レクチン処理による骨性アルカリフォスファターゼ(ALP Ⅲ)の分離定量法

Abstract

For decades, ALP had been interpreted as a marker enzyme of metabolic bone diseases and an isoenzyme specific for the osteoblast been widely known as ALP Ⅲ, too. Moreover, current tremendous advances in dialysis technology project an impact for the clinical significance of ALP Ⅲ, because of the advent of a new characteristic clinical entity formed renal osteodystrophy (ROD) which had been one of the inevitable complications in long term hemodialysis patients. So far, however, many conventional methods failed to separate clearly ALP Ⅲ from the liver specific ALP (ALP Ⅱ) and present determination of ALP Ⅲ by no means satisfied the clinical demand due to the lack of a direct quantitative method. On the other hand, lectin method, reported originally in 1984 by Rosalki, proved it to be possible to measure ALP Ⅲ directly as well as quantatively. So, the author performed this study to confirm more precisely several conditions of procedure of the lectin method, using established osteoblastic cell line ROS 17/2 as a marker, as well as the availability of its clinical application, and obtained the following results ; 1) The optimum concentration of Wheat-Germ lectin binded fully to ALP Ⅲ was 139 μmol/ml in distilled water. 2) The concentration of Triton X-100 preventing biliary ALP from ALP-lectin complex was 20%. 3) ALP Ⅲ activity incubated with lectin overnight at 4℃ after incubation for 30 min. at 37℃ was higher (mean±S.D, 24.0±15.2%) than that incubated with lectin for 30 min. at 37℃. 4) ALP Ⅲ was clearly separated from the liver fraction using affinity electrophoresis on polyacrylamide gel disc and isoelectric focusing on agarose gel. 5) ALP Ⅲ activity in precipitate showed good correlation (r=0.998) with the differential between total ALP activity and ALP Ⅱ activity in supernate.