title: Quantitation of skeletal alkaline phosphatase isoenzyme activity in canine serum.
abstract: Pursuing the hypothesis that quantitation of skeletal alkaline phosphatase (ALP) activity in canine serum would provide an index of the rate of bone formation, we compared three methods for isoenzyme-specific identification of skeletal ALP activity in canine serum: heat inactivation, wheat germ agglutinin (WGA) precipitation, and concanavalin A (ConA) precipitation. ALP isoenzyme activities were extracted from canine bone, intestine, and liver, diluted into heat-inactivated canine serum (i.e., serum without ALP activity), and used as calibrators of ALP isoenzyme activities. Differential sensitivity to inhibition by 10 mM L-homoarginine was used to distinguish intestinal ALP activity from hepatic and skeletal ALP activities (i.e., 9, 80, and 72% inhibition, respectively). To allow resolution of skeletal ALP activity from hepatic ALP activity, we tested two established methods (heat inactivation and WGA precipitation) and a novel method, ConA precipitation. The organ-derived skeletal and hepatic ALP isoenzyme activities were used to compare these three methods with respect to linearity, isoenzyme separation, and precision. All three methods were linear, but the WGA and ConA methods afforded greater isoenzyme separation and precision. The relative extent of isoenzyme separation (i.e., the difference in percentage remaining skeletal and hepatic ALP isoenzyme activities) averaged 23, 40, and 47% remaining ALP activity for the heat, WGA, and ConA methods, respectively. However, when these methods were applied to the quantitation of skeletal ALP activity in sera from 10 young and 10 adult beagles, the WGA method was found to be unacceptable because most of the results fell outside the range of the WGA assay calibrators (i.e., greater than 100% skeletal ALP activity). The heat and ConA methods showed that the amount of skeletal ALP activity in the beagle sera decreased with age, both as ALP activity per liter and as percentage of total serum ALP activity (p less than 0.001 for each). Skeletal ALP activity levels determined by ConA were correlated with values determined by heat inactivation (r = 0.87, p less than 0.001) but not with WGA-determined levels (r = 0.26). Intestinal ALP activity was detected in only 1 of these 20 sera. We conclude that ConA precipitation can be used for quantitation of skeletal ALP activity in beagle serum.
authors.affiliation: Department of Medicine, Loma Linda University, California.
authors.forename: J R
authors.initials: JR
authors.lastname: Farley
authors.forename: S L
authors.initials: SL
authors.lastname: Hall
authors.forename: C
authors.initials: C
authors.lastname: Ritchie
authors.forename: S
authors.initials: S
authors.lastname: Herring
authors.forename: C
authors.initials: C
authors.lastname: Orcutt
authors.forename: B E
authors.initials: BE
authors.lastname: Miller
mesh_terms: D000469:Alkaline Phosphatase / Q000097:blood*; D000704:Analysis of Variance; D000818:Animals; D001842:Bone and Bones / Q000201:enzymology*; D011232:Chemical Precipitation; D003208:Concanavalin A; D004285:Dogs; D006358:Hot Temperature; D007527:Isoenzymes / Q000097:blood; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet; D014909:Wheat Germ Agglutinins
chemical_list: D007527:Isoenzymes; D014909:Wheat Germ Agglutinins; D003208:Concanavalin A; D000469:Alkaline Phosphatase