Carbohydrate Metabolism in Leukocytes

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JOURNAL OF BACTERIOLOGY, May 1967, p. 1657-1661 Vol. 93, No. 5 Copyright ( 1967 American Society for Microbiology Printed in U.S.A. Carbohydrate Metabolism in Leukocytes VII.

1 JOURNAL OF BACTERIOLOGY, May 1967, p Vol. 93, No. 5 Copyright ( 1967 American Society for Microbiology Printed in U.S.A. Carbohydrate Metabolism in Leukocytes VII. Metabolism of Glucose, Acetate, and Propionate by Human Plasma Cells RUNE L. STJERNHOLM Department of Biochemistry, Western Reserve University, Cleveland, Ohio Received for publication 30 January 1967 Plasma cells obtained from the peripheral blood of a patient with multiple myeloma was incubated in serum and Krebs-Ringer bicarbonate buffer with 14Clabeled glucose, acetate, and propionate. Glucose utilization by these cells amounted to 0.5,mole per hr per 108 cells and was mainly via the Embden-Meyerhof pathway, and only 6% or less traversed the hexose monophosphate shunt. The presence of Krebs cycle activity was demonstrated by direct isolation of several labeled intermediates after incubation with either "C-acetate or "C-propionate. The distribution of 14C in lactate, succinate, fumarate, malate, aspartate, and glutamate indicate a complete Krebs cycle. Acetate was metabolized via the Krebs cycle to the extent of 0.15,tmoles per hr per 108 cells, and the rate of propionate utilization was 0.17,moles per hr per 108 cells. Intermediary metabolism in leukocytes has been the object of intensive study for the past 15 years. The presence of the Embden-Meyerhof pathway and the pentose cycle in various types of leukocytes from different species have been demonstrated by radio tracer technique and enzyme studies. A recent review by Cline eloquently summarizes several aspects of metabolism in white blood cells (5). In the present study, the metabolism by human plasma cells has been investigated in vitro with "4C-glucose, "4C-acetate, and '4C-propionate. The utilization of these substrates were determined, the extent of the pentose cycle was calculated, and the presence of a Krebs cycle was demonstrated in these cells. MATERIALS AND METHODS Leukocytes were obtained from a patient who was suffering from multiple myeloma and who had a peripheral white blood cell count of 40,000 per mm' containing more than 80% plasma cells. Dextran was used for acceleration of red cell sedimentation and heparin was added as an anticoagulant. The leukocyte-rich plasma was centrifuged for 5 min at 120 X g. The supernatant fluid which contained platelets and remaining red cells was decanted, and the sedimented leukocytes were washed by suspension in 0.9% NaCl and then centrifuged at 60 X g for 5 min. Cell counts were obtained with an AO Spencer Hemocytometer, and differential counts were made of the final preparation on stained smears. All preparations were made in siliconized glassware and at room temperature. Radioactive materials were obtained from the New 1657 England Nuclear Corp., Boston, Mass. They were checked by paper chromatography for contamination and were chemically degraded to establish radiochemical purity. All experiments were carried out in siliconized 50-ml Erlenmeyer flasks provided with a center well. The main compartment contained the white cell preparation suspended in autologous serum and Krebs- Ringer bicarbonate buffer. The vessels were closed with serum caps and flushed with a gas mixture of 95% 02 and 5% CO2. The reaction was conducted at 37 C in a shaker with a stroke volume of 5 cm (80 strokes per min). After incubation, 2 ml of 2 N NaOH was injected in the center well and 1 ml of 6 N H2SO4 into the main compartment to terminate the reaction. The liberated CO2 was allowed to diffuse into the NaOH for 12 hr at room temperature. The incubation mixture was then certrifuged for 10 min at 10,000 X g. The sediment was washed three times by suspension in 0.1 N H2SO4 followed by centrifugation. The combined supernatant solutions were extracted with ether continuously for 48 hr to obtain the extractable organic acids. After neutralization, the ether was evaporated and the salts chromatographed on a Dowex-1 formate column (4). Individual organic acids were further purified by chromatography on an acid Celite column (15). Amino acids and neutral compounds (carbohydrates) were obtained from the residue of the ether extraction. They were separated by passing the solution through Dowex-50 (H+ form) which removed cations and amino acids. The latter were recovered by elution with 1.5 N ammonia. Evaporation of the ammonia followed by chromatography on Whatman 3 MM paper strips in a phenol solvent saturated with water gave several bands of amino acids. Radioactivity in these bands was moni-

1658 STJERNHOLM J. BACTE'RIOL. tored with a paper strip counter. The identity of the amino acids was determined by spraying ninhydrin onto duplicate strips with known amino acids.

