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CLIN. CHEM. 24/2, 200-203(1978) Determination of Pyruvate Oxidation Rate and Citric Acid Cycle Activity in Intact Human Leukocytes and Fibroblasts Hans L. WIllems,1 Veerkamp2 Ted F. M. de Kort,1 Frans J. M. Trijbels,1 We measured pyruvate oxidation in Intact leukocytes and fibroblasts by measuring 14C02 production. The optimal pyruvate concentration appeared to be higher than that usually applied. Activities remained constant during the incubation and were proportional to the amount of tissue protein added. Mean values (± SD) were 2.8 ± 0.9 nmol/h per 106 cells and 37 ± 14 nmol/h per mg of protein for leukocytes and fibroblasts, respectively, uvate oxidation; and 2.1 ± 0.8 nmol/h for [1-14CJpyr- per 106 cells and 18 ± 7 nmol/h per mg of protein, respectively, ‘4C] pyruvate oxidation. We compared oxidation pyruvate and 2-oxoglutarate by intact cells for [2- rates with those of of isolated mitochondria. The ratio of 14C02 production vs. activity of mitochondrial marker enzyme demonstrated that the rate of pyruvate in intact cells, but oxidation can adequately be assayed that the permeability of the cell membrane is rate-limiting in the oxidation of 2-oxoglutarate. No significant oxidation of other intermediates of the citric acid cycle was found, presumably owing to a low rate of transport of these substances across the cell membrane. ‘ Keyphreases: pyruvate oxidation use of intact cells to measure enzyme activity transport across the cell membrane 2-oxoglutarate pediatric chemistry . lactic acidemia AddItional During the last years there has been increasing interest in determining enzyme activities in cultured fibroblasts and leukocytes. Though enzyme defects can be established in liver, kidney, or muscle biopsy materials, these studies are rather limited. Frequently only fibroblasts are available for enzyme assays. Because leukocytes can easily be obtained, they are also an important source for the study of enzyme activity. We have preferred to work with intact cells, so as to approach physiological conditions insofar as possible. Use of intact cells has the advantage that the intracellular relationships are intact. Moreover, the cytoplasm and the intra-mitochondrial content are diluted on sonication, which necessitates addition of cofactors to the incuba- Departments of Pediatrics,1 and Biochemistry,2 Nijmegen, Nijmegen, The Netherlands. Received Oct. 11, 1977; accepted Dec. 1, 1977. 200 CLINICAL CHEMISTRY, Vol. 24, No. 2, 1978 University of Leo A. H. Monnens,’ and Jacques H. tion medium. Our procedure is superior to most of the previous methods because deficiencies of cofactors may be established as well. Our aim in the present investigation was to study the contribution of transport across the cell membrane in the assay of pyruvate oxidation with [1-14C]pyruvate and the citric acid cycle activity with [2-14C]pyruvate and [1-’4CJ2-oxoglutarate in intact leukocytes and fibroblasts. Materials and Methods Leukocytes One volume of 50 g/liter dextran solution (T500; Pharmacia, Uppsala, Sweden) containing, per liter, 7 g of NaCl and 15 g of disodium ethylenediaminetetraacetate) was added to five volumes of blood. Blood samples were obtained after an overnight fast from children (1-10 years), all hospitalized for disorders not interfering with pyruvate metabolism. The total leukocyte count was in the range 5.10#{176} to 10.10#{176} per liter. The differential leukocyte count was: 60-70% neutrophils, 20-30% lymphocytes, and 2-10% monocytes. After 30 mm at 4 #{176}C the supernatant plasma-containing leukocytes, some erythrocytes, and platelets-was centrifuged for 8 mm at 300 X g. The supernatant plasma, which contained most of the platelets, was discarded. The pellet was