γ-Glutamyltransferase reference material: An example of International cooperation

19

Clinica Chimica Acta, 173 (1988) 19-26 Elsevier

CCA 03965

y-Glutamyltransferase reference material: an example of international cooperation Gerard Siest, Fraqoise Schiele, Jocelyne Muller and Maria Wellman Centre du Medicament

et Laboratoire du Centre de Midecine

(Received

PrLuentive, UA-CNRS

9 May 1987; revision received and accepted

Key worak y-Glutamyltransferase;

Reference

material;

597, Nancy (France)

11 July 1987)

Standardization;

Enzyme

activity

The different steps in the preparation of a certified reference material for y-glutamyltransferase are described. A preparation of pig kidney y-glutamyltransferase, partly hydrolyzed with papain, was prepared and lyophilized in a matrix containing bovine serum albumin. The between-ampoule variability of y-glutamyltransferase was estimated to be 0.6%. The predicted degradation at - 20” C is 0.00% per year. The certification procedure involved 15 participating laboratories. The certified y-glutamyltransferase catalytic concentration of the reconstituted material, using the IFCC proposed method, is 86.8 U/l with a 0.95 confidence interval of +2.1 U/l.

Introduction

The problem of the method dependence and lack of comparability of enzyme activity measurements has been addressed by national and international societies for clinical chemistry. These bodies recommended reliable method assay incorporating current knowledge. The potential applications of purified reference materials in clinical enzymology are mainly: testing the transferability of a method (reference method) among laboratories; defining the relationships between the reference method and nationally recommended methods which are similar in principle but differ in specific conditions; verifying the activities of secondary enzyme preparations produced in large quantities for everyday quality control [l].

Correspondence to: Dr. F. Schiele, Organisation Regionale de Examens de Sante, Centre de Medecine Preventive, 2, avenue du Doyen Parisot, B.P. 7-54501 Vandoeuvre-les-Nancy Cedex, France.

0009-8981/88/$03.50

0 1988 Elsevier Science Publishers

B.V. (Biomedical

Division)

20

Suitable enzyme reference materials should have defined stability of catalytic activity, and an activity in any selected method which is assured within defined limits. Catalytic properties should be as close as possible to those of the human serum enzyme. Matrix effects should be known and controlled. Although purity criteria for enzyme materials cannot be laid down as for simpler substances, enzyme reference materials should be defined sufficiently to allow preparation of successive batches with consistent properties [l]. In 1980, the Community Bureau of Reference (BCR) of the European Communities (EC) established a working group of enzymologists to prepare and evaluate reference preparations of enzymes of diagnostic importance. Justification of the choice of y-glutamyltransferase y-Glutamyltransferase (GGT, EC 2.3.2.2) was chosen as the first enzyme to be studied, essentially because of its known stability and properties and because of its widespread use in clinical laboratories. In the foetus, liver GGT activity is high, but after birth it decreases rapidly to adult values. In adult mammals, GGT is mainly found in the kidney, pancreas, testis, mammary gland, intestine, brain and liver, where it is present as an amphiphilic membrane-bound enzyme [2]. Detergents may solubilize the enzyme by binding to the hydrophobic, membrane-binding domain of the enzyme, forming a micellar-like complex, the ‘heavy’ or amphiphilic form of enzyme. Treatment with proteases such as papain, bromelain or trypsin removes the hydrophobic domain, thus forming a soluble, hydrophilic, ‘light’ form of the enzyme. Both the heavy and the light forms are present in serum [3]. In clinical chemistry and biochemical pharmacology, GGT is a useful index for exclusion of hepatic disorders and drug effects (including alcohol). Most hepatobiliary disorders can lead to an increase in serum GGT activity; these include hepatobiliary cholestasis, cirrhosis, alcoholic steatosis, cardiac insufficiencies with hepatic involvement or chronic hepatitis. Measurement of GGT is of particular value in the detection of hepatic metastases of various carcinomas [4-61. Serum GGT activity is also increased after administration of such inducers as phenobarbital, alcohol or a great number of xenobiotics [7-91. In summary, GGT measurement is used in primary preventive medicine, in the detoxication of alcoholic subjects, in the investigation of enzyme induction and, in the detection and monitoring of hepatobiliary disease (particularly cholestasis). Consequently, GGT measurement in many clinical laboratories is the second most frequent test, after alanine and aspartate aminotransferases (ALT, AST). Different steps in the preparation of a certified reference material The working procedure followed by the BCR for the preparation reference materials involves three steps: preliminary studies; feasibility preparation of a reference material for certification.

