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Sensitive assay of carnitine palmitoyl transferase activity in tissue homogenates with a modified spectrophotometric method for enzymatic carnitine determination
Chnica Chimica Acta, 135 (1983) 247-251 Elsevier
247
CCA 02712
Brief technical
note
Sensitive assay of carnitine palmitoyl transferase activity in tissue homogenates with a modified spectrophotometric method for enzymatic carnitine determination Thomas
Deufel * and Otto H. Wieland
Institut fiir Klinische Chemie, Stiidtisches (Received
Krankenhaw
Miinchen - Schwabing, Munich (FRG)
May 25th; revision August
2nd, 1983)
Introduction Carnitine palmitoyl transferase (CPT, EC 2.3.1.21) catalyses the reversible transfer of long chain acyl groups from acyl-coenzyme A to L-carnitine. Determination of CPT activity in tissues, especially muscle biopsy specimens, has gained clinical relevance, as CPT deficiency can be a cause of recurrent myoglobinuria [l]. Most assays require the use of radiolabelled compounds [2]. Spectrophotometric methods, measuring either release of free coenzyme A-SH groups from palmitoyl-coenzyme A, or formation of the palmitoyl-coenzyme A thioester bond [3,4], are of limited value in crude tissue homogenates because of their low sensitivity and interference by unspecific acyl-coenzyme A hydrolases. We describe a new sensitive assay of CPT activity in crude tissue homogenates which does not involve the use of radioisotopes. It is based on the photometric determination of free L-carnitine released from palmitoyl-carnitine during palmitoyl-transfer to coenzyme A (reaction 1).
palmitoyl-L-carnitine
+ CoA-SH
CPT + palmitoyl-CoA
+ r_-carnitine
(1)
For carnitine determination the enzymatic method of Marquis and Fritz [5] (reactions 2, 3) has been modified to eliminate free SH groups that would otherwise interfere with the assay. Thus, carnitine can be measured photometrically in the
* Correspondence to: Dr. T. Deufel, Inst. f. Klin. Chemie, Stadt. Krankenhaus Mtinchen-Schwabing, Kolner Platz 1, D-8000 Miinchen 40, FRG. Abbreuiations: CPT, carnitine palmitoyl transferase (EC 2.3.1.21); CAT, carnitine acetyl transferase (EC 2.3.1.7) DTNB, 5,5’-dithiobis-(2-nitrobenzoate); TNB, 5-thio-2-nitrobenzoate (absorption at 412 nm); HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid; BSA, bovine serum albumin: NCP, non-collagenous protein. 0009-8981/83/$03.00
0 1983 Elsevier Science Publishers
B.V.
248
presence L-carnitine CoA-SH
of CoA-SH. + acetyl-CoA-SH
CAT + acetyl-L-carnitine
+ DTNB + CoAS-NB
+ CoA-SH
(2) (3)
+ TNB + H +
Materials and methods Acetyl coenzyme A (sodium salt), palmitoyl-L-carnitine, carnitine acetyl transferase (CAT) and N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (HEPES) were supplied by Sigma Chemical Co., St. Louis, MO, USA; 5,5’-dithiobis-2-nitrobenzoate (DTNB) by Serva, Heidelberg, FRG; catalase: Boehringer, Mannheim, FRG; bovine serum albumin (BSA): Behring, Marburg, FRG; and perhydrol (TM) (30% hydrogen peroxide) and all other chemicals of analytical grade: Merck, Darmstadt, FRG. CAT (500 kU/l) was centrifuged at 10000 x g, 4”C, for 1 min immediately before use, the clear supernatant, containing ammonium sulphate, was discarded, and the pellet resuspended in a 20-fold volume of distilled water. Acetyl-coenzyme A (12 mmol/l) was dissolved in distilled water and stored frozen in small fractions at -20°C. The solutions were only refrozen once after thawing. Palmitoyl-L-carnitine (6 mmol/l) was dissolved in distilled water and stored frozen at -20°C. The concentrations of both free and esterified carnitine (after hydrolysis in 0.2 mol/l KOH at 56’C, 15 min) were always checked before use. CPT assay An assay mixture prepared freshly before use contained 20 mmol/l coenzyme A, which was omitted and replaced by Hz0 for sample blanks, 5 mmol/l EDTA, 100 mmol/l HEPES/NaOH, pH 7.5, and 25 g/l BSA. In Eppendorf plastic reaction cups 10 ~1 tissue homogenate (corresponding to approximately 0.5 mg wet muscle tissue) were added to 20 ~1 assay mixture. The reaction was started by addition of 10 ~1 palmitoyl-L-carnitine, cups were sealed, thoroughly mixed on a vortex mixer, and incubated for 30 min on an Eppendorf thermostat set to 37°C. For each homogenate a sample blank without coenzyme A was run in parallel. The reaction was stopped with 200 ~1 perchloric acid, 0.6 mol/l, and the cups were placed on ice for 15 min. After centrifugation at 10000 x g the clear supernatant was carefully neutralised with solid potassium hydrogen carbonate, using minimum amounts of n-octanol to prevent excessive foaming. The samples were placed on ice for another 15 min, and recentrifuged for 2 min at 10000 x g. 50 I_L~ of this neutralised extract were used in the carnitine assay described below. Carnitine assay DTNB (2.7 mmol/l)
