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The use of N-ethylmaleimide m the colorimetric assay of activated creatine kinase with a tetrazolium dye
Clinica Chimica Acta, 130 (1983) 245-250 Elsevier
245
CCA 2491
Brief technical
note
The use of N-ethylmaleimide in the calorimetric assay of activated creatine kinase with a tetrazolium
Jaroslav Department
Racek
of Cltnical Biochemistry,
* and Pave1 Slabjr
Faculty Hospital, 305 99 Plzeti (Czechoslovakia)
(Received September 17th. 1982; revision January 17th. 1983)
Creatine kinase (ATP : creatine N-phosphotransferase, EC 2.7.3.2) is a commonly used enzyme for diagnosis and monitoring of acute myocardial infarction. When assaying its activity in serum the method of Rosalki is usually used ([l] Table I, eq. a-c). Reduction of NADP+ to NADPH can be followed spectrophotometrically by observing the increase of absorbance at 340 nm. Calorimetric estimation in visible light using the reducing effect of NADPH hitherto has been difficult because of interference by the strong reducing agents added to the assay in order to activate creatine kinase. Our work overcomes this problem. Materials and methods The serum creatine kinase activity was determined in 42 patients with myocardial infarction. Each serum was tested by the standard ultraviolet assay and the colorimetric method developed by us. (A) Standard method The ultraviolet kinetic
test kit ‘Spinchem’
CK Reagent
(Smith Kline Instruments,
* To whom correspondence should be addressed. Abbreoiations: ADP, adenosine-5’-diphosphate; ATP, adenosine-5’-triphosphate; CK, creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2); G-6-PD, glucose-6-phosphate dehydrogenase (D-ghcase-6-phosphate : NADP 1-oxidoreductase, EC 1.1.1.49);HK, hexokinase (ATP : D-hexose 6-phosphotransferase, EC 2.7.1 .I); NAD+, nicotinamide-adenine dinucleotide; NADH, the reduced form of NAD+; NADP+, nicotinamide-adenine dinucleotide phosphate; NADPH, the reduced form of NADP+; NBT, 2,2’-di( p-nitrophenyl)-5,5’-diphenyl-3,3’-(3,3’-dimethoxy-4,~-diphenylene)ditetr~o~um chloride (nitro blue tetrazolium); NEM, N-ethylmaleimide; PMS, N-methylphenazinium methyl sulphate (phenazine methosulphate); PMSH,, the reduced form of PMS; R-SH, a compound with an active sulphhydryl group; UV, ultraviolet.
0009-8981/83/$03.00
0 1983 Elsevier Science Publishers B.V.
246 TABLE 1 REACTIONS ACTIVITY
TAKING
PLACE
(a) creatine phosphate + ADP
DURING
CK
*
HK
--*
(b) ATP + glucose
DETERMINATION
OF THE
CREATINE
KINASE
creatine + ATP ADP + glucose-6-phosphate
(c) glucose-6-phosphate t NADP+ G-~pD 6-phosphogluconate + NADPH + H+ (d) NADPH + H + + PMS + (e) PMSH, +NBT -+
NADP+ + PMSH > formazan + PMS
+
(f) R-SH+ NEM (8) 2 R-SH + NBT
-+
formazan + R-S-S-R
Sunnyvale, CA 94086, USA) was used. The composition of the reagent solution was as follows: creatine phosphate 52 mmol/l, ADP 2 mmol/l, magnesium aspartate 9 mmol/l, adenosine monophosphate 6 mmol/l, NADP+ 2 mmol/l, glucose 19 mmol/l, glutathione 28 mmol/l, HK 500 pkat/l, G-6-PD 167 pkat/l, tris(hydroxymethyl)aminomethane buffer 44 mmol/l, pH 6.8. Test procedure 1.0 ml of reagent solution was pipetted into each cuvette and preincubated to 37°C. Then 20 ~1 of sample (serum) was added; a timer was started. The absorbance was measured during incubation at 37’C exactly at 2.5, 3.5 and 4.5 min after the initiation of the reaction. An ‘Eskalab’ spectrophotometer Alpha (Smith Kline Instruments) set at 340 nm was used. The absorbance change in 1 min was multiplied by a factor 137 to obtain creatine kinase activity in pkat/l. (B) Calorimetric
