Importance of assay conditions in visualization and quantitation of serum alkaline phosphatase isoenzymes separated by electrophoresis

• Published in: Scandinavian journal of clinical and laboratory investigation Vol. 59; no. 8; pp. 593 - 605
• Main Authors: Hipólito-Reis, C, Dias, P O, Martins, M J
• Format: Journal Article
• Language: English
• Published: Oslo Informa UK Ltd 1999; Taylor & Francis; Scandinavian University Press
• Subjects: Alkaline Phosphatase - analysis; Alkaline Phosphatase - blood; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Buffers; Electrophoresis, Cellulose Acetate; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Hydrolases; Isoenzymes - analysis; Isoenzymes - blood; Kinetics; Male; Naphthalenes - metabolism; Organophosphates - metabolism; Organophosphorus Compounds - metabolism; Rats; Rats, Wistar; Substrate Specificity; Alkaline phosphatase; Biochemical analysis; Rat; Enzyme; Isozyme; Rodentia; Phosphoric monoester hydrolases; Esterases; Buffer solution; Operating conditions; Substrate; Vertebrata; Mammalia; Enzymatic activity; Animal; Clinical biology; Analytical method; Reagents; pH; Hydrolases; Electrophoresis; Qualitative analysis; Comparative study; Quantitative analysis
• ISSN: 0036-5513; 1502-7686
• DOI: 10.1080/00365519950185085

The importance of separation and identification of serum alkaline phosphatase (ALP; E.C. 3.1.3.1) fractions/isoenzymes has been frequently reported. Each serum ALP fraction/isoenzyme quantitation has both practical and theoretical importance. In the present work, serum was collected from Wistar rats and, in identical experimental conditions, total serum ALP activity and serum ALP electrophoretic fractions/isoenzymes activities were quantified. Different results for both kinds of ALP activity were obtained when different buffers or mixture of these buffers (carbonate/bicarbonate; 2-amino-2-methyl-1-propanol/HCl; Veronal, sodium diethylbarbiturate/HCl), pH conditions (9.4 and 10.4) and substrates (alpha- and beta-naphthyl phosphates) were used. Higher total serum ALP activity was always observed with beta-naphthyl phosphate, independently of the buffer (or mixture of buffers) and pH used. Electrophoresis allowed the separation of two serum ALP fractions. Activity of both serum ALP electrophoretic fractions was always higher with beta-naphthyl phosphate, except with carbonate/bicarbonate pH 10.4. The effect of a change in pH was buffer- (or mixture of buffers) and substrate-dependent; the addition of a second buffer (to that previously used) was not always accompanied by an increase or decrease (of the same magnitude) in our results. The results obtained with different buffers (or mixture of buffers) were not identical with substrates and pH values. It is concluded that (i) from the same electrophoretic separation of serum ALP fractions/isoenzymes, different values for its activity can be obtained by changing the assay conditions used for ALP visualization (revelation, staining); (ii) the same assay conditions for quantitation of total serum ALP and serum ALP electrophoretic fractions/isoenzymes should be used; (iii) the choice of assay conditions should take into account the biochemical problem being studied in each case.