Optimization of adenosine deaminase assay by response surface methodology

The effect of four variables (adenosine, glutamate dehydrogenase, phosphate buffer, and pH) on the measured catalytic concentration of adenosine deaminase (EC 3.5.4.4) was studied by Response Surface Methodology (RSM). This multivariate methodology offers an empirical approach to the study of enzyme assays and allows to detect the interaction between different variables of the system. Response–surface data showed maximum adenosine deaminase catalytic concentration at pH 7.2, adenosine 20 mmol/l, phosphate buffer 200 mmol/l and glutamate dehydrogenase 850 μkat/l when pleural fluids were used as samples. Optimum conditions for a material containing purified adenosine deaminase from human erythrocytes differed only slightly from that obtained for the pleural fluid.     

Interlaboratory study of the IFCC method for alanine aminotransferase performed with use of a partly purified reference material.

We present the results of a study on performance of a reference material for alanine aminotransferase (ALT, EC 2.6.1.2) and the corresponding IFCC-approved method in an interlaboratory trial involving 13 laboratories. The ALT material was partly purified from pig heart (specific activity, 150 kU/g) and was essentially free of six potentially contaminating enzyme activities, including aspartate aminotransferase (EC 2.6.1.1). The partly purified ALT was lyophilized in a triethanolamine-buffered matrix, pH 6.4, containing bovine serum albumin and saccharose. Under these conditions, the predicted yearly loss of activity was 0.02% at 4 degrees C and < 0.01% at -20 degrees C. The final blank-corrected results of the accepted set of data gave a mean (SD) of 128.5 (5.1) U/L. The among-laboratory SD was 4.6 U/L and the within-laboratory SD was 2.0 U/L. The certified ALT catalytic concentration in the reconstituted material was 129 U/L with a 0.95 confidence interval of +/- 4 U/L.     

A continuous method for the estimation of adenosine deaminase catalytic concentration in pleural effusions with a Hitachi 705 discrete analyser

An assay of adenosine deaminase activity in pleural effusions is described. For the continuous determination of adenosine deaminase, the liberated ammonia is estimated by coupling the liberated NH3 with 2-oxoglutarate. The reaction is followed by the decrease of NADH absorbance at 340 nm. The assay was optimized for a Hitachi 705 analyser, with respect to pH, adenosine concentration and glutamate dehydrogenase activity. The assay is linear to an adenosine deaminase catalytic concentration of 110 U/l. Elevated adenosine deaminase activities are found in pleural effusions of patients with tuberculosis, empyema and mesothelioma. Although elevated adenosine deaminase activity in pleural effusion is not pathognomonic for tuberculosis, it may be valuable as a first screening parameter.     

New IFCC reference procedures for the determination of catalytic activity concentrations of five enzymes in serum: preliminary upper reference limits obtained in hospitalized subjects

Consensus among clinical chemists has dictated a change in reference temperature for enzyme catalytic concentrations from 30 to 37 degrees C. Consequently, International Federation of Clinical Chemistry (IFCC) reference procedures have been redefined at the latter temperature. Acceptance in practice of these new procedures requires well-established reference values and clinical decision limits, but the establishment of reference values is complex. Therefore, as a provisional approach and to facilitate early application of the new IFCC procedures, we report our experience gained with them in the transfer of values from the consensus methods used hitherto in Germany to the new procedures. The preliminary upper reference limits were determined for catalytic activity concentrations of the enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), gamma-glutamyltransferase (gamma-GT) and lactate dehydrogenase (LDH) in human sera. Since enzyme measurements are almost always made on sera from non-ambulant subjects, we have used hospital patients aged 17 years and older as the subjects of our study. The catalytic activity concentrations obtained by measurements with the German consensus methods for the respective enzyme were chosen in combination with additional enzymes of similar diagnostic relevance to classify patients' samples as part of the respective reference collective. Measurements for the determination of the upper reference limits were performed manually by use of the primary reference procedures at the measurement temperature 37 degrees C according to IFCC, and also by employing mechanized measurements adapted to the reference procedures. The upper reference limits were calculated as the 97.5th percentile of the reference collectives and determined separately for women and men: ALT: 34 U/l (female) and 45 U/l (male); AST: 31 U/l (female) and 35 U/l (male); CK: 145 U/l (female) and 171 U/l (male); gamma-GT: 38 U/l (female) and 55 U/l (male); LDH: 247 U/l (female) and 248 U/l (male).     

