Enzymatic Assay of Calcium in Serum with Phospholipase D (1999)

Various methods for determining calcium in body fluids have been reported. Atomic absorption spectrophotometry (AAS) (1) is a most reliable method; however, because the AAS instrument is expensive and maintenance is difficult, it is unsuitable for routine assay. Spectrophotometric methods by use of o -cresolphthalein complexone (CPC) (2)(3)(4)(5)(6), which is based on chelation, is now widely available to assay calcium in clinical laboratories. Several enzymatic methods that use porcine pancreatic α-amylase (EC 3.2.1.1) (7) or phospholipase D (PLD; EC 3.1.4.4) (8) to measure calcium have also been developed. These methods are based on the activation of the enzymes by calcium. On the other hand, Kimura et al. (9) reported a kinetic assay for calcium in serum that uses urea amidolyase (EC 6.3.4.6) based on the inhibition of the enzyme by calcium. Previously reported method for determining free calcium ions in serum using PLD required appropriate ionic strength for its reaction (8). We have established a new and stable enzymatic method for determining calcium in serum that uses PLD, stabilizing calcium status by adding a large excess of bovine albumin to the reaction system.

The proposed enzymatic method for determining calcium in serum is based on the following sequence of reactions: \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{Diacetylphosphatidylcholine{+} H}_{\mathrm{2}}\mathrm{O}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[ \begin{array}{l}\mathrm{Phospholipase D}\\{\rightarrow}\\\mathrm{Calcium}\end{array}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{Choline {+} acid}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{Choline {+} 2 O}\_{\mathrm{2}}\mathrm{ {+} H}\_{\mathrm{2}}\mathrm{O } \begin{array}{l}\mathrm{Choline oxidase}\\{\rightarrow}\end{array}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{ Betaine {+} 2 H}\_{\mathrm{2}}\mathrm{O}\_{\mathrm{2}}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{2 H}\_{\mathrm{2}}\mathrm{O}\_{\mathrm{2}}\mathrm{ {+} EMSE} \begin{array}{l}\mathrm{Peroxidase}\\{\rightarrow}\end{array}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{ Quinone (purple)}\] \end{document}

Diacetylphosphatidylcholine (DAPC) is used for the PLD reaction substrate. The reaction rate at which PLD hydrolyzes DAPC into choline and diacetylphosphatidic acid depends on the amount of calcium in serum. Choline oxidase (COD) produces betaine and H2O2 from choline and O2. Peroxidase (POD) produces quinone from H2O2 with 4-aminoantipyrine (4-AA) and N -ethyl- N -(3-methylphenyl)- N ′-succinyl-ethylenediamine (EMSE). The absorbance at 546 nm for the quinone dye is measured.

This proposed method and the conventional methods were performed with the Hitachi Model 7170 automated …