A simple spectrophotometric assay for long-chain acyl-coa dehydrogenase activity measurements in human skin fibroblasts

Transcription

1 Original Article Ann Clin Biochem 1993; 30: A simple spectrophotometric assay for long-chain acyl-coa dehydrogenase activity measrements in hman skin fibroblasts Lodewijk Ijlst and Ronald J A Wanders! From the Departments of Clinical Biochemistry and Ipediatrics, University Hospital Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands SUMMARY. Long-chain acyl-coa dehydrogenase (LCAD) deficiency is an atosomal recessive disorder of fatty acid metabolism characterized by hypoglycemia, mscle weakness and hepato- and cardiomegaly to varying extents. Analysis oforganic acids in rine sally reveals dicarboxylic acidria with elevated levels of adipic, sberic and sebacic acids as well as longer chain dicarboxylic acids. Correct diagnosis of sspected patients reqires measrement of LCAD in tisse or preferably, white blood cells and/or cltred skin fibroblasts. In this paper we present a simple spectrophotometric enzyme assay based on the se offerricenim hexaflorophosphate as electron acceptor. Under optimized conditions the method presented allowed neqivocal identification of LCAD-deficiency in fibroblast homogenates. Additional key phrases: fatty acids;fatty acid {j-oxidation; metabolism; inborn errors An increasing nmber of inherited diseases in man have recently been identified in which there is an impairment in the mitochondrial oxidation of fatty acids (see references 1-6 for review). Medimchain acyl-coa dehydrogenase is probably most freqent among these disorders. Correct diagnosis of patients reqires measrement of the activity of each of the individal mitochondrial {j-oxidation enzymes. For measrement of medim-chain acyl-coa dehydrogenase and other acyl-coa dehydrogenase activities for types of assay methods have been developed." The first is based on the transfer of electrons to the physiological acceptor, electron transfer flavoprotein (TF), nder anaerobic conditions. Oxidized TF is strongly florescent. Accordingly, the acyl-coa dehydrogenase activity can be assessedby following the decrease of florescence of oxidized TF being redced by electrons directly derived from the acyl CoA dehydrogenase enzyme protein." The second method is based pon the se of artificial electron acceptors sch as dichlorophenol-indophenol (DCPIP) sing either phenazine methoslphate or TF as mediator. 8 The third method employs gas chromatography/mass spectrometry to measre Correspondence: Dr R J A Wanders. the formation of the prodct, enoyl-coa.9-14 In the forth method the formation of tritiated water from specifically labelled [2,3-3H] acyl-coa esters is measred. IS In the last few years the TF-based assay has become the method of choice for correct assessment of acyl-coa dehydrogenase activity especially in fibroblasts. A drawback of this method, however, is that TF is not commercially available and has to be prified from pig heart which is laborios, time consming and costly. Recently Lehman and coworkers" described a spectrophotometric assay for measrement of medim-chain acyl-coa dehydrogenase in fibroblasts which makes se of a ferricenim ion as electron acceptor. laborating on this work we now report that the activity of long-chain acyl CoA dehydrogenase can be measred reliably in homogenates of cltred skin fibroblasts sing ferricenim as terminal electron acceptor. The method described is simple, ses no special reagents and reqires only a spectrophotometer. MATRIALS AND MTHODS Materials Ferricenim hexaflorophosphate was prepared from ferrocene and sodim hexaflorophosphate, 293

2 294 [jist and Wanders obtainedfrom Aldrich, as described by Lehman and coworkers." Palmitoyl-CoA was from Boehringer GMBH (Mannheim, FRG) and octanoyl-coa was obtained from Sigma (St Lois, Mo, USA). The protein concentration was measred with bicinchoninic acid (BCA) obtained from Sigma sing hman serm albmin as standard. All other chemicals were of analytical grade. nzyme activity measrements The activity of acyl-coa dehydrogenase was measred spectra-photometrically by following the redction of the ferricenim ion at 300 nm sing an extinction coefficient of 4300 L mol- 1 ern - 1. The reaction medim (0'25 ml final volme) contained the following standard components: 100 mmolll sodim phosphate bffer ph 7' 2, O' 2 mmolll ferricenim hexaflorophosphate, 0'5 mmolll N-ethylmaleimide, 0 1 mmolll DTA, 0'1 % (w/v) Triton X-100 and fibroblast homogenate (0'1-0'16 mg protein). Following a preincbation period of 2 min reactions were started by addition of octanoyl-coa, palmitoyl CoA or water (for blank measrements). The acyl-coa esters were added at a final concentration of 0 5 mmolll nless otherwise indicated. Absorbance measrements were made on a Cobas Bio centrifgal analyser (Hoffman-La Roche) for at least 10 min at a temperatre of 37 C. Absorbance was read every 10 s. Reaction rates were calclated from the initial linear portions of the absorbance verss time plots. Preparation of fibroblast homogenates Fibroblasts were