Automated Determination of Creatine Kinase ... - Clinical Chemistry

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We measured creatine kinase (CK, EC activity in serum with a new reagent system utilizing thermostable glucokinase (EC Automated ...

CLIN. CHEM. 37/3,452-454 (1991)

Automated Determination of Creatine Kinase Activity in Serum with Use of Thermostable Glucokinase IkuhiroMaeda,Sadao Hayashl,NobuyuklAmino,and KIyoshiMlyaI We measured creatine kinase (CK, EC activity in Reagents for measuring CK activity were supplied in kit form (latroteck CK rate A) by latron Laboratories, serum with a new reagent system utilizing thermostable Inc., Tokyo, Japan. The principle of this enzymatic glucokinase (EC Automated determinations method is similar to the GSCC method except that were performed with Toshiba’s Model TBA-80S Biochemthermostable glucokinase is used instead of hexokinase. ical Analyzer. Precision studies demonstrated within-run The final concentrations of several components were and between-run CVs of 0.4%-2.4% and 2.8%-3.1%, respectively. The response linearity was confirmed for CK slightly different from those in the GSCC method. Reagent 1 contained, per liter, 4000 U of glucokinase, 1250 activity up to 1000 U/L at 37 CK activities correlated U of glucose-6-phosphate dehydrogenase (EC, well (r = 0.997) with those obtained by the manual 2.0 mmol of adenosine diphosphate, 25.0 mmol of D-glumethod recommended by the German Society for Clinical cose, 2.5 mmol of NADP’, 6.2 mmol of adenosine monoChemistry (measuring at 37 #{176}C) involving hexokinase (EC phosphate, 12.5 mol of diadenosine 5-phosphate However, CK activities measured by our method (AP5A), and 25.0 mmol of N-acetylcysteine dissolved in were consistently higher than those of the hexokinase method at reaction temperatures of 30, 37, and 40 #{176}C.imidazole acetate buffer, pH 6.7. Reagent 2 contained These data indicate that the new method with thermo130.0 mmol of creatine phosphate and 52.0 mmol of magnesium acetate per liter of Tris buffer, pH 8.5. stableglucokinase isbetterthan thatwiththermo-unstaMoni-Trol I and II chemistry controls were purchased ble hexokinase fordeterminationof CK activity inserum. from American Dade, Miami, FL. Nescol-X and XA AdditionalKeyphrases:hexokinase method compared heat control sera were purchased from Nippon Shoji Kaisha stability variation, source of Ltd., Osaka, Japan. 6-Phosphogluconate, trisodium salt, was purchased from Sigma Chemical Co., St. Louis, MO. Various methods have been established for measuring In the manual method, 100 pL of the sample was creatine kinase (CK; EC activity in serum. The added to 2.0 mL of Reagent 1 and kept at 37#{176}C for 5 O#{149}

enzymatic method involving hexokinase (EC has been widely used for determining CK activity in serum since the first spectrophotometric method reported by Oliver (1) and recommended by the German Society for

Clinical Chemistry (08CC) (2). Hexokinase, however, is ideal reagent for detection of CK activity, because this enzyme is unstable in the liquid state and also is not thermostable. Recently, Kamei et al. (3) isolated the thermostable enzyme glucokinase (EC from Bacillus stearothermophilus and used this enzyme to measure glucose (4). Their reagent was reportedly far more stable than the reagent containing hexokinase (3, 4). Kondo et al. (5) have now used thermostable glucokinase for the manual measurement of CK activity in serum. Here we evaluate a new kit in which this enzyme is used for automated measurement of CK activity. not an

Materials and Methods The Model TBA-80S Biochemical Analyzer (Toshiba Co., Ltd., Tokyo, Japan), a discrete, multichannel analyzer, has a maximum throughput of 10 500 results per hour at a constant reaction temperature of 37 #{176}C. For the manual method, we used the ultraviolet-visible recording spectrophotometer UV-265 (Shimadzu Corp., Kyoto, Japan). Central Laboratory for Clinical Investigation,

Osaka University

Hospital, 1-1-50, Fukushima, Fukushima-ku, Osaka, 553, Japan. Received September 6, 1990; accepted December 21, 1990. 452

