGu, MeCa) had levels that entered the normal range. A similar variability was noted with the variant MSUD cases. Heat stability of the decarboxylase activity was investigated in an attempt to exclude a nonenzymatic process (fig. 3). There was a sharp drop in activity following 10 min of exposure to temperatures between 420C and 50TC. With leucine, activity disappeared at 60TC, and for a-ketoisocaproate, it had virtually disappeared at 80TC. Disruption of the cells by freeze-thaw caused a sharp reduction in activity with both substrates. The observations are consistent with enzyme catalyzed reactions. DISCUSSION
The major objective in these studies was to investigate the report by Wendel et al. [6] that skin fibroblasts from patients with MSUD can decarboxylate BCKA when presented in high concentration and that the decarboxylation activity; under these circumstances, was often of the same magnitude as that in normal fibrobtasts. We decided to use the technique established in this laboratory with which we have had many years experience and to select fibroblasts from patients that had been carefully classified clinically. The fibroblasts had been stored in our "bank" under liquid nitrogen. On initiating fibroblast cultures from frozen stocks, they were again assayed for the severity of their defect using as substrate the BCAA, a technique which we have shown to correlate closely with clinical manifestations [4]. No attempt was made to exactly duplicate Wendel's methods. There are significant differences in the two assay techniques. We have harvested skin fibroblasts after growth to confluence by
276
DANCIS ET AL.
I18
16
x
ShWi
1 4-
E 0 12-
0
0~~~~
E8~~~~~~~~~~~mO E8En
-
.
~ ~~
~
~
~
~
-
E
~ ~~AK
2; H^ -~ ~ ~ ~ ~ DoV o~~~~~~~~~~~~oG
Ketoisoca proic ( mM ) FIG. 2. -Fibroblasts were incubated for 120 mmn with a-ketoisocaproic acid- l-14C at different concentrations and the liberated CO2 was measured. Values for those points surpassing values on vertical ordinate are indicated on the graph (TeSo, ShWi). (See fig. 1 for key.) The results with classical and variant MSUD represent mean of 2-4 determinations per point; controls are single determinations.
mechanically scraping them into buffer and incubating in suspension approximately one million cells per assay for 90 min Wendel et al [7] seeded 10,000-20,000 cells into each well of microtiter plates, incubated them overnight, and assayed their enzyme activity for 4 hr while still in monolayer. Our results with a-ketoisocaproate are similar to those reported by Wendel et al. [6] with high levels of substrate (>5 mM). Fibroblasts from patients with classical MSUD exhibited decarboxylase activity, and the results of controls and mutants, both variant and classical MSUD, overlapped extensively (fig. 2). We did not use substrate concentrations of keto acid below 1.0 mM, because at these low levels, it is necessary to use substrates with high specific activity which yields a background activity which
MAPLE SYRUP URINE DISEASE
277
100 80
C._) cs
60
i
0
40
0
C)a) G
I 20
so~~~~~~1 0
Not 30 40 50 60 70 80 90 100 treated Preincubation
temperature
FIG. 3. -Fibroblasts were incubated in buffer for 10 min at indicated temperatures, and then decarboxylase activity was measured. L-leucine-1-14C, 1.5 mM (ma -); a-ketoisocaproate-l-14C, 15 mM we considered too high to be acceptable. Wendel reported results with substrate concentrations as low as 10-5 M and stated that differentiation between mutants and controls was satisfactory at 5 x 10-5 M.* Interpretation of results with intact cells in terms of enzyme kinetics is very hazardous, as acknowledged by Wendel et al. [7], because of the multiple factors that may influence BCKA decarboxylation such as transport of substrate, pool sizes, competing reactions, etc. Despite this concern, these investigators suggest that their observations could be explained by the presence of two BCKA dehydrogenases with different affinities for the substrate. They speculate that only the enzyme with high affinity is the enzyme that is mutant in MSUD. One patient with classical MSUD in their series (and one in ours) had reduced decarboxylase activity at both low and high concentrations and would remain unexplained if both enzymes were totally independent. Our data with leucine as substrate are not inconsistent with the hypothesis of Wendel et al. [7]. Plateauing of decarboxylase activity at substrate concentrations considerably below those obtained with the keto acid (figs. 1 and 2) suggest a limiting factor proximal to this step such as transport of amino acid into or within the cell and/or transamination rate. We did not determine the intracellular concentration of radioactive a-ketoisocaproic acid at the various concentrations of radioactive leucine in the medium. If the intracellular concentrations of KIC are lower than the medium leucine * In table 2 of Wendel's article [6], decarboxylase activity is presented with BCKA at 5 x 10-4 M. However, the activities correspond to those in his figure 2 at 5 x 10-5 M.