method developed by us
Solutions and their stability Solution 1: ‘Spinchem’ CK Reagent (SKI) was reconstituted as specified by the manufacturer; the composition of this solution did not differ from that used in the standard UV method. Solution 2: 0.16 mol/l N-ethylmaleimide (Calbiochem, Los Angeles, CA, USA): 56 mg NEM was dissolved in 2.8 ml distilled water. The solution is stable at 25°C for 2 weeks. Solution 3: 4.0 mmol/l nitro blue tetrazolium (Lachema Brno, Czechoslovakia), 1.6 mmol/l phenazine methosulphate (Lachema Brno): 16.2 mg NBT was dissolved in 5.0 ml distilled water. This solution is transferred to 2.5 mg PMS just before use. The final solution must be protected from light, and is stable at 4’C for 24 h; the decomposition of PMS can be recognized by a colour change from yellow to green. Solution 4: 0.1 mol/l hydrochloric acid.
247
Test procedure 500 ~1 of Solution 1 was pipetted into a cuvette and time allowed for the cuvette contents to reach 37°C. Then 10 ~1 of serum was added and mixed and incubated for 10 min exactly. The reaction was stopped with 100 ~1 of Solution 2 which simultaneously inhibited the rest of the CK-activator (glutathione). 200 ~1 of Solution 3 was added after at least 1 min. The colour development was stopped after 5 min with 1000 ~1 of Solution 4. The absorbance of the sample was read at 530 nm against distilled water as the blank by an ‘Eskalab’ spectrophotometer Alpha. The colour of the reaction product is stable for at least 2 h. Reagent blank (10 ~1 of 155 mmol/l sodium chloride instead of serum) and standard control serum with a declared value of CK activity was measured with each series of assays. We used the lyophilized control serum MONI-TROL@ I-E (Merz + Dade AG, Dtidingen, Switzerland) with a creatine kinase activity of about 1.6 pkat/l, i.e. near the upper limit of reference values. The CK activity in the sample was calculated as follows: a,(pkat/l)
* a,(pkat/l)
= $$-$ 5
0
absorbance; A,, reagent blank abwere: A,, sample absorbance; A,, standard sorbance; a,, CK activity in the sample; and a,, CK activity in the standard control serum. Results (1) Precision (a) Within-day precision was calculated from 25 replicate measurements on a pooled serum. The mean value of CK activity was 1.25 pkat/l, and the coefficient of variation was 6.5%. (b) Day-to-day precision was calculated from the data on a pooled serum over a 20-day period. The mean value of CK activity was found to be 1.28 p kat/l, and the coefficient of variation 7.9%. (2) Accuracy The CK activities in the control sera with declared values were measured in each batch of assays for 20 days. The following control sera were used: MONI-TROL@ II, ENZA-TROL@ (Merz + Dade AG), Precinorm@ E, Precipatha E (Boehringer Mannheim, FRG) Q-PAK I, Q-PAK II (Hyland, Div. Travenol Laboratories S.A., Lessines, Belgium). The mean coefficient of bias was 5.3%. (3) Sensitivity (a) Sensitivity was determined as the difference of absorbance per pkat/l of enzyme activity. The estimated value was 0.057. (b) The low limit of detection was established as the smallest CK activity that can be differentiated from the blank at a significance level of 5%. This value of enzyme activity was found to be 0.2 pkat/l.
248
(4) Linearity Increase of absorbance is linear to an activity of 15 pkat/l. necessary to reduce the sample size or the time of incubation.
