In the Field of Clinical Examination, the Measurement of Creatine

Home , Adenylate kinase, Creatine kinase

J. Clin. Biochem. Nutr., 3, 17-25, 1987

Bacterial Glucokinase as an Enzymic Reagent of Good Stability for Measurement of Creatine Kinase Activity

Hitoshi KONDO, * Takanari SHIRAISHI, Masao KAGEYAMA, Kazuhiko NAGATA, and Kosuke TOMITA

Research and Development Center, UNITIKA Ltd., Uji 611, Japan

(Received January 10, 1987)

Summary An enzymic reagent, that has long-term stability even in the liquid state, was successfully employed for the measurement of serum

creatine kinase (CK, EC 2.7.3.2) activity. The enzyme used was the thermostable glucokinase (GlcK, EC 2.7.1.2) obtained from the thermo-

phile Bacillus stearothermophilus. The reagent was found to be stable in solution for about one month at 6•Ž and for about one week at 30•Ž. This

substitution of glucokinase for the hexokinase of the most commonly used hexokinase-glucose-6-phosphate dehydrogenase (HK-G6PDH) method

results in a remarkable improvement of the method. The CK activity measured by the GlcK-G6PDH method was linear up to about 2,000 U/

liter at 37•Ž. The GlcK-G6PDH method was found to give a satisfactory

precision and reproducibility (coefficient of variation less than 2.17%). Over a wide range of CK activity, an excellent agreement was obtained between the GlcK-G6PDH and the HK-G6PDH methods. Furthermore

several coexistents and anticoagulants were found to have little effect on the measured value of CK activity by the GlcK-G6PDH method.

Key Words: creatine kinase activity, glucokinase, improved stability of reagent, creatine kinase determination, thermostable enzyme

In the field of clinical examination, the measurement of creatine kinase (CK, ATP : creatine phosphotransferase, EC 2.7.3.2) activity in serum is one of the important examinations usually employed for diagnosis of cardiac diseases such as myocardial infarction or muscular diseases such as progressive muscular dystrophy. Numerous methods have been so far reported for the measurement of CK activity in serum. The most common method used nowadays is the hexokinase

*To whom correspondence should be addressed .

17 18 H. KONDO et al.

(HK)-glucose-6-phosphate dehydrogenase (G6PDH) method, because it is based on the most reasonable principle and has satisfactory sensitivity and reproducibility. Even since the first report on the HK-G6PDH method by Oliver [1], various im- provements have been made [2-16]. For example, there have been studies on the compounds inhibiting adenylate kinase, which exists in blood and causes a negative error in the HK-G6PDH method [7, 8, 13, 16], on the compounds containing thiol groups for activation of CK activity [10, 12], on the compounds chelating metal ions [11, 14], and the like. As a result, the HK-G6PDH method has been established as the most reliable method for the measurement of CK activity in the clinical laboratory. However, the HK-G6PDH method still has the problem that the enzymic reagent used for measuring CK activity has poor stability in the liquid state. Hence, once the assay mixture is prepared, it cannot be used for a long period of time, presumably due to the instability of HK. Recently, Kamei et al. has reported the isolation of the thermostable enzyme glucokinase (G1cK, EC 2.7.1.2), which is similar to HK in the enzymic reaction, from the moderate thermophilic bacterium Bacillus stearothermophilus and its superiority to HK in the enzymic properties [17]. The thermostable G1cK was successfully applied to the reagent for measuring glucose in the biological fluids and the working solution of the reagent consisting of G1cK was reported to be far more stable than that of the reagent consisting of HK [18]. Based on these findings, the authors aimed to develop a stable reagent for measuring serum CK activity by using G1cK insteadd of HK, in order to overcome the above-mentioned disadvantage encountered in the HK-G6PDH method.

MATERIALSAND METHODS

Materials. G1cK was purified from B. stearothermophilus according to the method of Kamei et al. [17]. G6PDH from Leuconostoc mesenteroides, N-acetyl-L- cysteine (NAC), P1,P5-di(adenosine-5'-)pentaphosphate trilithium salt (Ap5A), disodium salt of AMP, ATP, and NADP+, potassium salt of ADP, and creatine phosphate disodium salt were purchased from Boehringer Mannheim GmbH, Mannheim. Magnesium acetate, imidazole, sodium oxalate, sodium citrate, and lithium lactate were purchased from Wako Pure Chemical Industries, Ltd., Osaka. Disodium salt of EDTA, glucose, reduced form of glutathione, sodium L-ascorbate, and sodium pyruvate were obtained from Ishizu Seiyaku Co., Ltd., Osaka. Heparin sodium salt, sodium fluoride, sodium urate, and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were also obtained from Nakarai Chemicals, Ltd., Kyoto. Hemoglobin of human origin (H 7379) was purchased from Sigma Chemical Co., St. Louis, Mo. Bilirubin control was obtained from Eiken Chemical Co., Ltd., Tokyo. The other chemicals used were obtained from Boehringer Mannheim GmbH, Mann- heim and Wako Pure Chemical Industries, Ltd., Osaka. The CK measuring kit (HK-G6PDH method, NAC activated) based on the

