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Colorimetric estimation of lactic dehydrogenase isoenzymes by urea inhibition
121
CLINICA CHIMICA ACTA
COLORIMETRIC ISOENZYMES
ESTIMATION BY
S. G. WELSHMAN
UREA
AND E.
OF
LACTIC
DEHYDROGENASE
INHIBITION
CAROL
RIXON
The Laboratories, Belfast City Hospital, Belfast (N. Ireland) (Received
September
zgth,
1967)
SUMMARY
A calorimetric method is described for the estimation dehydrogenase activity in serum. This isoenzyme is considered
of urea-stable lactic to be almost identical
with the heart-specific isoenzyme LDH,. Results suggest that the estimation of this isoenzyme as a percentage of total lactic dehydrogenase is valuable for the detection of myocardial infarction.
INTRODUCTION
Considerable on the lactic described an specific LDH enzyme in a
interest
has been shown recently
in the inhibitory
effect of urea
dehydrogenase isoenzymes occurring in liver and muscle1v2. Hardy3 has ultra-violet spectrophotometric method for the estimation of heartisoenzyme in serum based on the selective inhibition of the liver iso2.6 M urea solution. The effect of varying concentrations of urea and
pyruvate on lactic dehydrogenase isoenzyme activity has been studied by Konttinen and Lindy4 who found that high pyruvate concentrations increased the activity of the heart isoenzyme in the presence of urea while the liver isoenzyme was virtually destroyed. It is the purpose of this study to find the most suitable conditions for determining urea-stable LDH in serum and adapt the dinitrophenylhydrazine colorimetric procedure for the estimation of this isoenzyme. FINAL U.V.
METHOD
Reagents I. Potassium phosphate buffer, 0.1 M, pH 7.5: mix I 1 of 0.1 M di-potassium hydrogen phosphate and 150 ml of 0.1 M potassium dihydrogen phosphate. z. Urea-phosphate buffer: dissolve 15.43 g of urea in IOO ml of the 0.1 M potassium phosphate buffer. 3. Sodium pyruvate solution: dissolve 250 mg of sodium pyruvate in IOO ml of 0.1 M potassium phosphate buffer. 4. Reduced nicotinamide-adenine dinucleotide.
C&z. Chim. Acta, 1g (1968) 121-123
122
WELSHMAN,
RIXON
Procedure Add IO ml of the urea-phosphate buffer to I mg of NADH and mix well. Place 2.8 ml of this solution in a test tube and add 0.1 ml of serum. Xix and incubate for 20 min at 25”. Add 0.1 ml of the sodium pyruvate solution, transfer to cuvette immediately and measure the optical density at I-min intervals for 5 min using a wave length of 340 mp. Difference in OD/min x IO* = Wroblewski units/ml of urea-stable LDH. Estimate the total LDH by the same method using the 0.1 M potassium phosphate buffer without urea. FINAL COLORIMETRIC
METHOD
Reagents I. Urea-phosphate buffer: dissolve r4.4 g of urea in IOO ml of 0.1 M potassium phosphate buffer, pH 7.5. 2. Pyruvate substrate: dissolve IO mg of sodium pyruvate in IOO ml of the 0.1 M potassium phosphate buffer. 3. Dinitrophenylhydrazine reagent : dissolve zoo mg of z,4_dinitrophenylhydrazine in 85 ml of concentrated hydrochloric acid and make up to I 1 with water. 4. Sodium hydroxide solution, 0.4 N: dissolve 16 g of sodium hydroxide in water and make up to I 1. Procedwe Dilute 0.1 ml of serum with 0.5 ml of urea-phosphate buffer and allow to stand at room temperature for I h. Use 0.2 ml of the diluted serum to carry out the enzyme reaction and the calorimetric development as described by Cabaud and Wroblewski6. The preparation of a standard curve may be avoided by the use of commercially available reagents and procedures for the calorimetric estimation of lactic dehydrogenase. In this laboratory the Sigma procedure No. 500 has proved very satisfactory. RESULTS
AND DISCUSSION
Solutions of LDH, from pig heart and LDH, from rabbit muscle were examined to find the best condition for differentiating between the two isoenzymes using urea. It was found that a concentration of approximately 3 x IO-~ M pyruvate, 2.0 M urea and an incubation time of 20 min at 25” completely destroyed the LDH, while more than 85% of the LDH, remained unaffected. However, under these conditions the percentage of urea-stable LDH in normal sera was abnormally high. A control serum (Enza-trol} with known LDH, isoenzyme content was analysed at different concentrations of urea. A 2.4 M urea solution gave a level of urea-stable LDH which was almost identical with the known concentration of LDH, in the control serum. Sera from 40 normal persons were analysed by the final U.V. procedure. The normal values were 36-109 units (mean 65 units) and x9-360/, of the total LDH (mean 307/,). The conditions for the estimation of urea-stable LDH by the U.V. method were applied to a calorimetric technique using the dinitrophenylhydrazine reaction6TB. Serum, 0.1 ml, was added to 0.5 ml of the urea-phosphate buffer to give a final concentration of 2.4 M urea. The time and temperature of incubation required to