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C.

This paper is the eighth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and the certification of reference preparations. Other parts deal with: Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes; Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase; Part 3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase; Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase Part 6. Reference procedure for the measurement of catalytic concentration of gamma-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of gamma-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 37 degrees C. The procedure described here is deduced from the previously described 30 degrees C IFCC reference method. Differences are tabulated and commented on.     

Certification of an enzyme reference material for alkaline phosphatase (CRM 371)

We have produced a batch of lyophilized alkaline phosphatase (AP) for use as an enzyme reference material. The enzyme was partly purified from pig kidney to a specific activity of 400 U/mg of protein and is essentially free from contaminating enzyme activities. The kinetic properties of the preparation are very close to those of the enzyme present in human serum. The partly purified AP was lyophilized in a matrix containing bovine serum albumin (40 g/L), MgCl2, ZnCl2 and NaCl. The vial-to-vial variability with respect to the catalytic concentration of the final product was 0.008. The predicted annual relative loss of activity was less than 0.01% at -20 degrees C and 0.04% at 4 degrees C. This material was certified using the IFCC proposed method. The certification procedure involved 19 laboratories throughout the world. The certified alkaline phosphatase catalytic concentration in the reconstituted material was 254 U/L with a 0.95 confidence interval of +/- 6 U/L.     

Production and certification of an enzyme reference material for gamma-glutamyltransferase (CRM 319). Part 1: Preparation and characterization

We have produced a batch of lyophilized gamma-glutamyltransferase as enzyme reference material. The "light" enzyme form was purified from pig kidney to a relatively high specific activity (120 kU/g) and was essentially free of contaminating enzymes. The partly purified gamma-glutamyltransferase, lyophilized in a matrix containing bovine serum albumin (Fraction V, 60 g/L), yielded a batch of 4000 ampules and was stored at -20 degrees C. The vial-to-vial variability with respect to the catalytic concentration of the final product (CV 0.6%) and its stability (predicted loss of activity at -20 degrees C was less than 0.01% per year) were considered sufficient to allow the use of this preparation for a certification procedure. The behavior of the reference material in comparison with human serum samples was evaluated three ways: (a) by kinetic characteristics, (b) by the ratio of activities for duplicate determinations by different methods, and (c) by use of the reference material to convert values obtained by various methodologies to those by the IFCC proposed method. The material appeared to be commutable for the two methods studied. The difference in the ratios obtained for patients' samples and reference material was less than +/- 5%, and the recalculated values for patients' samples as determined with the reference material differed from values determined by the IFCC method by no more than 4.8%.     

Establishment of reference intervals for enzyme catalytic activity concentration measurements at 37 degrees C--a practical approach

The change from measuring enzyme catalytic activity concentrations from 25 degrees C to 37 degrees C in the German Federal Republic has led to the need for new reference ranges for defined patient groups and for healthy individuals. Up to now, these are only present as tentative values and are incomplete, especially for children. This article describes a method for deriving reference ranges from results obtained from measurement at 25 degrees C and 37 degrees C and the use of percentiles to establish values for 37 degrees C. A total of 1,111,378 data from 507,305 patients were used to establish reference ranges for the following 11 enzymes at 37 degrees C using the test kits from Roche Diagnostics measured on the Modular analyser: acid phosphatase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, cholinesterase, creatine kinase, creatine kinase - MB subunit, gamma glutamyl transpeptidase, glutamate dehydrogenase, lactate dehydrogenase and lactate dehydrogenase - isoenzyme 1. The computed reference ranges from the data used gave rise to reference ranges, some of which were in agreement with the data from the producer, some of which, however, showed deviations from the values given by the producer. Ranges for newborns, children and adolescents could be computed with the prerequisite that ranges for 25 degrees C were available and that these had been established and validated. This method of establishing reference ranges for catalytic enzyme activities can be used for all producers, providing the number of data used is sufficient to allow for valid statistical analysis.     

Column-chromatographic separation of isoenzymes of aspartate aminotransferase.