was dissolved
in 0.5 mmol/l
HEPES,
pH 7.5, and stored
249
frozen at -20°C. Immediately before use, an assay mixture was prepared by diluting perhydrol in a lo-fold volume of the DTNB/HEPES solution. 20 ~1 of this mixture were placed in a plastic reaction cup, and 50 ~1 of the neutralised muscle extract, 150 ~1 of distilled water, and finally 10 ~1 of acetyl coenzyme A solution were added. The samples were stirred with a plastic stirring rod which was left in the unsealed cups to facilitate the escape of oxygen formed. After 10 min at room temperature, when any yellow colour had disappeared, 10 ~1 of catalase, diluted in a 50-fold volume of distilled water, were added, and the open cups were left at room temperature for about 30 min till no more oxygen bubbles were seen. 10 ~1 of CAT solution were added, the cups were sealed and incubated at room temperature for 30 min. The absorption at 405 nm was measured in an Eppendorf photometer against a reagent blank, using water instead of neutralised perchloric acid extract. One unit is equivalent to 1 ymol r_-carnitine formed per minute at 37’C, calculated from and the dilution factors (24 in the CPT assay, and 5 in ( 405nm = 13.6 cm2 - pmol-’ the carnitine assay). Non-collagenous protein (NCP) was determined as described by Lilienthal et al [6], and protein measured by the method of Lowry et al ]7]. CPT was also measured as described previously [8] with a photometric forward method in detergent extracts of washed muscle protein pellets. Results Optimal reaction conditions From Fig. 1 it can be seen that the enzyme activity is maximal under the conditions described. Omission of BSA results in a marked decrease in reaction rate. (In contrast, we have observed that the forward assay is strongly inhibited by BSA concentrations higher than 1 g/l, probably due to binding of palmitoyl-CoA to BSA.) The reaction is linear up to 40 min, as shown in Fig. 2. In most cases, sample blanks were not different from reagent blanks without tissue. However, to avoid errors possibly caused by acyl carnitine deacylases present in tissues [2], we always used sample blanks with CoA-SH omitted. The carnitine found in blanks is due to impurity of the palmitoyl carnitine preparation. Endogenous free tissue carnitine would remain undetectable because of the high dilution of the homogenates in the assay. Reproducibiiity of assays In a series of 12 samples of human muscle obtained at autopsy and homogenised as described in the legend to Fig. 1, CPT activity measured as described above ranged from 10.7 to 12.1 U/g NCP, (X+ SD: 11.5 jr 0.42; CV 3.7%). CPT activity in human muscle biopsies CPT activity in 13 muscle biopsies obtained from patients with no obvious symptoms of muscle disease used as controls was 4.6 rfr 1.4 (range 2.8-9.4) U/g NCP. In comparison, values obtained with the forward photometric method, also at
250
I2 0
2- / o- .
0
2
4
I 6
E
1 10
COD, (mmcl/l)
Fig. 1. Reaction conditions for the CPT backward assay. Human skeletal muscle obtained at autopsy and frozen in liquid nitrogen was rapidly weighed, pulverised (Dismembrator, Braun, Melsungen. FRG), and homogenised in a 20-fold volume of 50 mmol/l HEPES, 1 mmol/l EDTA, pH 7.5. CPT assays were performed varying concentrations of coenzyme A (a), palmitoyl carnitine (b), and BSA (c). respectively. All other conditions were as described in the ‘Materials and methods’ section. Fig. 2. Time course of the CPT backward reaction. Assays reaction conditions described in the ‘Materials and methods’
of CPT activity were performed under the section with 10 pl homogenate (see Fig. 1).
37’C, were 1.6 + 0.5 (range 0.5-2.3) U/g NCP. This conforms with findings of other authors, that the backward reaction yields higher activities than the forward reaction (21. Comparing CPT activities in muscles from 10 controls and 28 patients with recurrent rhabdomyolysis. but normal CPT, both methods were found to correlate fairly well (r 0.75; slope 0.21; intercept 0.42).