suspended in 1 ml of buffered saline (40 mmol/liter phosphate buffer, pH 7.4, containing 0.11 mol of NaC1 per liter). The remaining erythrocytes were removed by applying an hypotonic osmotic shock by exposure to distilled water for 90 s, after which isotonicity was restored (1). After washing with saline, the final pellet was suspended in buffered saline and the cells were counted in a Coulter Counter. Mitochondria were prepared from a concentrated suspension of leukocytes (30.109/liter) in 0.25 mol/liter of sucrose. The cell membrane was disrupted with a French press at 3.5 X 106 Pa. The cell debris was removed by centrifugation (10 mm at 70 X g) and the mitochondria obtained by centrifugation for 13 mm at 8000 X g were washed once with 0.25 mol/liter of sucrose. Fibroblasts tainate dehydrogenase (EC 1.4.1.2) activity were measured in cells and mitochondria, both after freezing and Skin biopsy material was cut into small fragments thawing three times. These assays were performed acand cultured in TC-199 medium (Flow Laboratories, cording to Cooperstein and Lazarow (4) and Schmidt Irvine, Scotland) enriched with, per liter, 200 g of fetal (5), respectively. calf serum and 50 g of chicken embryo extract, suppleThe respiratory control index of mitochondria of fimented with 1 X 10 USP units of penicillin and 100 mg broblasts was measured in simplified Dow (6) medium of streptomycin. After trypsinization for 5 mm at 37 #{176}C containing 1 mmol of pyruvate and 1 mmol of malate (2.5 g of trypsin per liter; Difco, Detroit, Mich.; 1:250 per liter. ADP was added in a final concentration of 0.2 pancreas) the fibroblasts were washed with the phosmmol/liter. phate-buffered saline. The cells were counted under the microscope in a special glass counting chamber after Radiolabeled Materials dilution of the cell suspension. [1-’4C]Pyruvate, [2-14C]pyruvate, and [1-’4C]2-oxoFor isolation of mitochondria, fibroblasts were susglutarate were obtained from the Radiochemical Centre, pended in a medium containing, per liter, 0.27 mol of Aniersham, and stored at -20 #{176}C in small aliquots as the mannitol, 0.1 mmol of disodium ethylenediaminetedry sodium salts, under nitrogen. traacetate, 0.5 g of bovine serum albumin, and 10 mmol of tris(hydroxymethyl)aminomethane#{149}HC1 (pH 7.3). Results The cells (4.109/liter) were disrupted at 4 #{176}C with a Isolation of Leukocytes and Fibroblasts Potter-Elvejhem homogenizer and the mitochondria were isolated by differential centrifugation for 7 mm at The differential leukocyte count remained unchanged 770 X g and 10 mm at 10 000 X g. after isolation in comparison with that in the peripheral blood. Recovery of leukocytes in the final preparation Enzyme Assays averaged 50-60%; 6% of the original amount of platelets Leukocytes (5.106 to 10.106) were incubated in the was found in the final leukocyte suspension, which phosphate-buffered saline and either pyruvate or 2contained on the average equal amounts of erythrocytes oxoglutarate (0.5 mmol/liter), the final volume being 2.1 and leukocytes. ml. The specific activity of the used substrates was 1.0 After trypsinization, 2-5% of the fibroblasts were non-viable, as judged by trypan blue uptake. mCi/mol. Incubation was done in a shaking waterbath at 37 #{176}C in 20-ml glass scintillation vials, sealed with Isolation of Mitochondria rubber stoppers, containing two small tubes, one fitting inside the other (2). The reaction was stopped after 60 Leukocytes are relatively resistant to the usual homm by injecting 0.3 ml of 3 mol/liter of perchloric acid mogenization procedures (7). The best disruption of and the ‘4C02 produced was trapped in Hyamine hycells was obtained by a more vigorous method: use of a French press. The respiratory control index of the midroxide [p -(diisobutyl-cresoxyethoxyethyl)dimethylbenzylammonium hydroxide] during further incubation tochondria of leukocytes could not be established befor 30 mm at 37 #{176}C. The trapped ‘4C02 was measured cause of the low oxidative capacity. after adding 10 ml of scintillation fluid (4 g of Omnifluor Fibroblasts could be well homogenized with a Potper liter of toluene) in a liquid scintillation counter. For ter-Elvejhem homogenizer in isotonic medium. The respiratory control index of the isolated mitochondria the determination of the blank values the cells were was 4.0 with pyruvate/malate as substrates. omitted. Fibroblasts (5.10 to 1.106) were incubated under identical conditions as described for the substrate concentrations oxoglutarate (0.2 mmol/liter). The ml. Protein was assayed according for leukocytes except of pyruvate and 2final volume was 1.5 to Lowry et al. (3). Mitochondria Pyruvate oxidation by mitochondria isolated from fibroblasts and leukocytes was measured at 37 #{176}C in an incubation mixture containing, per liter, 50 mmol of potassium phosphate buffer (pH 7.4), 0.5 mmol of disodium ethylenediaminetetraacetate, 2 mmol of ADP, 75 mmol of KC1, 10 mmol of MgCl2, 1 mmol of the substrate (0.5 mCi/mol), and 0.2-0.4 mg of mitochondrial protein. The final volume was 0.5 ml. Incubation time was 60 mm. Leukocytes and fibroblasts yielded about 4 and 40 mg of mitochondrial protein per 10#{176} cells, respectively. The assay procedure Cytochrome was as described for c oxidase (EC 1.9.3.1) intact activity cells. and glu- Enzyme Assays 14CO2 production from [1-14C]pyruvate was lower without added cells than that in the presence of boiled cells. Nearly identical blank values were obtained either in the absence of cells or after inhibition of pyruvate oxidation with arsenite (4.4 mmol/liter). The presence of boiled cells appeared to give an incorrect blank value by a higher nonenzymatic decarboxylation of [1-’4C] pyruvate (8). The results (Figure 1) indicate that the reaction rate of the oxidation of [1-14C]pyruvate and [2-’4C]pyruvate by fibroblasts remained constant during 60 mm of incubation, and also was proportional to the amount of cellular protein. Similar results were obtained for lenkocytes. Table 1 shows control values for the pyruvate oxidation rate in leukocytes and fibroblast.s. The CV for the assay amounts to 7%, both for leukocytes and Iibroblasts. The Lineweaver-Burk plots with [1-’C]pyruvate as a substrate CLINICAL revealed CHEMISTRY. an apparent Vol. 24, No. Km 2, 1978 value 201 - nrnol Table 1. Pyruvate Oxidation In Human Leukocytes and Cultured Flbroblastsa I 1-4CJPyruvat. Leukocytes I mg protein nmol/ h #{149}-.!1-#{176}CJpyruvote 12-”CJ pyruiat. 100 80 [2-4CJPyruvate b 2.8 ± 0.9 range 1.4-4.1 60 2.1 ± 0.8 range 1.1-3.5 (n = 11) (n 12) = 40 Fibroblasts’ 37 ± 14 18 ± 7 range 19-6 1 (n = 10) range 12-30 (n = 5) a Values are given 20 ± SD. as the mean 15 106 Ieukocytes. nmol/h per mg of protein. b nmol/h per in leukocytes of 0.14 mmol/liter of citric and fibroblasts and acid cycle with its for fibroblasts activity of in intact intermediates 2. Pyruvate intact culttred rates of [1-14C]pyruvate fibroblasts incubated dehydrogenase cells in leukocytes c oxidase 2-Oxoglutarate dehydrogenase c oxidase Cytochrome was too low to be used 202 as a reliable Dehydrogenase and Leukocytes. Activity in Intact Cells and Leukocytes MitOchOndria Intact (nmollh 218 5 77 1242 6930 3060 11 820 Cells MitochOndria per mg of protein) 2.9 16.6 0.2 - 492 (X 10.7 - 1270 1O) 30.6 31.5 4.0 11.1 12.4 18.4 5.9 13.0 1.6 6.5 0.5 8.4 dehydrogenase dehydrogenase 2-Oxoglutarate Glutamate of time concen- dehydrogenase Cytochrome Glutamate periods enzyme Several clinical disorders are associated with increased concentrations of lactate or