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Usefulness of bacterial thermostable enzymes in clinical chemical analysis
Stability and Stabilization of Biocatalysts A. Ballesteros, F.J. Plou, J.L. Iborra and P.J. Hailing (Editors) 9 1998 Elsevier Science B.V. All rights reserved.
U s e f u l n e s s of b a c t e r i a l t h e r m o s t a b l e analysis
337
e n z y m e s in clinical chemical
K. Tomita a, K. Nomura ~, T. Miura" and H. Shibata b aCollege of Engineering, Kanto Gakuin University, 4834 Mutsuura, Kanazawa-ku, Yokohama 236-8501, Japan bHead Office, Iatron Laboratories, Inc., 1-11-4 Higashikanda, Chiyoda-ku, Tokyo 101-0031, Japan
Since enzymes have many excellent characteristics, e.g., high substrate and reaction specificities, they have become popular in clinical chemical analysis. However, many of the usual enzymes tend to lose their activities on storage, especially in a solution state, even at rather low temperature, which limits their uses remarkably. On the other hand, enzymes from thermophiles are known to be superior to usual enzymes in thermal and storage stabilities. The author et al. [1] have successfully prepared many thermostable enzymes from a thermophilic bacterium, Bacillus stearothermophilus (BS). Moreover, we have obtained several enzymes from a mesophilic bacterium, Zymomonas mobilis (ZM), and found they also have good thermal stability[2]. In this paper, we show their usefulness as tools in clinical chemical analysis, with focusing the application of two kinds of them, i.e., glucokinase (GlcK, EC 2.7.1.2) and alanine dehydrogenase (AlaDH, EC 1.4.1.1), to the determination of some components in a serum. 1. MATERIAL AND METHODS 1.1. Material Glck was prepared from BS cell or ZM cell. AlaDH was prepared from Escherichia coll C 600 cell carrying the plasmid-encoding BS AlaDH gene [3]. Glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49, from Leuconostoc mesenteroides) was obtained from Boehringer Mannheim GmbH, Mannheim, Germany. The other chemicals were commercial products of analytical grade. 1.2. Methoda The enzymatic activities of GlcK and AlaDH were measured at 30~ on the bases of the change in absorbance at 340 nm due to the formation of NADPH (GlcK coupled with G6PDH) or NADH (AlaDH) according to the usual method.
338
Four kinds of reagents for the determination of the components in biological fluids, mainly sera, were compounded from adequate reactants mentioned in the Sections of 2.3.-2.6. A sample was added to the reagent, incubated at 30~ (glucose determination) or 37~ (the others), and the formation of NADPH or NADH was spectrophotometricaUy measured at 340 nm. 2. RESULTS AND DISCUSSION 2.1. Properties of GlcK The main properties of BS GlcK [4] and ZM GlcK [2] were as follows. The molecular weights were determined to be 68,000 (BS) and 65,000 (ZM), and 32,000 (BS) and 33,000 (ZM) for the subunits. The optimal pHs for the enzymatic activities were 8.5 (BS) and 7.0-8.0 (ZM). BS GlcK was stable in a wide pH range over 8, and ZM GlcK was stable in a pH range from 6.0 to 8.0. Noteworthily, both GlcKs were fully active even after treatment for 60 rain at 60~ (BS) and 50-55~ in the buffer solution, whereas hexokinase (HK, EC 2.7.1.1) from yeast rapidly lost its activity at temperature above 35~ (Figure 1).
100 75 50 9~
25
~
0 0
25
50
75
100
Temperature (~
Figure 1. Thermal stabilities of both GlcKs dissolved in the buffer solution. Comparison with yeast HK. After incubation for 15 rain at different temperatures, the remaining enzymatic actim'ties were measured. 0 : BS GlcK; A: ZM GlcK; m: yeast HK.
Both GlcKs showed no activities when fructose, mannose and glucosamine were used as phosphoryl acceptors. On the other hand, both the mammarian GlcK and the yeast HK were fully active to these monosaccharides. The Km values were
339 determined to be 0.1 mM (BS) and 0.2 mM (ZM) for glucose and 0.05 mM (BS) and 0.5 mM (ZM) for ATP, being much lower than those of mammarian GlcK and the yeast HK. 2.2. Properties of AlaDH The molecular weight was determined to be 230,000, and 38,000 for the subunit. The optimal pH for the enzymatic activity was 10.4. AlaDH was stable over a wide pH range above 7, moreover, over a wide temperature range. AlaDH was fully active even after treatment for 60 rain at 70~ in the buffer solution. AlaDH showed no activity when L-leucine, L-isoleucine and L-valine were used as hydrogen donors. The Km values were determined to be 10.0 mM for L-alanine and 0.26 mM for NAD § These properties were similar to other AlaDH from mesophilic Bacillus sp., except for pH and thermal stabilities.