We describe a column-chromatographic method for separating the mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase in human serum. Bed height of the ion exchanger, pH, and salt concentrations in the eluting buffers are shown to be variables affecting the separation of the isoenzymes. Under the optimized conditions selected for this study, a 30% increase in volume was observed in one fraction, associated with changing the salt concentration of the eluting buffer and attributed to a contraction of the DEAE-Sephadex A-50. Elution profiles (enzyme activity vs. fraction number) were examined with highly purified mitochondrial and cytoplasmic isoenzymes of human origin in bovine serum albumin and human serum. Recovery of the enzyme in the eluted fractions averaged 102% (SD, 2.0%) for specimens prepared from the purified isoenzymes and 104% (SD, 10.7%) for 38 human serum specimens. The separation technique showed linearity to catalytic concentrations in excess of 200 U/liter (reaction temperature 30 degrees C) for each isoenzyme. Additional information is presented regarding among-day precision and the effect of specimen dilution.     

A continuous method for the determination of leucine aminopeptidase in human serum with L-leucinamide as substrate

A continuous procedure for the determination of leucine aminopeptidase is described. L-leucinamide is used as substrate and the liberated ammonia is determined with the glutamate dehydrogenase reaction. The enzyme is Mn2+-activated and 30 mumol/l MnCl2 is necessary for an optimal activity measurement. Influence of buffer type, buffer concentration and pH are reported together with the apparent Km values of leucine aminopeptidase for L-leucinamide and of glutamate dehydrogenase for 2-oxoglutarate. Amastatin, a potent inhibitor, inhibits the reaction of leucine aminopeptidase completely, whereas it has no inhibitory effect on the reaction with glutamate dehydrogenase. The normal reference interval for leucine aminopeptidase is 12-65 U/l at 37 degrees C. The properties of the enzyme are discussed.     

Regulation of the interaction of purified human erythrocyte AMP deaminase and the human erythrocyte membrane.

The binding of purified human erythrocyte AMP deaminase to human erythrocyte membranes and the effect of binding on enzyme catalytic activity was investigated. AMP deaminase binds preferentially and specifically to the cytoplasmic surface of the erythrocyte membrane. The binding is saturable, reversible, and responsive to alterations of pH, of ionic strength, and of ATP and AMP concentrations. A limited number (approximately equal to 2.2 X 10(4) per erythrocyte) of apparently homogeneous high affinity (Ka approximately equal to 2.6 X 10(7) M-1) binding sites is present. The stability of purified and endogenously bound AMP deaminase is markedly improved by the interaction with the membrane, whereas the catalytic activity of AMP deaminase is sharply reduced. AMP deaminase displaces membrane bound glyceraldehyde 3-phosphate dehydrogenase in roughly a dose-response manner. No evidence for binding of AMP deaminase to spectrin or band 3 (the G3PD binding protein) was found in sucrose gradients, however. The interaction of AMP deaminase with the erythrocyte membrane may play an important role in the regulation of cellular adenine nucleotide metabolism.     

Improved assay for alcohol dehydrogenase activity in serum by centrifugal analysis

We describe an improved method for determination of alcohol dehydrogenase (EC 1.1.1.1) activity in 60 microL of human serum, based on conversion of ethanol to acetaldehyde with simultaneous reduction of NAD+ in glycine NaOH buffer (pH 9.0) at 37 degrees C in a centrifugal analyzer. The final concentration of NAD+ was 10 mmol/L and ethanol was 20 mmol/L. The dilution curve was linear with enzyme activity up to 200 U/L, and results by this method correlated well with those by a manual method (N Engl J Med 279: 241-248, 1968). Within-run precision (CV) was 0.9 to 8.2% over the range of 4.5 to 88.1 U/L, and day-to-day precision was 5.4 to 5.6%. In sera from 198 healthy individuals, mean alcohol dehydrogenase activity was 1.6 (SD 1.2, range 0-5) U/L. To evaluate the clinical utility of determining alcohol dehydrogenase, we measured the activity of alanine aminotransferase and alcohol dehydrogenase in sera from 470 patients with various diseases in our hospital, and found that results for the two enzymes did not correlate well.     

Certification of the mass concentration of creatine kinase isoenzyme 2 (CK-MB) in the reference material BCR 608.

