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[9] Enzymic determination of lactic acid
[9]
ENZYMIC DETERMINATION OF LACTIC ACID
41
TRANSKETOLASE A340 1 -~i
M I ERASE
TIME
FIG. 2. Assay of D-xylulose 5-phosphate and D-ribulose 5-phosphate.
If the absorbance does not reach a final steady value (owing to traces of isomerase in the enzymes used) the recording is continued long enough to allow the final linear rate to be extrapolated back to the moment the epimerase was added (Fig. 2). The values obtained for the ribulose 5-phosphate and xylulose 5-phosphate in the sample are corrected for any absorbance changes obtained in a control run with the sample absent.
Notes 1. Values obtained are reproducible to within -----5% of the mean. 2. The determination of ribulose 5-phosphate by this procedure can be time-consuming but xylulose 5-phosphate reacts readily. If its reaction with transketolase appears sluggish, one may try the effect of reversing the order of addition of the sample and transketolase. 3. The total pentose phosphate in a sample may be determined by omitting ribose 5-phosphate from the reaction mixture and adding ribose5-phosphate ketol isomerase, epimerase, and transketolase together. Under these conditions 2 molecules of pentose phosphate gives rise to the oxidation of 1 molecule of NADH.
[9]
Enzymic Determination of Lactic Acid
By
JOHANNES EVERSE
Several methods have been described for the chemical determination of lactic acid concentrations in tissue extracts, sera, and other biological
42
ANALYTICAL METHODS
[9]
systems. 1,2 These methods are based on the chemical conversion of lactic acid to acetaldehyde, which is subsequently coupled to an aromatic compound to produce a colored product. The intensity of the color is determined spectrophotometrically. Although these methods yield satisfactory results in most cases, the reactions are not specific for lactate. Other hydroxyacids and aldehydes may give a similar reaction with the reagents, and erroneously high values may be obtained. Furthermore, it is generally necessary to deproteinize the solutions before a lactate determination can be carried out, and long incubation times may be required for full color development. The advantages of an enzymic determination are, first, that the enzyme is generally quite specific for its substrate, and few erroneous values are obtained. Second, enzymic reactions that can be followed speetrophotometrically m a y be carried out rapidly and inexpensively without deproteinization of the solutions.
Enzymic Method Principle. Lactate dehydrogenase catalyzes the reaction Lactate ~ DPN + ~ pyruvate T DPNH + H + reversibly, and the equilibrium constant at pH 7.0 is 4 X 100 in favor of lactate formation. Because of this unfavorable equilibrium, the oxidation of lactate catalyzed b y lactate dehydrogenase is usually carried out at relatively high pH values. Even under these conditions, however, one does not achieve a complete oxidation of all the lactic acid; in fact only about 50% of the lactate is oxidized at equilibrium, when a concentration as high as 2 mg/ml of D P N + is used. A much more complete oxidation of lactic acid may be achieved with the use of analogs of D P N ÷ that have a more positive redox potential. In the method that is described here the 3-acetylpyridine analog of D P N ÷ is used as the coenzyme. This analog has a redox potential of --0.240, as compared to --0.320 for D P N ÷. (The redox potential of pyruvate/lactare is --0.190.) Hence, in the presence of 3-acetylpyridine-DPN ÷ at a concentration of 2 mg/ml the oxidation of lactate is .98.5% complete at p H 10, if the lactate concentration is 5 ~g/ml or less. This appears to be sufficiently accurate for most routine lactate determinations. S. B. Baker, this series, Vol. 3 [42]. : A. H. Free and H. M. Free, in "Clinical Pathology of the Serum Electrolytes" (F. William Sunderman and F. William Sunderman, Jr., eds.), p. 107. Thomas, Springfield, Illinois, 1966.
