1612317
Enzyme 22: 330-335 (1977)
Citrate Synthase Activity in Human Skeletal Muscle 1 G. Haralambie Lehrstuhl für Leistungs- und Sportmedizin (Prof. Dr. J. Keul), Medizinische Universitätsklinik, Freiburg i.Br.
Key Words. Human muscle enzymes • Citrate synthase • NADP-isocitrate dehydrogenase Abstract. Citrate synthase activity in soluble human muscle extracts (KCl-containing triethanol amine buffer) amounts to 17.5 ± 6.97 U/g wet weight at 37 °C (n = 36 healthy male subjects). Double determinations, both using two procedures and with muscle samples divided into two pieces and analyzed separately, gave very good reproducibilities. The possible causes of the com paratively high values are discussed. Significant correlations of citrate synthase activity with NADP-linked isocitrate dehydrogenase (r = + 0.826) and hexose phosphate isomerase (r = —0.582) were found. The distribution of citrate synthase activity in the samples studied is not of the normal type but appears to be binomial.
With support from the ‘Bundesinstitut für Sport wissenschaften’, Cologne.
pathways involved in energy production in muscle, at rest or during exercise. The present paper is an attempt to contribute to a better understanding of the quantitative aspects of citrate synthase activity in skeletal muscle of normal human male subjects.
Subjects and Methods Muscle samples were obtained by surgery from male subjects aged 17-45 years. These had no history of muscle or other metabolic diseases. The subjects maintained their usual daily activity up to 2-5 days before the surgical intervention (fresh fracture or removal of metal plates or nails, at least 10 months after these had been applied).
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Numerous publications have dealt with en zyme activities in human skeletal muscle but several key enzymes of energy metabolism have only been scarcely investigated in this respect. Available data are, in part, of little information value because of the inaccurate selection of sub jects, inadequate methods and treatment of samples, etc. It is obviously of major impor tance for muscle physiologists to have informa tion on the ‘normal’ level of enzyme activities in this organ. This would give a better insight into the various aspects pertaining to pathol ogy, also allowing to speculate on metabolic
Haralambie
331
n Trunk M. transversus abdominis Upper extremity M. triceps brachii (c. radiale) M. triceps brachii (c. longum) M. deltoides Lower extremity M. glutaeus max. M. vastus lateralis quadricipitis M. vastus medialis quadricipitis M. rectus femoris quadricipitis M. tibialis anterior M. gastrocnemius medialis
2 2
2 3 2
13 4 1
5 2
After withdrawal, the small muscle pieces were immediately plunged in ice-cooled buffer (see below) and transported within minutes to the laboratory. The samples were dissected free from visible fat and con nective tissue; excess liquid was removed with filter paper. The resulted muscle pieces were weighed immediately and homogenized in 20 times their weight of buffer (W/V) 0.3 mol/1 triethanolamine, pH 7.6, containing 0.15 mol/1 KC1. Homogenization was performed using an Ultraturrax apparatus type 18/10 (Janke & Kunkel, Staufen/Breisach) for 6 periods of 10 sec and subsequently a teflon-head homogenizer (Braun, Melsungen) for the same dura tion. In order to avoid an increase in temperature during this treatment, the thin-walled plastic reagent glasses were kept for all this time in melting ice. The resulted homogenates were centrifuged for 20 min at 30,000 g. The clear supernatant was further diluted with buffer to a final dilution of 1:210 and the analyses were performed within 1 ’/, h from sampling. Table I shows the type and the number of the ex amined muscles. Citrate synthase (EC 4.1.3.7) was determined (a) with the 5,5'-dithio-bis-(2-nitrobenzoate) proce dure of Srere (14), further designed as ‘412-nm method’, and (b) as described in Bergmeyer (5): 0.05 or 0.1 ml of diluted muscle extract was incubated in tris-hydroxymethyl-aminomethane buffer 0.1 mol/1, pH 8.1 in presence of acetyl-Co A (170 Mmol/1) and
oxaloacetate (150 Mmol/1); total volume was 3 ml. The decrease in absorbance after addition of oxaloacetate was recorded at 232 nm. The corresponding results are further stipulated as ‘232-nm method’ values. The same buffer and substrate concentrations were used in both procedures. In order to obtain an optimal activity, various substrate concentrations were tested with both methods and 150 Mmol/1 was further used. Molecular extinction coefficients of 13.6 (14) and 4.5 X 106 cm2/mol (15) were taken, respectively, in the calculation of the enzyme activity. NADP-linked isocitrate dehydrogenase activity (EC 1.1.1.42) was determined according to Wolfson and Williams-Ashman (19). Hexose phosphate isomerase activity (EC 5.3.1.9) was analyzed as described by Schwartz et al. (13) in 0.1 mol/1 glycine buffer, pH 8.5, with 2.2 mmol/1 fructose-6-phosphate and 0.7 mmol/1 NAD. All analyses were performed at 37 °C using a double-beam Pye-Unicam SP-1800 recording spectro photometer (band width 0.9 nm). Changes in absorb ance were recorded against blank solutions contain ing reagents and muscle extract but no substrate. In several cases muscle samples were divided into two parts and analyzed separately. Results are given in international units (Mmol/min) per gram of muscle wet weight (mean values ± SD). If not otherwise specified, activities of citrate synthase discussed here are based on the ‘232-nm method’. In comparing the activity of two enzymes, linear regres sion equations were computed and the correlation coefficient r indicated (12).
Results The mean activity of citrate synthase in the studied muscle samples (n = 36 subjects) was 17.5 ± 6.97 U/g wet weight (37 °C). The distri bution of activity values in the studied group was not of the normal type (KolmogorovSmirnov one-sample test, p< 0.01) (12) but appeared to be binomial (fig. 1). In 21 samples the activity of citrate synthase was compared to that of NADP-isociträte dehydrogenase. The mean isocitrate dehydrogenase activity was 21.3 ± 9.38 U/g and the correlation coefficient
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Table I. Muscles investigated in this study
332
Haralambie
n
10 -I
30-1
•
206
-
E
4-
c
(Nl
ro csj
2
4.
10-
-
T 20 4 Citrate synthase, U/g
36
U/g 37 °C
T
T
T
10
20
30
412nm
1
2
25-
was r = + 0.826 (p < 0.001). In 15 samples the activity of citrate synthase was compared to that of hexose phosphate isomerase; a signifi cant negative correlation was found in this respect (r = —0.582; p < 0.025). Since, to our knowledge, the ‘232-nm method’ has not yet been applied to crude extracts of human muscle, a comparison be tween the results obtained with this method and those of the alternative ‘412-nm procedure’ was made. Figure 2 shows the good agreement between the two methods (n = 15; r = + 0.992; p< 0.001).
15-
5U/g 232nm
T
T
5
15
T
25
1 3
To facilitate the comparison between the present results and those of other authors, having determined citrate synthase activity at 25 °C, the temperature coefficient 37 °C/25 °C was empirically determined by measuring the activity at the two temperatures mentioned. A mean value of 1.56 ± 0.09 (Q10 = 1.3) was found. This figure is only slightly higher than the temperature coefficient Q10 of‘about 1.2’ described by Alp et al. (1) in warm-blooded animals for citrate synthase. Figure 3 presents the results obtained with muscle samples divided into two pieces and
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Fig. 1. Frequency distribution of citrate synthase activity in the investigated muscle samples (table I). Fig. 2. Comparison between two determination procedures of citrate synthase activity in muscle ex tracts. Abscissa: ‘412-nm method’ (14); ordinate: ‘232-nm method’ (5). The dotted line represents the ideal correlation x = y. Fig. 3. Citrate synthase activity in muscle samples divided into two pieces and analyzed separately (see text). Dotted line: x = y
Citrate Synthase in Human Muscle
333
Table II. Citrate synthase activity in human skeletal muscle
Number of subjects
Muscle
Activity U/g Working temperature, °C wet weight
Reference
NI 3
errector trunci tibialis anterior muscles of the extremities vastus lateralis quadricipitis ‘various muscles’ see table I
25 25 NI 25 NI 25 37
2 11 10
20 6
15 36
4.0 2.2
2.63 ± 1.26 7.2 ± 1.1 2.5 to 5.6 11.2
17.5 ± 6.97
4 7 this paper
NI = Not indicated. 1 This value was calculated from data obtained at 37 °C using a temperature coefficient 37 °C/25 °C of 1.56 (see Results).