J. Clin. Biochem. Nutr. DETERMINATION OF CREATINE KINASE ACTIVITY WITH GLUCOKINASE 19 recommendations by the German Society for Clinical Chemistry was also purchased from Boehringer Mannheim GmbH, Mannheim. Serum samples were obtained from our hospital. Fixed amounts of several coexistents and anticoagulants were mixed in advance with serum samples, and the resultant mixtures were used as the serum sample for the experiments on the effects of coexistents and anticoagulants on the measured value of CK activity. Determination of the activities of GIcK and G6PDH. The activity of G1cK was measured at pH 9.0 with the assay mixture containing 4 mM ATP, 12 mM glucose, 0.9 mM NADP+, about 0.7 U/mi G6PDH from yeast, and 20 mM magnesium chloride. The activity of G6PDH was also measured at pH 9.0 with the assay mixture containing 2.64 mM glucose-6-phosphate, 0.9 mM NADP+, and 40 mM magnesium chloride. One unit of G1cK or G6PDH activity was defined as the amount of G1cK or G6PDH that forms 1 pmol of glucose-6-phosphate or 6- phosphogluconolactone in 1 min at 30°C. Determination of the amounts of creatine phosphate, NADP+, and NAC. Crea- tine phosphate and NADP+ were spectrophotometrically measured according to the methods of Lamprecht et al. [19] and Klingenberg [20], respectively. NAC was measured with 0.25 mM DTNB in a 50 mM potassium phosphate buffer (pH 7.5) by using the molar extinction coefficient of the product (13.6 x 103 liter•mol-1• cm-1 at 412 nm). Determination of CK activity. The CK activity in the serum sample was measured at 37°C both with a single reagent which containing all components and with two-separate reagents in which components except for creatine phosphate were contained in the first reagent and creatine phosphate in the second reagent. Unless otherwise stated, the final concentrations of reagent components were as follows : 86 mM imidazole-acetate buffer (pH 6.7), 3.0 U/mi Gick, 1.0 U/ml G6PDH, 25 mM creatine phosphate, 1.5 mM ADP, 20 mM glucose, 2.0 mM NADP+, 20 mM NAC, 10 mM magnesium acetate, 2.0 mM EDTA, 5.0 mM AMP, and 10 pM Ap5A. In the single reagent method, 20 pl of the serum sample was added to 500 pl of the mixture, which had been kept at 37°C, and the reaction rate was immediately monitored at 340 nm with a Gilford 2600 Spectrophotometer. In the method utilizing two-separate reagents, 15-20 pl of the serum sample was added to 400 pl of the first reagent, followed by the addition of 100 pl of the second reagent to start the reaction by CK. The instruments used for the latter method were a Gilford 2600 Spectrophotometer and a Hitachi 105-50 Clinical Spectrophotometer.

RESULTS AND DISCUSSION

GlcK G6PDH method for the determination of CK activity The principle of the determination of CK activity in the GIcK-G6PDH method can be represented as follows : CK Creatine phosphate + ADP --- Creatine + ATP (1)

Vol. 3, No. 1, 1987 20 H. KONDO et al.

GJcK Glucose + ATP -~ Glucose-6-phosphate -I-ADP (2) G6PDH Glucose-6-phosphate + NADP+ -- 6-Phosphogluconolactone+NADPH+ H+ ( 3 ) G1cK from B. stearothermophilus is similar to HK in the enzymic reaction, but has the following advantages over it in addition to the high thermal stability [17] : (a) high substrate specificity to glucose, (b) low Michaelis constant for ATP, (c) good stability at room temperature, (d) no adenosine triphosphatase activity. Furthermore, we found the combination of G1cK obtained from B. stearothermophilus and G6PDH obtained from L. mesenteroides very useful in the measurement of CK activity, from the finding that the stability of G 6PDH was higher in the G1cK-G6PDH method than in the HK-G6PDH method.

Stability of the working solution For the practical application of a reagent, the stability of the working solution is one of the important factors. At first, we examined the stability of the working solution with the single reagent. The CK activity of given serum samples was found to be fully recovered when tested in a reagent mixture that had been stored for at least 2 days at 30°C. This reagent stability is considered to be superior to that found with the usual HK-G6PDH method (less than several hours), being mainly ascribed to the use of the thermostable G1cK. In fact, G1cK was found to be stable for about 8-10 days at 30°C in the reagent mixture. As the result of the determination of the residual amounts of G6PDH, NADP+, creatine phosphate, and NAC in the reagent mixture, creatine phosphate was found to be the most unstable component, with the residual amount of it being about 50% after storage for 8 days at 30°C. This leads to the possibility that an even more stable reagent for measurement of CK activity could be developed by the stabilization of creatine phosphate in the reagent mixture. We found that the stabilization of creatine phosphate was able to be achieved both by separating creatine phosphate from the other reagent components as a second reagent and by adjusting the pH of the second reagent to 8.5. In the second reagent, if the buffer was 25 mM Tris-acetate then when four parts of the first reagent was mixed with one part of the second reagent, the final pH remained unchanged at 6.7. The stability of the working solution with these two separate reagents was examined by storage both at 6°C and at 30°C, and the results are shown in Fig. 1. The CK activity in serum samples was found to be fully recovered when tested with reagents that had been stored for about 30 days at 6°C or about 7 days at 30°C. On the other hand, with the HK-G6PDH method the full CK activity was able to be measured for merely several days even when the reagents were kept at 6°C. It is also clear that no detectable increase in the reagent blank occurred as shown in Fig. 1 and that the lag time was constant (less than about 2 min) for the above periods. Since the two separate reagents described in this paper were confirmed to have long-term stability even in the liquid state, we ex-