We describe the certification of a mass concentration value in the already prepared creatine kinase-2 reference material (BCR 608). Creatine kinase-2 was purified from human heart. The purified enzyme was diluted in order to measure its protein concentration by the Doetsch method. A protein concentration value of 124.30+/-13.17 mg/l was assigned to the stock solution of purified creatine kinase-2. This stock solution was diluted in 25 mmol/l piperazine-N,N'-bis[2-ethanesulfonic acid] (PIPES) pH 7.2, containing 2 mmol/l ADP, 5 mmol/l 2-mercaptoethanol, 154 mmol/l sodium chloride and 50 g/l human albumin to obtain a stable liquid standard of known creatine kinase-2 mass concentration (80.36 microg/l). This standard was then used to recalculate the creatine kinase-2 mass concentration measured in the BCR 608 material by immunoassay. The mass concentration of creatine kinase-2 in samples of reconstituted BCR 608 was certified to be 93.30+/-9.65 microg/l.     

Establishing a reference system in clinical enzymology.

The goal of standardization for measurements of catalytic concentrations of enzymes is to achieve comparable results in human samples, independent of the reagent kits, instruments and laboratory where the procedure is carried out. To pursue this objective, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has launched a project to establish a reference system in clinical enzymology. This system is based on three hinges: a) extensively evaluated and carefully described reference procedures, b) certified reference materials and c) a network of reference laboratories operating in a highly controlled manner. The original IFCC-recommended procedures for alanine aminotransferase, aspartate aminotransferase, creatine kinase, gamma-glutamyltransferase, lactate dehydrogenase and alpha-amylase have been slightly modified to optimize them at 37 degrees C, with the definition of detailed operating procedures. A group of laboratories perform these procedures manually, with self-made reagents on carefully calibrated instruments. Partially purified and stabilized materials, prepared in the past by the Community Bureau of Reference, have been re-certified by these laboratories for alanine aminotransferase, creatine kinase, gamma-glutamyltransferase and lactate dehydrogenase activities. Using these materials and the manufacturer's standing procedures, industry can assign traceable values to commercial calibrators. Thus, clinical laboratories, which will use routine procedures with these validated calibrators to measure human specimens, can finally obtain values which are traceable to reference procedures.     

The effect of storage at different temperatures on cholinesterase activity in human serum

The effect of storage on the catalytic concentration of cholinesterase and on the reference values of cholinesterase in human serum were studied. When serum was stored at room temperature (20 degrees C), at 4 degrees C and at - 20 degrees C (one year) there was no change in catalytic activity. Even after nine times freezing and thawing (over nine weeks) the catalytic activity was unaffected. The average reference value was significantly higher for males than for females (3.11 +/- 0.57 vs 2.50 +/- 0.43 kU/l).     

Metrological traceability of values for catalytic concentration of enzymes assigned to a calibration material.

BACKGROUND: The metrological traceability of values for the catalytic concentration of several enzymes assigned to a calibration material has been assured by following the recently published International Standard ISO 18153. METHODS: A traceable value with a measurement uncertainty was assigned for the catalytic concentration of alanine aminotransferase, creatine kinase, gamma-glutamyltransferase and lactate dehydrogenase in two materials from different sources. These are all measurable quantities, with the primary reference measurement procedure described by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and a primary calibrator giving metrological traceability to the SI unit of measurement. The metrologically traceable calibration was validated by measuring human serum samples using the primary reference measurement procedure and a routine commercial measurement procedure calibrated with the traceable materials. RESULTS: Results showed that the primary reference procedure, selected manufacturers' procedures and the end-user's routine procedure for each enzyme have the same analytical specificity. Four of eight commercial calibrators tested were commutable, whereas the others had a very small difference in absolute terms, indicating that these materials would be useful for calibration. CONCLUSION: The implementation of a reference system for enzyme measurements was demonstrated that assures the traceability of patient results to SI units.     

Measurement of aspartate aminotransferase activity: effects of oxamate.

Oxamate, a potent inhibitor of lactate dehydrogenase, is shown also to inhibit aspartate aminotransferase activity, both in human serum and in purified isoenzymes of human origin. The inhibition was competitive with respect to 2-oxoglutarate for both isoenzymes. The apparent Ki was 29 mmol/L for the cytoplasmic enzyme and 17 mmol/L for the mitochondrial enzyme. Noncompetitive inhibition was found between oxamate and aspartate. At saturating concentrations of substrate (2-oxoglutarate greater than or equal to 15 mmol/L, L-aspartate greater than or equal 150 mmol/L) oxamate inhibited the mitochondrial enzyme but had less effect on the cytoplasmic isoenzyme. Oxamate at 40 mmol/L inhibited the enzyme in serum by 11 and 9% in assays containing 2-oxoglutarate at 6.7 and 15 mmol/L, respectively. This concentration of oxamate inhibited enzyme activity in serum by 5% more than did the same concentration of Cl- (itself an inhibitor). Oxamate (less than or equal to 30 mmol/L) had no measurable effect on the stability or activity of porcine malate dehydrogenase. Until the effects of its inhibitory properties are considered, addition of oxamate to suppress lactate dehydrogenase-mediated side reactions in the assay of aspartate aminotransferase cannot be recommended.     