Discussion Compared to available data on citrate syn thase activity in human skeletal muscle, the present results are considerably higher (table II). This is very probably due to several causes which will briefly be considered below. (1) Selection of the subjects. This aspect generally seems to have been given little atten tion. Thus, in some papers the mere mention of ‘healthy control subjects’ is made without further details (7, 10, 11). The present subjects were — when one considers the purpose of this investigation — practically healthy since they had neither any muscle disease, nor a dimin
ished functional activity of the corresponding muscles until a few days before surgery. On the other hand, the comparatively larger group studied certainly offers more information than data from six or three subjects (3, 11). In a recent publication, Bass et al. (4) described higher values of citrate synthase activity in the m. vastus lateralis quadricipitis of sport-practic ing subjects (9.2 ± 0.83 U/g) and of highperformance athletes (14.8 ± 0.56 U/g wet weight, 25 °C). Values found in the present work in the same muscle were higher than those of the former group of Bass et al. (4); this, in spite of the fact that the subjects studied here were not engaged in any physical preparation program. (2) Muscle type investigated. It is known from animal experiments that various muscles may exhibit considerable differences in citrate synthase activity according to their predomi nantly glycolytic or oxidative metabolic patterns (16, 18). Therefore, comparisons be tween data of various work teams must be made essentially on the same studied muscle(s). This is here possible for m. vastus lateralis quadricipitis (3) and m. tibialis anterior (11)
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analyzed separately. The good agreement be tween the activities thus measured may indicate that reliable results on enzyme activity are obtained with the applied extraction procedure. On the other hand, the data suggest that — in the case of citrate synthase — determinations carried out on 150—300 mg muscle pieces may be ‘representative’ enough to enable one to discuss on whole muscle as well.
(table I, II). In both cases the present results of 18.0 ± 7.74 and 12.2 ± 3.85 U/g, respectively, are considerably higher than those in the papers quoted above, when the temperature differ ences are considered. (3) Treatment of samples. In this investiga tion the preparation of the extracts and the analyses were carried out as rapidly as possible whereas other authors freezed the muscle pieces for variable durations (10, 11). Freezing thawing, however, appears to influence nega tively the activity of citrate synthase (1, 11). (4) Extraction procedure. Using the Ultraturrax homogenizator, Keul (8) obtained dog myocardium homogenates practically free of mitochondria. In this study a further procedure was used (see Methods) to ensure subcellular particle destruction. Mammalian tissue citrate synthase has been reported to be inhibited by higher concentra tions of K+, such as that used in the extraction buffer; this inhibition is, however, reversible (17). On the other hand, KC1 protects the enzyme from inactivation by thiol-blocking reagents. (5) Analytical method. It is difficult to dis cuss here the possible influence of the method used for the measurement of the enzymatic activity. First, none of the papers quoted in table I give any details on the method used for citrate synthase activity. Second, it appears from the respective quotations that older methods based on oxalate formation with malate, NAD and malate dehydrogenase as auxiliary enzyme (so-called ‘optical tests’ at 340 or 334 nm) have been applied. It can be hypothesized that the more ‘direct’ procedures used in the present investigation also are more adequate in this respect. However, no definite conclusion, is possible as long as direct compari sons between the various methods are not available.
Haralambie
In spite of the somewhat higher values found here, citrate synthase belongs to those enzymes having a comparatively low activity in human skeletal muscle. The same is valid for isocitrate dehydrogenase activity. It is thus possible to speculate on the key role of these enzymes during hypermetabolic states of muscle (e.g. physical activity). One can assume that the increase in blood lactate and ketone bodies, occurring at submaximal work intensities in physically untrained persons (6, 9) is related to the low capacity of muscle tissue to carry out the breakdown of acetyl rests from pyruvate and fatty acids. The increased citrate synthase activity in muscle of trained men (4) as well as the fact observed in this laboratory (unpubl.) that other enzymes of the citrate cyclus have relatively higher activities as compared to citrate synthase, may be arguments for the hypothesis