278
DANCIS ET AL.
levels, as we suspect, and if there are two BCKA decarboxylases of differing affinities, the low affinity enzyme would be relatively inactive in our system. The decarboxylation that is measured would reflect the activity of the high affinity enzyme. This high affinity enzyme is presumably the physiologically effective one, and the one that undergoes mutation in MSUD. Regardless of the theoretical explanation, the most important conclusion from our studies is that, using standard techniques with intact cells, the BCAA appears to be the preferred substrate. As we have noted before, in association with our experience with hypoxanthine phosphoribosyltransferase deficiencies, the use of intact fibroblasts with natural substrates more closely approaches in vivo conditions at the cellular level and may have an advantage as a clinical model [14]. The observation appears to be pertinent of MSUD and its variants, also. However, investigations of metabolic defects at the molecular level require simpler systems. Until the problem of isolating an active BCKA dehydrogenase is solved, it may be best to postpone further speculation concerning questions such as the presence of specific decarboxylases for each BCKA, enzyme kinetics, and identification of the mutant site of the dehydrogenase complex. SUMMARY
Branched-chain keto acid decarboxylase activity in skin fibroblasts from control subjects and from patients with classical and variant forms of maple syrup urine disease (MSUD) was measured with leucine and a-ketoisocaproic acid. When the keto acid was used as substrate in high concentrations (more than 5 mM), the three groups overlapped extensively, even classical cases of MSUD exhibiting decarboxylase activity. With leucine as substrate, decarboxylase activity plateaued at about 1.5 mM, and the three groups could be clearly differentiated. Classical cases of MSUD had minimal or no decarboxylase activity.
1. 2. 3. 4. 5.
6. 7.
8. 9.
REFERENCES DANCIS J, HUTZLER J, LEVITZ M: Metabolism of the white blood cells in maple syrup urine disease. Biochim Biophys Acta 43:342 - 343, 1960 DANCIS J, HUTZLER J, LEVITZ M: Detection of the heterozygote in maple syrup urine disease. J Pediatr 66:595-603, 1965 DANCIS J, HUTZLER J, Cox RP: Enzyme defect in skin fibroblasts in intermittent branched-chain ketonuria and in maple syrup urine disease. Biochem Med 2:407-411, 1969 DANCIS J, HUTZLER J, SNYDERMAN SE, Cox, RP: Enzyme activity in classical and variant forms of maple syrup urine disease. J Pediatr 81:312-320, 1972 DANNER DJ, ELSAS LJ II: Subcellular distribution and cofactor function of human branched chain a-ketoacid dehydrogenase in normal and mutant cultured skin fibroblasts. Biochem Med 13:7-22, 1975 WENDEL U, WENTRUP H, RUDIGER HW: Maple syrup urine disease: analysis of branched chain ketoacid decarboxylation in cultured fibroblasts. Pediatr Res 9:709-717, 1975 WENDEL U, WOHLER W, GOEDDE HW, LANGENBECK U, PASSARGE E, RUDIGER HW: Rapid diagnosis of maple syrup urine disease (branched chain ketoaciduria) by microenzyme assay in leukocytes and fibroblasts. Clin Chim Acta 45:433-441, 1973 SEEGMILLER JE, WESTALL RG: The enzyme defect in maple syrup urine disease. J Ment DeficRes 11:288, 1967 MEISTER A: Sodium a-ketoisocaproate. Biochem Prep 3:66-70, 1953
MAPLE SYRUP URINE DISEASE