CLINICAL CHEMISTRY, Vol. 37, No. 3, 1991

mm. After that, 500 L

of Reagent 2 was added and the

absorbance at 340 nm was read with the recording spectrophotometer. In the automated method, we used TBA-80S settings shown in Table 1. Three minutes after the adding of Reagent 1, Reagent 2 was added at position 11, and the absorbance of the reaction solution was read for 3 mm at 340 and 380 nm. Results The reaction time courses for analyses of four human sera are shown in Figure 1. Each reaction proceeded in a rectilinear fashion after Reagent 2 was added. To examine the potential contamination of glucokinase by 6-phos-

phogluconate dehydrogenase, we added 6-phosphogluconate to the reaction mixture at a final concentration of 10 mmol/L and looked for a change of absorbance at 340 nm. No increase of absorbance was observed. Estimates of withinand between-assays precision (CV) of this method with the TBA-80S were based on 10 determinations in the same run and 10 consecutive determinations on different days. Within-assay CVs were 2.44% at 75.9 U/L, 1.11% at 253.8 UIL, and 0.42% at 482.2 U/L (all at 37 #{176}C). Between-assay CVs were 2.79% at 37.3 UIL and 3.08% at 229.0 UIL, also at 37 #{176}C. Linear-regression analysis of the determined values (y) vs the calculated values (x) gavey = 1.000x-0.899 (r = 0.999) for purified water and high-CK activity serum diluted with heat-treated serum (from 0 U/L through 1000 U/L, n = 5, all at 37 #{176}C). When we determined CK


Table 1. Settings of the TBA-80S Analyzer for Determining 1.


2. 3.



100 0 340(1) 380(2)

Base Main wavelength

9. 10.

> 4-.





Subwavelength Blankrange




Abs Abs Abs


Abs range

Low High


Sample range

Low High


13. 14.


15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Decimal point R-lpump no. R-Ipump pos. R-Ivol. A-lIpump no. A-lIpump pos. R-lIvol. A-Ill pump no. A-Ill pump pos. A-Ill vol. Startrefresh Refresh




20 &L


6. 7. 8.


CK RUF (8) 0

Reference Samplevol.

4. 5.


0) >


C) Q

25 30 37 40 Reaction Temperature (#{176}C) Fig. 2. Effect of reactiontemperatureon relative activities: values obtained by the hexokinasemethoddividedby valuesobtainedby the method involving thermostable glucokinase (0), human serum A (75 U/L); (0), human serum B (159 U/L); (n), human serum C (468 U/L); all activities listed as determined at 30 ‘C with the hexokinase method

Low High

y 0 1

__ 100




+ 4.57 (r = 0.997). 2 summarizes the relative activities of values

determined by the hexokinase method vs values determined by the method with thermostable glucokinase at

25, 30, 37, or 40 #{176}C. In the recommended method of the


International Federation of Clinical Chemistry (6), CK activity was affected by the volume fraction of serum. Because we used the volume fraction of 0.0385 in the main experiment, we additionally measured CK activ-










activities in 74 human sera by the method involving thermostable glucokinase (y) and the method with hexokinase (x) at 37 #{176}C, the equation for regression line was

C -3

(V L.







8.8385 8.8195












a, 1.0 C.) C

-a I-












4 Reaction ternperature(



Fig. 3. (Top) Effect of the volume fraction of serum on the thermo0.01-

2 Time

4 (mm)


FIg. 1. Reaction time courses for CK determinations by the method involving thermostable glucokinase Serum 1, 2, 3,and 4 werehuman sera; the blank was purified distilled water

stableglucokinasemethod;(bottom) effect of reaction temperature on relative activity measured (results of the hexokinase method divided by results of the thermostable glucokinase method) (Top): Humanserum, CK 245 U/L (determinedat a volumefraction of 0.0435), was the sample. Bottom: human serumsamples A’ (0), B’ (0), C () had CX

activitiesof 82, 170,and 399 IJ/L respectively,when determinedat 30 ‘C with the hexokinase method

CLINICAL CHEMISTRY, Vol. 37, No. 3, 1991 453

ity by using the volume fraction of 0.0435, recommended by International Federation of Clinical Chemistry; almost the same results were obtained (Figure 3,

Unfortunately, the reaction temperature in TBA-80S is fixed at 37 #{176}C. Thus the temperature conversion factor

CK activities by the hexokinase method increasing reaction temperature, compared with those obtained by the method involving thermostable glucokinase (Figure 3, bottom).

from 37 #{176}C to 30#{176}C should be calculated to obtain an international enzyme unit. However, the conversion factor varies with temperature (Figure 2). In view of these facts, we prefer the method involving thermostable glucokinase to the method involving hexokinase for determining CK activity in serum.