that this latter enzyme has really a key role in this respect. A comparison of the citrate synthase activity with the activities of other oxidative or glyco lytic enzymes has often been made (e.g., 3, 4, 10). Nevertheless, this only appears possible if: (1) the activities are determined at the physi ological temperature of 37 °C, since the various enzymes of human muscle have considerably different temperature coefficients; (2) the determinations are carried out to give, as far as possible, ‘optimal’ values. This is important because the various factors discussed above under 1 to 4 certainly may influence in various ways the results of muscle enzyme analyses. Thus, using ‘non-optimal’ procedures, data may be obtained which are not adequate for comparison purposes. Presently it cannot be ascertained that all the methods used in this study are ‘optimal’ in the broadest acception of this term. However, the correlations found between the different enzyme activities investigated, as well as the
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334
335
Citrate Synthase in Human Muscle
References 1 Alp, P.; Newsholme, E., and Zammit, V.: Activity of citrate synthase and NADMinked and NADP*linked isocitrate dehydrogenase in muscle from vertebrates and invertebrates. Biochem. J. 154: 689-700 (1976). 2 Bass, A.; Brdiczka, D.; Eyer, P.; Hofer, S., and Pette, D.: Metabolic differentiation of distinct muscle types at the level of enzymatic organiza tion. Eur. J. Biochem. 10: 198-206 (1969). 3 Bass, A.; Vondra, K.; Rath, R., and Vitek, V.: M. quadriceps femoris of man, a muscle with unusual enzyme activity pattern of energy supplying metabolism in mammals. Pflügers Arch. 345: 249255 (1975). 4 Bass, A.; Vondra, K.; Rath, R.; Vitek, V.; Teisinger, J.; Mackovâ, E.; Sprynarovâ, S., and Malkovskâ, M.: Enzyme activity patterns of energy supplying metabolism in the quadriceps femoris muscle (vastus lateralis). Eur. J. Physiol. 361: 169-173 (1976). 5 Bergmeyer, H.-U.: Methoden der enzymatischen Analyse, 3. Aufl., vol. 1, pp. 471-472 (Verlag Chemie, Weinheim 1974). 6 Corbett, J.L.; Johnson, R.; Krebs, H.A.; Walton, J., and Williamson, D.: The effect of exercise on blood ketone-body concentrations in athletes and untrained subjects. J. Physiol. 201: 83-84P (1969). 7 Hofer, S.; Hofer, H.W.; Kuhn, E., and Pette, D.: Changes of hexokinase in the enzyme activity pattern of muscle in human myotonia congenita and in experimental myotonia of the rat. Klin. Wschr. 49: 968-971 (1971). 8 Keul, J.: Das Verhalten von Enzymaktivitäten im Myokard von Hunden nach experimenteller Aortenstenose; in Roskamm and Reindell, Das chronisch kranke Herz. Grundlagen der funktio nellen Diagnostik und Therapie, pp. 147-153, (Schattauer, Stuttgart 1973).
9 Keul, J.; Doll, E., and Keppler, D.: Energy metabo lism of human muscle, pp. 107-108 (Karger, Basel 1972). 10 Lujf, A. : Veränderungen von Muskel- und Serum enzymaktivitäten bei Erkrankung menschlicher Skelettmuskulatur. Wien. klin. Wschr. 85: suppl. 5, pp. 2-18 (1973). 11 Nolte, J.; Pette, D.; Bachmaier, B.; Kiefhaber, P.; Schneider, H., and Scriba, P.C.: Enzyme response to thyrotoxicosis and hypothyroidism in human liver and muscle: comparative aspects. Eur. J. clin. Invest. 2: 141-149 (1972). 12 Sachs, L.: Statistische Auswertungsmethoden, 3. Aufl. (Springer, Berlin 1972). 13 Schwartz, M.K.; Bethune, V.; Bach, D.L., and Woodbridge, J.E.: New assay for measuring phosphohexose isomerase (PHI) activity. Clin. Chem. 17: 656-657 (1971). 14 Srere, P.: Citrate synthase; in Lowenstein, Methods in enzymology, voi. 13, pp. 3-11 (Academic Press, New York 1969). 15 Stadman, E.R.: Preparation and assay of acyl coenzyme A and other thiol esters; in Colowick and Kaplan, Methods in enzymology, voi. 3, pp. 931-941 (Academic Press, New York 1957). 16 Terjung, R. : Muscle fiber involvement during train ing of different intensities and durations. Am. J. Physiol. 230: 946-950 (1976). 17 Weitzman, P. and Danson, M.: Citrate synthase; in Horecker and Stadman, Current topics in cellular regulation, voi. 10, pp. 161-204 (Academic Press, New York 1976). 18 Winder, W.; Baldwin, K.M., and Holloszy, J.O.: Enzyme involved in ketone utilization in different types of muscle: adaptation to exercise. Eur. J. Biochem. 47: 461-467 (1974). 19 Wolfson, S.K. and Williams-Ashman, H.: Isocitric and 6-phosphogluconic dehydrogenases in human serum. Proc. Soc. exp. Biol. Med. 96: 231-234 (1957).
Received: December 31, 1976 Dr. G. Haralambie, Medizinische Universitätsklinik, Hugstetterstr. 55, D-7800 Freiburg (FRG)
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good reproducibility (both when two methods were used and when a muscle sample was divided and the pieces analyzed separately) strongly suggest that reliable, ‘near-optimal’ results have been obtained. These might serve as a basis for further developments in this field.