of enzyme studies and

21

Preliminary studies Pig kidney GGT (light form), solubilized by papain, was chosen as a source of enzyme. This choice was based on both our studies [IO-121, and data available from the literature [13-151. A matrix containing bovine serum albumin (60 g/l) and 0.05 mol/l of pH 7.8 Tris-IX1 buffer was shown to be convenient for lyophilization, without effect on the kinetic properties of the enzyme 1121. Fe~i~i~it~ studies This important step involves the preparation of a pilot batch of the reference material, the characterization of the preparation (purity, kinetic properties, conta~ating enzymes), the short- and long-term stability studies and the interlaboratory trials. The pilot batch was prepared as elaborated during the preliminary studies, with some minor modifications. The preparation of partially purified pig kidney GGT (light form) had a specific activity of 112 U/mg protein. Activities of AST, ALT and lactate dehydrogenase (LD) were not detectable. Activities of leucine arylamidase and alkaline phosphatase represented 0.35% and 0.47% of the GGT activity, respectively. The preparation was lyop~~ed in the matrix, defined in preliminary studies. After reconstitution of the lyophilized samples the kinetic properties of the GGT were very close to those of the human serum enzyme. The predicted degradation at - 20°C was O.OlC/yr with an approximate 95% limit of < O.l%/yr (380 days of storage). This pilot batch was used for interlaboratory studies. To determine a value for the enzyme catalytic concentration in the reconstituted material, a collaborative study was undertaken involving 17 laboratories from 10 countries. The method proposed by the Inte~ational Federation of Clinical Chemistry (IFCC) was used 1161. Two successive inter-comparisons were made [17]. A first trial showed that working conditions need to be controlled very carefully, and led to the elaboration of a very detailed protocol for five steps of GGT activity determinations: (1) check of the inst~mentation (pH meters, temperature of measurement, pipetting); (2) reconstitution of the lyophilized reference material by weighing; (3) dete~nation of the molar lineic absorbance of 5-~no-2”~trobenzoate (product of the reaction [16]); (4) measurements of the catalytic concentration of GGT (IFCC method) [16]; and (5) calculation and expression of the results. The need for such a strict protocol can be noticed from the comparison of the results of the first and second interlaboratory trial (Table I). The reference preparation (pilot batch) was finally submitted to interlaboratory trials in order to establish its property of ‘co~utabi~ty’. This property, as defined by Fasce et al [18] is the ability of an enzyme material to show interassay variations comparable to those of the human serum enzyme. In this purpose, the relative activities by several methods, eg IFCC method and method recommended by the Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology 1191,were simultaneously determined for the reference preparation and a large number of patient sera. A high degree of commutability of the material was shown between the IFCC method on the one hand and other methods, in widespread routine use in laboratories, on the other hand.

22 TABLE

I

Comparison

of the results obtained

No. Labs Grand mean (U/l) SD of the mean (U/l) Within-lab SD (U/l) Between-lab SD (U/l)

between

the first and the second

round-robin

(feasibility

First round-robin

Second round-robin

10 219.4 14.5 4.5 14.4

10 229.0 10.3 4.0 10.1

Considering these results, a final batch of GGT on a large scale for certification.

reference

material

study)

was prepared

Preparation of the certified enzyme reference material * This part of the work consists of the preparation of the reference material (purification and characterization of the enzyme, dilution in the matrix, lyophilization and ampouling); the homogeneity and stability studies and the certification procedure. Preparation of the reference material Pig kidney GGT was purified on average 400-fold. The final preparation had a specific activity of 120 U/mg of protein and was tested for contaminating enzymes, for action of inhibitors and for purity by electrophoresis in acrylamide gels. Thereafter the purified enzyme was diluted in the matrix (bovine serum albumin (BSA) 60 g/l, Tris, HCI buffer 0.05 mol/l, pH 7.8) to yield a catalytic concentration of 100 f 20 U/l. After filtration through a sterile 0.45 pm filter, the diluted preparation was lyophilized in ampoules by the National Institute for Biological Standards and Controls (NIBSC, London) [20]. 4000 ampoules (1 ml) of lyophilized material were finally obtained. The different steps of the lyophilization procedure were rigorously controlled: (1) mass of the liquid preparation dispensed into the ampoules; (2) residual moisture content of the lyophilized material (0.035%) and (3) oxygen content in the gas of the ampoules (0.204%) after lyophihzation [21]. The catalytic properties of the lyophilized material were re-examined after reconstitution with 1.00 ml distilled water. The major characteristics tested were pH optimum and affinities for the donor and acceptor substrates. They were compared with those of human GGT in two pools of sera from healthy and pathological subjects (Table II). Homogeneity study The GGT activity in the reconstituted material from twenty ampoules, taken regularly in the filling sequence was measured in triplicate. The

* Since this manuscript F, Muller J, Cohnct y-glutamyltransferase campaign. Clin Chem

was prepared. two papers closely concerning this subject were published: Schiele E, Siest G. Production and certification of an enzyme reference material for (CRM 319). Part 1: Preparation and characterisation. Part 2: Certification 1987;33:1971-1977, 1978-1982.