kept at - 80 C and thawed on the day of analysis in a medim containing 100 mmolll sodim phosphate, 0 1 mmolll DTA and 0 1% (w/v) Triton X-100. Sspensions were sbseqently sbjected to sonication for two periods of 15 s at 20W with a time interval of 45 s nder continos cooling in an ice water bath. The protein concentration of the homogenates ranged from 3 to 4 mg/ml. Cell cltre conditions Hman skin fibroblasts from control individals and two patients with long-chain acyl-coa dehydrogenase deficiency were grown according to standard procedres as described before. I? RSULTS AND DISCUSSION In initial experiments we sed the acyl-coa dehydrogenase reaction medim as described by Lehman and coworkers" which incldes 100 mmolll sodim phosphate bffer (ph 7 2), 0 1 mmolll DTA, 0'5 mmolll N-ethylmaleimide, O' 2 mmolll ferricenim hexaflorophosphate and an acyl-coa concentration of 50l'mollL. Under these conditions both the octanoyl-coa and palmitoyl-coa dependent rates of redction of ferricenim hexaflorophosphate in fibroblasts homogenates were fond to be linear for only a limited period of time. We therefore decided to stdy the effect of increasing concentrations of octanoyl-coa and palmitoyl CoA, respectively, on the linearity of the enzyme assay with time. Figre 1a shows that with 50l'mollL octanoyl-coa as sbstrate the decrease in absorbance at 300 nm came to a stop after 5 min. Linearity with time was fond to be mch better at higher sbstrate concentrations. Indeed, at the highest acyl-coa concentration sed (500 I'mollL) linearity was maintained for at least IOmin (Fig. 1a). As shown in Fig. 1b similar observations were made sing palmitoyl CoA as sbstrate. These reslts show that acyl-coa dehydrogenase activities with octanoyl-coa and palmitoyl-coa as sbstrate were best measred at an acyl-coa <: rt'l C.. <: 0 IA -e 0...D <t 1 3 r e ~ C <: D IA.D <t iii i 10 Time (min) Time (min) (a) (bl i 15 FIGUR 1. The activity ofacyl-coa dehydrogenase in control hman skin fibroblasts sing octanoyl-coa (A) and palmitoyl-coa (B) as sbstrate as afnction ofthe acyl-coa concentration sed in the standard reaction medim: 50 JJRlol/L(e). }OO JJRlol/L (.).200JJRlol/L (0) and 500 mol/l (. )

3 Spectrophotometric assay for LeAD in skin fibroblasts 295 concentration of 500 /LmollL. Palmitoyl-CoA is not only a sbstrate for long-chain acyl-coa dehydrogenase bt also reacts with medim-chain acyl-coa dehydrogenase and the newly identified enzyme, very-long-chain acyl-coa dehydrogenase.!" The same applies to octanoyl-coa which is reactive with short-chain, medim-chain as well as long-chain acyl-coa dehydrogenase. I We therefore decided to repeat the experiments sing control fibroblasts as well as fibroblasts from a patient with an established deficiency of long-ehain acyl-coa dehydrogenase as determined by sing the TF-based assay method? (LCADactivity < 100/0 of control vales in both cases). xperiments were done in the absence and presence of Triton X-IOO (0,1% w/v). When octanoyl-coa was sed as sbstrate (Fig. 2a and c) it is clear that enzyme activity increased with increasing sbstrate concentrations in both control and LCAD-deficient fibroblasts. The rates observed were somewhat lower in the LCAD-deficient fibroblasts compared with control fibroblasts independent of whether Triton X-l00 was present (Fig. la) or not (Fig. Ic), When palmitoyl-coa was sed as sbstrate (Fig. 2b and d) there was a steady increase in apparent enzyme activity when the concentration ofpalrnitoyl-coa was increased althogh the kinetics were fond to be different nder the incbation condition of Fig. 2b and d, respectively. Indeed, in the absence of Triton X-IOO optimal rates in control fibroblasts were fond at 200 /LmollL palmitoyl-coa in contrast to the sitation of Fig. 2b in which Triton was present. Under the latter conditions activity was maximal only at elevated acyl-coa concentrations exceeding 500 /LmollL. More importantly, however, rates of palrnitoyl CoA dehydrogenation in LCAD-deficient fibroblasts (open symbols) were fond to be mch higher in the absence of Triton X-IOO (Fig. 2d) than when Triton X-IOO was present (Fig. 2b). Accordingly, we selected a medim containing Triton X-IOO (0'1 % w/v) and palrnitoyl-coa at a concentration of 500 /LmollL since this allowed optimal discrimination between control and LCAD-deficient fibroblasts. According to Lehman and coworkers's componds sch as co-enzyme-a containing a free SH-grop are capable of redcing the ferricenim ion directly giving rise to artefactal high rates of 6 ---(a) 6 (c) ~ 5 c:: { 4 o ]. 3 > : <[ I ~ :~ 2 <[ I ()(J 500 Octanayl-CoA cancentration (IlmaI/L) 15 (b) 15 C. C. c c::.~ 10 : 10 g c os ~ 5 ~.>.> 5 += +: <[ <[ g Octanoyl- CoAcancentration (IlmoI/Ll (d) Palmitoyl- CaA cancentratian (I1mall Ll Pclmitayl-CcA cancentration (Il malll) FIGUR 2. The activity ofoctanoyl-coa dehydrogenase (a, c) and palmitoyl-ciia dehydrogenase (b, d) at different acyl-coa concentrations in control (.) and long-chain acyl-coa dehydrogenase deficient (0) fibroblasts in the presence (a, b) and absence (c, d) of Triton X-lOO.

