New Enzymatic Assay Using Phospholipase D to Measure Total Calcium in Serum

Abstract

Various methods have been used to measure calcium in body fluids. Atomic absorption spectrophotometry (AAS) is the most reliable (1), but it requires special instrumentation. The most widely used method involves colorimetric detection of calcium complexes by o -cresolphthalein complexone (o-CPC) (2)(3) or arsenazo. With the o-CPC method, reagent stability and recoveries at low concentrations are poor, and magnesium interferes in the reaction (4). Newer methods using o-CPC (5)(6)(7) or other colorimetric agents (8)(9)(10) are not entirely satisfactory. Various enzymatic methods have been described, including methods using porcine pancreatic α-amylase (EC 3.2.1.1) (11), phospholipase D (PL-D; EC 3.1.4.4) (12)(13), and urea amidolyase (14). The first two are based on activation of enzymes by calcium, whereas the third is based on inhibition of the enzyme by calcium. The α-amylase method is reportedly inaccurate for patients with hyperamylasemia (11), and the other 2 methods each require 2 reaction steps (12). In this report, we describe a new, simple, specific enzymatic assay based on activation of PL-D. We investigated the assay characteristics and its suitability for use in routine laboratory tests. We obtained 126 serum samples from patients admitted to Shinshu University Hospital after receiving informed consent from the patients and approval by our institutional ethics committee. We obtained PL-D from Streptomyces chromofuscus [ (15); Asahi Kasei Pharma], bis( p -nitrophenyl) phosphate (BPNPP) from Kanto Chemical Co., Good’s buffer from Doujin Laboratories, and SRM 915 and 909a from NIST. The reagent sets for the o-CPC and α-amylase methods were from Serotec Co. Ltd. and Ono Pharmaceutical Co. Ltd., respectively. Other reagents were analytical grade (Wako Pure Chemical). The new method is based on increased PL-D-catalyzed hydrolysis of BPNPP by calcium ions, as follows. The p -nitrophenol released …