Simple non-radiochemical HPLC-linked method for screening for purine metabolism disorders using dried blood spot

BACKGROUND: Pathologies associated with rare inherited disorders affecting purine metabolic pathways range from renal failure to neurological dysfunction and immunodeficiency. The disorders are usually diagnosed by measuring enzyme activities in hemolysates. A non-radiochemical HPLC-linked method is described for simultaneous determination of the activities of hypoxanthine-guanine phosphoribosyltransferase (HPRT: E.2.4.2.8.), adenine phosphoribosyltransferase (APRT: E.2.4.2.7.), adenosine deaminase (ADA: E.3.5.4.4.) and purine nucleoside phosphorylase (PNP: E.2.4.2.1.) in dried blood spots. METHOD: 7-mm-diameter blood spots stored at 4 degrees C or room temperature were transferred to an Eppendorf tube and eluted with 500-microl 0.1 mol/l Tris-HCl buffer, pH 7.4. The eluate was added to substrate solutions and incubated at 37 degrees C. Reaction products were analysed by HPLC. RESULTS AND CONCLUSIONS: The enzyme activities tested in spot eluates were similar to those in erythrocyte lysates from the same subjects. None of the enzymatic activities tested were significantly affected by different storage temperatures. The main advantages of the proposed method are small blood volume required, easy sample collection and transfer, and accurate results. The method is therefore suitable for screening inborn errors of purine metabolism even in newborns.     

Bioluminescent assay for serum adenosine deaminase with immobilized bacterial luciferase

We describe a bioluminescence method for measuring adenosine deaminase activity in serum. The method involves use of batchwise enzyme reaction containing adenosine, alpha-ketoglutarate, glutamic dehydrogenase and NADH. The resulting solution is injected to the continuous-flow bioluminescence system. In the system, a bacterial luciferase and NAD(P)H:FMN oxidoreductase are covalently co-immobilized on Sepharose 4B. Carrier solution (pH 6.8) for bioluminescence reaction contains FMN and decanal. The continuous-flow light-emitting system, in which the reactor (flow cell packed with immobilized enzyme) is placed in front of a photomultiplier tube inside a photon counter, is versatile and simple. Concentration and response are linearly related from 1.2 to 92.5 pmol per injection of ammonia. The precision of the method is satisfactory (coefficient of variation 3.9-6.8%). We validated the technique by comparing results with conventional assay method (UV method). Normal values for adenosine deaminase activity of serum ranged from 7.0 to 22.0 U/l in agreement with those obtained by other method. The Sepharose 4B-immobilized enzymes are stable for more than one year. This assay system could be used as a routine clinical laboratory test in the diagnosis of liver damage.     

Preparation and characterization of reference materials for human pancreatic lipase: BCR 693 (from human pancreatic juice) and BCR 694 (recombinant).

There is a lack of certified reference material (CRM) for lipase catalytic activity. Consequently between-method comparability is very poor. The aim of this study was to produce two lipase CRMs, one from human pancreatic juice (BCR 693), and another using recombinant technologies (BCR 694). Lipase was purified from pancreatic juice, using column chromatography and isoelectric focusing. Recombinant lipase was produced with a transfected cell line and purified with column chromatography. Adding buffered bovine serum albumin and subsequent freeze-drying were used to stabilize both materials. A standardized titrimetric method was employed to compare their catalytic properties to those of two plasma pools of patients suffering from acute pancreatitis. About 5 kU (titrimetry, 37 degrees C) of each material were obtained. They were lyophilized without apparent modifications of their catalytic properties, which stayed identical to those exhibited by the enzyme present in patient's pools. Stability of both materials was estimated at several years when stored in a dry form at -20 degrees C. Both materials appear to have similar catalytic properties and stability and were found commutable as regards a reference method and a routine measurement procedure. An international certification campaign will be carried out to assign values to BCR 693 and BCR 694.