Enzyme 22: 330-335 (1977)
Citrate Synthase Activity in Human Skeletal Muscle 1 G. Haralambie Lehrstuhl für Leistungs- und Sportmedizin (Prof. Dr. J. Keul), Medizinische Universitätsklinik, Freiburg i.Br.
Key Words. Human muscle enzymes • Citrate synthase • NADP-isocitrate dehydrogenase Abstract. Citrate synthase activity in soluble human muscle extracts (KCl-containing triethanol amine buffer) amounts to 17.5 ± 6.97 U/g wet weight at 37 °C (n = 36 healthy male subjects). Double determinations, both using two procedures and with muscle samples divided into two pieces and analyzed separately, gave very good reproducibilities. The possible causes of the com paratively high values are discussed. Significant correlations of citrate synthase activity with NADP-linked isocitrate dehydrogenase (r = + 0.826) and hexose phosphate isomerase (r = —0.582) were found. The distribution of citrate synthase activity in the samples studied is not of the normal type but appears to be binomial.
With support from the ‘Bundesinstitut für Sport wissenschaften’, Cologne.
pathways involved in energy production in muscle, at rest or during exercise. The present paper is an attempt to contribute to a better understanding of the quantitative aspects of citrate synthase activity in skeletal muscle of normal human male subjects.
Subjects and Methods Muscle samples were obtained by surgery from male subjects aged 17-45 years. These had no history of muscle or other metabolic diseases. The subjects maintained their usual daily activity up to 2-5 days before the surgical intervention (fresh fracture or removal of metal plates or nails, at least 10 months after these had been applied).
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Numerous publications have dealt with en zyme activities in human skeletal muscle but several key enzymes of energy metabolism have only been scarcely investigated in this respect. Available data are, in part, of little information value because of the inaccurate selection of sub jects, inadequate methods and treatment of samples, etc. It is obviously of major impor tance for muscle physiologists to have informa tion on the ‘normal’ level of enzyme activities in this organ. This would give a better insight into the various aspects pertaining to pathol ogy, also allowing to speculate on metabolic
Haralambie
331
n Trunk M. transversus abdominis Upper extremity M. triceps brachii (c. radiale) M. triceps brachii (c. longum) M. deltoides Lower extremity M. glutaeus max. M. vastus lateralis quadricipitis M. vastus medialis quadricipitis M. rectus femoris quadricipitis M. tibialis anterior M. gastrocnemius medialis
2 2
2 3 2
13 4 1
5 2
After withdrawal, the small muscle pieces were immediately plunged in ice-cooled buffer (see below) and transported within minutes to the laboratory. The samples were dissected free from visible fat and con nective tissue; excess liquid was removed with filter paper. The resulted muscle pieces were weighed immediately and homogenized in 20 times their weight of buffer (W/V) 0.3 mol/1 triethanolamine, pH 7.6, containing 0.15 mol/1 KC1. Homogenization was performed using an Ultraturrax apparatus type 18/10 (Janke & Kunkel, Staufen/Breisach) for 6 periods of 10 sec and subsequently a teflon-head homogenizer (Braun, Melsungen) for the same dura tion. In order to avoid an increase in temperature during this treatment, the thin-walled plastic reagent glasses were kept for all this time in melting ice. The resulted homogenates were centrifuged for 20 min at 30,000 g. The clear supernatant was further diluted with buffer to a final dilution of 1:210 and the analyses were performed within 1 ’/, h from sampling. Table I shows the type and the number of the ex amined muscles. Citrate synthase (EC 4.1.3.7) was determined (a) with the 5,5'-dithio-bis-(2-nitrobenzoate) proce dure of Srere (14), further designed as ‘412-nm method’, and (b) as described in Bergmeyer (5): 0.05 or 0.1 ml of diluted muscle extract was incubated in tris-hydroxymethyl-aminomethane buffer 0.1 mol/1, pH 8.1 in presence of acetyl-Co A (170 Mmol/1) and
oxaloacetate (150 Mmol/1); total volume was 3 ml. The decrease in absorbance after addition of oxaloacetate was recorded at 232 nm. The corresponding results are further stipulated as ‘232-nm method’ values. The same buffer and substrate concentrations were used in both procedures. In order to obtain an optimal activity, various substrate concentrations were tested with both methods and 150 Mmol/1 was further used. Molecular extinction coefficients of 13.6 (14) and 4.5 X 106 cm2/mol (15) were taken, respectively, in the calculation of the enzyme activity. NADP-linked isocitrate dehydrogenase activity (EC 1.1.1.42) was determined according to Wolfson and Williams-Ashman (19). Hexose phosphate isomerase activity (EC 5.3.1.9) was analyzed as described by Schwartz et al. (13) in 0.1 mol/1 glycine buffer, pH 8.5, with 2.2 mmol/1 fructose-6-phosphate and 0.7 mmol/1 NAD. All analyses were performed at 37 °C using a double-beam Pye-Unicam SP-1800 recording spectro photometer (band width 0.9 nm). Changes in absorb ance were recorded against blank solutions contain ing reagents and muscle extract but no substrate. In several cases muscle samples were divided into two parts and analyzed separately. Results are given in international units (Mmol/min) per gram of muscle wet weight (mean values ± SD). If not otherwise specified, activities of citrate synthase discussed here are based on the ‘232-nm method’. In comparing the activity of two enzymes, linear regres sion equations were computed and the correlation coefficient r indicated (12).
