- Email: [email protected]
[66] Determination of α-keto acids
414
CITRIC ACID CYCLE
[~]
a-Keto acids are quantitatively decarboxylated by ceric sulfate 8~,33 or by hydrogen peroxide," and these procedures are useful in the determination of purity of a given a-keto acid preparation. Other procedures which may be valuable in certain instances include preparation of the sodium bisulfite derivative, nonenzymatic transamination to form the corresponding amino acid, and ultraviolet and infrared absorption studies. Certain a-keto acids, e.g., a-keto--~-methiolbutyric and f~-mercaptopyruvic, give specific color reactions also characteristic of the analogous amino acids. Finally, enzymatic and microbiological procedures may be useful for the characterization and quantitative determination of a-keto acids. For example, many a-keto acids are reduced by lactic dehydrogenase, 1~,35 and at least five of these" are reduced at rates of about the same order of magnitude as pyruvate. The yeast a-keto acid decarboxylase also exhibits a wide range of substrate specificity.14 32 C. Fromageot and P. Desnuelle, Biochem. Z. 279, 174 (1935). H. A. Krebs and W. A. Johnson, Enzymologia 4, 148 (1937). 34A. Meister, J. Biol. Chem. 200, 571 (1953). 35 A. Meister, J. Biol. Chem. 184, 117 (1950). 38a-Ketobutyrate, glyoxylate, ~-mercaptopyruvate, 5-hydroxypyruvate, and DL-aketo-~-hydroxybutyrate. aa
[66] Determination of a-Keto Acids
By THEODOREE. FRIEDEMANN Assay Methods Principle. The methods are based on the observations of Dakin and Dudley 1 that the ponitrophenylhydrazones of keto acids are soluble in, or are extracted from organic solvents by, Na2CO3 solution and yield brilliant red to purple colors on addition of strong alkali. Lu ~ adapted these properties of hydrazones to the determination of microgram quantities, using reaction with 2,4-dinitrophenylhydrazine, extraction of hydrazones by ethylacetate, re-extraction by 10% Na2CO3 solution, addition of N NaOH, and photoelectric determination of color density. In the procedures of Friedemann et al. 3,4 described below, the steps are 1 H. 2 G. 3 T. 4 T.
D. Dakin and H. W. Dudley, J. Biol. Chem. 15, 127 (1913). D. Lu, Biochem. J. 33, 249 (1939). E. Friedemann and G. E. Haugen, J. Biol. Chem. 147, 415 (1943). E. Friedemann, V. M. Kinney, and P. K. Keegan, unpublished.
[66]
DETERMINATION OF a-KETO ACIDS
415
essentially the same; however, the separate determination of keto acids is based on differences in the rates of reaction with 2,4-dinitrophenylhydrazine, on the distribution of the hydrazones in the aqueous phase and various solvents, and, to some extent, on differences in light absorption of the hydrazones in alkali. Thus, at 25 °, the monocarboxylic keto acids (and ketones and aldehydes, in general) react completely with the hydrazine within about 4 minutes, the dicarboxylic keto acids within about 20 minutes. The monocarboxylic acid hydrazones are preferentially extracted from acid aqueous solution by aromatic hydrocarbons; the dicarboxylic acid hydrazones by aliphatic and aromatic alcohols. Both groups of hydrazones are readily extracted by esters and ethers. Since a large part of the energy of metabolism in most living organisms is obtained from carbohydrate through the formation of pyruvic acid, this acid is the principal monocarboxylic keto acid in biological materials; also, since oxalacetic acid is unstable, yielding pyruvic acid and C02, a-ketoglutaric acid is the principal dicarboxylic acid, unless the sample is analyzed immediately. That these two are the predominating keto acids in blood and urine has been shown by chromatographic 5,6 and enzymatic 7,s procedures. Reagents
Protein precipitant, 10 % TCA solution. Prepare fresh solution each month. Store in the refrigerator. Acetic acid, 5% solution. Lloyd's reagent. Hydrazine reagent, 0.1 To solution. Dissolve 500 mg. of 2,4-dinitrophenylhydrazine in 500 ml. of 2 N HC1. Prepare fresh solution each month; store in the refrigerator. If the hydrazine is not readily and completely soluble, recrystallize from ethyl acetate. Solvents. Ethyl benzene, benzyl alcohol, and ethyl acetate are the recommended solvents in methods 1, 2, and 3. Benzene, n-butyl alcohol, and dibutyl ether may be substituted in the respective methods 1, 2, and 3 if the preferred solvents are not available. All solvents should be distilled after procurement. Dibutyt ether should be redistilled at frequent intervals and stored in the refrigerator in order to prevent the accumulation of peroxides. 5D. Seligson and B. Shapiro, Anal. Chem. 24, 754 (1952). 6E. Kulonen, E. Karp~n, and T. Ruokolainen, Scand. J. Clin. & Lab. Invest. 4, 189 (1952). 7 H. A. Krebs, Biochem. J. 32, 108 (1938). 8 p. E. Simola and F. E. Krusius, Z. physiol. Chem. 261, 209 (1939).
