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A New Method for Measuring Serum Pyruvate Kinase and Creatine Kinase Activities Using a Thermostable Glucokinase

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A New Method for Measuring Serum Pyruvate Kinase and Creatine Kinase Activities Using a Thermostable Glucokinase Jpn J Clin Chem 20 : 235-241, 1991. Technical Report A New Method for Measuring Serum Pyruvate Kinase and Creatine Kinase Activities Using a Thermostable Glucokinase Takanari Shiraishi*, Hitoshi Kondo*, Hiroyuki Tsubota* * Summary A new methodfor measuringthe activityof serum pyruvatekinase (PK:EC 2.7.1.40) was developed by using a thermostable glucokinase(Glck) from a thermophilicbacterium, Bacillus stearothermophilus.The ATP formed by the reaction of PK is finallyconverted to NADPHvia glucose-6-phosphateby the action of Glck and glucose-6-phosphatedehyd- rogenase. The change in absorbance at 340nmwas foundto be linearup to about 5000U/Iof PK. The within-runand day-to-daycoefficients of variationwere1.13% at 85.4U/Iand 2.08%at 45.7U/I,respectively. The influenceof variouscoexistents and anticoagulants,such as bilirubin, ascorbate, glucose,EDTA, sodium fluoride, etc., on the assay was negligible.The reagent was stable in solutionfor aboutone monthat 10°C. A methodfor simultaneouslymeasuring the activitiesof PKand creatine kinase (CK)in a single specimenwas also developed.This was based on the fact that the assay conditionsfor bothen- zymeswere similar.This methodwas foundto have a highdegree of precisionand a good cor- relationwith respective PK and CK assay methods.This simultaneousmeasurement may be usefulfor the accurate differentialdiagnosis of myocardialinfarction. Key words : pyruvate kinase, creatine kinase, glucokinase, simultaneous assay, myocardial infarction progressive muscular dystrophy").It is less Introduction influenced by external factors such as exercise than is seum creatine kinase Pyruvate kinase(PK:EC2.7.1.40)is an (CK:EC2.7.3.2)activity".PK assay is, eyzyme which catalyzes the conversion therefore,regarded as useful for more from phosphoenolpyruvate(PEP)to accurate diagnosis of myocardial infarction. pyruvate in the glycolytic pathway;this The use of three enzymatic methods has eyzyme exists in large amounts in cardiac been reported for serum PK assay: muscle,skeletal muscle,brain and liver. the hexokinase(HK)-glucose-6-phosphate Serum PK activity becomes elevated dehydrogenase(G6PDH)method".the primarily in myocardial infarction and lactate dehydrogenase method9)and the pyruvate oxidase(POP)-peroxidase(POD) methoe).When these methods are used, *Biochemistry Department there are a relatively low stability of the ,Research and Development Center,UNITIKA Ltd.,Kyoto reagent in the liquid state and a relatively * *Technical Section ,IATRON LABORATORIES, narrow dynamic range of measurement. Inc.,Chiba The present report deals with the application of a thermostable glucokinase Received on Mar.19,1991,Accepted on Aug.2,1991 (GlcK:EC2.7.1.2)from a thermophilic Jpn J Clin Chem Vol.20No.4Dec.1991 235 and P1,P5-di(adenosine-5'-)pentaphosphate (Ap5A)were purchased from Boehringer Mannheim Yamanouchi K.K.,Tokyo.The other chemicals used were obtained from Wako Pure Chemical Industries Ltd.,Osaka. Reagent kits for PK assay by the the HK- G6PDH and POP-POD methods were Fig.1Principles of the PK and CK assays. The following abbreviations are used: purchased from Dainippon Pharmaceutical G6P, glucose-6-phosphate ; 6PG, 6-phospho- Co.Ltd,Osaka and Kyowa Medex Co. gluconate. For the other abbreviations, see the Ltd.,Tokyo,respectively.The reagent kit text. for the CK assay by the GlcK-G6PDH method was obtained from Dia-Iatron Co. Ltd,Tokyo. Serum samples were obtained from bacterium,Bacillus stearothermophilus,to patients and healthy adults.Fixed amounts serum PK assay and to the development of of several coexistents and anticoagulants a simultaneous assay of PK and CK were mixed in advance with serum activities in a single specimen.This samples,and the resultant mixtures were simultaneous assay may be useful for the used.A mixture of PK from rabbit muscle differential diagnosis of myocardial and a normal control serum (obtained from infarction8). Ortho Diagnostic Systems Ltd.,Tokyo) was used in the experiment regarding the Principles of the Assay linearity of the assay. Fig.1shows the principles of the PK and Methods CK assays using the GlcK-G6PDH method. The GlcK and G6PDH assays were PK and CK catalyze the conversion from carried out similarly to those described in a PEP to pyruvate and that from creatine previous paper12).One unit of GlcK or phosphate(CrP)to creatine,respectively, G6PDH activity was defined as the amount forming ATP from ADP.The ATP thus of enzyme that formed one p mol of formed is finally converted to NADPH,via product in1min at30•Ž . glucose-6-phosphate,by the action of GlcK The PK and PK-CK simultaneous assays and G6PDH.PK and CK activities can then were carried out with the two solutions R1 be measured by determining the increase