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10. ADICKES F, ANDRESEN G: Series of normal aliphatic 83-hydroxy and a-keto acids, Justus Liebig's Annal der Chem 555:41-42, 1943 11. LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 12. DANCIS J, HUTZLER J, Cox RP: Evaluation of amino acid metabolism in maple syrup urine disease, in Tissue Culture, Methods and Applications, edited by KRUSE PF JR, PATTERSON MK JR, New York, Academic Press, 1973, pp 639-643 13. ZALESKI LA, DANCIS J, Cox RP, HUTZLER J, ZALESKI WA, HILL A: Variant maple syrup urine disease in mother and daughter. Can Med Assoc J 109:299-304, 1973 14. HOLLAND MJC, DILORENzo AM, DANCIS J, BALIS ME, Yu TF, Cox RP: Hypoxanthine phosphoribosyltransferase activity in intact fibroblasts from patients with X-linked hyperuricemia. J Clin Invest 57:1600-1605, 1976
Maple Syrup Urine Disease: Branched-Chain Keto Acid Decarboxylation in Fibroblasts as Measured with Amino Acids and Keto Acids JOSEPH DANCIS,1 JOEL HUTZLER,1 AND RODY P. Cox2.3
Maple syrup urine disease (MSUD) is an autosomal recessive disease in which the primary defect is that of oxidative decarboxylation of the branched-chain keto acids (BCKA). In the original demonstration of the metabolic defect, leukocytes were incubated with radioactive branched-chain amino acids (BCAA) labeled in the carboxyl carbon. There was a sharp reduction in the evolution of radioactive CO2 by leukocytes from patients with MSUD [1]. Appropriate studies eliminated a defect in transamination, the first step in the degradative pathway of the BCAA. The essentials of the original technique have been retained in this laboratory in all subsequent studies of MSUD, except for one in which the BCKA were used as substrate [2]. Extensive experience has demonstrated a correlation between results using BCAA as substrate and the clinical history, indicating that the assay provided a reasonably accurate measure of the patient's ability to degrade dietary BCAA [3, 4]. Several laboratories have preferred to use radioactive BCKA as substrate, thereby avoiding the interposed step of transamination [5-8]. Recently, Wendel et al. [6] have reported an extensive study of the kinetics of decarboxylation of radioactive BCKA by intact skin fibroblasts using a microassay developed in their laboratory. They reported that at low substrate concentrations (5 x 10-4 M) the metabolic defect in patients with MSUD was readily demonstrable. At high substrate concentrations (15 -160 x 10-4 M), significant decarboxylation was exhibited by fibroblasts from patients with MSUD, including those with the classical disease (with one exception), and there was extensive overlap in results between controls and mutants. In the present study we have reinvestigated the important and unexpected observations of Wendel et al. [6]. Subjects that had been carefully classified as classical or variant types of MSUD according to criteria previously established in this laboratory were selected for study. Skin fibroblasts were assayed concurrently with BCAA Received September 16, 1976; revised January 20, 1977. This work was supported by grants HD 04526 and AM 14528 from the National Institutes of Health and by the Samuel A. Berger Foundation. 1 Department of Pediatrics, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10006. 2 Department of Medicine and Pharmacology, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10016. 3 Division of Human Genetics, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10016. © 1977 by the American Society of Human Genetics. All rights reserved.
