Susumu Osawa

Enzymatic Characterization of an Amine Oxidase from Arthrobacter sp. Used to Measure Phosphatidylethanolamine

Ethanolamine oxidase was screened with the aim of using it to establish a novel enzymatic phosphatidylethanolamine assay. Ethanolamine oxidase activity was detected in the crude extract of Arthrobacter sp., and the enzyme was purified more than 15-fold in three steps with a 54% yield. SDS–PAGE revealed the presence of only one band, which migrated, with an apparent molecular mass of 70 kDa. Biochemical characterization of the enzyme showed phenylethylamine to be the preferred substrate, with the highest kcat/Km value. The primary structure, determined by sequencing the cloned gene, showed a high degree of identity to Cu-containing phenylethylamine oxidase (64%). When heterologously overexpressed in Escherichia coli, the enzyme exhibited only a trace of amine oxidase activity, but high levels of activity emerged after exposure to Cu2+, as is typical of recombinant copper amine oxidases. Preliminary application of this enzyme coupled with phospholipase D for determination of phosphatidylethanolamine is also described. This is the first enzymatic method for the measurement of phosphatidylethanolamine.

Nationwide multicenter study aimed at the establishment of common reference intervals for standardized clinical laboratory tests in Japan.

Abstract Background: The Japanese Association of Medical Technologists (JAMT) sought to establish common reference intervals (RIs) applicable nationwide in Japan for 27 serum constituent analytes for which certified reference materials are available and nine analytes frequently measured in routine tests. More than 100 laboratories certified for metrological traceability collaborated in the recruitment, sampling, and measurement of analytes for the establishment of RIs. No previous attempt has been made to establish RIs by such a large number of laboratories. The allowable limits of trueness and intermediate precision based on the JAMT criteria were applied to the reference values measured by these laboratories, and measured values within the allowance limits were used to establish RIs. Methods: Reference individuals included 5748 healthy volunteers aged 18–65 years who were engaged in medical care-related work based on the CLSI guidelines. After secondary exclusion of individuals in whom abnormal values were detected in basic routine test items and adjustment for the distribution of age and gender, 3371 reference individuals were chosen in the parametric determination of RIs. Employing the three-level nested ANOVA, between-laboratory, -region, -sex, and -age variations were evaluated. Results: No significant difference was noted in between-region variations in any item. Results of ANOVA revealed between-sex and -age variations in 14 and 15 analytes, respectively. Based on these results of variation, RIs were established with and without partition by sex. Conclusions: Since no between-region variation was detected in reference values among accuracy-certified core laboratories, RIs applicable nationwide were established.

Enzymatic assay of phosphatidylethanolamine in serum using amine oxidase from Arthrobacter sp.

BACKGROUND In human serum, as for phospholipids not containing choline, phosphatidylethanolamine (PE) exists approximately 5% in a whole phospholipid. PE is well known as one of the main components of biological membranes, and also plays important roles that contribute to apoptosis and cell signaling. However, it could not measure PE with other phospholipids due to a lack of choline in them. METHODS Using an amine oxidase (EC 1.4.3.6), from Arthrobacter species, a simple and rapid enzymatic assay for measurements of PE in serum was established. That assay used the Hitachi 7170 analyzer to evaluate the analytical performance. RESULTS The average within-run CVs were 0.38-1.27% (n=20) at 69-160 μmol/l. The correlation between values obtained with the present method (y) and the high-performance liquid chromatography (HPLC) method (x) was: y=0.944x+9.441 (r=0.977, S(y|x)=5.82, n=34). In addition, the reference interval of healthy subjects was 115±45 μmol/l. CONCLUSIONS This new enzymatic method shows a high specificity for serum PE and can be easily applied to an automated analyzer. The present method is available as a novel marker of changes in the clinical condition of serum phospholipids.