Results The mean activity of citrate synthase in the studied muscle samples (n = 36 subjects) was 17.5 ± 6.97 U/g wet weight (37 °C). The distri bution of activity values in the studied group was not of the normal type (KolmogorovSmirnov one-sample test, p< 0.01) (12) but appeared to be binomial (fig. 1). In 21 samples the activity of citrate synthase was compared to that of NADP-isociträte dehydrogenase. The mean isocitrate dehydrogenase activity was 21.3 ± 9.38 U/g and the correlation coefficient
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Table I. Muscles investigated in this study
332
Haralambie
n
10 -I
30-1
•
206
-
E
4-
c
(Nl
ro csj
2
4.
10-
-
T 20 4 Citrate synthase, U/g
36
U/g 37 °C
T
T
T
10
20
30
412nm
1
2
25-
was r = + 0.826 (p < 0.001). In 15 samples the activity of citrate synthase was compared to that of hexose phosphate isomerase; a signifi cant negative correlation was found in this respect (r = —0.582; p < 0.025). Since, to our knowledge, the ‘232-nm method’ has not yet been applied to crude extracts of human muscle, a comparison be tween the results obtained with this method and those of the alternative ‘412-nm procedure’ was made. Figure 2 shows the good agreement between the two methods (n = 15; r = + 0.992; p< 0.001).
15-
5U/g 232nm
T
T
5
15
T
25
1 3
To facilitate the comparison between the present results and those of other authors, having determined citrate synthase activity at 25 °C, the temperature coefficient 37 °C/25 °C was empirically determined by measuring the activity at the two temperatures mentioned. A mean value of 1.56 ± 0.09 (Q10 = 1.3) was found. This figure is only slightly higher than the temperature coefficient Q10 of‘about 1.2’ described by Alp et al. (1) in warm-blooded animals for citrate synthase. Figure 3 presents the results obtained with muscle samples divided into two pieces and
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Fig. 1. Frequency distribution of citrate synthase activity in the investigated muscle samples (table I). Fig. 2. Comparison between two determination procedures of citrate synthase activity in muscle ex tracts. Abscissa: ‘412-nm method’ (14); ordinate: ‘232-nm method’ (5). The dotted line represents the ideal correlation x = y. Fig. 3. Citrate synthase activity in muscle samples divided into two pieces and analyzed separately (see text). Dotted line: x = y
Citrate Synthase in Human Muscle
333
Table II. Citrate synthase activity in human skeletal muscle
Number of subjects
Muscle
Activity U/g Working temperature, °C wet weight
Reference
NI 3
errector trunci tibialis anterior muscles of the extremities vastus lateralis quadricipitis ‘various muscles’ see table I
25 25 NI 25 NI 25 37
2 11 10
20 6
15 36
4.0 2.2
2.63 ± 1.26 7.2 ± 1.1 2.5 to 5.6 11.2
17.5 ± 6.97
4 7 this paper
NI = Not indicated. 1 This value was calculated from data obtained at 37 °C using a temperature coefficient 37 °C/25 °C of 1.56 (see Results).