416
CITRIC ACID CYCLE
[66]
Sodium carbonate, 10 % solution of anhydrous salt. Filter and keep in a Pyrex container. Sodium hydroxide, 1.5 N solution. Keto acid standards. Redistill pyruvic acid (Eastman) in vacuo and immediately prepare 0.1 to 0.5 N stock solution. Weigh accurately 8 to 25 g. of the acid in a weighing bottle; transfer quantitatively to a 1-1. or 500-ml. volumetric flask, using boiled, cold distilled water. Check the purity of the acid by titration with standard alkali. Solutions have been stored in the refrigerator for more than a year without apparent deterioration, a-Ketoglutaric acid may be obtained from commercial sources. Recrystallize if not pure, as determined by titration with standard alkali. Transfer 100 to 500 mg. to a volumetric flask, using 5% acetic acid solution; dilute to the mark and store in the refrigerator. Prepare working standards, using 5% acetic acid for all dilutions. Collection of Samples and Preparation for Analysis. Draw blood from the vein with a minimum of stasis. Tap the syringe gently while holding it in a vertical position to remove the bubbles of gas; bring the plunger to the mark, and expel the contents through the needle into 5 vol. of TCA solution contained in a stoppered centrifuge tube. Mix the contents, and immmediately place in the refrigerator. Centrifugate, and analyze the clear supernatant solution. Collect urine in plastic or amber glass bottles containing enough 20 N H:S04 to give 0.05 to 0.1 N acidity at the end of the collection period. Urine contains considerable quantities of interfering earbonyl compounds, apparently derived from ingested foods, which can be removed by Lloyd's reagent. For every 10 ml. of the acidified urine add 0.75 g. of Lloyd's reagent; shake vigorously and filter immediately. Tissue suspensions, as used in in vitro studies of intermediary metabolic reactions, may be treated directly with TCA solution, which precipitates the proteins and stops the reactions. Tissues from living animals are frozen in liquid nitrogen; they are crushed 9 or ground; the cold samples are weighed and added to measured volumes of cold TCA solution. Weighed, fresh plant tissues are ground in measured volumes of cold TCA solution in the Waring blendor. The tissue suspensions are cleared by eentrifugation. General Procedure. Transfer 3.0 ml. of sample extract to each of two 18 X 200-ram. test tubes. For each series of not more than ten tubes, prepare three reagent blanks containing 3.0 ml. of TCA or acetic acid solution as in the sample extract. Incubate all tubes 10 minutes in a bath or pan of water maintained at 25 ___ 2 °. At accurately timed intervals, t j. B. Graeser, J. E. Ginsberg, and T. E. Friedemann, J. Biol. Chem. 104, 149 (1934).
[66]
DETERMII~IATION
OF a - K E T O ACIDS
417
say 30 seconds, add in turn 1.0 ml. of hydrazine solution to each tube. Continue incubation at 25 ° for the exact time indicated below, at which time add the exact volume of appropriate solvent from a buret to each tube at the timed intervals. Mix immediately by aerating the contents for 2 minutes with a rapid stream of air or nitrogen through a Wright capillary pipet in each tube (10-ram. o.d. glass tubing; upper portion 4 inches long; lower portion 8 inches long, drawn to fine capillary with square-cut tip; both ends flamed). Remove the capillary pipets. Centrifugate at low speed to facilitate separation of phases. Ethyl benzene and ethyl acetate separate readily without centrifugation. Attach rubber bulbs to capillary pipets, and remove the aqueous reaction phase. Give a violent circular motion to the tube to dislodge any remaining solution. (If considered necessary, add 1 ml. of water, mix contents by circular motion of tube, and centrifugate.) Remove the remaining aqueous phase. Add 6.0 ml. of Na2C03 solution from a pipet. Remove the rubber bulbs from the capillary pipets, and again mix the contents by rapid aeration for 2 minutes. Remove the capillary pipets; centrifugate if necessary. Insert rapidly a 5-ml. pipet through the solvent layer, and gently blow out any solvent which may have entered. Transfer 5.0 ml. of the ioa:er carbonate extract to a colorimeter tube, held in the bath or pan of water at 25 °. At definite intervals, say 30 seconds, add 5.0 ml. of NaOH solution in turn to each colorimeter tube. A red to orange-red color is produced. At the end of 5 minutes, at the same time intervals between each tube, determine the color density, after setting the galvanometer reading to 100 by means of the reagent blanks. Use a photoelectric colorimeter provided with a filter having maximal transmittance at 420 rag; if a spectrophotometer is used, set the instrument at wavelength 435 mg. METHOD 1. Determination of pyruvic acid and other m onocarboxylic keto acids. Proceed as above. Incubate the sample extract and hydrszine solution for exactly 5 minutes; then add 3.0 ml. of ethyl benzene. METHOD 2. Determination of a-ketoglutaric acid and other dicarboxylic keto acids. Proceed as above. Incubate for exactly 25 minutes; then add 8.0 ml. of benzyl alcohol. METHOD 3. Determination of total keto acids. Proceed as above. Incubate the sample extract and hydrazine solution for exactly 25 minutes; then add 8.0 ml. of ethyl acetate. Calculations
