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

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 41600 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 degrees C and 0.04% at 4 degrees C. The certified value for adenosine deaminase catalytic concentration in the reconstituted reference material is (2.55+/-0.09) microkat/l when measured by the method that uses adenosine as substrate and glutamate dehydrogenase as auxiliary enzyme at 37 degrees 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.

Production and certification of an enzyme reference material for creatine kinase isoenzyme 2 (CRM 608).

We describe the preparation of a lyophilized material containing purified human creatine kinase 2 (CK-MB), and the certification of its catalytic concentration. The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control, or for calibration of the creatine kinase 2 catalytic concentration measurements. The enzyme was purified from human heart by ethanol precipitation and chromatography successively on DEAE-Sephacel and Blue-Sepharose. The purified enzyme had a specific activity of 998.4 U/mg and was > 99% pure on polyacrylamide gel electrophoresis. The material was examined for several possible contaminating enzymes, which were found to be absent. The purified creatine kinase 2 had two subunits (B and M) with molecular masses of 43,650 and 41,700 g/mol, respectively, and an isoelectric point at pH 5.8. The material was prepared by diluting the purified creatine kinase 2 in a matrix containing 25 mmol/L PIPES buffer, pH 7.2, 2 mmol/L ADP, 5 mmol/L 2-mercaptoethanol, 154 mmol/L sodium chloride and 50 g/L human serum albumin, dispensing it into vials and freeze-drying. The batch was shown to be homogeneous. The loss of enzyme activity on storage at -20 degrees C is predicted to be less than 0.18% per annum on the basis of accelerated degradation studies. The catalytic concentration of creatine kinase in samples of the reconstituted material is certified to be 67.2+/-1.8 U/L (1.12+/-0.03 microkat/L) when measured, at 30 degrees C, by the Recommended Method of the International Federation of Clinical Chemistry.

Production and certification of an enzyme reference material for pancreatic alpha-amylase (CRM 476).

We describe the preparation of a lyophilized material containing purified human pancreatic alpha-amylase and the certification of its catalytic concentration. The enzyme was purified from human pancreas by ammonium sulphate precipitation and chromatography successively on DEAE-Sephacel, CM-Sepharose and Sephadex G-75. The purified enzyme had a specific activity of 52.9 kU/g protein and was > 99% pure on polyacrylamide gel electrophoresis. Only trace amounts of lipase and lactate dehydrogenase were detected in the purified fraction. The purified pancreatic alpha-amylase had a molar mass of 57,500 g/mol and an isoelectric point at 7.1. The material was prepared by diluting the purified alpha-amylase in a matrix containing PIPES buffer 25 mmol/l, pH 7.0, sodium chloride 50 mmol/l, calcium chloride 1.5 mmol/l, EDTA 0.5 mmol/l and human serum albumin 30 g/l, dispensing in ampoules and freeze-drying. The ampoules were homogeneous and the yearly loss of activity on the basis of accelerated degradation studies was less than 0.01% at -20 degrees C. The certified value for alpha-amylase catalytic concentration in the reconstituted reference material is 555 U/l +/- 11 U/l when measured by the specified method at 37 degrees C. The material can be used to verify the comparability of results from laboratories, for intra-laboratory quality control or for calibration of alpha-amylase catalytic concentration measurements.

Fluorometric study of serum in patients with chronic renal failure.

Sera from 84 uremic patients (53 on conservative treatment and 31 on hemodialysis) were studied spectrofluorometrically in comparison with 20 normal subjects and 11 jaundiced patients. Serum fluorescence in patients with chronic renal failure was significantly higher than in normals and patients with jaundice. Two types of serum fluorescence spectra (A and B) were predominant (93%) and all the patients who were free of any medication for more than two months exhibited the type A spectrum. The intensity of fluorescence was in parallel with the serum creatinine levels. Dialysis experiments of uremic serum in vitro showed that the substance(s) responsible for its fluorescence is dialyzable. Moreover hemodialysis caused a significant decrease in the intensity of serum fluorescence in dialyzed patients. Administration of pyridoxine intravenously caused quite different results in normal subjects and uremic patients. Namely normals presented a transient (for several minutes) increase in serum fluorescence whereas uremics showed a higher and more permanent increase which lasted for several days.