2 1658 STJERNHOLM J. BACTE'RIOL. tored with a paper strip counter. The identity of the amino acids was determined by spraying ninhydrin onto duplicate strips with known amino acids. Each radioactive amino acid was eluted from the paper and recrystallized to constant specific activity after addition of carrier. The water eluate from the Dowex-50 H+ column contained neutral compounds and anions. The latter were removed by passing the solution through a column of Duolite A-4 OH-. The carbohydrates in the neutral fraction were absorbed on activated charcoal and then recovered by elution with dilute ethyl alcohol (16). Carbohydrates were chromatographed in butanol-pyridine-water (10:3:3) on Whatman 3 MM paper strips. Lipids were extracted from the original sediment according to Folch et al. (7). The residue from the lipid extraction was considered as proteins and glycogen. The contents of the lipid and protein fractions were not investigated further. Acetate was degraded by the Schmidt reaction (12), lactate by the method of Katz et al. (9), malate as outlined by Wood et al. (18), methylmalonate as described by Stjernholm et al. (14), and fumarate was reduced to succinate and degraded as described by Wood et al. (18). Glutamate was degraded as described by Mosbach et al. (11), and aspartate was converted to malate and then degraded as above. Radioactivity was determined with a Tri-Carb liquid scintillation spectrometer (Packard Instrument Co., Inc., Downers Grove, Ill.) or a gas-phase counter. Equivalence between the two counters was obtained by employing conversion factors. RESULTS The incorporation of radioactivity into the products from "4C-glucose is shown in Table 1. The glucose utilized was calculated from these values and found to be 0.47,moles, 0.45,umoles, and 0.51,umoles per hr per 108 cells with glucose- 1-14C, glucose-6-"4c and glucose-2-'4c, respectively. The ether-extractable acids were composed almost entirely of lactate and acetate. The distribution of "4C in these acids is shown in Table 2. When glucose-1-'4c and glucose-6-'4c were the labeled substrates, the methyl carbon (C-3) of lactate contained 95 and 99% of the '4C, respectively, which is as expected when the glucose is metabolized via the Embden-Meyerhof pathway. With glucose-2-"4c, there was some "4C in both C-3 and C-1 of the lactate, and the 14C distribution pattern in the lactate indicates that the plasma cell metabolized glucose to a small extent via the pentose cycle. Several methods are available for a quantitative determination of the proportion of glucose metabolized via the pentose cycle. These methods have been discussed extensively by Wood, Katz, and Landau (17). One model is based on the yields of radioactive CO2 obtained from incubation with glucose-1-"4c and glucose-6-"4c. TABLE 1. Incorporation of 14C into products isolated 4C-glucose Metabolite Expt la Expt 2' Glucose- Glucose- Glucose- 1-14C 6-14C 2-14C (dpm) (dpm) (dpm) Respiratory CO2 32,600 3,400 9,000 Ether-extractable 90, , ,000 organic acids Lipids 5,000 6,000 10,000 Amino acids 29,000 24,000 40,000 Residue 10,000 10,000 20,000 a In experiment 1, each flask contained 32,umoles of glucose (1,uc) and 2.55 X 108 cells suspended in 7 ml of autologous serum. The differential count gave 90% plasma cells, 2% large lymphocytes, and 8% segmented cells. In experiment 2, the flask contained 23 MAmoles of glucose (2 uc) and 500 X 108 cells suspended in 5 ml of autologous serum and 10 ml of Krebs-Ringer-bicarbonate buffer. The differential count gave 94% plasma cells, 3% monocytes, and 3% segmented cells. Incubation time was for 2 hr at 37 C. The patient had been treated with nitrogen mustard derivatives intermittently, but was without treatment for several months at the time of the experiments. The time lapse between experiment 1 and 2 was 11 months. TABLE 2. Distribution of 14C in lactate and acetate produced by plasma cells incubated with labeled glucosea Lactate Acetate Labeled substrate (%) So) C-i C-2 C-3 C-1 C-2 Glucose-1-_4C Glucose-6-4C Glucose-2-4C a The recovery of 14C in the degradations ranged from 92 to 98%. When the pentose cycle was calculated according to equation 4 in the study by Wood et al. (17) from the data recorded in our Table 1, a value of 6% was obtained. A second method (17) utilizes the randomization of 14C into C-1 and C-3 of fructose-6-phosphate when the substrate is glucose-2-_4c. It can be assumed that the labeling of lactate and acetate reflect the labeling pattern in the hexose monophosphates. With the data in our Table 2 and by use of equation 7 from the study by Wood et al. (17), it was found that 1.8 and 3.5% of glucose-2-'4c were utilized via the pentose cycle. The variations observed