pyruvate (or both) in blood or cerebrospinal fluid (or both). The mean clinical features of patients suffering from these disorders are mental retardation, muscular hypotonia, and convulsions (1). Measuring pyruvate oxidation rate in leukocytes and fibroblasts can be helpful in the diagnosis of children suffering from lactic acidemia. Leukocytes and fibroblasts contain the enzymatic equipment for pyruvate oxidation (9-11), such as pyruvate dehydrogenase (1), the citric acid cycle (12), and the electron transport system (13-15). The oxidation rates of [1-’4C]pyruvate and [2-’4C]pyruvate we found for intact leukocytes and fibroblasts (Table 1) much exceed those previously published (1, 16, 17), perhaps because of the higher substrate concentration we used. In other investigations (1, 16, 17) pyruvate concentration was below the Km value. Pyruvate is oxidized in fibroblasts more rapidly than Ratio Pyruvate and [2-14C]pyruvate for various Discussion 38 dehydrogenase Cytochrome c oxidase Glutamate dehydrogenase dehydrogenase dehydrogenase CLINICAL CHEMISTRY, Vol. 24, No. 2, 1978 0.5 protein marker. Activity Pyruvate 0.4 trations (incubation time, 1 h) Flbroblasts 2-Oxoglutarate 03 mg (using 1.0 mg of protein) and with increasing activity Dehydrogenase and 2-Oxoglutarate Mltochondria of Flbroblasts Intact Pyruvate 0.2 as substrates was almost impossible. Citric acid cycle activity could be only demonstrated by using [2-14C]pyruvate as substrate (Table 1). No measurable activity was found with [U-14C]malate, [1,5-’4C]and [6-”C]citrate, [1,4-’4C]succinate, or [5-’4C]2-oxoglutarate as substrates. The oxidation rate with [1-’4C]2-oxoglutarate and [1-14C]glutamate as substrates was low in intact cells. Table 2 compares the oxidation rates of [1-’4C]pyruvate and [1-’4CJ2-oxoglutarate by intact leukocytes and fibroblasts and by their mitochondria. Cytochrome c oxidase and glutamate dehydrogenase were measured as mitochondrial markers. The ratio of pyruvate dehydrogenase activity to activity of mitochondrial markers is in the same order of magnitude for intact cells and mitochondria. The discrepancy of the ratio for pyruvate dehydrogenase to glutamate dehydrogenase between intact cells and mitochondria might be due to leakage of glutamate dehydrogenase out of the mitochondrial matrix. The ratio for 2-oxoglutarate dehydrogenase activity was, however, much higher in mitochondria than in intact cells. Cytochrome c oxidase Table 0.1 60 mm Fig. 1. Oxidation for leukocytes 0.11 mmol/liter. Determination 45 30 in leukocytes. The oxidative pathway in leukocytes is of minor importance (18). The generation of ATP quantitatively depends less on oxidative phosphorylation than on glycolysis (7). The approximately equal ‘4C02 production from [1-’4C]pyruvate and [2-’4C]pyruvate with intact leukocytes and fibroblasts (Table 1) contrasts with the results presented by Cederbaum et al. (16) and with the observations on rat adipose tissue (19). Our data suggest that in leukocytes and fibroblasts acetyl-CoA produced from pyruvate is almost completely oxidized in the citric acid cycle under the conditions we used. No reliable measurements of citric acid cycle activity could be performed with intermediates of the citric acid cycle as substrates in intact leukocytes and fibroblasts. This might be due to a low activity of the citric acid cycle or to the limited transport of these substrates across the cell membrane or the mitochondrial membrane. The experiments with [2-’4C]pyruvate as substrate show that in vitro measurement of the overall citric acid cycle activity is feasible. Moreover, earlier studies (9, 12) demonstrated a definite citric acid cycle activity in both types of cells. The low rate of transport of 2-oxoglutarate across the cell membrane will cause a difference in its rate of oxidation between intact cells and mitochondna. We conclude that pyruvate oxidation can be reliably measured in intact leukocytes and fibroblasts with use of [1-14C]pyruvate. Concomitant assay of ‘4C02 production from [2-’4C]pyruvate can provide an indication for the total activity of the citric acid cycle, but the individual steps of this cycle can only be determined by 2. Fox, R. M., A simple incubation Biochem. 