PARTIAL BREAKDOWN OF GLYCATED ALKALINE-PHOSPHATASES MEDIATED BY REACTIVE OXYGEN SPECIES

The lower levels of serum alkaline phosphatase (AP) activity found in patients with diabetes mellitus apparently originate from the selectiv e disappearance or decrease in bone AP activity in the circulation. He nce, we investigated in vitro the effect of glycation on the activitie s of five AP isozymes. Aseptic incubation with 25 mmol/L of D-glucose and APs rapidly reduced bone and placental AP activities before those of liver, kidney and intestinal enzymes. The resulting bone and placen tal AP molecules were clearly glycated, according to the result of ami nophenylboronic acid affinity chromatography. Furthermore, Western blo tting analysis revealed that the placental AP molecule was fragmented, and its partial cleavage took place at Ala(154) on the AP molecule. S ince glycation of serum proteins causes the generation of reactive oxy gen species, the effects of reactive oxygen species on placental AP ac tivity were assayed, and the results indicated that hydroxyl radicals might be a major factor for the specific inactivation of AP activities . The reduction in AP activity by incubation with glucose in vitro was reversed by the further addition of catalase. Furthermore, ferrous io n with hydrogen peroxide, which generates hydroxyl radicals, had an in hibitory effect on AP activities. These findings suggest that the redu ced AP activity in diabetic patients might result from partial cleavag e of the bone AP molecule by reactive oxygen species induced by glycat ion. (C) 1998 Elsevier Science B.V. All rights reserved.