Above this value, it is
(5) Znfluence of hemolysis Moderate hemolysis does not effect the test, but severely hemolyzed produce a false elevation of measured CK activity.
samples
may
(6) Specificity The specificity of the calorimetric test is not different from that of UV method because the first step is identical. The colour development could be probably influenced by some reducing agents present occasionally in serum. Therefore we tested the influence of some reducing substances which may be observed in serum in significant concentrations. Glucose, uric acid and ketones (acetone and acetoacetic acid) are not able to reduce tetrazolium salts. Ascorbic acid, however, can convert these chromogens into coloured formazans. We have found that the absorbance elevation due to the presence of ascorbic acid in serum is very small: viz. 0.0016 for an ascorbic acid concentration of 68 pmol/l, a value rarely exceeded in serum. This elevation causes a positive error of 2.86% at a creatine kinase activity of 1.0 pkat/l and 0.29% at 10.0 pkat/l. These changes (more often even smaller because of the lower ascorbic acid level in serum of most people) have no clinical significance. Therefore it is not necessary to use a serum blank and only a reagent blank is required. (7) Correlation between CK activity measured by the calorimetric Fig. 1 compares the results obtained by both the methods.
5
Fig. 1. Comparison
10
and UV methods Agreement is good.
15 I~katlll
of the calorimetric
with the
uv
method
of measuring
CK activity.
249
Least-squares regression analysis of 42 samples showed the slope of the line to be 1.13 1 which indicates a proportional error of 13.1%. From the value of the intercept, a constant error is estimated at -0.225 pkat/l. The correlation coefficient is 0.985. Thus, for a value of 10.0 pkat/l in UV test, the calorimetric test would give an average value of 11.1 pkat/l. At an activity of 1.72 pkat/l both methods give the same results. Discussion The conversion of NAD+ to NADH (resp. NADP+ to NADPH) and vice versa is a commonly used reaction when determining the enzyme activities or substrate concentrations in UV-spectrophotometry. NADH as a reducing agent reacts with tetrazolium salts and the development of the coloured formazan can be measured in visible light. This principle has been applied to aminotransferases [2,3], various dehydrogenases [3-81, glucose and some other substances [9- 111, but not activated CK, because of the strong reducing agents used as enzyme activators. Free sulphhydryl groups ( - SH) of these activators also cause reduction of tetrazolium salts (Table I, eq. g), and thus interfere in this test by increasing the results. Therefore, it is necessary to remove the unconsumed activator after the reactions a-c, Table I. We used N-ethylmaleimide. This compound reacts additionally with the SH-groups of both the activator and enzyme (Table I, eq. I). The following rapid inactivation of all the SH-groups removes the activator and stops the enzyme reaction (Table I, eq. a). After this step the colour reagent can be added. The product of the subsequent reactions (Table I, eq. d and e) is the coloured formazan, the concentration of which is proportional to CK activity. Thus all compounds with SH-groups can be inactivated [12] and N-ethylmaleimide can be used for removing all the commonly used CK-activators (glutathione, N-acetylcysteine, dithiothreitol etc.). The same approach could be adopted for CK-isoenzymes assay based on both the immunochemical and electrophoretic principles. Besides nitro blue tetrazolium, other tetrazolium salts may be used [13]. Phenazine methosulphate, as an electron mediator between NADPH and the tetrazolium dye, can be substituted by I-methoxy-phenazine methosulphate which appears more photochemically stable [8]. The use of the method described is convenient for the clinical CK activity assay, especially in small laboratories without an UV-spectrophotometer. References 1 Rosalki SB. An improved procedure for serum creatine phosphokinase determinations. J Lab Clin Med 1967; 69: 696-701. 2 Lippi U, Guid G. A new calorimetric ultramicromethod for serum glutamic-oxalacetic and glutamicpyruvic transaminase determination. Clin Chim Acta 1970; 28: 43 l-437. 3 Whitaker JF. A general calorimetric procedure for the estimation of enzymes which are linked to the NADH/NAD+ system. Clin Chim Acta 1969; 24: 23-37.
