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

The importance of separation and identification of serum alkaline phosphata se (ALP; E.C. 3.1.3.1) fractions/isoenzymes has been frequently reported. E ach serum ALP fraction/isoenzyme quantitation has both practical and theore tical importance. In the present work, serum was collected from Wistar rats and, in identical experimental conditions, total serum ALP activity and se rum ALP electrophoretic fractions/isoenzymes activities were quantified. Di fferent results for both kinds of ALP activity were obtained when different buffers or mixture of these buffers (carbonate/bicarbonate; 2-amino-2-meth yl-1-propanol/HCl; Veronal, sodium diethylbarbiturate/HCl), pH conditions ( 9.4 and 10.4) and substrates (alpha- and beta-naphthyl phosphates) were use d. Higher total serum ALP activity was always observed with beta-naphthyl p hosphate, 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-na phthyl 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 ac companied 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, diff erent values for its activity can be obtained by changing the assay conditi ons used for ALP visualization (revelation, staining); (ii) the same assay conditions for quantitation of total serum ALP and serum ALP electrophoreti c fractions/isoenzymes should be used; (iii) the choice of assay conditions should take into account the biochemical problem being studied in each cas e.