Shigeru Ueda

A reference material for traceability of aspartate aminotransferase (AST) results

Abstract Standardization of aspartate aminotransferase (AST) determination is highly desirable for inter-laboratory comparison. Serum AST mean values for 20 patients suffering from viral hepatitis showed an inter-laboratory (n=13) variation of 9.4%. Part of this variation was due to two laboratories using procedures without pyridoxal-5′-phosphate. A traceable AST value was assigned to an enzyme calibrator (EC) through the appropriate International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) primary reference procedure. The EC was commutable for seven routine methods. Common calibration using the EC reduced the inter-laboratory coefficient of variation (CV=5.9% ) and allowed retention of a common reference interval for a set of routine procedures. Calibration made superfluous the expression of results in multiples of the upper reference limit, which increased inter-laboratory variation (CV=18.5%). Furthermore, for 92% of patients, calibration with the EC allowed the correction of misclassifications when taking into account the reference interval of the reference procedure. Use of this EC could be proposed to complete the AST reference system.

1,2-Dioleoylglycerol method for pancreatic lipase catalytic activity in serum

Abstract Background: There is a need for a pancreatic lipase (LIP) reference assay to provide an accurate base to which routine methods can be traceable. Methods: This study developed a novel LIP assay method in which 1,2-dioleoylglycerol (DODG) is the substrate and LIP activity is measured in a coupled enzymatic reaction from the increase in absorbance at 340 nm with production of NADPH. Results: With this method, LIP activity was linear up to 440 U/L (8-times expected upper limit of physiological concentration). When assayed manually, the between-laboratory variation for six samples surveyed at five laboratories was 3.80–26.4% (CV) for samples containing about 20–290 U/L LIP activity; when assayed using an automated analyzer, the range was 1.86–4.86% (four laboratories). Interference by >5 mmol/L glycerol and low specificity with post-heparin samples were noted, but in practice these are avoidable. Precision analyzed by automated assay of 49 samples twice in random order produced a covariance of 2.27 U/L, which is comparable to routine methods, and good correlations were obtained with five routine methods. Conclusions: Although further studies are required, the DODG method may be likely applicable as one candidate reference method.

Novel enzymatic method for assaying Lp-PLA2 in serum

BACKGROUND Measurement of lipoprotein-associated phospholipase A2 (Lp-PLA2) can be used as an adjunct to traditional cardiovascular risk factors for identifying individuals at higher risk of cardiovascular events. This can be performed by quantification of the protein concentration using an ELISA platform or by measuring Lp-PLA2 activity using platelet-activating factor (PAF) analog as substrate. Here, an enzymatic Lp-PLA2 activity assay method using 1-O-Hexadecyl-2-acetyl-rac-glycero-3-phosphocholine (rac C16 PAF) was developed. METHODS The newly revealed substrate specificity of lysoplasmalogen-specific phospholipase D (lysophospholipase D (LysoPLD)) was exploited. Lp-PLA2 hydrolyzes 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16 PAF) to 1-O-Hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPAF). LysoPLD acted on LysoPAF, and the hydrolytically released choline was detected by choline oxidase. RESULTS Regression analysis of Lp-PLA2 activity measured by the enzymatic Lp-PLA2 activity assay vs. two chemical Lp-PLA2 activity assays, i.e. LpPLA2 FS and PLAC® test, and ELISA, gave the following correlation coefficients: 0.990, 0.893 and 0.785, respectively (n = 30). CONCLUSION Advantages of this enzymatic Lp-PLA2 activity assay compared with chemical Lp-PLA2 methods include the following; (i) only requires two reagents enabling a simple two-point linear calibration method with one calibrator (ii) no need for inhibitors of esterase-like activity in serum.

Enzymatic assay method for measuring mizoribine levels in serum.

A sensitive and specific method for assaying serum mizoribine levels that can be applied to general automatic clinical analyzers was developed. Regression analysis of the enzymatic assay (y) vs. the HPLC method (x) produced the following relation: y=0.964x+0.090 (n=262, Sy, x=6.37 ng/mL).

Development of an enzyme cycling method by a purine nucleoside phosphorylase for assaying inorganic phosphate

We have developed a novel enzymatic cycling method that uses purine nucleoside phosphorylase (PNP) (EC 2.4.2.1) from Bacillus sp. to measure inorganic phosphate. The method utilizes the reversibility of the PNP reaction, in which the forward and reverse reactions are catalyzed in the presence of an excess amount of inosine and guanine, respectively, a principle similar to that previously demonstrated with creatine kinase (CK). Real-time detection was accomplished by coupling the reaction with commercially available xanthine dehydrogenase (EC 1.17.1.4) in the presence of NAD+. The efficiency of the cycling reaction per unit of enzyme (U ml−1) was remarkably higher than the CK.

Improvement and evaluation of a 1,2-dioleoylglycerol method for measuring pancreatic lipase catalytic activity in serum

*Corresponding author: Shigeru Ueda, Asahi Kasei Pharma Corporation, 632-1, Mifuku, Izunokuni-shi, Shizuoka, Japan, Phone: +81 558 768593, Fax: +81 558 767149, E-mail: ueda.sc@om.asahi-kasei.co.jp Yoshiaki Iizuka: Tobu College of Medical Technology, Saitama, Japan Keita Yamashita: Tsukuba Medical Center Hospital, Ibaraki, Japan Shin-Ichi Sakasegawa: Asahi Kasei Pharma Corporation, Shizuoka, Japan Toshiro Hanada: Wako Pure Chemical Industries, Ltd., Osaka, Japan Wataru Tani: Reference Material Institute for Clinical Chemistry Standards, Kanagawa, Japan Hiroshi Adachi: Alfresa Pharma Corporation, Osaka, Japan Riichi Haga: Mitsui Memorial Hospital, Tokyo, Japan Mari Yamaguchi: Roche Diagnostics K.K., Tokyo, Japan Wataru Kurotani: Kainos Laboratories, Inc., Tokyo, Japan Mitsuo Sekiguchi and Susumu Osawa: Chiba Institute of Science, Chiba, Japan Shigemi Hosogaya: Tokyo University of Technology, Tokyo, Japan Dongchon Kang: Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan