The Measurement of Enzyme Activities in the Resting Human Polymorphonuclear Leukocyte — Critical Estimate of a Method

Abstract

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.

Methods for samples preparation in proteomic research

“…For preparation of the sample, ultrasonic disintegrators are successfully used to homogenize cells, after cell culturing or after isolation from the organism. For example, after detailed studies on the usefulness of ultrasonic homogenization on the leukocytes, Fauth et al confirmed that this type of homogenization did not affect enzymatic activity of 13 investigated enzymes [88]. However, the procedure may lead to the disruption of non-covalently bound molecular clusters (like multienzyme complexes) [89].…”

Pyruvate: immunonutritional effects on neutrophil intracellular amino or alpha-keto acid profiles and reactive oxygen species production

“…via glycolysis in which one molecule of glucose breaks down into two molecules of pyruvate or reversible transamination reactions, etc. ), but also unites several key metabolic processes (Agam and Gutman 1972; Curi et al 1989, 1988; Fauth et al 1993 and 1990; Frei et al 1975; Fuchs et al 1994; Homem de Bittencourt et al 1993; Ing et al 1997; Newsholme et al 1987; Wu et al 2005 and 2003). …”

“…In eukaryotic cells, for example, pyruvate is converted into acetyl-CoA, releasing NADH and carbon dioxide in a process called pyruvate decarboxylation (Fauth et al 1993 and 1990; Fink 2008, 2007a, b, 2004; Willems et al 1978). This oxidative conversion into acetyl-CoA, which is the main input for a series of reactions named after Hans Adolf Krebs (awarded the 1953 Nobel Prize) is usually catalysed by the pyruvate dehydrogenase complex as part of aerobic respiration (Krebs 1940; 1938).…”

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.…”