Discussion Kamei et al. (3) reported the following advantages of glucokinase from B. stearothermophilus: Firstly, the Km of this glucokinase for glucose is low and its Km for ATP is lower than that of hexokinase. Secondly, it is stable over a wide temperature range. Regarding the first point, we confirmed that there was no lag time in the reaction time course and observed no blank reaction because glucokinase has no ATPase activity. Thus CK activity was measurable in a short time. Regarding the second point, CK activities by the method with thermostable glucokinase were higher by 10% than those by the method with hexokinase at reaction temperatures of 30, 37, or 40 #{176}C. This might be caused by thermounstable hexokinase. The International Federation of Clinical Chemistry has prescribed the reaction temperature in enzyme activity determination at 30#{176}C (6).

References 1. Oliver IT. A spectrophotometric method for the determinationof creatine phosphokinase and myokinase. Biochem J 1955;61: 116-22. 2. Recommendations of German Society for Clinical Chemistry. Standardization of methods for the estimation of enzyme activities in biological fluids. J Clin Chem Clin Biochem 1977;15: 225-60. 3. Kamei S, Tomita K, Nagata K, et al. A stable glucokinase from a thermophilic bacterium. I. Purification and some properties. J Clin Biochem Nutr 1987;3:1-9. 4. Toinita K, Kamei 5, Nagata K, et al. A stable glucokinase from a thermophilic bacterium. II. Application to the glucose determination. J Clin Biochem Nutr 1987;3:11-6. 5. Kondo H, Shiraishi T, Kageyama M, Nagata K, Tomita K. Bacterial glucokinase as an enzymic reagent of good stability for measurement of creatine kinase activity. J Clin Biochem Nutr 1987;3:17-25. 6. Expert Panel on Enzymes; Committee on Standards (IFCC). IFCC methods for the measurement of catalytic concentration of enzymes; Part 1. General considerations concerning the determination of the catalytic concentration of an enzyme in the blood serum or plasma of man. Clin Chim Acta 1979; 98:163F-74F.

top). Finally, decreased


CLIN. CHEM. 37/3, 454-458 (1991)

Genetic Screening of Newborns for Sickle Cell Disease: Correlation of DNA Analysis with Hemoglobin Electrophoresis KrIsten J. Skogerboe,’Sheyla F. West,1 Magdalena 0. MurllIo,1 MIchael W. Glass,2 Santosh Shaunak,2 and Jonathan F.Taft’

Although DNA analysis based on the polymerase chain reaction (PCR) offers potential advantages for screening newborns for sickle cell disease, few data are available concerning the reliability of PCR-based tests for such screening.

We describe

a protocol for detecting the A, S, and C alleles of the /3-globin gene in dried blood from

phenylketonuria screening cards. This method is based on PCR and detection with allele-specific oligonucleotide probes. Results of a blind comparison

of PCR analysis of

the dried blood with hemoglobin electrophoresis of wholeblood samples agreed for 80 of 81 samples. The single discrepancy is probably not attributable to a failure of the PCR method, but rather to limitations of the electrophoresis method. The PCR method should be a highly accurate means of detecting /3-globin alleles in routine genetic screening with dried blood already collected for (e.g.) pheriylketonuria screening. ‘Department of Laboratory Medicine SB-b, University of Washington, Seattle, WA 98195, and2 Washington State Department of Health Laboratory, 1610 N.E. 150th St., Seattle WA 98155. Received November 5, 1990; accepted January 10, 1991. 454

CLINICAL CHEMISTRY, Vol. 37, No. 3, 1991

AddftionalKeyphrases:allele-specific oligonucleotides dotblot analysis cord blood electrophoresis compared fi-globin alleles

heritable disorders

The polymerase chain reaction (PCR) (1, 2) has been used in combination with allele-specific oligonucleotide (ASO) probes (3) for diagnosis of several genetic diseases caused by point mutations, including sickle cell disease (4). Mass neonatal screening for sickle cell disease is important because early identification and treatment of infants with sickle celldisease improves survival (5, 6). The small amount of adult hemoglobin produced by neonates makes detection of clinically of hemoglobin an analytical challenge.



DNA techniques complement traditional electrophoretic methods, providing the ability to detect alleles of the 3-globin gene directly and to confirm results via a different methodology. DNA methods may also offerseveralpotential advantages for newborn sickle cell screening programs, such as greater reliability, specimen stability, and amenability to automation and large-scale screening. Initial studies have demonstrated the technical feasibility DNA amplification from Guthrie card specimens

of for

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