Leukocyte Energy Metabolism III. Anaerobic and Aerobic ATP Production and Related Enzymes

Abstract

We have measured anaerobic and aerobic energy metabolism in leukocytes from human and animal blood, using several methods. We have taken special care to correct our results for an unavoidable contamination with thrombocytes, which has been neglected in all previous work. White cells were isolated by a method combining sedimentation through Dextran with low speed centrifugations. A pellet of thrombocytes was prepared in parallel from the same blood. Mitochondria were isolated by differential centrifugation in 0.25 M sucrose. Firstly, we have specially studied two cytosolic glycolytic enzymes, phosphoglycerate kinase and pyruvate kinase, both of which catalyze a reaction producing ATP. These two enzymes are very active in leukocytes. Other nucleotides, such as CDP, GDP and UDP can also be phosphorylated. Secondly, we have measured the activity of several mitochondrial enzymes, the most active being α-glycerophosphate oxidase. Using an enzymatic test, we have measured the amount of ATP produced in oxidative phosphorylation; in these experiments it was necessary to consider the activity of adenylate kinase as well as correcting for thrombocyte contamination. Several metabolites in direct or indirect relation with the Krebs cycle were used as oxidizable substrates: glutamate, succinate and α-glycerophosphate gave the best results. Except for amytal, inhibitors of oxidative phosphorylation also inhibit ATP production in leukocyte mitochondria. In conclusion, it can be said that oxidative phosphorylation exists in leukocytes, but that the phosphorylation steps in glycolysis produce 1,000 to 10,000 times more ATP than aerobic energy metabolism.

Pyruvate-Dependent Changes in Neutrophil Amino- and Alpha-Keto Acid Profiles or Immunity: Which Mechanisms Are Involved?

“…And in fact, the energy output is high, because the transition of one molecule of pyruvate via the TCA cycle, together with oxidative phosphorylation, produces 15 molecules of ATP as well as NADPH, which can for example be further metabolized by membranous NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) as well [9] [10] [42]- [44]. Under physiological conditions, the complex is inactive and only activated during respiratory burst.…”

Impaired Phagocytosis in Leukocytes from Newborn Infants

“…The leukocytes are isolated by a previously described technique [4]. 15 ml of blood were obtained from the umbilical cord of newborn infants delivered at term, with no signs of fetal distress, neonatal asphyxia or perinatal infections.…”

“…PGK and PK are measured in leukocyte cytosol as previously described [4]. For the measurement of glycolysis the samples containing intact leukocytes in KRBAGA medium [2] are gassed for 10 min with Na and incubated for 30 min at 37°C under constant shaking.…”

“…After deproteinization with 0.6 mol/1 perchloric acid, the amounts of glucose [10] and lactate [9] are measured in the supernatant. All results are corrected for an unavoidable thrombocyte contamination [4].…”

An Approach to Direct Metabolic Studies in Man

“…Thus, the biochemical studies of leuco cytes often allow recognition of lesions affecting other tissues. It is also possible to isolate subcellular fractions of white cells, such as granules [2,4] or mitochondria [14,18], which can provide much information about normal or pathological intracellular processes. For example, the isolation of leuco cyte mitochondria in hyperthyroidism permitted the demonstration of a significant decrease of aerobic ATP production in these mitochondria, compared with mitochondria from normal subjects [13].…”