251
Discussion CPT in human muscle biopsies is mostly measured using radioisotopes, as in the isotope exchange method or the radioactive forward method [l]. Both methods require at least as much tissue as the one described here, and, even more important, involve the drawbacks inherent in the use of tritiated or “C-labelled substrates. The CPT activities of human muscle found with our assay are of the same order of magnitude as those reported in the literature [1,9]. Exact comparison of results is impossible because of the different experimental procedures and assay conditions employed in different laboratories. The carnitine method used for this assay is also suitable for determination of free and short chain acyl carnitine in plasma, urine and tissues [8]. Acknowledgement This study was supported
by the Deutsche
Forschungsgemeinschaft
(DFG).
References 1 Di Mauro S, Melis Di Mauro P. Muscle CPT deficiency and myoglobinuria. Science 1973; 180: 329-331. 2 McGarry JD, Leatherman GF, Foster DW. Camitine palmitoyltransferase I. J Biol Chem 1978; 253: 4128-4136. 3 Saggerson ED. Camitine acyltransferase activities in rat liver and heart measured with palmitoyl-CoA and octanoyl-CoA. Biochem J 1982; 202: 397-405. 4 Solberg HE. Different camitine acyltransferases in calf liver. Biochim Biophys Acta 1972; 280: 422-433. 5 Marquis NR, Fritz IB. Enzymological determination of free carnitine concentrations in rat tissues. J Lipid Res 1964; 5: 184-187. 6 Lilienthal JL, Zierler KL, Folk BP, Buka R, Riley M. A reference base and system for analysis of muscle constituents. J Biol Chem 1950; 182: 501-508. 7 Lowry DH, Rosebrough NY, Farr AL, Randall FJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275. 8 Pongratz D, Htibner G, Deufel T, Wieland 0, Pongratz E, Liphardt R. Klinische, morphologische und biochemische Befunde bei Camitinmangelmyopathien. Klin Wschr 1979; 57: 927-936. 9 Scholte HR, Jennekens FGI, Bouvy JJBJ. Carnitine palmitoyltransferase II deficiency with normal camitine palmitoyltransferase I in skeletal muscle and leucocytes. J Neurol Sci 1979; 40: 39-51.
247
CCA 02712
Brief technical
note
Sensitive assay of carnitine palmitoyl transferase activity in tissue homogenates with a modified spectrophotometric method for enzymatic carnitine determination Thomas
Deufel * and Otto H. Wieland
Institut fiir Klinische Chemie, Stiidtisches (Received
Krankenhaw
Miinchen - Schwabing, Munich (FRG)
May 25th; revision August
2nd, 1983)
Introduction Carnitine palmitoyl transferase (CPT, EC 2.3.1.21) catalyses the reversible transfer of long chain acyl groups from acyl-coenzyme A to L-carnitine. Determination of CPT activity in tissues, especially muscle biopsy specimens, has gained clinical relevance, as CPT deficiency can be a cause of recurrent myoglobinuria [l]. Most assays require the use of radiolabelled compounds [2]. Spectrophotometric methods, measuring either release of free coenzyme A-SH groups from palmitoyl-coenzyme A, or formation of the palmitoyl-coenzyme A thioester bond [3,4], are of limited value in crude tissue homogenates because of their low sensitivity and interference by unspecific acyl-coenzyme A hydrolases. We describe a new sensitive assay of CPT activity in crude tissue homogenates which does not involve the use of radioisotopes. It is based on the photometric determination of free L-carnitine released from palmitoyl-carnitine during palmitoyl-transfer to coenzyme A (reaction 1).
palmitoyl-L-carnitine
+ CoA-SH
CPT + palmitoyl-CoA
+ r_-carnitine
(1)
For carnitine determination the enzymatic method of Marquis and Fritz [5] (reactions 2, 3) has been modified to eliminate free SH groups that would otherwise interfere with the assay. Thus, carnitine can be measured photometrically in the
* Correspondence to: Dr. T. Deufel, Inst. f. Klin. Chemie, Stadt. Krankenhaus Mtinchen-Schwabing, Kolner Platz 1, D-8000 Miinchen 40, FRG. Abbreuiations: CPT, carnitine palmitoyl transferase (EC 2.3.1.21); CAT, carnitine acetyl transferase (EC 2.3.1.7) DTNB, 5,5’-dithiobis-(2-nitrobenzoate); TNB, 5-thio-2-nitrobenzoate (absorption at 412 nm); HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid; BSA, bovine serum albumin: NCP, non-collagenous protein. 0009-8981/83/$03.00
0 1983 Elsevier Science Publishers
B.V.
