Alkaline phosphatase activity of glutaraldehyde-treated bovine pericardium used in bioprosthetic cardiac valves

Abstract

Bioprosthetic valves fail frequently because of pathological mineralization, a process that begins in cell remnants of the glutaraldehyde (GLUT) fixed tissue. Other pathological cardiovascular calcification and physiological mineralization in skeletal/dental tissues are both largely initiated in cell-derived membranous structures (often called 'matrix vesicles'), and the enzyme alkaline phosphatase (AP) likely has an important function in the pathogenesis of mineral nucleation. This study tested the hypothesis that AP might also be present in and contribute to calcification of bioprosthetic valves. AP activity of fresh and GLUT-treated bovine pericardium was measured by the conversion of p-nitrophenyl phosphate to p-nitrophenol. Following 24 hours in 0.6% HEPES-buffered GLUT and storage for 2 weeks in 0.2% GLUT, considerable AP hydrolytic activity remained in GLUT-treated tissue relative to that of fresh tissue (V(max), 24 vs. 45 μmol reaction product/min/mg tissue protein, respectively), although binding was somewhat reduced (K(m), 1.9 x 103 vs. 1.4 x 103 μM substrate, respectively). Enzyme reaction product was demonstrated in both fixed and fresh tissue by light microscopic histochemical studies, confirming the biochemical results. Reaction product was noted along membranes of vascular endothelial cells and intestinal fibroblasts, the sites of early calcific deposits in bioprosthetic valves, by ultrastructural examination of GLUT-treated tissue. We conclude that GLUT-treated bovine pericardium retains much of the hydrolytic activity of AP, an enzyme associated with normal skeletal and pathological cardiovascular and noncardiovascular mineralization, and suggest that further examination of the mechanistic role of this enzyme may stimulate new approaches for slowing or preventing calcification of bioprosthetic tissue.