Enzyme reference materials availability

In 1960, at the general assembly of the International Pharmaceutical Federation (FIP), the obsolescence of various national pharmacopeial methods for assaying pharmaceutical enzymes was demonstrated. An international commission on pharmaceutical enzymes was created to deal with this unsatisfactory situation and develop improved assay methods and guidelines for the preparation of pharmaceutical enzyme reference materials. The Center for Standards has a coordination function in organizing collaborative enzyme assays between academic, industrial, and national pharmaceutical control laboratories and in distributing FIP pharmaceutical enzyme standards. Since 1960, many FIP assay methods and standard preparations have been adopted by national and international pharmacopeias, such as the European Pharmacopoeia. The ultimate goal is to provide official, preferentially nonempirical, standardized assay methods by which comparison of commercially available pharmaceutical enzymes is made possible. The most desirable situation would be an international uniformity of enzyme standards and assay methods, which would allow physicians and clinicians to unambiguously compare the potencies of commercially available enzyme products. 

The requirements for, preparation of, and application of enzyme reference materials have been discussed extensively (134, 135, 151, 152, 153, 154, 155, 156, 157, 158). Cooperation among clinical enzvmologists in Europe and the United States over the last several years has resulted in the availability of only a few enzyme reference materials. SRM 8430 from NIST is a preparation of human erythrocyte aspartate aminotransferase in a human albumin matrix (144) certified reference material (CRM) 319 from the Community Bureau of Reference (BCR) of the Commission of the European Communities is a preparation of porcine y-glutamvltransferase in a bovine serum matrix (159). The working group of the BCR is in the process of establishing protocols and evaluating... 

Though various methods for the determination of enzyme activity are used routinely in clinical laboratories, many of them are covered by patents, and the components of their test kits are not commercially available. In clinical chemistry, reference methods and certified enzyme reference materials (enzyme calibrators) for determination of activities of enzymes in human blood serum are recommended by international commissions (ISOTC/212, International Federation of Clinical Chemistry IFCC, Institute for Reference Materials and Measurements EU, etc.) at present for improved accuracy and establishment of traceability chains of the enzyme measurement system. By using these reference materials, the inaccuracy (imprecision between laboratories) has been minimized to within several per cent in several enzymes, such as lactate dehydrogenase (LDH) (EC 1.1.1.27), y-glutamyltrans-peptidase (y-GT) (EC 2.3.2.2), alanine aminotransferase (ALT) (EC 2.6.1.2), creatine kinase (CK) (EC 2.7.3.2), alkaline phosphatase (ALP) (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2), and a-amylase (AMY)... 

The first four materials (IRMM/IFCC-452, 453, 454, 455) are expected to be released during 2000. Projects on the certification of reference materials for cardiac marker (myoglobin) and total protein concentration in serum are under discussion. Even so the number of available CRMs for clinical chemistry and occupational toxicology is still limited. This has to do with the complexity of physiological compounds (e.g. proteins), the instabihty (e.g. enzymes), or the volatility (e.g. solvents). 

In order to measure the exact amount of a specific protein (analyte) by IHC signal intensity, a critical requirement is the availability of a standard reference material (present in a known amount by weight) that can be used to calibrate the assay (IHC stain). It is then possible to determine the amount of test analyte (protein) by a translation process from the intensity of IHC signals. In this respect it is helpful to consider the IHC stain as a tissue based ELISA assay (Enzyme Linked ImmunoSorbent Assay), noting that ELISA is used in the clinical laboratory as a standard quantitative method for measuring protein by weight in fluids, by reference to a calibrating reference standard. 

It is relevant to ask how often the routine measurement procedures currently used in laboratory medicine provide results that are traceable to high-level calibrators and reference measurement procedures (Lequin personal communication). It turns out that primary reference measurement procedures and primary calibrators are only available for about 30 types of quantity such as blood plasma concentration of bilirubins, cholesterols and sodium ion. International reference measurement procedures from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and corresponding certified reference material from BCR are available for the catalytic activity concentration of a few enzymes such as alkaline phosphatase and creatine kinase in plasma. For another 25 types of quantity, such... 

CK-MB can be measured in numerous ways. Immunoassays developed in recent years have improved on the analytical and clinical sensitivity and specificity of the earlier immunoinhibition and immunoprecipitation assays. These assays now (1) measure CK-MB directly and provide mass measurements, (2) are easily automated, and (3) provide rapid results (<30 minutes). Mass assays reliably measure low CK-MB concentrations in both samples with low total enzyme activity (<100 U/L) and with high total enzyme activity (>10,000 U/L). Furthermore, no interferences from other proteins have been documented. The majority of commercially available immunoassays that use monoclonal anti-CK-MB antibodies are the same as those listed in Table 5-2 for cardiac troponin assays. Excellent concordance has been shown between mass concentration and activity assays. A primary reference material is commercially available to assist in harmonization. If used for assay standardization, then this material allows... 

Two kits are available from Englyst Carbohydrate to help ensure that accurate analytical results are obtained. The kit for the colorimetric procedure contains color reagent and a second kit contains a solution of allose as internal standard for the GC procedure. Both kits contain the required enzymes, sugar solutions, and reference materials and are rigorously tested in-house. 

The role of reversed micelles in the manufacture of fine chemicals with enzymes also needs to be assessed and analysed. An outstanding example is lipase catalysed interesterification to produce cocoa butter substitute from readily available cheap materials (Luisi, 1985). This example of reversed micelles is sometimes referred to as a colloidal solution of water in organic systems. A number of water insoluble alkaloids, prostanoids, and steroids have been subjected to useful transformations (Martinek et al., 1987). Peptide synthesis has also been conducted. The advantages of two liquid phases are retained to a very great extent the amount of water can be manipulated to gain advantages from an equilibrium viewpoint. 

Many target organic molecules are too bulky to enter or leave zeolites, l- or this category the ordered mesoporous materials, like MCM-41, MCM-48 and SBA-15, became available. The latter material can even accommodate enzymes. Quite recently the use of these ordered mesoporous materials in catalysis has been reviewed [14]. The review covers no less than 447 references. Another recent review [15] deals with alkylation, hydrogenation and oxidation by mesoporous materials. 

Linearity of a method should be established or a series of standards selected for use with non-linear-method calibration. This can be checked by preparing and analyzing serial dilutions of aqueous reference standard solutions, quality control materials, enzyme solutions, or commercially available materials for demonstrating linearity (again, these are designed for use in human medicine) and comparing the determined values with the theoretical values calculated for the dilutions. The serial dilutions used for linearity checks can also help establish the analytical sensitivity when defined as the minimal detectable change from one concentration to another. 

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