272
MAPLE SYRUP URINE DISEASE
273
or BCKA. The results with leucine confirmed our previous experience, demonstrating its usefulness in establishing the clinical diagnosis. In contrast, the studies with a-ketoisocaproic acid at substrate concentrations of 1 mM and above were similar to the observations of Wendel et al. [6], showing limited correlation with the clinical diagnosis. MATERIALS AND METHODS
Radioactive Substrates L-leucine- 1-14C was purchased from New England Nuclear (Cambridge, Mass.). Purity was checked by ascending chromatography in n-butanol:acetic acid:water, 60:15:25 (v/v/v) and phenol:water, 80:20 (w/v). L-leucine was added to the radioactive amino acid to make a final solution of 20 mM in 0.1 M NaCl, 0.5 Ac/Amole. The pH was adjusted to 3 with HCl. Sodium a-ketoisocaproate- 1- 14C was prepared from L-leucine- 1- 14C by oxidation with L-amino acid oxidase [9]. The keto acid was checked for purity by ascending chromatography in n-butanol:acetic acid:water, 60:15:25, in n-butanol:water:concentrated ammonia, 14:2: 1, and by high voltage electrophoresis in 1 M acetic acid. The keto acid was diluted to a 200 mM solution, 0.05 ucL/imole using sodium-a-ketoisocaproate prepared synthetically [10]. The pH was approximately 3 without further adjustment. Substrates were stored at -20'C in a 50 ml Erlenmeyer flask with a well containing 0.1 ml 1 N NaOH. The flasks were thawed 30 min prior to use and agitated on a Dubnoff water bath at 20°C to trap volatile radioactive materials. The pH of the solution was checked frequently and maintained at 3. Tissue Culture Biopsies of skin were grown in monolayer culture in Waymouth's medium containing 10% fetal calf serum and antibiotics (penicillin 50 U, streptomycin 50 ,ug, and kanamycin 30 ,ug per ml). For many subjects, the fibroblasts had been maintained in liquid nitrogen for several months to several years. These were thawed, grown to confluence and harvested 4 to 10 days after the last medium change.
Enzyme Assays Fibroblasts were collected with a rubber policeman and incubated in 0.35 ml of solution containing 0.25 ml Krebs-Ringer phosphate buffer, pH 7.1, 0.005 ml thiamine HCl, 100 mg/ml and radioactive substrate. When necessary, pH was readjusted to 7.1 with 1 N NaOH. Incubations were for 120 min at 35°C with '4C02 collected in a center well charged with 0.1 ml 1 N NaOH. At the end of the incubation, the NaOH was transferred to a scintillation cocktail to determine radioactivity. The cells were digested and protein determined by the method of Lowry et al. [11]. The procedure has been previously described in detail [12]. Heat Stability Cells were harvested into buffer and heated on a water bath for 10 min at indicated temperatures and then assayed for decarboxylase activity. Source of Fibroblasts Fibroblasts from six control subjects were selected to provide an equal sex distribution and an age range that would cover that represented by the patients with MSUD. Many of the patients with MSUD have been previously reported. Of these, TeHe, CaGu, and MeCa represent classical cases as previously defined; ElHa and ThAd are variants [4]. BeRo is the mother of NiRo and is also a variant [13]. GM612 was received from the Genetic Mutant Cell Repository, Camden, N.J., and described as a classical case. DaMa was referred by Dr. David L. Valle, Johns Hopkins University School of Medicine and AnKl, by Dr. A. M. 0. Veale, University of Auckland, New Zealand. The former is consistent with a classical case by clinical history and
274
DANCIS ET AL.