2.3. Determination of glucose In clinical chemical analysis, the determination of glucose is a very important factor for the diagnosis of diabetes mellitus. Although the method using HK as the key enzyme (HK method) is recommended as the reference method for glucose determination by several organizations, e.g., American Association for Clinical Chemistry[5], the properties of HK are much inferior to those of GlcK as described above. The application of BS GlcK to the glucose determination (GlcK method) [6] was
1t,511tl
2500 N 211110 o 15110
5OO
0
10
20
30
40
Time(day) Figure 2. Storage stability of the reagent solution for the determination of glucose. Comparison of GlcK method and HK method. After storage for several ten days at room temperature, each reagent solution was used for the determination of glucose in a serum sample. O: GlcK method; m: HK method.
340 attempted according to the principle 9 Glucose is phosphorylated by GlcK in the presence of ATP to yield glucose 6-phosphate (G6P); G6P is converted to 6phosphogluconate (6PG) by G6PDH in the presence of NADP, which results in the formation of NADPH; finally, glucose is determined as the change in absorbance at 340 nm. The assay had a high degree of precision, i.e., coefficient of variation (CV) was 0.7-1.3% (within-run reproducibility) and 1.1-2.3% (between-run reproducibility ), and was linear up to about 8000 mg 1"1. A good correlation was observed between GleK method and HK method for both plasma and urine samples. The outstanding superiority of GlcK method to HK method was the storage stability of the reagent solution. The solution based on GlcK method was found to be stable at least for about one month at room temperature (Figure 2) [7] and 21 months at 10~ [8] , whereas that based on HK method was stable for only a few days at room temperature (Figure 2) [7]. ZM GlcK was also applied to glucose determination [2], and gave as excellent results as BS GlcK. As for the stability of the reagent solution, ZM GlcK gave a little inferior results to BS GlcK, but much superior to HK. 2.4. Determination of creatine kin~ae activity The determination of creatine kinase (CK, EC 2.7.3.2 ) activity is much in use for the diagnosis of myocardial infarction or progressive muscular dystrophy. The current method based on using HK (HK method) involves the same disadvantage in the stability of the reagent solution as that shown above. The new method [9] was also developed by using BS GlcK based on the principle: CK acts on creatine phosphate in the presence of ADP to yield ATP; followed by two reactions coupled with GlcK and G6PD, respectively, ATP causes a formation of NADH. The assay had a high degree of precision, i.e., CV was 1.12.2% (within-run reproducibility), and was linear up to about 2000 U 1-'. It was noteworthy that the reagent solution was found to be stable at least for one week at room temperature and for 13 months at 10 ~ [8]. The conventional HK method gave only one day stability at room temperature. 2.5. Determination of magnesium ion The determination of magnesium ion is a matter of significance for the diagnosis of renal diseases and gastrointestinal disorders. The colorimetric method, in which Xylidyl Blue is utilized as a detector of magnesium ion, is currently used, but this method has a defect in specificity. The atomic absorption spectrophotometry is looked upon as the most reliable method, but requires an expensive instrument. The reagent for magnesium ion determination [10] was successftflly composed with using GlcK on the basis of the paper of Tabata et a]. [11]. The principle of the assay was based on the fact that magnesium ion-ATP complex acts as a substrate of GlcK, as shown in Figure 3. The concentration of magnesium ion is then determined as an increasing rate in absorbance at 340 nm due to NADPH turnover. This simple and rapid spectrophotometric method was found to have a good correlation with the atomic
341 absorption spectrophotometry and to be very little affected by several cations in a serum such as K § Na § Zn s+, Ca s§ NH4+ and so on. The present method was recognized as to be more specific to magnesium ion than the Xylidyl Blue method. The assay had a high degree of precision, i.e., CV was 0.85-1.7% (within-run reproducibility) and 1.25-2.15% (between-run reproducibility), and was linear up to about 100 mg 1-'. This linearity range was considerably wider than that of the Xylidyl Blue method. The present reagent had an excellent stability at least for about one month at 10~ in liquid form. It should be emphasized that this method is the world's first practical enzymatic method for measuring metal ion. GlcK Glucose
~
Mg- ATP ATP
~
G6P M g - ADP Mga+ ~
""~ADP
G6PDH G6P + NADP +
6PG + NADPH + H §
Figure 3. The schematic principle of GlcK method for the determination of magnesium ion.
