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Transamination and Oxidative Decarboxylation Rates of Branched-Chain 2-0X0 Acids in Cultured Human Skin Fibroblasts

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Transamination and Oxidative Decarboxylation Rates of Branched-Chain 2-0X0 Acids in Cultured Human Skin Fibroblasts 003 1-3998/88/230 I -0040$02.00/0 PEDIATRIC RESEARCH Vol. 23, No. 1, 1988 Copyright O 1988 International Pediatric Research Foundation, Inc. Prinfed in (I.S.A. Transamination and Oxidative Decarboxylation Rates of Branched-Chain 2-0x0 Acids in Cultured Human Skin Fibroblasts PETER SCHADEWALDT, WOLFGANG RADECK, HANS-WERNER HAMMEN, AND UDO WENDEL Institut fur Physiologische Chernie II [P.S., W.R., H- W.H.]and Kinderklinik C /U. W.], Universitiit Diisseldorf; Moorenstr. 5, 0-4000 Diisseldorf; FRG ABSTRACT. Transamination and oxidative decarboxyla- acids derived from L-leucine and/or L-valine have been used as tion of branched-chain L-amino acid derived 2-0x0 aqids in substrates. In few studies, (S)- or unspecified (R,S)-3-methyl-2- cultured human skin fibroblasts from normal subjects and oxopentanoate have been used as we11 (16- 19). However, (R)-3- from a patient with maple syrup urine disease (variant methyl-2-oxopentanoate metabolism has not been examined. form) were comparatively studied in incubations with 1- The aim of the present study was to get further insight into L- I4C-labeled substrates (1 mmol/liter). With normal cells, allo-isoleucine metabolism in man. We therefore studied 1) I4CO2release ranged from about 11 to 3 nmol/90 min/mg oxidative decarboxylation rates of all four branched-chain 2-0x0 of cell protein in the order 3-methyl-2-oxo['4CJbutanoate acids in intact skin fibroblasts derived from normal subjects and > (S)-3-methyl-2-oxo[vpentanoate > 4-methyl-2- from a patient with variant MSUD and 2) 2-0x0 acid transami- ~xo['~C]pentanoate> (R)-3-methyl-2-oxo[~pentanoate. nation, since amination of (R)-3-methyl-2-oxopentanoate is the Formation of the corresponding branched-chain amino[I4C] only way for L-allo-isoleucine formation in vivo. acids was substantially higher than I4CO2 production (around 10-fold) and similar with L-valine, L-isoleucine, MATERIALS AND METHODS and L-leucine. L-Allo-isoleucine production [from (R)-3- methyl-2-oxopentanoate] was significantly lower. With ma- Chemicals. L-[l-14C]leucine(2.1 GBq/mmol), L-[l-14C]valine ple syrup urine disease fibroblasts, comparable transami- (2.0 GBq/mmol), and sodium [l-14C]pyruvate(340 MBq/mmol) nation rates were observed. Related to the findings with were from New England Nuclear, Dreieich, KI4CN (370 MBq/ normal cells, I4CO2release from each substrate was differ- mmol), NaHI4CO3 (2.1 GBq/mmol), and [methyl-'4C]toluene ently reduced and apparent residual branched-chain 2-0x0 standard were obtained from Amersham-Buchler,Braunschweig. acid dehydrogenase complex activity with 3-methyl-2-0~- Enzymes and coenzymes were from Boehringer, Mannheim, obutanoate, 4-methyl-2-oxopentanoate, (S)-, and (R)-3- Acylase I (EC 3.5.1.14) was from Sigma, Deisenhofen, and methyl-2-oxopentanoate amounted to 12, 13, 22, and SO%, Dowex 50 WX8 (50-100 mesh) from Serva, Heidelberg. All respectively. (Pediatr Res 23: 40-44, 1988) other chemicals were purchased in the highest available purity from Merck, Darmstadt, or Sigma. Purity of branched-chain 2- Abbreviations 0x0 acids was checked by automatic amino acid analysis and by a modification of the HPLC method described previously (20). MSUD, maple syrup urine disease A Nucleosil 5CI8 column (250 X 4 mm, Macherey-Nagel, Diiren, HPLC, high-performance liquid chromatography guard column Lichrosorb RP-18, 4 x 4 mm, Merck) and 33% PBS, phosphate-buffered saline CH3CN at 1.3 ml/min were used. The respective quinoxalinol derivatives were prepared as described previously (21). Purity was as specified by the suppliers. Unlabeled racemic (R,S)-3- methyl-2-oxopentanoate was optically inactive. In MSUD, the oxidative decarboxylation of branched-chain Branched-chain 2-0x0 acids. 2-0x0 acids from L-[l 14C]leucine 2-0x0 acids is impaired by a deficiency in branched-chain 2-0x0 and L-[l-14C]valinewere prepared as described previously (22). acid dehydrogenase activity (EC 1.2.4.4). Due to the metabolic 1-I4C-labeled (S)- (1 4.1 MBq/mmol) and (R)-3-methyl-2-0x0- block and normal aminotransferase activities, branched-chain pentanoate (9.2 MBq/mmol) were synthesized from the radio- amino as well as the corresponding 2-0x0 acids accumulate in active labeled N-acetyl derivatives of D-allo-isoleucine and D- MSUD (1-3). (S)-3-methyl-2-oxopentanoate, the transamination isoieucine, respectively (23, 24). Purity (>97%) and the specific product of L-isoleucine, can undergo racemization in vivo (4, 5). radioactivities of the products were examined by HPLC analysis The main metabolic fate of (R)-3-methyl-2-oxopentanoate thus as described above. Polarimetrically, impurities were not detect- formed appears to be