Development of a new measurement method for serum calcium with chlorophosphonazo-III

Background Although serum calcium has been measured using the o-cresolphthalein complexone (oCPC) method in the clinical laboratory, this method still has some problems regarding linearity and reagent stability. We developed a new measurement procedure using chlorophosphonazo-III (CPZ-III: 2,7-bis (4-chloro-2-phosphonophenylazo) -1,8- dihydroxy-3, 6-naphthalenedisulphonic acid, disodium salt) as a chelator with an acid medium for serum calcium measurement. The present method showed better linearity and reagent stability compared with the oCPC method. Methods Characteristics were studied in optimized conditions measuring wavelength by absorption spectra analysis, and interference of protein and metals with Mg2+, Fe2+, Cu2+ and Zn2+. The method was applied to an automated analyser (7170; Hitachi High Technologies Corp). The measurement performance was evaluated for accuracy, precision, recovery rate, linearity and reagent stability with a comparison study against atomic absorption spectrophotometry (AAS). Results The within-run and between-run variations (coefficient of variation [CV]) were 0.92–1.01% and 0.75–1.43%, respectively. The linearity was 0–7.0 mmol/L. The comparison study obtained y = 1.002x (AAS) – 0.10, Sy/x = 0.18 mmol/L, n = 50. Reagent stability was at least 20 d at 4°C without daily calibration. Conclusion The new calcium measurement method in serum was demonstrated to have reliable and acceptable performances as a routine test in clinical laboratory.

Multianalyte Conventional Reference Material (MacRM): A Useful Tool for Nationwide Standardization of Laboratory Measurements for Medical Care—A Model Study in Japan

BACKGROUND The Japanese Committee for Clinical Laboratory Standards (JCCLS) has developed a multianalyte conventional reference material (MacRM) for nationwide standardization of laboratory measurements. METHODS To prepare the MacRM, pooled sera were obtained from healthy Japanese individuals. Target values of the pooled sera for 30 analytes were assigned on the basis of the measurement results of 45 certified clinical laboratories whose calibration was verified by measuring certified reference materials (CRMs) provided by the National Institute of Standards and Technology, the Institute for Reference Materials and Measurements, and JCCLS. Commutability of MacRM was assessed by comparison with results for 150 individual inpatients at Fukuoka University Chikushi Hospital. Survey samples were prepared by essentially the same method for MacRM but without target values. The survey samples were used to assess agreement among 165 laboratories that used various assay kits and platforms calibrated with the MacRM. RESULTS The commutability of MacRM was confirmed for 30 analytes with sera from 150 individual patients. The imprecision (CV) of measurements of survey samples (high and low concentrations) among the 165 laboratories was 0.4%-10.0%. Twenty-six of 30 analytes were within the goals for interinstitutional allowable bias. An aliquot of MacRM stored frozen at -80 °C remained stable for ≥4 years. CONCLUSIONS The MacRM was successfully applied as a calibrator to achieve nationwide standardization for 30 analytes measured by 165 laboratories that used various methods from different manufacturers.

Development of a simple indocyanine green measurement method using an automated biochemical analyser

Background The indocyanine green retention rate is important for assessing the severity of liver disorders. In the conventional method, blood needs to be collected twice. In the present study, we developed an automated indocyanine green method that does not require blood sampling before intravenous indocyanine green injections and is applicable to an automated biochemical analyser. Methods The serum samples of 471 patients collected before and after intravenous indocyanine green injections and submitted to the clinical laboratory of our hospital were used as samples. The standard procedure established by the Japan Society of Hepatology was used as the standard method. In the automated indocyanine green method, serum collected after an intravenous indocyanine green injection was mixed with the saline reagent containing a surfactant, and the indocyanine green concentration was measured at a dominant wavelength of 805 nm and a complementary wavelength of 884 nm. Results The coefficient of variations of the within- and between-run reproducibilities of this method were 2% or lower, and dilution linearity passing the origin was noted up to 10 mg/L indocyanine green. The reagent was stable for four weeks or longer. Haemoglobin, bilirubin and chyle had no impact on the results obtained. The correlation coefficient between the standard method (x) and this method (y) was r=0.995; however, slight divergence was noted in turbid samples. Conclusion Divergence in turbid samples may have corresponded to false negativity with the standard procedure. Our method may be highly practical because blood sampling before indocyanine green loading is unnecessary and measurements are simple.

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