4 296 Ij/st and Wanders TABL 1. Reslts of acyl-co.a dehydrogenase activity measrements in fibroblast homogenates from control individals and two patients with an established deficiency oflong-chain acyl-coa dehydrogenase Measrement Octanoyl-CoA dehydrogenase activity (nmol/min mg protein) Palmitoyl-CoA dehydrogenase activity (nmol/min mg protein) Palmitoyl-CoA dehydrogenase/octanoyl-coa dehydrogenase-ratio Sorce of fibroblasts Control (n = 22) Patient 1 (n = 5) Patient 2 (n = 2) 5 03 (1'41) 6 54 (1'67) 3,70-3, (3'90) 3 66 (1'14) 2' 88-3 '13 2'53 (0'80) 0'55 (0'05) 0'78-0,82 Reslts are expressed as mean (SO) with the nmber of separate observations between parentheses reaction. Since free coenzyme A is a contaminant of commercial acyl-coa esters and may be formed via hydrolysis of acyl-coa esters dring incbation, N-ethylmaleimide was added to the standard incbation medim16 to avoid this potential problem. Since N-ethylmaleimide also reacts with free SH-grops present in proteins it was essential to investigate the effect of N ethylmaleimide on the LCAD-activity in control and mtant fibroblasts. Sbseqent experiments showed that N-ethylmaleimide did inhibit the rate of palmitoyl-coa dependent redction of ferricenim somewhat. However, at a concentration of 250 JLmoIlL, which is likely to be sfficient to trap any SH-containing componds potentially present in the medim, inhibition was only slight amonting to less than IO% in control fibroblasts (reslts not shown). Based on these reslts we selected a concentration of O 25 mmoill N-ethylmaleimide for inclsion in the standard reaction medim. Using these optimized conditions we measred acyl-coa dehydrogenase activities in fibroblasts from control sbjects as well as from two LCADdeficient patients (Table 1). The reslts show that long-chain acyl-coa dehydrogenase measred sing the assay method described in this paper was, indeed, deficient in fibroblasts from the two LCAD-deficient patients. This was reflected in strongly abnormal palmitoyl-coa verss octanoyl-coa dehydrogenase activity ratios. In smmary, we have described a simple spectrophotometric assay method for measrement of long-chain acyl-coa dehydrogenase in fibroblast homogenates sing ferricenim hexaflorophosphate which is very easy to prepare." The method allowed clear discrimination between control and LCAD-deficient fibroblasts althogh a larger series of analyses in additional LCAD-deficient patients is reqired to establish the tre reliability ofthe method. This is especially important since residal activities appear to be somewhat higher with the present method as compared to the TF-based assay.! After sbmission of the present manscript two papers appeared on the se of ferricenim for acyl-coa dehydrogenase activity measrements. 19,20 In agreement with the reslts described here residal activities were also fond to be mch higher in the hands of these athors. In fact, Taylor and coworkers'? conclded that the ferricenim-based assay shold not be sed for medim-chain acyl-coa dehydrogenase activity measrements in fibroblasts becase residal activities are so high, amonting to 500/0 of control vales in fibroblast homogenates from established MCAD-deficient patients. The reslts of Scholte and coworkers/? and orselves (npblished observations) agree with this view, sggesting that the ferricenim-based assay may be sed only for long-chain acyl-coa dehydrogenase activity measrements ntil the nderlying basis for the high levels of residal activity in MCAD-deficient fibroblasts is resolved. Acknowledgements The athors grateflly acknowledge Mrs let van der Gracht for expert preparation of the manscript. RFRNCS Vianey-Liad C, Divry P, Gregersen