eNewsletter 2013 January - February
Activities of the IFCC Committee on Reference Systems of Enzymes (C-RSE)
Dr Ferruccio Ceriotti - C-RSE Chair
The most relevant "term of reference" of the C-RSE is the development of enzyme reference measurement procedures. In the past years, under the chairmanship first of Lothar Siekmann and then of Gerhard Schumann, the reference measurement procedures developed by IFCC in the eighties at 30 °C for AST, ALT, gamma-GT, CK, LDH, alfa-amylase and ALP were revised to adapt them at the measurement temperature of 37 °C. Apart from the change in measurement temperature, a series of other modifications were performed with the aim of providing reference methods directly applicable to routine analytical systems without the need of any modification. The main scope of this operation was to allow the definition of an uninterrupted traceability chain from the highest metrological level (for enzymes represented by the reference measurement procedure) to the results of the patients' samples. These reference measurement procedures constitute the basis, through the use of commutable calibration material, for the harmonization of the results in clinical enzymology. A second task of the committee is the collaboration with the Institute for Reference Materials and Measurements (IRMM) of the European Community for the assignment of target values to secondary reference materials (Certified Reference Materials - CRM-) through the coordination of a network of reference laboratories. Reference materials and reference laboratories represent the second and third constituent of the Reference Measurement System for the measurement of enzyme catalytic activities. Unfortunately not all the manufacturers still took advantage of this reference measurement system and the consequently the standardization of results in clinical enzymology is still highly insufficient.
Among the enzymes currently measured in clinical laboratories the only one not yet standardized is pancreatic lipase. Currently the activity of the Committee is mainly directed towards the definition of a reference measurement procedure for this enzyme. The situation of pancreatic lipase is somewhat different from the previous enzymes because no method was previously endorsed by IFCC as reference. Pancreatic lipase is a peculiar enzyme that exhibits its catalytic activity at the oil-water interface. The method previously proposed as reference (Tietz (1)) used a pH-stat procedure difficult to perform and a substrate which preparation is very difficult to standardize; so the Committee decided to work on photometric methods employing soluble substrates. Two method principles are currently under evaluation: one uses as substrate 1,2-dioleoylglycerol (DODG) a diglyceride from which, with the help of a monoglyceride lipase as auxiliary enzyme, pancreatic lipase liberates free glycerol that is quantified by a series of auxiliary enzymes through the measurement of NADPH absorbance. The second method uses as substrate 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin (DGGR). The pancreatic lipase cleaves the glutaric acid resorufin ester that spontaneously releases resorufin that is read at 572 nm. Several multicenter experiments were performed to test the two methods, but unfortunately both present some weakness. In particular the specificity for pancreatic lipase and the reproducibility in the substrate preparation (and consequently the transferability among reference laboratories) are the most challenging aspects. The members of the Committee are working to try to solve these problems that seem more demanding than the initial (optimistic) evaluation.
Committee members
- Robert Rej (USA)
- F. Javier Gella (Spain)
- Jaen-Mark Lessinger (France)
- Shigeru Ueda (Japan)
Publications
1. Tietz NW, Astles JR, Shuey DF. Lipase activity measured in serum by a continuous-monitoring pH-stat technique - an update. Clin Chem 1989;35:1688-93.