2.6. Determination of,/-glu~myltransferase activity Determination of~/-glutamyltransferase (v .GT, EC 2.3.2.2) activity is routinely employed in the diagnosis of hepatic diseases. The colorimetrlc method, in which ~/-glutamyl 4-nitroanilide (,/-Glu4NA) is utilized as a substrate for ,/-glutamyl donor, is most commonly used in current, but ~/-Glu4NA has a serious problem, i.e., poor solubility in water. The present method [12] was ultraviolet spectrophotometric one, in which the readily soluble substrate ~/-glutamyl dipeptide was used as the ~/-glutamyl donor and thermostable amino acid dehydrogenase as the attxiliary enzyme. In the preliminary experiment, ~/-glutamylalanine (~/-GluAla) was found to be the most specific and sensitive donor substrate. This method is consisted of two enzymatic reactions: First, ~/-GluAla releases L-alanine by the action of~/-GT in the presence of glycylglycine(GlyGly); second, L-alanine is converted to pyruvate by AlaDH in the presence of NAD, accompanied by NADH formation. NADH is continuously monitored at 340 nm. The present method is strictly stoichiometric, since one NADH is produced from one ~/-GluAla by every ~/-GT action. The assay had a high degree of precision, i.e., CV was 0.8-2.8% (within-run reproducibility) and about 2.2% (between-run reproducibility), and was linear up to about 700 U 1-1. The present method well correlated to the ,/-Glu4NA method. Although this method had about one-third
342 of the sensitivity to the T -Glu4NA method, it was adequate for the practical assay. The storage of the reagent solution did not affect the measuring activity at least over about 30 days at 10~ 3. CONCLUSIONS The bacterial thermostable enzymes, GlcK and AlaDH, had high degrees of stability, especially thermal and storage stab'dities, which made them useful in clinical chemical analysis. The other properties were also well suited for the analysis, i.e., low Km values, high substrate specificities and so on. These enzymes were successfully applied to the establishment of new enzymatic methods which gave long term stability of the reagent solutions as well as rapid, accurate and reliable results.
REFERENCES 1. K.Tomita, K.Nagata and K.Okada, Ann. N.Y. Acad. Sci., 672 (1992) 178. 2. K.Tomita and K.Nomura, Ann. N.Y. Acad. Sci., 750 (1995) 338. 3. Y.Sakamoto, S.Nagata, N.Esaki. H.Tanaka and K.Soda, J. Ferment. Bioeng., 69 (1990) 154. 4. S.Kamei, K.Tomita, K.Nagata, H.Okuno, T.Shiraishi, A.Motoyama, A. Ohkubo and M.Yamanaka, J. Clin. Biochem. Nutr., 3 (1987)1. 5. P.Duncan, J.Neese, D.Bayse and C.Stewart, Clin. Chem., 20 (1974) 882. 6. K.Tomita, S.Kamei, K.Nagata, H.Okuno, T.Shiraishi, A.Motoyama, A. Ohkubo and M.Yamanaka, J. Clin. Biochem. Nutr,. 3 (1987) 11. 7. K.Tomita, K.Nagata, H.Kondo, T.Shiraishi, H, Tsubota, H.Suzuki and H.Ochi, Ann. N. Y. Acad. Sci., 613 (1990) 421. 8. K.Tomita, K.Nomura, H.Kondo, K.Nagata and H.Tsubota, J. Pharm.Biomed. Anal., 13 (1995) 477. 9. H.Kondo, T.Shiraishi, M.Kageyama, K.Nagata and K.Tomita, J.Clin.Biochem. Nutr., 3 (1987) 17 10. T. Shiraishi, H. Suzuki, H.Ochi, K. Kawahara, H. Kondo, K. Nagata and K.Tomita, Jap. J. Clin. Chem., (in Japanese) 20 (1991) 37. 11. M.Tabata, T.Kido, M.Totani and T.Murachi, Agric. Biol. Chem., 50 (1986) 1909. 12. H.Kondo, M.Hashimoto, K.Nagata, K.Tomita and H.Tsubota, Clin. Chim. Acta, 207 (1992) 1.