enzyme level, and the latter was classified by clinical history and this study as a variant with a low tolerance for the BCAA. FeHu was referred by Dr. Selma Snyderman of this institution. RESULTS
In previous reports from this laboratory using BCAA as substrate, decarboxylase activity was reported as the ratio of CO2 evolution to the incorporation of the radioactive amino acid into cell protein. The latter established a baseline of metabolic activity against which to measure decarboxylase activity. The results were reproducible and provided a fairly sharp definition of the magnitude of the metabolic defect. In the present study, the MSUD fibroblasts had passed through many additional generations after storage, and the rate of incorporation of radioactive amino acids into protein was lower and more erratic. For that reason, and for better comparison with the experiments with BCKA, the results are now expressed as nmoles CO2 evolved per mg cell protein. The results with leucine are presented in figure 1. Each point for the MSUD cases, classical and variant, represent the mean of two to four determinations. Variability Te So Ba St
1.6-
'.4-
.12 C31
Kr St
\La 0Wi
E .0
Do Ve
0 0.8D 0 0
0.14
0.2
~~ ,4
~
WI ~~~~~~~Sh
x
-
~ ~ ~~~..Th Ad
~ ~ ~
~~~~~
~El Ho n~ Fr Hu, Do Ma
0.1 0.5
1.5
4.5
1GM612
Leucine conc. (mM) FIG. 1.-Fibroblasts were incubated for 120 min with leucine-1-14C at different concentrations (1.5 ); variants (-- -); classical mM), and the liberated radioactive CO2 was measured. Controls ( MSUD (--- -). The five classical MSUD cases had values close to background and are represented by one line. The results with classical and variant MSUD represent mean of 2-4 determinations per point; controls are single determinations.
MAPLE SYRUP URINE DISEASE
275
about the means were relatively small. Radioactivity liberated by fibroblasts from classical MSUD never exceeded 0.1 nmole per mg. Maximal decarboxylase activity was approached or reached at levels of substrate concentration of 1.5 mM leucine. Consistent with previous reports is the ability to distinguish among the classical, variant, and control subjects [3, 4]. There was some minor irregularity in BCAA decarboxylase activity of fibroblasts from several variants when compared to the severity of their disease. For example, ElHa fibroblasts had less activity than previously reported which may reflect the higher passage (more advanced age) of her cells. Exact subclassification among variants is inherently difficult, requiring maximal control of the assay procedure, because of the small differences in decarboxylase activity among the subgroups. The results with a-ketoisocaproate are presented in figure 2. Variability about the means of replicate determinations for the classical and variant cases of MSUD was much broader than observed with leucine. The range for some points was as broad as 100%, though it was generally less than 50%. Maximal decarboxylase activity was approached or reached at 15 mM, 10 times the substrate concentration for leucine. Using this level to compare the decarboxylase activity among the various subjects, we find the MSUD fibroblasts functioning at a generally lower level than the controls. Seven of nine MSUD cases liberated 4 nmoles or less of radioactive CO2 per mg protein per 120 min as compared to 2 of seven controls. However, there were many striking inconsistencies between the results with a-ketoisocaproate and the clinical history of the classical cases of MSUD. Only DaMa remained at very low levels. All other classical MSUD cases had easily detectable decarboxylase activity, and two (CaGu, MeCa) had levels that entered the normal range. A similar variability was noted with the variant MSUD cases. Heat stability of the decarboxylase activity was investigated in an attempt to exclude a nonenzymatic process (fig. 3). There was a sharp drop in activity following 10 min of exposure to temperatures between 420C and 50TC. With leucine, activity disappeared at 60TC, and for a-ketoisocaproate, it had virtually disappeared at 80TC. Disruption of the cells by freeze-thaw caused a sharp reduction in activity with both substrates. The observations are consistent with enzyme catalyzed reactions. DISCUSSION
The major objective in these studies was to investigate the report by Wendel et al. [6] that skin fibroblasts from patients with MSUD can decarboxylate BCKA when presented in high concentration and that the decarboxylation activity; under these circumstances, was often of the same magnitude as that in normal fibrobtasts. We decided to use the technique established in this laboratory with which we have had many years experience and to select fibroblasts from patients that had been carefully classified clinically. The fibroblasts had been stored in our "bank" under liquid nitrogen. On initiating fibroblast cultures from frozen stocks, they were again assayed for the severity of their defect using as substrate the BCAA, a technique which we have shown to correlate closely with clinical manifestations [4]. No attempt was made to exactly duplicate Wendel's methods. There are significant differences in the two assay techniques. We have harvested skin fibroblasts after growth to confluence by
276
DANCIS ET AL.
