The measurement of enzyme activities in the resting human polymorphonuclear leukocyte--critical estimate of a method.

As a system for study, the isolated human polymorphonuclear leukocyte combines the advantages of a quasi-non-invasive preparation with a nearly complete complement of enzymes of carbohydrate and energy metabolism. However, small sample volumes and, in some cases, very low enzyme activities make high demands on sample processing, storage, and performance of continuous measurements, if the enzyme activities are to be measured with acceptable reproducibility. In the presented study several aspects of homogenization, storage, and continuous measurement were scrutinized, to identify critical steps and consider ways of optimizing the method. Polymorphonuclear leukocytes were separated from the blood of healthy subjects by sedimentation and density gradient centrifugation. After ultrasonic homogenization, 13 enzymes of glycolysis and gluconeogenesis, the tricarboxylic acid cycle, and glycogen metabolism were determined photometrically. The variation of several conditions showed: 1. The duration of exposure to ultrasound for the homogenization of polymorphonuclear leukocytes has no influence over a wide range of time. 2. Addition of the detergents Triton X-100 and deoxycholic acid, as well as the SH-group protector dithiothreitol, to the homogenizing medium increased the measured activities of only a few enzymes. 3. Considerable inaccuracy was encountered when the suspension was divided into parts for homogenization with different additives; such splitting of the suspension should therefore be performed only when necessary, as in the determination of reference values (e.g. protein or DNA content of the cell suspension). 4. Twenty four-fold determination of enzyme activities from one homogenate resulted in precisions between 4.5% (citrate synthase) and 14.4% (transketolase), which is satisfactory for the low activities (as low as 1 U/l) in the homogenate. 5. The reproducibility of enzyme activities, measured in homogenates of polymorphonuclear leukocytes from different blood samples drawn simultaneously, was only slightly worse than that of the continuous measurement method itself. Thus, the precision of the measurement of enzyme activity seems to be the main determinant of the overall method. In conclusion, the described procedure of separation, homogenization, and enzyme measurement in human polymorphonuclear leukocyte meets the requirements of biochemical or clinical trials and can be recommended for clinical metabolic studies.

[Maximal turnover rates of glycolysis enzymes and of the citrate cycle of separated granulocytes in the postoperative period]. / Maximale Umsatzraten an Enzymen der Glykolyse und des Zitratzyklus von separierten Granulozyten in der postoperativen Phase.

The human granulocyte is easy to obtain and shows a nearly complete enzymatic equipment. It therefore represents an interesting model for in-vitro studies of metabolic disorders under various clinical conditions. In the presented study, the activities of several enzymes of glycolysis and citric cycle are measured in granulocytes separated from surgical patients (n = 10). Blood samples of 20 to 40 ml were drawn 6.5 +/- 4.8 hours after termination of surgical procedure. All patients were artificially respirated and nourished intravenously according to the results of indirect calorimetry. Hexokinase (HK), pyruvate kinase (PK), lactate dehydrogenase (LDH), and isocitrate dehydrogenase (IDH) were measured photometrically in the cell homogenate. The values were compared to those determined in a group of healthy, not-anesthetized persons, nourished and studied identically (n = 12). In granulocytes separated from patients following major surgery we found increased activities of HK (29.8 vs. 24.1 mU/mg protein in controls), LDH (2,484 vs. 1,868 mU/mg protein, p less than 0.01) and IDH (41.5 vs. 35 mU/mg protein, p less than 0.05), and a reduced activity of PK (1,623 vs. 2,265 mU/mg protein, p less than 0.01). Assuming that the alterations in enzyme activities of isolated granulocytes reflect metabolic alterations of the whole organism to a certain extent, the results can be interpreted as a decreased induction of PK by insulin, an increase of lactate recycling via Cori cycle (LDH), and a stimulated substrate flux in citric cycle (IDH). The separated human granulocyte is recommended as a model of posttraumatic metabolic disorders. It should be taken into consideration for studies leading to further improvement of nutrition during posttraumatic glucose mal-utilization.