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.
Fauth
U
U
Klinik für Anästhesiologie der Johannes Gutenberg-Universität Mainz.
Schlechtriemen
T
T
Heinrichs
W
W
Puente-Gonzalez
I
I
Halmágyi
M
M
D002499:Centrifugation, Density Gradient; D002952:Citric Acid Cycle; D004798:Enzymes / Q000097:blood*; D006003:Glycogen / Q000097:blood; D006019:Glycolysis; D006600:Hexosephosphates / Q000097:blood; D006801:Humans; D009504:Neutrophils / Q000201:enzymology*; D010783:Photometry; D015203:Reproducibility of Results; D013194:Staining and Labeling
D004798:Enzymes; D006600:Hexosephosphates; D006003:Glycogen