U s e f u l n e s s of b a c t e r i a l t h e r m o s t a b l e analysis
337
e n z y m e s in clinical chemical
K. Tomita a, K. Nomura ~, T. Miura" and H. Shibata b aCollege of Engineering, Kanto Gakuin University, 4834 Mutsuura, Kanazawa-ku, Yokohama 236-8501, Japan bHead Office, Iatron Laboratories, Inc., 1-11-4 Higashikanda, Chiyoda-ku, Tokyo 101-0031, Japan
Since enzymes have many excellent characteristics, e.g., high substrate and reaction specificities, they have become popular in clinical chemical analysis. However, many of the usual enzymes tend to lose their activities on storage, especially in a solution state, even at rather low temperature, which limits their uses remarkably. On the other hand, enzymes from thermophiles are known to be superior to usual enzymes in thermal and storage stabilities. The author et al. [1] have successfully prepared many thermostable enzymes from a thermophilic bacterium, Bacillus stearothermophilus (BS). Moreover, we have obtained several enzymes from a mesophilic bacterium, Zymomonas mobilis (ZM), and found they also have good thermal stability[2]. In this paper, we show their usefulness as tools in clinical chemical analysis, with focusing the application of two kinds of them, i.e., glucokinase (GlcK, EC 2.7.1.2) and alanine dehydrogenase (AlaDH, EC 1.4.1.1), to the determination of some components in a serum. 1. MATERIAL AND METHODS 1.1. Material Glck was prepared from BS cell or ZM cell. AlaDH was prepared from Escherichia coll C 600 cell carrying the plasmid-encoding BS AlaDH gene [3]. Glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49, from Leuconostoc mesenteroides) was obtained from Boehringer Mannheim GmbH, Mannheim, Germany. The other chemicals were commercial products of analytical grade. 1.2. Methoda The enzymatic activities of GlcK and AlaDH were measured at 30~ on the bases of the change in absorbance at 340 nm due to the formation of NADPH (GlcK coupled with G6PDH) or NADH (AlaDH) according to the usual method.
338
Four kinds of reagents for the determination of the components in biological fluids, mainly sera, were compounded from adequate reactants mentioned in the Sections of 2.3.-2.6. A sample was added to the reagent, incubated at 30~ (glucose determination) or 37~ (the others), and the formation of NADPH or NADH was spectrophotometricaUy measured at 340 nm. 2. RESULTS AND DISCUSSION 2.1. Properties of GlcK The main properties of BS GlcK [4] and ZM GlcK [2] were as follows. The molecular weights were determined to be 68,000 (BS) and 65,000 (ZM), and 32,000 (BS) and 33,000 (ZM) for the subunits. The optimal pHs for the enzymatic activities were 8.5 (BS) and 7.0-8.0 (ZM). BS GlcK was stable in a wide pH range over 8, and ZM GlcK was stable in a pH range from 6.0 to 8.0. Noteworthily, both GlcKs were fully active even after treatment for 60 rain at 60~ (BS) and 50-55~ in the buffer solution, whereas hexokinase (HK, EC 2.7.1.1) from yeast rapidly lost its activity at temperature above 35~ (Figure 1).
100 75 50 9~
25
~
0 0
25
50
75
100
Temperature (~
Figure 1. Thermal stabilities of both GlcKs dissolved in the buffer solution. Comparison with yeast HK. After incubation for 15 rain at different temperatures, the remaining enzymatic actim'ties were measured. 0 : BS GlcK; A: ZM GlcK; m: yeast HK.
Both GlcKs showed no activities when fructose, mannose and glucosamine were used as phosphoryl acceptors. On the other hand, both the mammarian GlcK and the yeast HK were fully active to these monosaccharides. The Km values were
339 determined to be 0.1 mM (BS) and 0.2 mM (ZM) for glucose and 0.05 mM (BS) and 0.5 mM (ZM) for ATP, being much lower than those of mammarian GlcK and the yeast HK. 2.2. Properties of AlaDH The molecular weight was determined to be 230,000, and 38,000 for the subunit. The optimal pH for the enzymatic activity was 10.4. AlaDH was stable over a wide pH range above 7, moreover, over a wide temperature range. AlaDH was fully active even after treatment for 60 rain at 70~ in the buffer solution. AlaDH showed no activity when L-leucine, L-isoleucine and L-valine were used as hydrogen donors. The Km values were determined to be 10.0 mM for L-alanine and 0.26 mM for NAD § These properties were similar to other AlaDH from mesophilic Bacillus sp., except for pH and thermal stabilities.