in and R2,respectively(TableI).In the PK absorbance at340nm due to the formation assay,R1 contained GlcK,G6PDH,ADP, of NADPH. NADP,glucose,etc.,and R2PEP.The The principles of the PK and CK assays reaction was started with R2.In the PK- are the same,except for the use of one of CK simultaneous assay,R1contained the substrates. This indicates that a GlcK,G6PDH,ADP,NADP,glucose, simultaneous assay of PK and CK activities PEP,etc.,and R2CrP.PK activity was can be designed,if such parameters as determined with R1,followed by the optimum pH and reagent composition are addition of R2to start the CK reaction. adequately established. The activity observed after the addition of R2 corresponded to the total activity of PK Materials and Methods and CK in the sample.The CK activity could therefore be calculated as the Materials difference between the two observed GlcK was purified from B.stearo- values.One unit of PK or CK activity was thermophilus,according to the method of defined as the amount of enzyme that Kamei et aril.G6PDH from Leuconostoc formed one p mol of product in 1 min at mesenteroides,the disodium salts of AMP, 37•Ž. ATP,NADP and CrP,the potassium salts Assays were carried out at37•Ž with of ADP and PEP,N-acetylcysteine(NAC) Shimadzu UV-265 Spectrophotometer and 236 TableIReagent constituentsfor the PK assay®and the PK-CKsimultaneous assay® were:assay code,Rate A-14-24;sample volume,8ƒÊl;R1volume,320ƒÊ1;R2 volume,80ƒÊ1;wavelength,450/340nm; K factor,8199.Those for the PK-CK simultaneous assay were:assay code,Rate B-14-24,B-40-50;sample volume,8p1; R1volume,320ƒÊ1;R2volume,80p1; wavelength,450/340nm;K factor,6592 for PK and8199for(PK+CK). Results Construction of reagents for the PK assay The reagent composition was determined so that each component could give the Fig.2Effects ofG1cK,G6PDH maximum PK activity.Fig.2shows an ,PEP,ADP and NADP concentrations on the PK example of the plots of relative PK activity activity. against the concentrations of reagents. The PK assay was carried out at pH6 .7and TableI°summarizes the concentrations of 37•Ž.The ordinate represents the relative PK reagents thus determined forR1and R2. activity(about150U/1) .The abscissa PK showed its maximum activity at a wide represents the final concentrations ofG1cK G , 6PDH,PEP,ADP and NADP in the pH range of6.5-7.2.The final pH of the reaction mixture.Symbols:•› reagent mixture was set at6.7. ,G1cK;A, G6PDH;•›,PEP;•ž,ADP;•¢ ,NADP. Evaluation of reagents for the PK assay It was found that the lag time of Hitachi7150Automatic Analyzer . measurement was less than about 3min Chemistry parameters for the PK assay (refer to the time course of PK reaction in with the Hitachi 7150 Automatic Analyzer Fig.6).Fig.3 shows that the measured PK Jpn J Clin Chem Vol.20No.4Dec.1991 237 Fig.4Correlation between theGlcK-G6PDH method and the HK-G6PDH method for the measurement of serum PK activity. The ordinate represents the measured PK activity determined by theG1cK-G6PDH method and the abscissa that determined by the HK-G6PDH method. The linear Fig.3Dependence of the measured PK regression equation with 51 serum samples activity on dilution. was determined to be y=1.04x+6.29(r= 0.992). activity was linear up to about5000U/1. degree of correlation was also found to the The coefficient of variation(CV)in the POP-POD method (with 41 serum within-run reproducibility was1.13%at a samples,y=1.13x+10.4and r=0.996). mean PK activity of84.4U/1(n=10).The The stability of the workingsolution in CV at a mean PK activity of45.7U/1was the PK assay may be one of the most 2.08%in the day-to-day assays over a ten- important factors in a practical use.Fig.5 day period. shows that its storage over one month at The PK assay was not influenced by 10•Ž gave no changes in PK activity,lag several coexistents such as glucose(0- time and reagent blank. 10g/1),ascorbate(0-200mg/1),lactate(0- 200mg/1),urate(0-200mg/1)and bilirubin Development of the PK-CK simulta- (0-200mg/1),and by anticoagulants such as neous assay EDTA(2g/1),sodium citrate(5g/1),sodium As described above,the pH for the PK fluoride(10g/1) and heparin(200mg/1). assay was set at6.7,which was the same as However,it was slightly interfered by the optimum pH value for the CKassay12). hemoglobin in a positive direction,whereas In addition,the assay conditions for PK and by sodium oxalate(5g/1)in a negative CK were similar.Differences between the direction.The effect by hemoglobin may PK and CK assays were the optimal be mainly due to the PK present in concentrations of magnesium acetate, erythrocytes. potassium acetate,ADP and G6PDH.It Fig.4shows that present method was confirmed,however,that these precisely correlated to the HK-G6PDH differences did not cause a serious effect on method in serum PK assay.The same the PK and CK assays.Table I ® 238 Fig.5Storage stability of the reagents in a liquid state atlot.•› ,•›andA represent measured PK activity,lag time of measurement and reagent blank measured at340nm(0D340),respectively.
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