I18
16
x
ShWi
1 4-
E 0 12-
0
0~~~~
E8~~~~~~~~~~~mO E8En
-
.
~ ~~
~
~
~
~
-
E
~ ~~AK
2; H^ -~ ~ ~ ~ ~ DoV o~~~~~~~~~~~~oG
Ketoisoca proic ( mM ) FIG. 2. -Fibroblasts were incubated for 120 mmn with a-ketoisocaproic acid- l-14C at different concentrations and the liberated CO2 was measured. Values for those points surpassing values on vertical ordinate are indicated on the graph (TeSo, ShWi). (See fig. 1 for key.) The results with classical and variant MSUD represent mean of 2-4 determinations per point; controls are single determinations.
mechanically scraping them into buffer and incubating in suspension approximately one million cells per assay for 90 min Wendel et al [7] seeded 10,000-20,000 cells into each well of microtiter plates, incubated them overnight, and assayed their enzyme activity for 4 hr while still in monolayer. Our results with a-ketoisocaproate are similar to those reported by Wendel et al. [6] with high levels of substrate (>5 mM). Fibroblasts from patients with classical MSUD exhibited decarboxylase activity, and the results of controls and mutants, both variant and classical MSUD, overlapped extensively (fig. 2). We did not use substrate concentrations of keto acid below 1.0 mM, because at these low levels, it is necessary to use substrates with high specific activity which yields a background activity which
MAPLE SYRUP URINE DISEASE
277
100 80
C._) cs
60
i
0
40
0
C)a) G
I 20
so~~~~~~1 0
Not 30 40 50 60 70 80 90 100 treated Preincubation
temperature
FIG. 3. -Fibroblasts were incubated in buffer for 10 min at indicated temperatures, and then decarboxylase activity was measured. L-leucine-1-14C, 1.5 mM (ma -); a-ketoisocaproate-l-14C, 15 mM we considered too high to be acceptable. Wendel reported results with substrate concentrations as low as 10-5 M and stated that differentiation between mutants and controls was satisfactory at 5 x 10-5 M.* Interpretation of results with intact cells in terms of enzyme kinetics is very hazardous, as acknowledged by Wendel et al. [7], because of the multiple factors that may influence BCKA decarboxylation such as transport of substrate, pool sizes, competing reactions, etc. Despite this concern, these investigators suggest that their observations could be explained by the presence of two BCKA dehydrogenases with different affinities for the substrate. They speculate that only the enzyme with high affinity is the enzyme that is mutant in MSUD. One patient with classical MSUD in their series (and one in ours) had reduced decarboxylase activity at both low and high concentrations and would remain unexplained if both enzymes were totally independent. Our data with leucine as substrate are not inconsistent with the hypothesis of Wendel et al. [7]. Plateauing of decarboxylase activity at substrate concentrations considerably below those obtained with the keto acid (figs. 1 and 2) suggest a limiting factor proximal to this step such as transport of amino acid into or within the cell and/or transamination rate. We did not determine the intracellular concentration of radioactive a-ketoisocaproic acid at the various concentrations of radioactive leucine in the medium. If the intracellular concentrations of KIC are lower than the medium leucine * In table 2 of Wendel's article [6], decarboxylase activity is presented with BCKA at 5 x 10-4 M. However, the activities correspond to those in his figure 2 at 5 x 10-5 M.