2.3. Determination of glucose In clinical chemical analysis, the determination of glucose is a very important factor for the diagnosis of diabetes mellitus. Although the method using HK as the key enzyme (HK method) is recommended as the reference method for glucose determination by several organizations, e.g., American Association for Clinical Chemistry[5], the properties of HK are much inferior to those of GlcK as described above. The application of BS GlcK to the glucose determination (GlcK method) [6] was
1t,511tl
2500 N 211110 o 15110
5OO
0
10
20
30
40
Time(day) Figure 2. Storage stability of the reagent solution for the determination of glucose. Comparison of GlcK method and HK method. After storage for several ten days at room temperature, each reagent solution was used for the determination of glucose in a serum sample. O: GlcK method; m: HK method.
340 attempted according to the principle 9 Glucose is phosphorylated by GlcK in the presence of ATP to yield glucose 6-phosphate (G6P); G6P is converted to 6phosphogluconate (6PG) by G6PDH in the presence of NADP, which results in the formation of NADPH; finally, glucose is determined as the change in absorbance at 340 nm. The assay had a high degree of precision, i.e., coefficient of variation (CV) was 0.7-1.3% (within-run reproducibility) and 1.1-2.3% (between-run reproducibility ), and was linear up to about 8000 mg 1"1. A good correlation was observed between GleK method and HK method for both plasma and urine samples. The outstanding superiority of GlcK method to HK method was the storage stability of the reagent solution. The solution based on GlcK method was found to be stable at least for about one month at room temperature (Figure 2) [7] and 21 months at 10~ [8] , whereas that based on HK method was stable for only a few days at room temperature (Figure 2) [7]. ZM GlcK was also applied to glucose determination [2], and gave as excellent results as BS GlcK. As for the stability of the reagent solution, ZM GlcK gave a little inferior results to BS GlcK, but much superior to HK. 2.4. Determination of creatine kin~ae activity The determination of creatine kinase (CK, EC 2.7.3.2 ) activity is much in use for the diagnosis of myocardial infarction or progressive muscular dystrophy. The current method based on using HK (HK method) involves the same disadvantage in the stability of the reagent solution as that shown above. The new method [9] was also developed by using BS GlcK based on the principle: CK acts on creatine phosphate in the presence of ADP to yield ATP; followed by two reactions coupled with GlcK and G6PD, respectively, ATP causes a formation of NADH. The assay had a high degree of precision, i.e., CV was 1.12.2% (within-run reproducibility), and was linear up to about 2000 U 1-'. It was noteworthy that the reagent solution was found to be stable at least for one week at room temperature and for 13 months at 10 ~ [8]. The conventional HK method gave only one day stability at room temperature. 2.5. Determination of magnesium ion The determination of magnesium ion is a matter of significance for the diagnosis of renal diseases and gastrointestinal disorders. The colorimetric method, in which Xylidyl Blue is utilized as a detector of magnesium ion, is currently used, but this method has a defect in specificity. The atomic absorption spectrophotometry is looked upon as the most reliable method, but requires an expensive instrument. The reagent for magnesium ion determination [10] was successftflly composed with using GlcK on the basis of the paper of Tabata et a]. [11]. The principle of the assay was based on the fact that magnesium ion-ATP complex acts as a substrate of GlcK, as shown in Figure 3. The concentration of magnesium ion is then determined as an increasing rate in absorbance at 340 nm due to NADPH turnover. This simple and rapid spectrophotometric method was found to have a good correlation with the atomic
341 absorption spectrophotometry and to be very little affected by several cations in a serum such as K § Na § Zn s+, Ca s§ NH4+ and so on. The present method was recognized as to be more specific to magnesium ion than the Xylidyl Blue method. The assay had a high degree of precision, i.e., CV was 0.85-1.7% (within-run reproducibility) and 1.25-2.15% (between-run reproducibility), and was linear up to about 100 mg 1-'. This linearity range was considerably wider than that of the Xylidyl Blue method. The present reagent had an excellent stability at least for about one month at 10~ in liquid form. It should be emphasized that this method is the world's first practical enzymatic method for measuring metal ion. GlcK Glucose
~
Mg- ATP ATP
~
G6P M g - ADP Mga+ ~
""~ADP
G6PDH G6P + NADP +
6PG + NADPH + H §
Figure 3. The schematic principle of GlcK method for the determination of magnesium ion.