The procedure for calculating the. individual acids is illustrated by the following data and formulas based on analysis of standard solutions, using the Evelyn colorimeter with filter 420. Thus, the following respective calculated color densities per micromole of pyruvic and ~-ketoglutaric acids
418
CITRIC ACID CYCLE
[6~]
were observed: by method 1, 2.037 and 0.149; by method 2, 0.181 and 1.862; by method 3, 2.119 and 1.919. If D1, D2, and Da represent the observed densities by methods 1, 2, and 3, and P and G represent micromoles of pyruvic and a-ketoglutaric acids in 3 ml. of the sample extract taken for analysis, then D1 = 2.037P ~ 0.149(7 D2 = 0.181P ~- 1.862G D3 = 2.119P -{- 1.919G By using any two of the methods and by solving the two respective equations for P and G, the individual acids may be evaluated. However, the most accurate results are obtained with methods 1 and 2 which provide sharp separation of the hydrazones.
Concentrations in Blood and Urine The methods have yielded the following results. The blood from ten moderately active subjects contained 0.86 to 1.63 rag. of pyruvic acid per 100 ml., with an average of 1.204 mg.; it contained 0.10 to 0.32 mg. of ~-ketoglutaric acid, with an average of 0.19 mg. The 24-hour urine contains an average of 20 __+ 8 mg. of pyruvic acid and an average of 33 _ 15 mg. of a-ketoglutaric acid.
[67] Preparation and Assay of Oxalacetic Acid B y SAMUEL P. BESSMAN
Preparation Reactions
2 Na diethyloxalacetate ~- H~SO~ --* 2 Diethyloxalacetate W Na2S04 Diethyloxaiacetate W 2 HOH ~ Oxalacetic acid ~ 2 ethanol Method. 1-8 One hundred grams (0.48 mole) of sodium diethyloxalacetate is dissolved in 500 ml. of 10-8 M disodium ethylenediaminetetraacetate (Na2-EDTA). When solution is complete, the mixture is placed in a liter separatory funnel and concentrated sulfuric acid is added dropwise until acid to congo red paper. Diethyloxalacetate separates as a heavy oil. Sufficient ether is added with shaking to bring all the oil into
1L. S. Simon, Compt. rend. 137, 855 (1903). 2p. p. Cohen, J. Biol. Chem. 136, 565 (1940). 3L. O. Krampitz and C. H. Werkman, Bioehem. J. 35, 595 (1941).
CITRIC ACID CYCLE
[~]
a-Keto acids are quantitatively decarboxylated by ceric sulfate 8~,33 or by hydrogen peroxide," and these procedures are useful in the determination of purity of a given a-keto acid preparation. Other procedures which may be valuable in certain instances include preparation of the sodium bisulfite derivative, nonenzymatic transamination to form the corresponding amino acid, and ultraviolet and infrared absorption studies. Certain a-keto acids, e.g., a-keto--~-methiolbutyric and f~-mercaptopyruvic, give specific color reactions also characteristic of the analogous amino acids. Finally, enzymatic and microbiological procedures may be useful for the characterization and quantitative determination of a-keto acids. For example, many a-keto acids are reduced by lactic dehydrogenase, 1~,35 and at least five of these" are reduced at rates of about the same order of magnitude as pyruvate. The yeast a-keto acid decarboxylase also exhibits a wide range of substrate specificity.14 32 C. Fromageot and P. Desnuelle, Biochem. Z. 279, 174 (1935). H. A. Krebs and W. A. Johnson, Enzymologia 4, 148 (1937). 34A. Meister, J. Biol. Chem. 200, 571 (1953). 35 A. Meister, J. Biol. Chem. 184, 117 (1950). 38a-Ketobutyrate, glyoxylate, ~-mercaptopyruvate, 5-hydroxypyruvate, and DL-aketo-~-hydroxybutyrate. aa
[66] Determination of a-Keto Acids
By THEODOREE. FRIEDEMANN Assay Methods Principle. The methods are based on the observations of Dakin and Dudley 1 that the ponitrophenylhydrazones of keto acids are soluble in, or are extracted from organic solvents by, Na2CO3 solution and yield brilliant red to purple colors on addition of strong alkali. Lu ~ adapted these properties of hydrazones to the determination of microgram quantities, using reaction with 2,4-dinitrophenylhydrazine, extraction of hydrazones by ethylacetate, re-extraction by 10% Na2CO3 solution, addition of N NaOH, and photoelectric determination of color density. In the procedures of Friedemann et al. 3,4 described below, the steps are 1 H. 2 G. 3 T. 4 T.