250 4 Babson AL, Phillips GE. A rapid calorimetric assay for serum lactic dehydrogenase. Clin Chim Acta 1965; 12: 210-215. 5 Bergmeyer HN. Methoden der enzymatischen Analyse, 2nd ed, Vol. 1. Berlin: Akademie Verlag, 1970: 538. 6 Van Der Helm VJ. A simplified method of demonstrating lactic dehydrogenase isoenzymes in serum. Clin Chim Acta 1962; 7: 124-128. I Nachlas MM, Margulies SI, Goldberg JD, Seligman AM. The determination of lactic dehydrogenase with a tetrazolium salt. Anal Biochem 1960; 1: 317-326. 8 Nakamura S, Arimura K, Ogawa K, Yagi T. Use of I-methoxy-5-methylphenazinium methyl sulfate ( I-methoxyPMS) in the assay of some enzymes of diagnostic importance. Clin Chim Acta 1980; 101: 321-326. 9 Carroll JJ, Smith N, Babson AL. A calorimetric serum glucose determination using hexokinase and glucose-6-phosphate dehydrogenase. Biochem Med 1970; 4: 17 I- 180. 10 Coburn HJ, Carroll JJ. Improved manual and automated calorimetric determination of serum glucose with use of hexokinase and glucose-6-phosphate dehydrogenase. Clin Chem 1973; 19: 127- 130. 11 Wright WR, Rainwater JC, Tolle LD. Glucose assays systems: Evaluation of a calorimetric hexokinase procedure. Clin Chem 1971; 17: 1010-1015. 12 Okamura M. An improved method for determination of L-ascorbic acid and L-dehydroascorbic acid in blood plasma. Clin Chim Acta 1980; 103: 259-268. 13 Burlina A. Improved electrophoretic separation of creatine kinase isoenzymes. Clin Chem 1980; 26: 317-320.
245
CCA 2491
Brief technical
note
The use of N-ethylmaleimide in the calorimetric assay of activated creatine kinase with a tetrazolium
Jaroslav Department
Racek
of Cltnical Biochemistry,
* and Pave1 Slabjr
Faculty Hospital, 305 99 Plzeti (Czechoslovakia)
(Received September 17th. 1982; revision January 17th. 1983)
Creatine kinase (ATP : creatine N-phosphotransferase, EC 2.7.3.2) is a commonly used enzyme for diagnosis and monitoring of acute myocardial infarction. When assaying its activity in serum the method of Rosalki is usually used ([l] Table I, eq. a-c). Reduction of NADP+ to NADPH can be followed spectrophotometrically by observing the increase of absorbance at 340 nm. Calorimetric estimation in visible light using the reducing effect of NADPH hitherto has been difficult because of interference by the strong reducing agents added to the assay in order to activate creatine kinase. Our work overcomes this problem. Materials and methods The serum creatine kinase activity was determined in 42 patients with myocardial infarction. Each serum was tested by the standard ultraviolet assay and the colorimetric method developed by us. (A) Standard method The ultraviolet kinetic
test kit ‘Spinchem’
CK Reagent
(Smith Kline Instruments,
* To whom correspondence should be addressed. Abbreoiations: ADP, adenosine-5’-diphosphate; ATP, adenosine-5’-triphosphate; CK, creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2); G-6-PD, glucose-6-phosphate dehydrogenase (D-ghcase-6-phosphate : NADP 1-oxidoreductase, EC 1.1.1.49);HK, hexokinase (ATP : D-hexose 6-phosphotransferase, EC 2.7.1 .I); NAD+, nicotinamide-adenine dinucleotide; NADH, the reduced form of NAD+; NADP+, nicotinamide-adenine dinucleotide phosphate; NADPH, the reduced form of NADP+; NBT, 2,2’-di( p-nitrophenyl)-5,5’-diphenyl-3,3’-(3,3’-dimethoxy-4,~-diphenylene)ditetr~o~um chloride (nitro blue tetrazolium); NEM, N-ethylmaleimide; PMS, N-methylphenazinium methyl sulphate (phenazine methosulphate); PMSH,, the reduced form of PMS; R-SH, a compound with an active sulphhydryl group; UV, ultraviolet.