248
presence L-carnitine CoA-SH
of CoA-SH. + acetyl-CoA-SH
CAT + acetyl-L-carnitine
+ DTNB + CoAS-NB
+ CoA-SH
(2) (3)
+ TNB + H +
Materials and methods Acetyl coenzyme A (sodium salt), palmitoyl-L-carnitine, carnitine acetyl transferase (CAT) and N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (HEPES) were supplied by Sigma Chemical Co., St. Louis, MO, USA; 5,5’-dithiobis-2-nitrobenzoate (DTNB) by Serva, Heidelberg, FRG; catalase: Boehringer, Mannheim, FRG; bovine serum albumin (BSA): Behring, Marburg, FRG; and perhydrol (TM) (30% hydrogen peroxide) and all other chemicals of analytical grade: Merck, Darmstadt, FRG. CAT (500 kU/l) was centrifuged at 10000 x g, 4”C, for 1 min immediately before use, the clear supernatant, containing ammonium sulphate, was discarded, and the pellet resuspended in a 20-fold volume of distilled water. Acetyl-coenzyme A (12 mmol/l) was dissolved in distilled water and stored frozen in small fractions at -20°C. The solutions were only refrozen once after thawing. Palmitoyl-L-carnitine (6 mmol/l) was dissolved in distilled water and stored frozen at -20°C. The concentrations of both free and esterified carnitine (after hydrolysis in 0.2 mol/l KOH at 56’C, 15 min) were always checked before use. CPT assay An assay mixture prepared freshly before use contained 20 mmol/l coenzyme A, which was omitted and replaced by Hz0 for sample blanks, 5 mmol/l EDTA, 100 mmol/l HEPES/NaOH, pH 7.5, and 25 g/l BSA. In Eppendorf plastic reaction cups 10 ~1 tissue homogenate (corresponding to approximately 0.5 mg wet muscle tissue) were added to 20 ~1 assay mixture. The reaction was started by addition of 10 ~1 palmitoyl-L-carnitine, cups were sealed, thoroughly mixed on a vortex mixer, and incubated for 30 min on an Eppendorf thermostat set to 37°C. For each homogenate a sample blank without coenzyme A was run in parallel. The reaction was stopped with 200 ~1 perchloric acid, 0.6 mol/l, and the cups were placed on ice for 15 min. After centrifugation at 10000 x g the clear supernatant was carefully neutralised with solid potassium hydrogen carbonate, using minimum amounts of n-octanol to prevent excessive foaming. The samples were placed on ice for another 15 min, and recentrifuged for 2 min at 10000 x g. 50 I_L~ of this neutralised extract were used in the carnitine assay described below. Carnitine assay DTNB (2.7 mmol/l)
was dissolved
in 0.5 mmol/l
HEPES,
pH 7.5, and stored
249
frozen at -20°C. Immediately before use, an assay mixture was prepared by diluting perhydrol in a lo-fold volume of the DTNB/HEPES solution. 20 ~1 of this mixture were placed in a plastic reaction cup, and 50 ~1 of the neutralised muscle extract, 150 ~1 of distilled water, and finally 10 ~1 of acetyl coenzyme A solution were added. The samples were stirred with a plastic stirring rod which was left in the unsealed cups to facilitate the escape of oxygen formed. After 10 min at room temperature, when any yellow colour had disappeared, 10 ~1 of catalase, diluted in a 50-fold volume of distilled water, were added, and the open cups were left at room temperature for about 30 min till no more oxygen bubbles were seen. 10 ~1 of CAT solution were added, the cups were sealed and incubated at room temperature for 30 min. The absorption at 405 nm was measured in an Eppendorf photometer against a reagent blank, using water instead of neutralised perchloric acid extract. One unit is equivalent to 1 ymol r_-carnitine formed per minute at 37’C, calculated from and the dilution factors (24 in the CPT assay, and 5 in ( 405nm = 13.6 cm2 - pmol-’ the carnitine assay). Non-collagenous protein (NCP) was determined as described by Lilienthal et al [6], and protein measured by the method of Lowry et al ]7]. CPT was also measured as described previously [8] with a photometric forward method in detergent extracts of washed muscle protein pellets. Results Optimal reaction conditions From Fig. 1 it can be seen that the enzyme activity is maximal under the conditions described. Omission of BSA results in a marked decrease in reaction rate. (In contrast, we have observed that the forward assay is strongly inhibited by BSA concentrations higher than 1 g/l, probably due to binding of palmitoyl-CoA to BSA.) The reaction is linear up to 40 min, as shown in Fig. 2. In most cases, sample blanks were not different from reagent blanks without tissue. However, to avoid errors possibly caused by acyl carnitine deacylases present in tissues [2], we always used sample blanks with CoA-SH omitted. The carnitine found in blanks is due to impurity of the palmitoyl carnitine preparation. Endogenous free tissue carnitine would remain undetectable because of the high dilution of the homogenates in the assay. Reproducibiiity of assays In a series of 12 samples of human muscle obtained at autopsy and homogenised as described in the legend to Fig. 1, CPT activity measured as described above ranged from 10.7 to 12.1 U/g NCP, (X+ SD: 11.5 jr 0.42; CV 3.7%). CPT activity in human muscle biopsies CPT activity in 13 muscle biopsies obtained from patients with no obvious symptoms of muscle disease used as controls was 4.6 rfr 1.4 (range 2.8-9.4) U/g NCP. In comparison, values obtained with the forward photometric method, also at
250
I2 0
2- / o- .