$It is quite obvious, however, that the metabolism of glucose by the plasma cell via the pentose cycle was very minor. The neutral fraction from incubation with 14Cglucose was radioactive.$

3 VOL. 93, 1967 CARBOHYDRATE METABOLISM IN LEUKOCYTES 1659 with the two methods are probably within experimental error. It is quite obvious, however, that the metabolism of glucose by the plasma cell via the pentose cycle was very minor. The neutral fraction from incubation with 14Cglucose was radioactive. Paper chromatography of it gave only one radioactive band, which corresponded to glucose. Elution and crystallization of this glucose followed by degradation with Leuconostac mesenteroides (2) showed that no randomization had occurred in the residual sugar. The incorporation of acetate-1-14c into products of plasma cells is shown in Table 3. Respiratory CO2 and amino acids acquired the highest radioactivity. A small amount of ]4C was recovered in the ether-extractable organic acids other than acetic acid, proteins, and lipids. From the values recorded in Table 3, it can be calculated that acetate was utilized by human plasma cells to the extent of 0.15,umoles per hr per 108 cells. It is possible to determine that there is a complete Krebs cycle by isolating intermediates of the cycle and determining the labeling patterns after incubation with various 14C substrates known to be metabolized by the cycle. Acetate-l- 14C is converted to citric acid by the condensing enzyme from acetyl-coenzyme A and oxalacetate. After further metabolism via aconitate and isocitrate, the resulting a-ketoglutarate should contain more 14C in C-5 than in C-1. If glutamate is in equilibrium with a-ketoglutarate, this relationship should be reflected in the carboxyl groups of this amino acid. a-ketoglutarate is converted to succinate and fumarate, which are symmetrical molecules, and then to TABLE 3. Incorporation of 14C into products isolated acetate-_l 4Ca Metabolite AMt Of 14C Percentage (dpm) of total 14C recovered Respiratory CO2 4,800, Ether-extractable or- 608, ganic acidsb Amino acids 2,300, Lipids 200, Proteins 576, a The flask contained 23 j,moles of glucose, 2,umoles of acetate-1_14c (5 jac), and 5.0 X 108 cells suspended in 5 ml of donor serum and 10 ml of Krebs-Ringer-bicarbonate buffer. Incubation was for 2 hr at 37 C. The differential count was identical with that listed in Table 1 under experiment 2. b The ether-extractable organic acids have been corrected for residual acetate. malate and oxalacetate. The carboxyl groups of these acids therefore should be equally labeled, and aspartate should reflect this distribution. Lactate likewise reflects the labeling pattern in pyruvate. Six intermediates isolated after incubation with acetate-1-'4c were purified and degraded (Table 4). The degradation patterns support the concept that the plasma cell posseses a complete Krebs cycle. Additional evidence for the presence of a Krebs cycle was obtained by incubation of plasma cells with propionate-3-14c. Propionate enters the Krebs cycle via methylmalonyl-coenzyme A (6) and isomerization to succinyl coenzyme A which is further metabolized in the cycle (1). The incorporation of propionate-3-14c into products of plasma cells is shown in Table 5. Ether-extractable acids contained 44% of the incorporated radioactivity. The respiratory CO2 and amino acids fractions containing 14C in glutamate and TABLE 4. Distribution of 14C in ether-extractable organic acids and amino acids isolated from plasma cells incubated with acetate-i-14c Metabe Total 14C Distribution of 14C (%) Metabolite Toaldpm C-5 C4 C-3 C-2 C-1 Lactate , Aspartate.. 1,000, Malate... 80, Fumarate... 32, Succinate , Glutamate... 1,300, Citrate... 46X000 -a -a -a -a -a a Not degraded. TABLE 5. Incorporation of 14C into products isolated propionate-3-l4ca Metabolite Amt of Percentage of Metabolite ~ 14C (dpm) total 14C recovered Respiratory CO2 1,000, Ether extractable 1,700, organic acidsb Amino acids 1 000, Lipids 9, Proteins 160, a The flask contained 23,moles of glucose, 10,umoles of propionate-3-14c (10 jac), and 5.0 X 108 cells suspended in 5 ml of donor serum and 10 ml of Krebs-Ringer-bicarbonate buffer. Incubation was for 2 hr at 37 C. b The ether-extractable acids have been corrected for residual propionate.