COLORIMETRY
OF
LDH
ISOENZYMES
123
estimate the urea-stable LDH was investigated. The concentration of this isoenzyme in five sera was first determined by the U.V. urea inhibition technique and then estimated by the calorimetric technique at temperatures ranging from 15’ to 37’ and also at r5-min intervals for a total period of go min. At temperatures greater than 30’ the urea inactivated all the lactic dehydrogenase. Below 25” the effect of urea inhibition ceased after 60 min when the urea-stable LDH activity was identical with the values found by the U.V. method. Thirty sera with total LDH values from 210 units to 3040 units were estimated by the U.V. and calorimetric techniques. In all cases, the results produced by the two methods were in close agreement. Serum from normal controls and patients with increased total LDH were estimated by the calorimetric method and the results are shown in Table I. The percentage concenTABLE
I
CONCENTRATIONSOF UREA-STABLE No. of subjects Normal controls Myocardial infarction Miscellaneous diseases
40 56 36
LDH
IN SERUM Urea-stable
BYTHECOLORIMETRIC LDH
units/ml 61 (36-105) 780 (rgo-zaoo) 130
(9-250)
Percentage total LDH
PROCEDURE
l7f
30 (‘g-36) 55 (42~75) 22 (6-36)
tration of urea-stable LDH in cases of myocardial infarction show a distinctly higher level than in the normal controls. The group of miscellaneous diseases with elevated total LDH produce a normal or reduced percentage of the urea-stable isoenzyme. This group did not include diseases involving increased breakdown of erythrocytes. In four cases of untreated pernicious anaemia the percentage of this isoenzyme ranged from 65582.
REFERENCES I 2 3 4 5 6
D. T. PLUMMER, J, H. WILKINSONAND W. A. WITHYCOMBE, Biochem.J., I. A. BRODY, Nature, 201 (1964) 685. S. M. HARDY, Nature, 206 (1965) 933. A. KONTTINEN AND S. LINDY, Clin. Chim. Acta, 16(1967) 377. P. G. CABAUD AND F. WROBLEWSKI, Am. J. Clin. Pathol., 30 (1958) 234. Sigma Technical Bulletin No. 500. Clin. Chim.
Acta,
89 (1963)
19 (1968)
48 p,
121-123
CLINICA CHIMICA ACTA
COLORIMETRIC ISOENZYMES
ESTIMATION BY
S. G. WELSHMAN
UREA
AND E.
OF
LACTIC
DEHYDROGENASE
INHIBITION
CAROL
RIXON
The Laboratories, Belfast City Hospital, Belfast (N. Ireland) (Received
September
zgth,
1967)
SUMMARY
A calorimetric method is described for the estimation dehydrogenase activity in serum. This isoenzyme is considered
of urea-stable lactic to be almost identical
with the heart-specific isoenzyme LDH,. Results suggest that the estimation of this isoenzyme as a percentage of total lactic dehydrogenase is valuable for the detection of myocardial infarction.
INTRODUCTION
Considerable on the lactic described an specific LDH enzyme in a
interest
has been shown recently
in the inhibitory
effect of urea
dehydrogenase isoenzymes occurring in liver and muscle1v2. Hardy3 has ultra-violet spectrophotometric method for the estimation of heartisoenzyme in serum based on the selective inhibition of the liver iso2.6 M urea solution. The effect of varying concentrations of urea and
pyruvate on lactic dehydrogenase isoenzyme activity has been studied by Konttinen and Lindy4 who found that high pyruvate concentrations increased the activity of the heart isoenzyme in the presence of urea while the liver isoenzyme was virtually destroyed. It is the purpose of this study to find the most suitable conditions for determining urea-stable LDH in serum and adapt the dinitrophenylhydrazine colorimetric procedure for the estimation of this isoenzyme. FINAL U.V.
METHOD
Reagents I. Potassium phosphate buffer, 0.1 M, pH 7.5: mix I 1 of 0.1 M di-potassium hydrogen phosphate and 150 ml of 0.1 M potassium dihydrogen phosphate. z. Urea-phosphate buffer: dissolve 15.43 g of urea in IOO ml of the 0.1 M potassium phosphate buffer. 3. Sodium pyruvate solution: dissolve 250 mg of sodium pyruvate in IOO ml of 0.1 M potassium phosphate buffer. 4. Reduced nicotinamide-adenine dinucleotide.