0
2
4
I 6
E
1 10
COD, (mmcl/l)
Fig. 1. Reaction conditions for the CPT backward assay. Human skeletal muscle obtained at autopsy and frozen in liquid nitrogen was rapidly weighed, pulverised (Dismembrator, Braun, Melsungen. FRG), and homogenised in a 20-fold volume of 50 mmol/l HEPES, 1 mmol/l EDTA, pH 7.5. CPT assays were performed varying concentrations of coenzyme A (a), palmitoyl carnitine (b), and BSA (c). respectively. All other conditions were as described in the ‘Materials and methods’ section. Fig. 2. Time course of the CPT backward reaction. Assays reaction conditions described in the ‘Materials and methods’
of CPT activity were performed under the section with 10 pl homogenate (see Fig. 1).
37’C, were 1.6 + 0.5 (range 0.5-2.3) U/g NCP. This conforms with findings of other authors, that the backward reaction yields higher activities than the forward reaction (21. Comparing CPT activities in muscles from 10 controls and 28 patients with recurrent rhabdomyolysis. but normal CPT, both methods were found to correlate fairly well (r 0.75; slope 0.21; intercept 0.42).
251
Discussion CPT in human muscle biopsies is mostly measured using radioisotopes, as in the isotope exchange method or the radioactive forward method [l]. Both methods require at least as much tissue as the one described here, and, even more important, involve the drawbacks inherent in the use of tritiated or “C-labelled substrates. The CPT activities of human muscle found with our assay are of the same order of magnitude as those reported in the literature [1,9]. Exact comparison of results is impossible because of the different experimental procedures and assay conditions employed in different laboratories. The carnitine method used for this assay is also suitable for determination of free and short chain acyl carnitine in plasma, urine and tissues [8]. Acknowledgement This study was supported
by the Deutsche
Forschungsgemeinschaft
(DFG).
References 1 Di Mauro S, Melis Di Mauro P. Muscle CPT deficiency and myoglobinuria. Science 1973; 180: 329-331. 2 McGarry JD, Leatherman GF, Foster DW. Camitine palmitoyltransferase I. J Biol Chem 1978; 253: 4128-4136. 3 Saggerson ED. Camitine acyltransferase activities in rat liver and heart measured with palmitoyl-CoA and octanoyl-CoA. Biochem J 1982; 202: 397-405. 4 Solberg HE. Different camitine acyltransferases in calf liver. Biochim Biophys Acta 1972; 280: 422-433. 5 Marquis NR, Fritz IB. Enzymological determination of free carnitine concentrations in rat tissues. J Lipid Res 1964; 5: 184-187. 6 Lilienthal JL, Zierler KL, Folk BP, Buka R, Riley M. A reference base and system for analysis of muscle constituents. J Biol Chem 1950; 182: 501-508. 7 Lowry DH, Rosebrough NY, Farr AL, Randall FJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275. 8 Pongratz D, Htibner G, Deufel T, Wieland 0, Pongratz E, Liphardt R. Klinische, morphologische und biochemische Befunde bei Camitinmangelmyopathien. Klin Wschr 1979; 57: 927-936. 9 Scholte HR, Jennekens FGI, Bouvy JJBJ. Carnitine palmitoyltransferase II deficiency with normal camitine palmitoyltransferase I in skeletal muscle and leucocytes. J Neurol Sci 1979; 40: 39-51.