[9]
ENZYMIC DETERMINATION
OF LACTIC ACID
43
Reagents Borate buffer, 0.01 M, pH 9.2, which is prepared by dissolving 0.38 g of sodium borate (Na~B407.10H~O) in 100 ml of water 3-Acetylpyridine-DPN÷, 20 mg/ml, dissolved in water Lactate dehydrogenase, beef heart, 5 mg/ml The solution containing the unknown amount of lactic acid is diluted with water until the estimated concentration of lactic acid is between 20 and 50 t~g/ml. Procedure. Transfer into a 1-ml cuvette: 0.8 ml of 0.01 M borate buffer, 0.1 ml of 3-acetylpyridine-DPN÷ solution, 0.01 ml of lactate dehydrogenase solution, and 0.1 ml of the diluted lactic acid solution. After the reaction has reached equilibrium, the optical density of the solution is determined at 363 nm. The reference cuvette contains the same ingredients as the assay cuvette, except that water has been added instead of the enzyme. The lactate concentration (~g/ml) of the original solution may be calculated from the following equation, assuming that the original solution was a-fold diluted to bring the lactate concentration within the measurable range. X = [(A in optical density) (10a) (90)]/9.1 in which 90 represents the molecular weight of lactic acid, and 9.1 is the millimolar extinction coefficient of 3-acetylpyridine-DPNH at 363 nm. General Remarks
The enzymic method for the determination of lactic acid has been used in our laboratory for several years; it yields highly satisfactory results if the manipulations are properly performed. Two aspects of the method need special attention in order to prevent the occurrence of erroneous values. The absorbance at 363 nm that is observed during a lactic acid determination is due to the formation of 3-acetylpyridine-DPNH. The oxidized coenzyme does not absorb light at 363 nm. However, commercial preparations of 3-acetylpyridine-DPN÷ sometimes produce a slight absorbance at 363 nm at alkaline pH values. It is therefore necessary to use the coenzyme analog in the reference cuvette, as described in the procedure, in order to compensate for any absorbance that is of a nonenzymic origin. One should also be aware of the fact that a certain amount of lactate is usually present on the skin. Erroneously high values for lactate may be found if one mixes the contents of the cuvette by hand without cover-
44
ANALYTICAL METHODS
[9]
ing the cuvette first with a clean piece of plastic film. The presence of lactate on the skin requires that solutions do not come in contact with the skin, or with any material that previously has been touched by uncovered hands. The reaction in the cuvette should reach equilibrium within 2 or 3 min, and the optical density at 363 nm may be determined at that time. The reduced coenzyme is fairly stable under the assay conditions, but it is advisable to take the measurements within 5 min after the equilibrium is attained, in order to minimize any spurious nonspecific reactions. It is advisable to keep the conditions as constant as possible for maximum reproducibility.
ENZYMIC DETERMINATION OF LACTIC ACID
41
TRANSKETOLASE A340 1 -~i
M I ERASE
TIME
FIG. 2. Assay of D-xylulose 5-phosphate and D-ribulose 5-phosphate.
If the absorbance does not reach a final steady value (owing to traces of isomerase in the enzymes used) the recording is continued long enough to allow the final linear rate to be extrapolated back to the moment the epimerase was added (Fig. 2). The values obtained for the ribulose 5-phosphate and xylulose 5-phosphate in the sample are corrected for any absorbance changes obtained in a control run with the sample absent.
Notes 1. Values obtained are reproducible to within -----5% of the mean. 2. The determination of ribulose 5-phosphate by this procedure can be time-consuming but xylulose 5-phosphate reacts readily. If its reaction with transketolase appears sluggish, one may try the effect of reversing the order of addition of the sample and transketolase. 3. The total pentose phosphate in a sample may be determined by omitting ribose 5-phosphate from the reaction mixture and adding ribose5-phosphate ketol isomerase, epimerase, and transketolase together. Under these conditions 2 molecules of pentose phosphate gives rise to the oxidation of 1 molecule of NADH.
[9]
Enzymic Determination of Lactic Acid
By
JOHANNES EVERSE
Several methods have been described for the chemical determination of lactic acid concentrations in tissue extracts, sera, and other biological
42
ANALYTICAL METHODS
[9]
systems. 1,2 These methods are based on the chemical conversion of lactic acid to acetaldehyde, which is subsequently coupled to an aromatic compound to produce a colored product. The intensity of the color is determined spectrophotometrically. Although these methods yield satisfactory results in most cases, the reactions are not specific for lactate. Other hydroxyacids and aldehydes may give a similar reaction with the reagents, and erroneously high values may be obtained. Furthermore, it is generally necessary to deproteinize the solutions before a lactate determination can be carried out, and long incubation times may be required for full color development. The advantages of an enzymic determination are, first, that the enzyme is generally quite specific for its substrate, and few erroneous values are obtained. Second, enzymic reactions that can be followed speetrophotometrically m a y be carried out rapidly and inexpensively without deproteinization of the solutions.