C&z. Chim. Acta, 1g (1968) 121-123
122
WELSHMAN,
RIXON
Procedure Add IO ml of the urea-phosphate buffer to I mg of NADH and mix well. Place 2.8 ml of this solution in a test tube and add 0.1 ml of serum. Xix and incubate for 20 min at 25”. Add 0.1 ml of the sodium pyruvate solution, transfer to cuvette immediately and measure the optical density at I-min intervals for 5 min using a wave length of 340 mp. Difference in OD/min x IO* = Wroblewski units/ml of urea-stable LDH. Estimate the total LDH by the same method using the 0.1 M potassium phosphate buffer without urea. FINAL COLORIMETRIC
METHOD
Reagents I. Urea-phosphate buffer: dissolve r4.4 g of urea in IOO ml of 0.1 M potassium phosphate buffer, pH 7.5. 2. Pyruvate substrate: dissolve IO mg of sodium pyruvate in IOO ml of the 0.1 M potassium phosphate buffer. 3. Dinitrophenylhydrazine reagent : dissolve zoo mg of z,4_dinitrophenylhydrazine in 85 ml of concentrated hydrochloric acid and make up to I 1 with water. 4. Sodium hydroxide solution, 0.4 N: dissolve 16 g of sodium hydroxide in water and make up to I 1. Procedwe Dilute 0.1 ml of serum with 0.5 ml of urea-phosphate buffer and allow to stand at room temperature for I h. Use 0.2 ml of the diluted serum to carry out the enzyme reaction and the calorimetric development as described by Cabaud and Wroblewski6. The preparation of a standard curve may be avoided by the use of commercially available reagents and procedures for the calorimetric estimation of lactic dehydrogenase. In this laboratory the Sigma procedure No. 500 has proved very satisfactory. RESULTS
AND DISCUSSION
Solutions of LDH, from pig heart and LDH, from rabbit muscle were examined to find the best condition for differentiating between the two isoenzymes using urea. It was found that a concentration of approximately 3 x IO-~ M pyruvate, 2.0 M urea and an incubation time of 20 min at 25” completely destroyed the LDH, while more than 85% of the LDH, remained unaffected. However, under these conditions the percentage of urea-stable LDH in normal sera was abnormally high. A control serum (Enza-trol} with known LDH, isoenzyme content was analysed at different concentrations of urea. A 2.4 M urea solution gave a level of urea-stable LDH which was almost identical with the known concentration of LDH, in the control serum. Sera from 40 normal persons were analysed by the final U.V. procedure. The normal values were 36-109 units (mean 65 units) and x9-360/, of the total LDH (mean 307/,). The conditions for the estimation of urea-stable LDH by the U.V. method were applied to a calorimetric technique using the dinitrophenylhydrazine reaction6TB. Serum, 0.1 ml, was added to 0.5 ml of the urea-phosphate buffer to give a final concentration of 2.4 M urea. The time and temperature of incubation required to
COLORIMETRY
OF
LDH
ISOENZYMES
123
estimate the urea-stable LDH was investigated. The concentration of this isoenzyme in five sera was first determined by the U.V. urea inhibition technique and then estimated by the calorimetric technique at temperatures ranging from 15’ to 37’ and also at r5-min intervals for a total period of go min. At temperatures greater than 30’ the urea inactivated all the lactic dehydrogenase. Below 25” the effect of urea inhibition ceased after 60 min when the urea-stable LDH activity was identical with the values found by the U.V. method. Thirty sera with total LDH values from 210 units to 3040 units were estimated by the U.V. and calorimetric techniques. In all cases, the results produced by the two methods were in close agreement. Serum from normal controls and patients with increased total LDH were estimated by the calorimetric method and the results are shown in Table I. The percentage concenTABLE
I
CONCENTRATIONSOF UREA-STABLE No. of subjects Normal controls Myocardial infarction Miscellaneous diseases
40 56 36
LDH
IN SERUM Urea-stable
BYTHECOLORIMETRIC LDH
units/ml 61 (36-105) 780 (rgo-zaoo) 130
(9-250)
Percentage total LDH
PROCEDURE
l7f
30 (‘g-36) 55 (42~75) 22 (6-36)
tration of urea-stable LDH in cases of myocardial infarction show a distinctly higher level than in the normal controls. The group of miscellaneous diseases with elevated total LDH produce a normal or reduced percentage of the urea-stable isoenzyme. This group did not include diseases involving increased breakdown of erythrocytes. In four cases of untreated pernicious anaemia the percentage of this isoenzyme ranged from 65582.
REFERENCES I 2 3 4 5 6
D. T. PLUMMER, J, H. WILKINSONAND W. A. WITHYCOMBE, Biochem.J., I. A. BRODY, Nature, 201 (1964) 685. S. M. HARDY, Nature, 206 (1965) 933. A. KONTTINEN AND S. LINDY, Clin. Chim. Acta, 16(1967) 377. P. G. CABAUD AND F. WROBLEWSKI, Am. J. Clin. Pathol., 30 (1958) 234. Sigma Technical Bulletin No. 500. Clin. Chim.
Acta,
89 (1963)
19 (1968)
48 p,
121-123