| Popis: |
We describe a simple method for separation and quantification of the bone and liver isoenzymes of alkaline phosphatase (AP). Bone isoenzyme is clearly separated from the liver one by specific binding to wheat-germ lectin and subsequently retarded during electrophoretic separation. We modified lectin-affinity electrophoresis on cellogel membranes described by Rosalki and Foo (1984). Barbital buffer pH=9.2 and electrophoresis temperature of 25 C are giving the best resolution. Visualisation using Fast Violet B salt is suitable for densitometric quantification. Using method of Hausamen et al. (1967) we determined total AP and quantified bone and liver AP by heat-inactivation, also. Precision in series (CV≤5%), reproducibility (CV≤5%) and correlation with heat-inactivation method (r=0.87, r=0.84 for bone and liver isoenzymes, respectively) were found for lectin-affinity electrophoresis. We established referent values in sera from healthy people: 87 adults (age 21-81: 42 men, 45 women) and 21 children (age 1-14). Total AP and bone and liver isoenzymes (mean±1.96SD U/l) for men (142.5±45.5, 69.6±37.8, 72.9±26.7, respectively), women (122.3±40.5, 66.2±33.5, 56.2±23.9, respectively) and children (467.0±172.7, 397.3±137.4, 70.0±58.8, respectively) were determined. We examined differences in total and isoenzymes activities related to age of subjects. Lectin-affinity electrophoresis is a rapid, precise, reproducible and suitable method for use in the diagnostic laboratory. |