transamination to L-allo-isoleucine which able. is regularly found in the patients blood (6-10). Cell culture. Human fibroblasts were obtained from skin biop- In most studies on kinetics and regulation of branched-chain sies of two normal subjects (strain RE and WE, mixed at equal 2-0x0 acid metabolism in human tissues, e.g. with homogenates portions for the assays) and of a patient (strain JA) with a variant of heart and skeletal muscle (1 l), mitochondria1 preparations form (intermittent type) of MSUD. Cells were multiplied in (12), and cultured intact and broken cells (1 3- 15), only 2-0x0 monolayer culture as described (17). After 10-20 passages, fibro- blasts at confluency were harvested by trypsinization, suspended Received May 29, 1987; accepted September 1, 1987. Correspondence Dr. Peter Schadewaldt, Institut fur Physiologische Chemie 11, in culture medium, washed twice with NaCl solution (0.154 moll Universitit Diisseldorf, Moorenstr. 5, D-4000 Dusseldorf, West Germany. Sup- liter), and resuspended in PBS, Dulbecco's modification (PBS), ported by Grant We 614/6-1 from the Deutsche Forschungsgemeinschaft. with bovine serum albumin (1.5 g/liter) and D-glucose (12.4 METABOLISM OF BRANCHED-CHAIN 2-OX0 ACIDS 4 1 mmol/liter). 0.4 ml of a suspension adjusted to about 2 x lo6 cells/ml was inserted into each assay. Mycoplasma contamina- tion tests (25) were negative. Incubations. I4CO2-tight 22-ml incubation flasks (1 1) were used to incubate fibroblasts (0.3-0.5 mg of cell protein) in a final volume of 0.9 ml PBS containing bovine serum albumin (0.07%, w/v), D-glucose (5.5 mmol/liter), and 2-oxo[ 1-I4C]acids as indi- cated. The reaction was started by addition of cell suspension and was stopped by injection of 0.5 ml sulfosalicylic acid (lo%, w/v). In blanks, cells were omitted. All samples were run in duplicate. Assays. I4CO2released during incubation was completely ab- sorbed in 0.5 ml ethanolamine-ethylenglycol(1:2,v/v) and meas- ured by liquid scintillation counting (1 1). I4CO2 recovery was checked with NaHI4CO3 solutions and counting efficiency by internal standardization with [14C]toluene. For the estimation of free amino acids, 0.1 ml DL-norleucine solution (0.1 mmol/liter) was then added to the medium and precipitated protein removed by centrifugation. One ml of the supernatant was freeze-dried and the residue dissolved in 0.5 ml LiOH solution (0.25 mol/liter, 2% 2.2'-thiodiethanol, w/v). The concentration of and radioactivity in amino acids were deter- mined on an automatic amino acid analyzer in runs with and without ninhydrin, respectively. Fig. 1. Time dependence of L-leucine production in incubations of For the determination of 14C-radioactivity in acid insoluble cultured human skin fibroblasts with 4-methyl-2-oxopentanoate (I material, the precipitate was thoroughly washed (5 x 1 ml) with mmol/liter). Cells derived from normal subjects were incubated at 37" C 3% sulfosalicylic acid (w/v), containing all branched-chain for 2-120 min. Sample content of free leucine was determined and amino (10 mmol/liter) and 2-0x0 acids (1 mmol/liter), by soni- corrected for the content in appropriate blanks with unincubated cells. cation and centrifugation and dissolved in 1 ml hydroxide of In the inset, the rate of leucine production is plotted against the incuba- hyamine 10x (Packard Instruments, Frankfurt). tion time. The respective rates were calculated from the differences of Protein concentrations (2.1 x 106 cells were equivalent to 1 leucine content and of the min elapsed between two successive incubation mg of cell protein) were estimated by the Lowry procedure (26), times. using bovine serum albumin as a standard. Calculations. Metabolic rates were calculated from the appro- priate 14C02liberation on the basis of the specific radioactivity of the CI-atom of the respective 2-oxo[14C]acid in the medium. Results are means + SD of the independent experiments per- formed on different days. Statistically significant differences were determined by Student's paired t test. RESULTS Time and concentration dependence. In incubations of normal fibroblasts in the absence of 2-0x0 acids, free amino acids (with the exception of aspartate) accumulated at gradually enhanced rates. The increase of leucine, valine, and isoleucine during O- 30 min of incubation amounted to 2.0 + 0.3, 2.7 + 0.4, and 0.9 E L + 0.3 nmol/mg of cell protein, and during 90-120 min to 3.7 2 0.4, 3.1 +. 0.4, and 1.5 + 0.5 nmol/mg of cell protein, respec- tively. The time course of leucine accumulation in incubations with 4-methyl-2-oxopentanoate (1 mmol/liter) is shown in Figure 1. The rate of formation declined up to 30 min of incubation and 0 C then remained constant up to the end of the experiment. With 0 - S- I~-'~c]KMV R- i 1-"C] KMV the other three branched-chain 2-0x0 acids, comparable effects on the accumulation of the corresponding branched-chain amino acids were found (data not shown).
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