0009-8981/83/$03.00
0 1983 Elsevier Science Publishers B.V.
246 TABLE 1 REACTIONS ACTIVITY
TAKING
PLACE
(a) creatine phosphate + ADP
DURING
CK
*
HK
--*
(b) ATP + glucose
DETERMINATION
OF THE
CREATINE
KINASE
creatine + ATP ADP + glucose-6-phosphate
(c) glucose-6-phosphate t NADP+ G-~pD 6-phosphogluconate + NADPH + H+ (d) NADPH + H + + PMS + (e) PMSH, +NBT -+
NADP+ + PMSH > formazan + PMS
+
(f) R-SH+ NEM (8) 2 R-SH + NBT
-+
formazan + R-S-S-R
Sunnyvale, CA 94086, USA) was used. The composition of the reagent solution was as follows: creatine phosphate 52 mmol/l, ADP 2 mmol/l, magnesium aspartate 9 mmol/l, adenosine monophosphate 6 mmol/l, NADP+ 2 mmol/l, glucose 19 mmol/l, glutathione 28 mmol/l, HK 500 pkat/l, G-6-PD 167 pkat/l, tris(hydroxymethyl)aminomethane buffer 44 mmol/l, pH 6.8. Test procedure 1.0 ml of reagent solution was pipetted into each cuvette and preincubated to 37°C. Then 20 ~1 of sample (serum) was added; a timer was started. The absorbance was measured during incubation at 37’C exactly at 2.5, 3.5 and 4.5 min after the initiation of the reaction. An ‘Eskalab’ spectrophotometer Alpha (Smith Kline Instruments) set at 340 nm was used. The absorbance change in 1 min was multiplied by a factor 137 to obtain creatine kinase activity in pkat/l. (B) Calorimetric
method developed by us
Solutions and their stability Solution 1: ‘Spinchem’ CK Reagent (SKI) was reconstituted as specified by the manufacturer; the composition of this solution did not differ from that used in the standard UV method. Solution 2: 0.16 mol/l N-ethylmaleimide (Calbiochem, Los Angeles, CA, USA): 56 mg NEM was dissolved in 2.8 ml distilled water. The solution is stable at 25°C for 2 weeks. Solution 3: 4.0 mmol/l nitro blue tetrazolium (Lachema Brno, Czechoslovakia), 1.6 mmol/l phenazine methosulphate (Lachema Brno): 16.2 mg NBT was dissolved in 5.0 ml distilled water. This solution is transferred to 2.5 mg PMS just before use. The final solution must be protected from light, and is stable at 4’C for 24 h; the decomposition of PMS can be recognized by a colour change from yellow to green. Solution 4: 0.1 mol/l hydrochloric acid.