4 1660 STJERNHOLM J. BACTERIOL. TABLE 6. Distribution of 14C in ether-extractable organic acids and amino acids isolated from, plasma cells incubated with propionate-3-"4c Metabolitea Total "C (dpm) Distribution of 14C (%l) ---- C-5 CA4 C-3 C-2 C-1 Lactate... 1,100, Aspartate 400, Malate. 180, Fumarate... 65, Succinate... 85, Glutamate , Methylmalonate.. 6, a Citrate (160,000 dpm) was isolated but not degraded. An unidentified compound (80,000 dpm) was found in the malate fraction from the Dowex-1 formate column. The two metabolites were separated on the Celite column. Attempts to identify the unknown acid failed. aspartate were equally labeled and accounted for more than 50% of the labeled metabolites. A small amount of 14C was found in the proteins and an insignificant amount of 14C was recovered in the lipids. From the values in Table 5, it was calculated that propionate was metabolized by the plasma cells to the extent of 0.17,umoles per hr per 108 cells. Seven intermediates were isolated, purified, and degraded (Table 6). As expected, methylmalonate reflects the propionate and was labeled exclusively in C-3. The almost equal labeling of C-2 and C-3 of malate, aspartate, and lactate also reflects the pathway of propionate via succinate. The occurrence of 4C in the carboxyl groups of the dicarboxylic acids indicates that the metabolites were recycled in the Krebs cycle. The absence of significant radioactivity in C-4 and C-5 of the glutamate shows that very little, if any, acetate was derived from the labeled propionate. DISCUSSION The opportunity to study intermediary metabolism in plasma cells is indeed rare, and this investigation may be the first attempt. A few plasma cells commonly appear in the peripheral blood of patients with multiple myeloma. The plasma cells obtained in this study were isolated from a patient who exhibited an overwhelmingly predominant plasmacytosis and who also showed hyperglobulinemia and Benco-Jones proteinuria. The glycolytic rates of plasma cells and lymphocytes are almost equal, amounting to 0.5,umoles per hr per 108 cells. Thus, Hedeskov and Esmann (8) reported a similar value for peripheral blood lymphocytes. The glucose utilization is low in comparison to that of neutrophils, the difference being almost 10-fold (13). The presence of neutrophils in the plasma cell preparations (Table 1; 8 and 3% in experiments 1 and 2, respectively) will undoubtedly influence the glucose utilization. The true values are probably lower than reported here. The presence of a pentose cycle is indicated from the difference in 14C content of the respiratory CO2 observed with glucose-1-14c and glucose-6-"4c. This is further supported by the 14C distribution pattern observed in the lactate when glucose-2-"4c was the substrate. Calculations by two different methods show, however, that the pentose cycle activity in the plasma cell is very small. One of the most neglected areas in considering intermediary metabolism of the leukocytes is the role of the Krebs cycle, which is associated with the mitochondria. Histochemically, the cytoplasm of the plasma cell shows numerous mitochondria, which may vary in shape and size, but are usually smaller than those of lymphocytes. A number of enzyme activities have been measured in several types of leukocytes (3), but no attempts have been made to show that the intact cycle is operating. It is seldom possible to isolate all of the intermediates of the Krebs cycle, because many of them are present only in catalytic amounts. In the experiments with acetate-1-"4c and propionate-3-14c, six and seven intermediates, respectively, were isolated and degraded. The 14C distribution pattern in these metabolites give unequivocal proof of a complete Krebs cycle. Metabolism of acetate and propionate via the Krebs cycle expressed in terms of micromoles per hour per 108 cells indicates that Krebs cycle activity is substantial when compared to the glycolytic pathway. The energy requirement for the plasma cell seems to be derived almost equally from glycolysis and the Krebs cycle coupled with a cytochrome system. It is noted from the values in Table 1 that 0.5 Amoles of glucose are utilized by 108 plasma cells per hr. Substrate phosphorylation during glycolysis would provide 1,umole of adenosine triphosphate per hr per 108 cells. The respiratory CO2 observed with acetate-1-14c probably reflects a minimal rate of the Krebs cycle, but would yield about 0.6 moles of adenosine triphosphate per hr per 101 cells via oxidative phosphorylation. Recent studies suggest that the plasma cells are responsible for the synthesis of immunoglobulins (10). Plasma cells contain a Golgi apparatus and an abundance of endoplasmic reticulum, both of which are required for protein synthesis. Unless there is a change in glycolysis