41,578 (1971). using carbohydrate in a patient with familial intermittent lactic acidosis and pyruvate dehydrogenase deficiency. Pediat. Res. 10, 713 (1976). 17. Falk, R. E., Cederbaum, S.D., Blass, J. P., et a!., Effects of a ketogenic diet in two brothers with pyruvate dehydrogenase deficiency. Pediatrics 58,713 (1976). mitochondria or disrupted cells. Prolonged trypsinization of fibroblasts may increase the cellular uptake of di- and tricarboxylic acids (12) but may disturb cellular metabolism. We suggest that use of intact leukocytes, and especially fibroblasts, may be appropriate to detect defects in pyruvate oxidation clinical- 3. 193, 265 method 189,665 5. Schmidt, matischen Clin. Invest. Uhlendorf, B. W., A defect an intermittent movement in pyruvate disorder. J. R. J., Chern. Lazarow, and A., A microspectrophotometric of cytochrome oxidase. J. Biol. Chem. 6. Max S. R., Garbus, J., and Wehman, H. J., Simple procedure rapid isolation of functionally intact mitochondria from human rat skeletal muscle. Anal. Biochem. 46, 576 (1972). 7. Kirschner, R. H., Getz, G. S., and Evans, chondria. Function and biogenesis. Enzyme A. E., Leucocyte 13, 56 (1972). for and mite- 8. Oldhani, K. G., Radiochernical enzyme assays: Factors affecting their sensitivity and the selection of optimum conditions for assays. Int. J. AppI. Radiat. Isot. 21, 421 (1970). 9. Jemelin, aerobic Biol. M., and and Clin. aerobic 11,289 Frei, 11. Stjernholm, drate J., Leucocyte energy production and related ATP (1970). 10. Sun, A. S., Aggarwal, human cells: Aging in (1975). B., and, culture. R. L., Burns, metabolism by leukocytes. 12. Blass, J. P., Schulman, herited defect congenital affecting lactic 13. Castelli, A., system in human (1973). 14. Pious, lytic acidosis. Packer, enzymes. Biochem. C. P., and Enzyme J: Clin. metabolism L., Enzyme Arch. levels Biophys. III. AnEnzymol. of normal 170, 1 Hohnadel, J. H., Carbohy13,7 (1972). J. D., Young, tricarboxylic the D. S., and Horn, E., An inacid cycle in a patient with 51, 1845 (1972). Invest. DeSole, P., Gozzo, M. L., et al., Electron transport leukocyte mitochondria. Ital. J. Biochem. 22, 167 D. A., Independent enzymes in human regulation fibroblasts. of cytochrornes J. Cell. Physiol. and glyco79, 423 (1972). 15. Evans, A. E.,and Getz, G. S., Mitochondrial electron transport enzymes in normal and leukemic human leukocytes. Blood 31, 710 (1968). 16. 18. Cederbaum, Blass, (1977). J., and in a child with 49,423 (1970). Randall, J. Biol. E., Glutamate dehydrogenase. In Met hoden der EnzyAnalyse. H. U. Bergmeyer, Ed., Verlag Chemie GmbH, 1962, p 752. Weinheim, References J. P., Avigan, S. J., for the determination (1951). of pyruvate let-enriched 1. Blass, Anal. (1951). Cooperstein, ly. decarboxylase collection. 0. H., Rosebrough, N. J., Farr, A. L., and measurement with the Folin phenol reagent. Lowry, Protein 4. for ‘CO2 flask S. D., Blass, j. P., Cederbaum, ketoglutarate preparations J. P., Minkoff, S. D., and Kark, N., H. A. P., Rapid dehydrogenase and from blood. et al., Sensitivity Clin. to diagnosis deficiencies in plate. Chim. Acta 75, 21 19. Hanson, R. W., Patel, M. S., Jornain-Baurn, M., and Ballard, F. J., Role of mitochondria in metabolism of pyruvate and lactate by rat adipose tissue. Metabolism 20, 27 (1971). CLINICAL CHEMISTRY, Vol. 24, No. 2, 1978 203