247
Test procedure 500 ~1 of Solution 1 was pipetted into a cuvette and time allowed for the cuvette contents to reach 37°C. Then 10 ~1 of serum was added and mixed and incubated for 10 min exactly. The reaction was stopped with 100 ~1 of Solution 2 which simultaneously inhibited the rest of the CK-activator (glutathione). 200 ~1 of Solution 3 was added after at least 1 min. The colour development was stopped after 5 min with 1000 ~1 of Solution 4. The absorbance of the sample was read at 530 nm against distilled water as the blank by an ‘Eskalab’ spectrophotometer Alpha. The colour of the reaction product is stable for at least 2 h. Reagent blank (10 ~1 of 155 mmol/l sodium chloride instead of serum) and standard control serum with a declared value of CK activity was measured with each series of assays. We used the lyophilized control serum MONI-TROL@ I-E (Merz + Dade AG, Dtidingen, Switzerland) with a creatine kinase activity of about 1.6 pkat/l, i.e. near the upper limit of reference values. The CK activity in the sample was calculated as follows: a,(pkat/l)
* a,(pkat/l)
= $$-$ 5
0
absorbance; A,, reagent blank abwere: A,, sample absorbance; A,, standard sorbance; a,, CK activity in the sample; and a,, CK activity in the standard control serum. Results (1) Precision (a) Within-day precision was calculated from 25 replicate measurements on a pooled serum. The mean value of CK activity was 1.25 pkat/l, and the coefficient of variation was 6.5%. (b) Day-to-day precision was calculated from the data on a pooled serum over a 20-day period. The mean value of CK activity was found to be 1.28 p kat/l, and the coefficient of variation 7.9%. (2) Accuracy The CK activities in the control sera with declared values were measured in each batch of assays for 20 days. The following control sera were used: MONI-TROL@ II, ENZA-TROL@ (Merz + Dade AG), Precinorm@ E, Precipatha E (Boehringer Mannheim, FRG) Q-PAK I, Q-PAK II (Hyland, Div. Travenol Laboratories S.A., Lessines, Belgium). The mean coefficient of bias was 5.3%. (3) Sensitivity (a) Sensitivity was determined as the difference of absorbance per pkat/l of enzyme activity. The estimated value was 0.057. (b) The low limit of detection was established as the smallest CK activity that can be differentiated from the blank at a significance level of 5%. This value of enzyme activity was found to be 0.2 pkat/l.
248
(4) Linearity Increase of absorbance is linear to an activity of 15 pkat/l. necessary to reduce the sample size or the time of incubation.
Above this value, it is
(5) Znfluence of hemolysis Moderate hemolysis does not effect the test, but severely hemolyzed produce a false elevation of measured CK activity.
samples
may
(6) Specificity The specificity of the calorimetric test is not different from that of UV method because the first step is identical. The colour development could be probably influenced by some reducing agents present occasionally in serum. Therefore we tested the influence of some reducing substances which may be observed in serum in significant concentrations. Glucose, uric acid and ketones (acetone and acetoacetic acid) are not able to reduce tetrazolium salts. Ascorbic acid, however, can convert these chromogens into coloured formazans. We have found that the absorbance elevation due to the presence of ascorbic acid in serum is very small: viz. 0.0016 for an ascorbic acid concentration of 68 pmol/l, a value rarely exceeded in serum. This elevation causes a positive error of 2.86% at a creatine kinase activity of 1.0 pkat/l and 0.29% at 10.0 pkat/l. These changes (more often even smaller because of the lower ascorbic acid level in serum of most people) have no clinical significance. Therefore it is not necessary to use a serum blank and only a reagent blank is required. (7) Correlation between CK activity measured by the calorimetric Fig. 1 compares the results obtained by both the methods.
5
Fig. 1. Comparison
10
and UV methods Agreement is good.
15 I~katlll
of the calorimetric
with the
uv
method
of measuring
CK activity.