Enzymic Method Principle. Lactate dehydrogenase catalyzes the reaction Lactate ~ DPN + ~ pyruvate T DPNH + H + reversibly, and the equilibrium constant at pH 7.0 is 4 X 100 in favor of lactate formation. Because of this unfavorable equilibrium, the oxidation of lactate catalyzed b y lactate dehydrogenase is usually carried out at relatively high pH values. Even under these conditions, however, one does not achieve a complete oxidation of all the lactic acid; in fact only about 50% of the lactate is oxidized at equilibrium, when a concentration as high as 2 mg/ml of D P N + is used. A much more complete oxidation of lactic acid may be achieved with the use of analogs of D P N ÷ that have a more positive redox potential. In the method that is described here the 3-acetylpyridine analog of D P N ÷ is used as the coenzyme. This analog has a redox potential of --0.240, as compared to --0.320 for D P N ÷. (The redox potential of pyruvate/lactare is --0.190.) Hence, in the presence of 3-acetylpyridine-DPN ÷ at a concentration of 2 mg/ml the oxidation of lactate is .98.5% complete at p H 10, if the lactate concentration is 5 ~g/ml or less. This appears to be sufficiently accurate for most routine lactate determinations. S. B. Baker, this series, Vol. 3 [42]. : A. H. Free and H. M. Free, in "Clinical Pathology of the Serum Electrolytes" (F. William Sunderman and F. William Sunderman, Jr., eds.), p. 107. Thomas, Springfield, Illinois, 1966.
[9]
ENZYMIC DETERMINATION
OF LACTIC ACID
43
Reagents Borate buffer, 0.01 M, pH 9.2, which is prepared by dissolving 0.38 g of sodium borate (Na~B407.10H~O) in 100 ml of water 3-Acetylpyridine-DPN÷, 20 mg/ml, dissolved in water Lactate dehydrogenase, beef heart, 5 mg/ml The solution containing the unknown amount of lactic acid is diluted with water until the estimated concentration of lactic acid is between 20 and 50 t~g/ml. Procedure. Transfer into a 1-ml cuvette: 0.8 ml of 0.01 M borate buffer, 0.1 ml of 3-acetylpyridine-DPN÷ solution, 0.01 ml of lactate dehydrogenase solution, and 0.1 ml of the diluted lactic acid solution. After the reaction has reached equilibrium, the optical density of the solution is determined at 363 nm. The reference cuvette contains the same ingredients as the assay cuvette, except that water has been added instead of the enzyme. The lactate concentration (~g/ml) of the original solution may be calculated from the following equation, assuming that the original solution was a-fold diluted to bring the lactate concentration within the measurable range. X = [(A in optical density) (10a) (90)]/9.1 in which 90 represents the molecular weight of lactic acid, and 9.1 is the millimolar extinction coefficient of 3-acetylpyridine-DPNH at 363 nm. General Remarks
The enzymic method for the determination of lactic acid has been used in our laboratory for several years; it yields highly satisfactory results if the manipulations are properly performed. Two aspects of the method need special attention in order to prevent the occurrence of erroneous values. The absorbance at 363 nm that is observed during a lactic acid determination is due to the formation of 3-acetylpyridine-DPNH. The oxidized coenzyme does not absorb light at 363 nm. However, commercial preparations of 3-acetylpyridine-DPN÷ sometimes produce a slight absorbance at 363 nm at alkaline pH values. It is therefore necessary to use the coenzyme analog in the reference cuvette, as described in the procedure, in order to compensate for any absorbance that is of a nonenzymic origin. One should also be aware of the fact that a certain amount of lactate is usually present on the skin. Erroneously high values for lactate may be found if one mixes the contents of the cuvette by hand without cover-
44
ANALYTICAL METHODS
[9]
ing the cuvette first with a clean piece of plastic film. The presence of lactate on the skin requires that solutions do not come in contact with the skin, or with any material that previously has been touched by uncovered hands. The reaction in the cuvette should reach equilibrium within 2 or 3 min, and the optical density at 363 nm may be determined at that time. The reduced coenzyme is fairly stable under the assay conditions, but it is advisable to take the measurements within 5 min after the equilibrium is attained, in order to minimize any spurious nonspecific reactions. It is advisable to keep the conditions as constant as possible for maximum reproducibility.