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levels, as we suspect, and if there are two BCKA decarboxylases of differing affinities, the low affinity enzyme would be relatively inactive in our system. The decarboxylation that is measured would reflect the activity of the high affinity enzyme. This high affinity enzyme is presumably the physiologically effective one, and the one that undergoes mutation in MSUD. Regardless of the theoretical explanation, the most important conclusion from our studies is that, using standard techniques with intact cells, the BCAA appears to be the preferred substrate. As we have noted before, in association with our experience with hypoxanthine phosphoribosyltransferase deficiencies, the use of intact fibroblasts with natural substrates more closely approaches in vivo conditions at the cellular level and may have an advantage as a clinical model [14]. The observation appears to be pertinent of MSUD and its variants, also. However, investigations of metabolic defects at the molecular level require simpler systems. Until the problem of isolating an active BCKA dehydrogenase is solved, it may be best to postpone further speculation concerning questions such as the presence of specific decarboxylases for each BCKA, enzyme kinetics, and identification of the mutant site of the dehydrogenase complex. SUMMARY
Branched-chain keto acid decarboxylase activity in skin fibroblasts from control subjects and from patients with classical and variant forms of maple syrup urine disease (MSUD) was measured with leucine and a-ketoisocaproic acid. When the keto acid was used as substrate in high concentrations (more than 5 mM), the three groups overlapped extensively, even classical cases of MSUD exhibiting decarboxylase activity. With leucine as substrate, decarboxylase activity plateaued at about 1.5 mM, and the three groups could be clearly differentiated. Classical cases of MSUD had minimal or no decarboxylase activity.
1. 2. 3. 4. 5.
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REFERENCES DANCIS J, HUTZLER J, LEVITZ M: Metabolism of the white blood cells in maple syrup urine disease. Biochim Biophys Acta 43:342 - 343, 1960 DANCIS J, HUTZLER J, LEVITZ M: Detection of the heterozygote in maple syrup urine disease. J Pediatr 66:595-603, 1965 DANCIS J, HUTZLER J, Cox RP: Enzyme defect in skin fibroblasts in intermittent branched-chain ketonuria and in maple syrup urine disease. Biochem Med 2:407-411, 1969 DANCIS J, HUTZLER J, SNYDERMAN SE, Cox, RP: Enzyme activity in classical and variant forms of maple syrup urine disease. J Pediatr 81:312-320, 1972 DANNER DJ, ELSAS LJ II: Subcellular distribution and cofactor function of human branched chain a-ketoacid dehydrogenase in normal and mutant cultured skin fibroblasts. Biochem Med 13:7-22, 1975 WENDEL U, WENTRUP H, RUDIGER HW: Maple syrup urine disease: analysis of branched chain ketoacid decarboxylation in cultured fibroblasts. Pediatr Res 9:709-717, 1975 WENDEL U, WOHLER W, GOEDDE HW, LANGENBECK U, PASSARGE E, RUDIGER HW: Rapid diagnosis of maple syrup urine disease (branched chain ketoaciduria) by microenzyme assay in leukocytes and fibroblasts. Clin Chim Acta 45:433-441, 1973 SEEGMILLER JE, WESTALL RG: The enzyme defect in maple syrup urine disease. J Ment DeficRes 11:288, 1967 MEISTER A: Sodium a-ketoisocaproate. Biochem Prep 3:66-70, 1953
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10. ADICKES F, ANDRESEN G: Series of normal aliphatic 83-hydroxy and a-keto acids, Justus Liebig's Annal der Chem 555:41-42, 1943 11. LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 12. DANCIS J, HUTZLER J, Cox RP: Evaluation of amino acid metabolism in maple syrup urine disease, in Tissue Culture, Methods and Applications, edited by KRUSE PF JR, PATTERSON MK JR, New York, Academic Press, 1973, pp 639-643 13. ZALESKI LA, DANCIS J, Cox RP, HUTZLER J, ZALESKI WA, HILL A: Variant maple syrup urine disease in mother and daughter. Can Med Assoc J 109:299-304, 1973 14. HOLLAND MJC, DILORENzo AM, DANCIS J, BALIS ME, Yu TF, Cox RP: Hypoxanthine phosphoribosyltransferase activity in intact fibroblasts from patients with X-linked hyperuricemia. J Clin Invest 57:1600-1605, 1976