249
Least-squares regression analysis of 42 samples showed the slope of the line to be 1.13 1 which indicates a proportional error of 13.1%. From the value of the intercept, a constant error is estimated at -0.225 pkat/l. The correlation coefficient is 0.985. Thus, for a value of 10.0 pkat/l in UV test, the calorimetric test would give an average value of 11.1 pkat/l. At an activity of 1.72 pkat/l both methods give the same results. Discussion The conversion of NAD+ to NADH (resp. NADP+ to NADPH) and vice versa is a commonly used reaction when determining the enzyme activities or substrate concentrations in UV-spectrophotometry. NADH as a reducing agent reacts with tetrazolium salts and the development of the coloured formazan can be measured in visible light. This principle has been applied to aminotransferases [2,3], various dehydrogenases [3-81, glucose and some other substances [9- 111, but not activated CK, because of the strong reducing agents used as enzyme activators. Free sulphhydryl groups ( - SH) of these activators also cause reduction of tetrazolium salts (Table I, eq. g), and thus interfere in this test by increasing the results. Therefore, it is necessary to remove the unconsumed activator after the reactions a-c, Table I. We used N-ethylmaleimide. This compound reacts additionally with the SH-groups of both the activator and enzyme (Table I, eq. I). The following rapid inactivation of all the SH-groups removes the activator and stops the enzyme reaction (Table I, eq. a). After this step the colour reagent can be added. The product of the subsequent reactions (Table I, eq. d and e) is the coloured formazan, the concentration of which is proportional to CK activity. Thus all compounds with SH-groups can be inactivated [12] and N-ethylmaleimide can be used for removing all the commonly used CK-activators (glutathione, N-acetylcysteine, dithiothreitol etc.). The same approach could be adopted for CK-isoenzymes assay based on both the immunochemical and electrophoretic principles. Besides nitro blue tetrazolium, other tetrazolium salts may be used [13]. Phenazine methosulphate, as an electron mediator between NADPH and the tetrazolium dye, can be substituted by I-methoxy-phenazine methosulphate which appears more photochemically stable [8]. The use of the method described is convenient for the clinical CK activity assay, especially in small laboratories without an UV-spectrophotometer. References 1 Rosalki SB. An improved procedure for serum creatine phosphokinase determinations. J Lab Clin Med 1967; 69: 696-701. 2 Lippi U, Guid G. A new calorimetric ultramicromethod for serum glutamic-oxalacetic and glutamicpyruvic transaminase determination. Clin Chim Acta 1970; 28: 43 l-437. 3 Whitaker JF. A general calorimetric procedure for the estimation of enzymes which are linked to the NADH/NAD+ system. Clin Chim Acta 1969; 24: 23-37.
250 4 Babson AL, Phillips GE. A rapid calorimetric assay for serum lactic dehydrogenase. Clin Chim Acta 1965; 12: 210-215. 5 Bergmeyer HN. Methoden der enzymatischen Analyse, 2nd ed, Vol. 1. Berlin: Akademie Verlag, 1970: 538. 6 Van Der Helm VJ. A simplified method of demonstrating lactic dehydrogenase isoenzymes in serum. Clin Chim Acta 1962; 7: 124-128. I Nachlas MM, Margulies SI, Goldberg JD, Seligman AM. The determination of lactic dehydrogenase with a tetrazolium salt. Anal Biochem 1960; 1: 317-326. 8 Nakamura S, Arimura K, Ogawa K, Yagi T. Use of I-methoxy-5-methylphenazinium methyl sulfate ( I-methoxyPMS) in the assay of some enzymes of diagnostic importance. Clin Chim Acta 1980; 101: 321-326. 9 Carroll JJ, Smith N, Babson AL. A calorimetric serum glucose determination using hexokinase and glucose-6-phosphate dehydrogenase. Biochem Med 1970; 4: 17 I- 180. 10 Coburn HJ, Carroll JJ. Improved manual and automated calorimetric determination of serum glucose with use of hexokinase and glucose-6-phosphate dehydrogenase. Clin Chem 1973; 19: 127- 130. 11 Wright WR, Rainwater JC, Tolle LD. Glucose assays systems: Evaluation of a calorimetric hexokinase procedure. Clin Chem 1971; 17: 1010-1015. 12 Okamura M. An improved method for determination of L-ascorbic acid and L-dehydroascorbic acid in blood plasma. Clin Chim Acta 1980; 103: 259-268. 13 Burlina A. Improved electrophoretic separation of creatine kinase isoenzymes. Clin Chem 1980; 26: 317-320.