23

TABLE Catalytic

II characteristics

Optimal pH 30 o C K, GluCNA a (mmol/l) K,

GlrgtV b (mmoV1)

a GluCNA: b Glygly:

of the lyophihzed

GGT

reference

material

Lyophihaed GGT reference material

Human

Low level

pools High level

8.3 + 0.2 1.12 31.0

7.9+0.2 0.87 17.7

7.9 f 0.2 0.8 17.2

y-L-~ut~yl-3-~boxy-4-~tro~~de Glycyl~ycine.

results were submitted to a two way analysis of variance. A between ampoule variation of about 0.6% was found [21]. Stability study An accelerated degradation study was performed as described by Tydemand and Kirkwood [22]. Ampoules were stored at - 20°C, +4’C, 20°C 37’C, 45OC and 56°C. GGT activities were measured after storage for 30, 184, 234, 307 and 643 days. After storage of 643 days, the predicted annual loss of activity was 0.00% at - 20 o C and 0.12% at + 4” C 1211. However, the results showed statistically highly significant (p < 0.001) deviations from the assumed Arrhenius model. Certification procedure All parameters that had in the feasibility studies been demonstrated to influence the results of GGT measurements were rigorously

_)

3

85 .

Certified

LABORATORIES

Fig. 1. GGT catalytic concentration in reconstituted 0.95 confidence intervals (0) and for the certified interval).

material (U/l). GGT catalytic

value

Bar graphs for laboratory means and concentration (0) (0.95 confidence

24

controlled. This included the instrumentation, the quality of reagents, the procedure for reconstitution of the lyophilized preparation and the analytical procedure. Participants were provided with ampoules of the candidate GGT material and with all reagents, including 5-amino-2-nitrobenzoic acid for the determination of molar lineic absorbance. They were asked to do duplicate measurements of GGT activity in each reconstituted ampoule. Measurements were to be carried out on different days, a different ampoule being freshly reconstituted and analyzed every 3 days. Fifteen laboratories participated in this trial: two from Germany, France, the United Kingdom and Belgium and one from Italy, Denmark, The Netherlands, Switzerland, the United States and Japan. The results obtained from all of participating laboratories are summarized in Fig. 1 (means and 0.95 confidence intervals). The certified GGT catalytic concentration of the reconstituted material, calculated from the accepted set of results of nine laboratories, was 86.8 U/l with a 0.95 confidence interval of k 2.1 U/l [21,23].

Conclusion

The GGT reference material will become available to interested parties through the BCR as Reference Material CRM 319. The BCR working group is now establishing protocols for the preparation and evaluation of other reference materials. Owing to their clinical importance, preparations of creatine kinase, alkaline phosphatase, alanine aminotransferase, lactate dehydrogenase (isoenzyme l), prostatic acid phosphatase will be given priority. BCR has also started to genetic engineering for the preparation of reference materials (Table III). Some of these projects will be developed in cooperation with our American colleagues.

TABLE

III

Current

status in the preparation

of enzyme

reference

material

Preliminary study y GGT

Completed

1983

Creatine

kinase

Completed

1984

Alkaline

phosphatase

Completed

1985

Alanine aminotransferase Lactate dehydrogenase isoenzyme 1 Prostatic acid phosphatase Genetic engineering approach

In progress In progress

1986-1987 1986-1987

In progress In progress

1986-1987 1986-1987

(European

Community,

BCR)