J. Cliii. Biochem. Nutr. DETERMINATION OF CREATINE KINASE ACTIVITY WITH GLUCOKINASE 21

(A)

()

Fig. 1. Stability of the reagent in a liquid state following storage at 30°C (A) or 6°C (B~. Stability of the reagents was examined by measuring CK activity in serum. s and c represent the measured CK activity by the G1cK-G6PDH method and the HK-G6PDH method based on the recommendations by the German Society for Clinical Chemistry, respectively. A and A represent the reagent blank measured at 340 nm (OD340) 1n the G1cK-G6PDH method and the HK-G6PDH method, respectively.

Fig. 2. Dependence of the measured value of CK activity on the dilution. The serum sample was diluted by the addition of saline. amined several basic properties of the reagents for measurement of CK activity in serum.

Evaluation o f the reagent for measuring CK activity The linearity of the measured CK activity was examined at the volume fraction of serum sample in the assay mixture of 1/33.3. Figure 2 indicates that the CK activity in serum was able to be accurately measured up to about 2,000 U/

Vol. 3, No. 1, 1987 22 H. KONDO et a1. liter at 37°C. This linear relationship indicates the satisfactory performance of this method and indicates its practical use. Table 1 shows the precision of the measurement at the volume fraction of serum sample in the assay mixture of 1/33.3. The deviation was found to be within the permissible range for practical use, as judged from the coefficient of variation of 2.17% at the normal range of CK activity (mean CK activity of 51.3 U/liter) and 1.11% at the higher CK activity (mean CK activity of 205.1 U/liter). Effect of several coexistents on the measured value of CK activity was examined at the volume fraction of serum sample in the assay mixture of 1/33.3. Glucose (0-1,000 mg/dl), hemoglobin (0-500 mg/dl), bilirubin (0-20 mg/dl), lactate (0-250 mg/dl), reduced form of glutathione (0-25 mg/dl), and pyruvate (0-250 mg/dl) had no effect on the measured value of CK activity. However, the measured CK activity was slightly affected toward a negative deviation by urate (0-25 mg/ dl) and ascorbate (0-25 mg/dl). Thus, in this G1cK-G6PDH method the measured

Table 1. Within-run reproducibility of the G1cK-G6PDH method.

a Standard deviation; b Coefficient of variation.

Table 2. Effect of anticoagulants on the measured value of CK activity.

J. Clin. Bioehem. Nutr. DETERMINATION OF CREATINE KINASE ACTIVITY WITH GLUCOKINASE 23

Fig. 3. Correlation between CK activities as measured by the G1cK-G6PDH method and the HK-G6PDH method. Twenty-nine serum samples were used in this experiment.

CK activity was found to be little affected by several substances that may coexist in the serum. Table 2 represents the effect of anticoagulants on the measured CK activity. EDTA, heparin, and sodium oxalate had no effect on the measured value of CK activity, but sodium citrate and sodium fluoride led to some negative error in the measured value. It was confirmed that the latter two had no effect on the coupling enzymes G1cK and G6PDH. The measured CK activity by the G1cK-G6PDH method was compared with that by the HK-G6PDH method based on the recommendations by the German Society for Clinical Chemistry, both procedures being run at 37°C. Figure 3 illus- trates the excellent agreement between the two methods. The calculated linear regression equation and correlation coefficient were Y=1.008 •X- 3.133 and r- 0.999, respectively.

Usefulness of the GIcK--G6PDH method in the measurement of serum CK activity Although both the single and the two separate reagents have been widely used in the clinical laboratory, the HK-G6PDH method has poor stability in the liquid state during the storage as mentioned above. The greatest advantage of the present GIcK-G6PDH method is the long-term stability of the reagents even in the liquid state both with the single reagent mixture and with the two separate reagents. The stability of the present single reagent was improved by 4-5 times than that obtained by the single reagent based on the HK-G6PDH method. Fur- thermore, the reagent solutions in the case of the two separate reagents were stable for about one month in a refrigerator and about one week at room temperature. This excellent stability seems to be due to mainly both the use of the thermostable G1cK and the improvement of the reagent type. The G1cK-G6PDH method is not

Vol. 3, No. 1, 1987 24 H. KONDO et al. only accurate and reliable, but also little affected by several coexistents in the serum. Thus, it is evident that the GIcK-G6PDH method is obviously useful for clinical determination of serum CK activity.

REFERENCES

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