Production and certification of an enzyme reference material for adenosine deaminase 1 (BCR 647)

• Published in: Clinica chimica acta Vol. 306; no. 1; pp. 79 - 89
• Main Authors: Bota, Alexandre, Gella, F.-Javier, Profilis, Christos, Férard, Georges, Hadjivassiliou, Anthony G, Hørder, Mogens, Schiele, Françoise, Segura, Rosa, Canalias, Francesca
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 01-04-2001; Elsevier
• Subjects: Adenosine deaminase; Adenosine Deaminase - metabolism; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Catalysis; Enzyme measurements; Enzyme Stability; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Humans; Hydrolases; Reference material; Reference Standards; Standardisation; Standardisation; Enzyme measurements; BCR, Community Bureau of Reference; DGKC, German Society for Clinical Chemistry; PAGE, polyacrylamide gel electrophoresis; Reference material; IFCC, International Federation of Clinical Chemistry and Laboratory Medicine; Adenosine deaminase; SDS, sodium dodecyl sulphate; ADA, adenosine deaminase; IRMM, Institute for Reference Materials and Measurements; EHNA, erythro-9-(2-hydroxy-3-nonyl)adenine; Human origin; Biochemical analysis; Enzyme; Standardization; Certification; EC 3.5.4.4; Enzymatic activity; Clinical biology; Production; Hydrolases; Quantitative analysis
• ISSN: 0009-8981; 1873-3492
• DOI: 10.1016/S0009-8981(01)00399-0

Background: We describe the preparation of a lyophilised reference material containing purified human adenosine deaminase 1 and the certification of its catalytic concentration.
Methods: The enzyme was purified from human erythrocytes.
Results: The enzyme was >99% pure on polyacrylamide gel electrophoresis. Only trace amounts (<0.4%) of alanine aminotransferase, aspartate aminotransferase and
l-lactate dehydrogenase were detected in the purified fraction. The purified adenosine deaminase had a molar mass of 41
600 g/mol and an isoelectric pH at 4.7, 4.85 and 5.0. The material was prepared by diluting the purified adenosine deaminase in a matrix containing 50 mmol/l Tris–HCl buffer pH 7.4 and 30 g/l human serum albumin; dispensing in vials and freeze-drying. The batch was homogeneous and the predicted loss of adenosine deaminase activity per year on the basis of accelerated degradation studies was 0.006% at −20°C and 0.04% at 4°C. The certified value for adenosine deaminase catalytic concentration in the reconstituted reference material is (2.55±0.09) μkat/l when measured by the method that uses adenosine as substrate and glutamate dehydrogenase as auxiliary enzyme at 37°C.
Conclusions: The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control, or for calibration of the adenosine deaminase catalytic concentration measurements.