Discussion Compared to available data on citrate syn thase activity in human skeletal muscle, the present results are considerably higher (table II). This is very probably due to several causes which will briefly be considered below. (1) Selection of the subjects. This aspect generally seems to have been given little atten tion. Thus, in some papers the mere mention of ‘healthy control subjects’ is made without further details (7, 10, 11). The present subjects were — when one considers the purpose of this investigation — practically healthy since they had neither any muscle disease, nor a dimin
ished functional activity of the corresponding muscles until a few days before surgery. On the other hand, the comparatively larger group studied certainly offers more information than data from six or three subjects (3, 11). In a recent publication, Bass et al. (4) described higher values of citrate synthase activity in the m. vastus lateralis quadricipitis of sport-practic ing subjects (9.2 ± 0.83 U/g) and of highperformance athletes (14.8 ± 0.56 U/g wet weight, 25 °C). Values found in the present work in the same muscle were higher than those of the former group of Bass et al. (4); this, in spite of the fact that the subjects studied here were not engaged in any physical preparation program. (2) Muscle type investigated. It is known from animal experiments that various muscles may exhibit considerable differences in citrate synthase activity according to their predomi nantly glycolytic or oxidative metabolic patterns (16, 18). Therefore, comparisons be tween data of various work teams must be made essentially on the same studied muscle(s). This is here possible for m. vastus lateralis quadricipitis (3) and m. tibialis anterior (11)
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analyzed separately. The good agreement be tween the activities thus measured may indicate that reliable results on enzyme activity are obtained with the applied extraction procedure. On the other hand, the data suggest that — in the case of citrate synthase — determinations carried out on 150—300 mg muscle pieces may be ‘representative’ enough to enable one to discuss on whole muscle as well.
(table I, II). In both cases the present results of 18.0 ± 7.74 and 12.2 ± 3.85 U/g, respectively, are considerably higher than those in the papers quoted above, when the temperature differ ences are considered. (3) Treatment of samples. In this investiga tion the preparation of the extracts and the analyses were carried out as rapidly as possible whereas other authors freezed the muscle pieces for variable durations (10, 11). Freezing thawing, however, appears to influence nega tively the activity of citrate synthase (1, 11). (4) Extraction procedure. Using the Ultraturrax homogenizator, Keul (8) obtained dog myocardium homogenates practically free of mitochondria. In this study a further procedure was used (see Methods) to ensure subcellular particle destruction. Mammalian tissue citrate synthase has been reported to be inhibited by higher concentra tions of K+, such as that used in the extraction buffer; this inhibition is, however, reversible (17). On the other hand, KC1 protects the enzyme from inactivation by thiol-blocking reagents. (5) Analytical method. It is difficult to dis cuss here the possible influence of the method used for the measurement of the enzymatic activity. First, none of the papers quoted in table I give any details on the method used for citrate synthase activity. Second, it appears from the respective quotations that older methods based on oxalate formation with malate, NAD and malate dehydrogenase as auxiliary enzyme (so-called ‘optical tests’ at 340 or 334 nm) have been applied. It can be hypothesized that the more ‘direct’ procedures used in the present investigation also are more adequate in this respect. However, no definite conclusion, is possible as long as direct compari sons between the various methods are not available.
Haralambie
In spite of the somewhat higher values found here, citrate synthase belongs to those enzymes having a comparatively low activity in human skeletal muscle. The same is valid for isocitrate dehydrogenase activity. It is thus possible to speculate on the key role of these enzymes during hypermetabolic states of muscle (e.g. physical activity). One can assume that the increase in blood lactate and ketone bodies, occurring at submaximal work intensities in physically untrained persons (6, 9) is related to the low capacity of muscle tissue to carry out the breakdown of acetyl rests from pyruvate and fatty acids. The increased citrate synthase activity in muscle of trained men (4) as well as the fact observed in this laboratory (unpubl.) that other enzymes of the citrate cyclus have relatively higher activities as compared to citrate synthase, may be arguments