2.6. Determination of,/-glu~myltransferase activity Determination of~/-glutamyltransferase (v .GT, EC 2.3.2.2) activity is routinely employed in the diagnosis of hepatic diseases. The colorimetrlc method, in which ~/-glutamyl 4-nitroanilide (,/-Glu4NA) is utilized as a substrate for ,/-glutamyl donor, is most commonly used in current, but ~/-Glu4NA has a serious problem, i.e., poor solubility in water. The present method [12] was ultraviolet spectrophotometric one, in which the readily soluble substrate ~/-glutamyl dipeptide was used as the ~/-glutamyl donor and thermostable amino acid dehydrogenase as the attxiliary enzyme. In the preliminary experiment, ~/-glutamylalanine (~/-GluAla) was found to be the most specific and sensitive donor substrate. This method is consisted of two enzymatic reactions: First, ~/-GluAla releases L-alanine by the action of~/-GT in the presence of glycylglycine(GlyGly); second, L-alanine is converted to pyruvate by AlaDH in the presence of NAD, accompanied by NADH formation. NADH is continuously monitored at 340 nm. The present method is strictly stoichiometric, since one NADH is produced from one ~/-GluAla by every ~/-GT action. The assay had a high degree of precision, i.e., CV was 0.8-2.8% (within-run reproducibility) and about 2.2% (between-run reproducibility), and was linear up to about 700 U 1-1. The present method well correlated to the ,/-Glu4NA method. Although this method had about one-third
342 of the sensitivity to the T -Glu4NA method, it was adequate for the practical assay. The storage of the reagent solution did not affect the measuring activity at least over about 30 days at 10~ 3. CONCLUSIONS The bacterial thermostable enzymes, GlcK and AlaDH, had high degrees of stability, especially thermal and storage stab'dities, which made them useful in clinical chemical analysis. The other properties were also well suited for the analysis, i.e., low Km values, high substrate specificities and so on. These enzymes were successfully applied to the establishment of new enzymatic methods which gave long term stability of the reagent solutions as well as rapid, accurate and reliable results.
REFERENCES 1. K.Tomita, K.Nagata and K.Okada, Ann. N.Y. Acad. Sci., 672 (1992) 178. 2. K.Tomita and K.Nomura, Ann. N.Y. Acad. Sci., 750 (1995) 338. 3. Y.Sakamoto, S.Nagata, N.Esaki. H.Tanaka and K.Soda, J. Ferment. Bioeng., 69 (1990) 154. 4. S.Kamei, K.Tomita, K.Nagata, H.Okuno, T.Shiraishi, A.Motoyama, A. Ohkubo and M.Yamanaka, J. Clin. Biochem. Nutr., 3 (1987)1. 5. P.Duncan, J.Neese, D.Bayse and C.Stewart, Clin. Chem., 20 (1974) 882. 6. K.Tomita, S.Kamei, K.Nagata, H.Okuno, T.Shiraishi, A.Motoyama, A. Ohkubo and M.Yamanaka, J. Clin. Biochem. Nutr,. 3 (1987) 11. 7. K.Tomita, K.Nagata, H.Kondo, T.Shiraishi, H, Tsubota, H.Suzuki and H.Ochi, Ann. N. Y. Acad. Sci., 613 (1990) 421. 8. K.Tomita, K.Nomura, H.Kondo, K.Nagata and H.Tsubota, J. Pharm.Biomed. Anal., 13 (1995) 477. 9. H.Kondo, T.Shiraishi, M.Kageyama, K.Nagata and K.Tomita, J.Clin.Biochem. Nutr., 3 (1987) 17 10. T. Shiraishi, H. Suzuki, H.Ochi, K. Kawahara, H. Kondo, K. Nagata and K.Tomita, Jap. J. Clin. Chem., (in Japanese) 20 (1991) 37. 11. M.Tabata, T.Kido, M.Totani and T.Murachi, Agric. Biol. Chem., 50 (1986) 1909. 12. H.Kondo, M.Hashimoto, K.Nagata, K.Tomita and H.Tsubota, Clin. Chim. Acta, 207 (1992) 1.