VOL. 93, 1967 CARBOHYDRATE METABOLISM IN LEUKOCYTES 1661 or Krebs cycle activity during antibody formation, it seems reasonable to assume that high energy phosphates required for protein synthesis

5 VOL. 93, 1967 CARBOHYDRATE METABOLISM IN LEUKOCYTES 1661 or Krebs cycle activity during antibody formation, it seems reasonable to assume that high energy phosphates required for protein synthesis may be derived from either or both pathways. ACKNOWLEDGMENTS This work was supported in part by Public Health Service grants AM-06366, 5-K3-AM , and GM from the National Institutes of Health. I wish to thank Sam Zito for expert assistance, Harland G. Wood and Lars G. Ljungdahl for valuable discussions, and Robert W. Kellermeyer who selected the patient for this stldy. LITERATURE CITED 1. BECK, W. S., AND S. OCHOA Metabolism of propionic acid in animal tissues. IV. Further studies on the enzymatic isomerization of methylmalonyl coenzyme A. J. Biol. Chem. 232: BERNSTEIN, I. A., AND H. G. WOOD Determination of isotopic carbon patterns in carbohydrate by bacterial fermentation, p In S. P. Colowick and N. 0. Kaplan [ed.j, Methods in enzymology, vol. 4. Academic Press, Inc., New York. 3. BLUM, K. U Enzympathologie der Blutzellen. Blut 8: BUSCH, H., R. B. HURLBURT, AND V. R. POTTER Anion exchange chromatography of acids of the citric acid cycle. J. Biol. Chem. 196: CLINE, M. J Metabolism of the circulating leukocyte. Physiol. Rev. 45: FLAVIN, M., P. J. ORTIZ, AND S. OCHOA Metabolism of propionic acid in animal tissues. Nature 176: FOLCH, J., M. LEES, AND G. H. S. STANLEY A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: HEDESKOV, C. J., AND V. ESMANN Respiration and glycolysis of normal human lymphocytes. Blood 28: KATZ, J., S. ABRAHAM, AND I. L. CHAIKOFF Analytical procedures using a combined combustion-diffusion vessel. Anal. Chem. 27: MELLORS, P. C., AND L. KORNGOLD The cellular origin of human immunoglobulins. J. Exptl. Med. 118: MOSBACH, E. H., E. F. PHARES, AND S. F. CAR- SON Degradation of isotopically labeled citric, a-ketoglutaric and glutamic acids. Arch. Biochem. 33: PHARES, E. F Degradation of labeled propionic and acetic acids. Arch. Biochem. 33: SBARRA, A. T., AND M. L. KARNOVSKY The biochemical basis of phagocytosis. I. Metabolic changes during the ingestion of particles by polymorphonuclear leukocytes. J. Biol. Chem. 234: STJERNHOLM, R. L., R. E. NOBLE, AND D. KOCH- WESER Formation of methylmalonyl- CoA and succinyl-coa by extracts of Mycobacterium smegmatis. Biochim. Biophys. Acta 64: Swni, H. E., AmD L. 0. KRAMPITZ Acetic acid oxidation by Escherichia coli: evidence for the occurrence of a tricarboxylic acid cycle. J. Bacteriol. 67: WHISTLER, R. L., ANm D. F. DURSO Chromatographic separation of sugars on charcoal. J. Am. Chem. Soc. 72: WOOD, H. G., J. KATZ, AND B. R. LANDAU Estimation of pathways of carbohydrate metabolism. Biochem. Z. 338: WOOD, H. G., R. L. STJERNHOLM, AND F. W. LEAVER The role of succinate as a precursor of propionate in the propionic acid fermentation. J. Bacteriol. 72:

Activation of Fatty Acid Synthesis in Cell-free

JOURNAL OF BACTERIOLOGY, Jan. 1968, p. 157-161 Copyright ( 1968 American Society for Microbiology Vol. 95, No. 1 Printed in U.S.A. Activation of Fatty Acid Synthesis in Cell-free Extracts of Saccharomyces