Search

Background: Many different methods have been used to assay amylase activity, using nitrophenylated oligosaccharides as substrate; however, the hydrolysis steps in these methods are complex.Methods: We developed a new continuously monitoring assay for amylase activity in biological fluids, using 2-chloro-4-nitrophenyl-4-O-β-d-galactopyranosylmaltoside (GalG2CNP) as the substrate; this assay was used with anti-human salivary amylase monoclonal antibodies for specific determination of the pancreatic isoenzyme. Amylase converted GalG2CNP into β-d-galactopyranosylmaltose and 2-chloro-4-nitrophenol, which was measured at 405 nm.Results: GalG2CNP was cleaved between 2-chloro-4-nitrophenol and β-d-galactopyranosylmaltose and did not undergo transfer reactions. The within-assay CVs (n = 20) for total amylase (T-AMY) and pancreatic amylase (P-AMY) were 0.6–1.6% and 0.5–2.5%, respectively; and day-to-day CVs (n = 10) for T-AMY and P-AMY were 0.8–3.7% and 0.6–4.1%, respectively. T-AMY and P-AMY activities in serum or urine obtained by the proposed method correlated well with those determined by the 2-chloro-4-nitrophenyl 4-O-β-d-galactopyranosyl-β-maltotetraoside method or the modified IFCC method.Conclusions: This novel assay for T-AMY and P-AMY measures both activities stoichiometrically, directly, and easily, and may be suitable for routine procedures.

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} \[\mathrm{Choline\ {+}\ diacetylphosphatidic\ acid}\] \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} \[\mathrm{2\ H}\_{\mathrm{2}}\mathrm{O}\_{\mathrm{2}}\mathrm{\ 4-aminoantipyrine\ {+}\ EMSE}\ \begin{array}{l}\mathrm{Peroxidase}\\{\rightarrow}\end{array}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{\ Quinone\ dye\ (purple)}\] \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 …

We describe a new kinetic assay for determining urea in serum or urine with use of urease (EC 3.5.1.5) and leucine dehydrogenase (EC 1.4.1.9). The latter enzyme is suitable for the kinetic assay of NH4+ because itsKmvalue for NH4+ at pH 8.75 is large (∼500 mmol/L). Interference from endogenous NH4+ in serum or urine is obviated by subtraction of the assayed endogenous NH4+value in a sample blank. For serum, within-assay CVs (n = 10) were 0.39–0.58%; day-to-day CVs (n = 10) were 1.56–2.30%. In urine, within-assay CVs (n = 10) were 0.86–1.15%. Analytical recovery of urea (0.893–71.4 mmol/L) added to patients’ sera (urea 6.14 mmol/L) was 99.2–105.2%. The calibration curve for serum was linear through zero for urea concentrations up to 142.9 mmol/L and for urine up to 714.3 mmol/L. No influences of added ammonium ion, bilirubin, hemoglobin, ascorbic acid, or Intralipid were observed. The regression equations for this method (y) and conventional methods (x = Determiner-LUN for serum assays, Serotec UUR-R for urine) were: y = 1.016x − 0.12 mmol/L (r = 0.999, Sy|x = 0.34 mmol/L, n = 100) for sera, and y = 1.070x − 12.6 mmol/L (r = 0.998, Sy|x = 7.41 mmol/L, n = 100) for urine.