for the hypothesis that this latter enzyme has really a key role in this respect. A comparison of the citrate synthase activity with the activities of other oxidative or glyco lytic enzymes has often been made (e.g., 3, 4, 10). Nevertheless, this only appears possible if: (1) the activities are determined at the physi ological temperature of 37 °C, since the various enzymes of human muscle have considerably different temperature coefficients; (2) the determinations are carried out to give, as far as possible, ‘optimal’ values. This is important because the various factors discussed above under 1 to 4 certainly may influence in various ways the results of muscle enzyme analyses. Thus, using ‘non-optimal’ procedures, data may be obtained which are not adequate for comparison purposes. Presently it cannot be ascertained that all the methods used in this study are ‘optimal’ in the broadest acception of this term. However, the correlations found between the different enzyme activities investigated, as well as the
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References 1 Alp, P.; Newsholme, E., and Zammit, V.: Activity of citrate synthase and NADMinked and NADP*linked isocitrate dehydrogenase in muscle from vertebrates and invertebrates. Biochem. J. 154: 689-700 (1976). 2 Bass, A.; Brdiczka, D.; Eyer, P.; Hofer, S., and Pette, D.: Metabolic differentiation of distinct muscle types at the level of enzymatic organiza tion. Eur. J. Biochem. 10: 198-206 (1969). 3 Bass, A.; Vondra, K.; Rath, R., and Vitek, V.: M. quadriceps femoris of man, a muscle with unusual enzyme activity pattern of energy supplying metabolism in mammals. Pflügers Arch. 345: 249255 (1975). 4 Bass, A.; Vondra, K.; Rath, R.; Vitek, V.; Teisinger, J.; Mackovâ, E.; Sprynarovâ, S., and Malkovskâ, M.: Enzyme activity patterns of energy supplying metabolism in the quadriceps femoris muscle (vastus lateralis). Eur. J. Physiol. 361: 169-173 (1976). 5 Bergmeyer, H.-U.: Methoden der enzymatischen Analyse, 3. Aufl., vol. 1, pp. 471-472 (Verlag Chemie, Weinheim 1974). 6 Corbett, J.L.; Johnson, R.; Krebs, H.A.; Walton, J., and Williamson, D.: The effect of exercise on blood ketone-body concentrations in athletes and untrained subjects. J. Physiol. 201: 83-84P (1969). 7 Hofer, S.; Hofer, H.W.; Kuhn, E., and Pette, D.: Changes of hexokinase in the enzyme activity pattern of muscle in human myotonia congenita and in experimental myotonia of the rat. Klin. Wschr. 49: 968-971 (1971). 8 Keul, J.: Das Verhalten von Enzymaktivitäten im Myokard von Hunden nach experimenteller Aortenstenose; in Roskamm and Reindell, Das chronisch kranke Herz. Grundlagen der funktio nellen Diagnostik und Therapie, pp. 147-153, (Schattauer, Stuttgart 1973).
9 Keul, J.; Doll, E., and Keppler, D.: Energy metabo lism of human muscle, pp. 107-108 (Karger, Basel 1972). 10 Lujf, A. : Veränderungen von Muskel- und Serum enzymaktivitäten bei Erkrankung menschlicher Skelettmuskulatur. Wien. klin. Wschr. 85: suppl. 5, pp. 2-18 (1973). 11 Nolte, J.; Pette, D.; Bachmaier, B.; Kiefhaber, P.; Schneider, H., and Scriba, P.C.: Enzyme response to thyrotoxicosis and hypothyroidism in human liver and muscle: comparative aspects. Eur. J. clin. Invest. 2: 141-149 (1972). 12 Sachs, L.: Statistische Auswertungsmethoden, 3. Aufl. (Springer, Berlin 1972). 13 Schwartz, M.K.; Bethune, V.; Bach, D.L., and Woodbridge, J.E.: New assay for measuring phosphohexose isomerase (PHI) activity. Clin. Chem. 17: 656-657 (1971). 14 Srere, P.: Citrate synthase; in Lowenstein, Methods in enzymology, voi. 13, pp. 3-11 (Academic Press, New York 1969). 15 Stadman, E.R.: Preparation and assay of acyl coenzyme A and other thiol esters; in Colowick and Kaplan, Methods in enzymology, voi. 3, pp. 931-941 (Academic Press, New York 1957). 16 Terjung, R. : Muscle fiber involvement during train ing of different intensities and durations. Am. J. Physiol. 230: 946-950 (1976). 17 Weitzman, P. and Danson, M.: Citrate synthase; in Horecker and Stadman, Current topics in cellular regulation, voi. 10, pp. 161-204 (Academic Press, New York 1976). 18 Winder, W.; Baldwin, K.M., and Holloszy, J.O.: Enzyme involved in ketone utilization in different types of muscle: adaptation to exercise. Eur. J. Biochem. 47: 461-467 (1974). 19 Wolfson, S.K. and Williams-Ashman, H.: Isocitric and 6-phosphogluconic dehydrogenases in human serum. Proc. Soc. exp. Biol. Med. 96: 231-234 (1957).
Received: December 31, 1976 Dr. G. Haralambie, Medizinische Universitätsklinik, Hugstetterstr. 55, D-7800 Freiburg (FRG)
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good reproducibility (both when two methods were used and when a muscle sample was divided and the pieces analyzed separately) strongly suggest that reliable, ‘near-optimal’ results have been obtained. These might serve as a basis for further developments in this field.