Feasibility study

Preparation of reference material

Completed 1983-1984 In progress 1985-1987 In progress 1986-1987

Completed

1986

25

Acknowledgements

The coordination by the Community Bureau of Reference (Drs. H. Marchandise, E. Colinet) is Commission of the European Communities, Brussels, Belgium acknowledged. We would also like to acknowledge the Working Group on Enzyme and the following other participants: H. Brettschneider, D. Calam, F. Ceriotti, E. Colinet, P. Foucart, J. Frei, J.C.M. Hafkenscheid, M. Horder, A. Lauwers, M. Mathieu, D.W. Moss, J. Muller, J. Okuda, R. Rej, S.B. Rosa&, F. Schiele, E. Schmidt, L.M. Shaw, G. Siest, J.H. Stromme and I. Verhamme. References 1 Moss DW, Schiele F, Siest G. Colinet E. Reference materials for clinical enzymology: the work of the Community Bureau of Reference of the European Community. Clin Chem 1986;32:556-558. 2 Huseby NE. Separation and characterization of human gamma-glutamyltransferases. Clin Chim Acta 1981;111:39-45. 3 Huseby NE. Hydrophilic form of gamma-glutamyltransferase: proteolytic formation in liver homogenates and its estimation in serum. Clin Chim Acta 1982;124:113-121. 4 Shaw LM. Keeping pace with a popular enzyme: GGT. Diagnostic Med 1982;5:63-78. 5 Braun JP. Soula C, Rico AG, Benard P. Burgat Sacaze V. g~ma-Glutamyltransf~rase et cancer. Ann Biol Clin 1981;39:53-59. 6 Goldberg DM. Structural, functional and clinical aspects of g~a-~ut~yltransferase. Crit Rev Clin Lab Sci 1980;12:1-58. 7 Eckardt MJ, Ryback RS, Rawlings RR, Graubard BI. Biochemical diagnosis of alcoholism. A test of the discriminating capabilities of gamma-glutamyltranspeptidase and mean corpuscular volume. JAMA 1981;246:2707-2710. 8 Lamy J, Baglin MC, Ferrant JP. Weill J. Diminution de la gamma-glutamyltranspeptidase strique des ethyliques ii la suite du sevrage. Clin Chim Acta 1974;56:169-173. 9 Siest G, Ratanasavanh D, Bagrel D. Tazi A, Galteau MM. Place de la gamma-glutamyltransftrase parmi les proddts indirects de l’evaluation du fonctionnement des enzymes du mttabolisme du medicament. Ann Biol Clin 1980;39:67-73. 10 Petitclerc C, Schiele F, Bagrel D, Mahassen A, Siest G. Kinetic properties of gamma-glutamyltransferase from human liver. Clin Chem 1980;26:1688-1693. 11 Schieie F, Artur Y, Bagrel D, Petitclerc C, Siest G. M~surement of g~a-glutamyltransferase in clinical chemistry: kinetic basis and st~d~disation propositions. Clin Chim Acta 1981;112:187-195. 12 Schiele F, Marechal J, Galteau MM. Purified gala-~utamyltransferase as an enzyme reference material. In: Siest G, Heusghem C, eds. gala-Glutamyltransferases: Advances in biochemical pharmacology, 3rd series, Paris: Masson, 1982:139-145. 13 Shaw LM, Petersen-Archer LE, London, SW, March E. Electrophoretic, kinetic and immunoinhibition properties of gamma-glutamyltransferase from various tissues compared. Clin Chem 1980;26:1523-1527. 14 Tate SS, Ross ME. Human kidney gamma-glutamyltransferase. Catalytic properties, subunit structure and localization of the gamma-glutamyl binding site and the light subunit. J Biol Chem 1977; 252~6042-6045. 1.5 Hughey RP. Curthoys NP. Comparison of the size and physical properties of gamma-glutamyltranspeptidase purified from rat kidney following solubilization with papain or with Triton X100. J Biol Chem 1976:251:7863-7870. 16 Shaw LM, Strmnme JH. London JL, Theodorsen L. Intemation~ Federation of Clinical Chemistry: Expert Panel on IFCC Methods for the measurement of catalytic concentration of enzymes. Part 4. IFCC method for gala-glut~yltr~sferase. J Clin Chem Clin Biochem 1983;21:633-646.

26 17 Schiele F, Muller J, Moss DW. Siest Cl, Colinet E and the Working Group on Enzyme of the Community Bureau of Reference of the European Communities. Transferability study for the IFCC reference method for measurement of gamma-glutamyltransferase activity using a reference enzyme material (abstract). 5th International Congress of Clinical Enzymology, Jerusalem, Israel, September 1985. 18 Fame Jr CF. Rej R, Copeland WH, Vanderlinde RE. A discussion of enzyme reference materials: applications and specifications. Clin Chem 1973;19:5-9. 19 The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology. Recommended method for determination of gamma-glutamyltransferase in blood. Stand J Clin Lab Invest 1976;36:119-125. 20 Campbell PJ. International biological standards and reference preparations. J Biol Stand 1974:2:249-267. 21 Commission of the European Communities. Community Bureau of Reference. The certification of the catalytic concentration of gamma-glutamyltransferase in a reconstituted lyophilized material (CRM 319). Brussels: Community Bureau of Reference. Directorate General for Research, Science and Development. Commission of the European Communities, 1986, Report EUR 10628. 22 Rirkwood TBL, Tydeman MS. Design and analysis of accelerated degradation tests for the stability of biological standards. II. A flexible computer program for data analysis used for the production of biological standards and reference preparations. J Biol Stand 1984;12:207-214. 23 Marchandise H, Colinet E. Assessment of methods of assigning certified values to reference materials. Fresenius Z Anal Chem 1983;316:669-672.