N, Mathie M. The inborn errors of mitochondrial fatty acid oxidation. J Inher Metab Dis 1987; 10 (sp pi I): Stanley CA. New genetic defects in mitochondrial fatty acid oxidation and carnitine deficiency. Adv Pediatr 1987; 34: Dran M, Wadman SK. Chemical diagnosis of inherited disorders of fatty acid metabolism and ketogenesis. nzyme 1987; 38: Politt RJ. Disorders of mitochondrial (j-oxidation. Prenatal and early postnatal diagnosis and their relevance to Reye's syndrome and sdden infant death. J Inher Metab Dis 1989; 12 (sppl I): Ann Clin Biochem 1993: JO

5 Spectrophotometric assay for LeAD in skin fibroblasts Roe CR, Coates PM. Acyl-CoA dehydrogenase deficiencies. In: Scriver CR, Beadet AL, Sly WS, Valle D, eds. The Metabolic Basis ofinherited Disease 6th edn. New York: McGraw Hill, 1989; Bennett MJ. The laboratory diagnosis of inborn errors of mitochondrial fatty acid ~-oxidation. Ann C/in Biochem 1990; 27: Frerman F, Goodman S1. Florimetric assay of acyl-coa dehydrogenases in normal and mtant hman fibroblasts. Biochem Med 1985; 33: Rhead WJ, Amendt BA, Fritchman KS, Felts SJ. Dicarboxylic acidria: deficient [1_ 14C] octanoate oxidation and medim-chain acyl-coa dehydrogenase in fibroblasts. Science 1983; 72: Kolvraa S, Gregersen N, Christensen, Hoboth N. In vitro fibroblast stdies in a patient with C6-CIGdiearboxylic acidria: evidence for a defect in general acyl-coa dehydrogenase in fibroblasts. Clin Chim Acta 1982; 126: Divry P, Vianey-Liad C, Cotte J. Gas chromatography-mass spectrometry (GC-MS) diagnosis of two cases of medim-chain acyl-coa dehydrogenase deficiency. J Inher Metab Dis 1984; 7: Bogneres PF. Rocchiccioli F, Kolvraa S, Hadchod M, Lala-KeralyJ, Chassain JL, et al. Medim-chain acyl-coadehydrogenasedeficiencyin two siblingswith a Reye-like syndrome. J Pediatr 1985; 106: Niezen-Koning K, Chapman T, Mlder I, Smit GPA, Reijngod DJ, Berger R. Determination of medim chain acyl-coa dehydrogenase activity in cltred skin fibroblasts sing mass spectrometry. Clin Chim Acta 1991; 199: Dran M, Cletjens CBJM, Ketting D, Dorland L, de Klerk JBC, van Sprang FJ, et al. Diagnosis of medim-chain acyl-coa dehydrogenase deficiency in lymphocytes and liver by a gas-chromatographic method: the effect of oral riboflavin spplementation. Pediatr Res 1992; 31: Niezen-KoningK, Wanders RJA, Nagel GT, IjIst L, Heymans HSA. A new, simple assay for acyl-coa dehydrogenase in cltred skin fibroblasts sing stable isotopes and GC/MS. Biochim Biophys Acta 1992; 1180: Amendt BA, Rhead WJ. Catalytic defect of medimchain acyl-coenzyme A dehydrogenase deficiency. Lack of cofactor responsiveness and biochemical heterogeneity in eight patients. J Clin Invest 1985; 76: Lehman TC, Hale D, Bhala A, Thorpe C. An acyl-coenzyme A dehydrogenase assay tilizing the ferricenim ion. Anal Biochem 1990; 186: Wanders RJA, van Roermnd CWT, van Wijland MJA, Heikoop J, Schtgens RBH, van den Bosch H, et al. Peroxisomal fatty acid ~-oxidation in relation to the accmlation of very long chain fatty acids in peroxisomal disorders. J Clin Invest 1987;80: Izai K, Uchida Y, Orii T, Yamamoto S, Hashimoto T. Novel fatty acid ~-oxidation enzymes in rat liver mitochondria: prification and properties of verylong-chain acyl-coenzyme A dehydrogenase. J BioI Chem 1992; 267: Taylor RW, Jackson S, Porfarzam M, Bartlett K, TrnbIl DM. Measrement of acyl-coa dehydrogenase activity in cltred skin fibroblasts and blood platelets. J Inher Metab Dis 1992; 15: Scholte HR, Ross JD, Blom W, Boonman AMC, van Diggelen OP, Hall CL, et al. Assessment of deficiencies of fatty acyl-coa dehydrogenases in fibroblasts, mscle and liver. J Inher Metab Dis 1992; 15: Accepted for pblication 17 December 1992