D. Dakin and H. W. Dudley, J. Biol. Chem. 15, 127 (1913). D. Lu, Biochem. J. 33, 249 (1939). E. Friedemann and G. E. Haugen, J. Biol. Chem. 147, 415 (1943). E. Friedemann, V. M. Kinney, and P. K. Keegan, unpublished.
[66]
DETERMINATION OF a-KETO ACIDS
415
essentially the same; however, the separate determination of keto acids is based on differences in the rates of reaction with 2,4-dinitrophenylhydrazine, on the distribution of the hydrazones in the aqueous phase and various solvents, and, to some extent, on differences in light absorption of the hydrazones in alkali. Thus, at 25 °, the monocarboxylic keto acids (and ketones and aldehydes, in general) react completely with the hydrazine within about 4 minutes, the dicarboxylic keto acids within about 20 minutes. The monocarboxylic acid hydrazones are preferentially extracted from acid aqueous solution by aromatic hydrocarbons; the dicarboxylic acid hydrazones by aliphatic and aromatic alcohols. Both groups of hydrazones are readily extracted by esters and ethers. Since a large part of the energy of metabolism in most living organisms is obtained from carbohydrate through the formation of pyruvic acid, this acid is the principal monocarboxylic keto acid in biological materials; also, since oxalacetic acid is unstable, yielding pyruvic acid and C02, a-ketoglutaric acid is the principal dicarboxylic acid, unless the sample is analyzed immediately. That these two are the predominating keto acids in blood and urine has been shown by chromatographic 5,6 and enzymatic 7,s procedures. Reagents
Protein precipitant, 10 % TCA solution. Prepare fresh solution each month. Store in the refrigerator. Acetic acid, 5% solution. Lloyd's reagent. Hydrazine reagent, 0.1 To solution. Dissolve 500 mg. of 2,4-dinitrophenylhydrazine in 500 ml. of 2 N HC1. Prepare fresh solution each month; store in the refrigerator. If the hydrazine is not readily and completely soluble, recrystallize from ethyl acetate. Solvents. Ethyl benzene, benzyl alcohol, and ethyl acetate are the recommended solvents in methods 1, 2, and 3. Benzene, n-butyl alcohol, and dibutyl ether may be substituted in the respective methods 1, 2, and 3 if the preferred solvents are not available. All solvents should be distilled after procurement. Dibutyt ether should be redistilled at frequent intervals and stored in the refrigerator in order to prevent the accumulation of peroxides. 5D. Seligson and B. Shapiro, Anal. Chem. 24, 754 (1952). 6E. Kulonen, E. Karp~n, and T. Ruokolainen, Scand. J. Clin. & Lab. Invest. 4, 189 (1952). 7 H. A. Krebs, Biochem. J. 32, 108 (1938). 8 p. E. Simola and F. E. Krusius, Z. physiol. Chem. 261, 209 (1939).
416
CITRIC ACID CYCLE
[66]
Sodium carbonate, 10 % solution of anhydrous salt. Filter and keep in a Pyrex container. Sodium hydroxide, 1.5 N solution. Keto acid standards. Redistill pyruvic acid (Eastman) in vacuo and immediately prepare 0.1 to 0.5 N stock solution. Weigh accurately 8 to 25 g. of the acid in a weighing bottle; transfer quantitatively to a 1-1. or 500-ml. volumetric flask, using boiled, cold distilled water. Check the purity of the acid by titration with standard alkali. Solutions have been stored in the refrigerator for more than a year without apparent deterioration, a-Ketoglutaric acid may be obtained from commercial sources. Recrystallize if not pure, as determined by titration with standard alkali. Transfer 100 to 500 mg. to a volumetric flask, using 5% acetic acid solution; dilute to the mark and store in the refrigerator. Prepare working standards, using 5% acetic acid for all dilutions. Collection of Samples and Preparation for Analysis. Draw blood from the vein with a minimum of stasis. Tap the syringe gently while holding it in a vertical position to remove the bubbles of gas; bring the plunger to the mark, and expel the contents through the needle into 5 vol. of TCA solution contained in a stoppered centrifuge tube. Mix the contents, and immmediately place in the refrigerator. Centrifugate, and analyze the clear supernatant solution. Collect urine in plastic or amber glass bottles containing enough 20 N H:S04 to give 0.05 to 0.1 N acidity at the end of the collection period. Urine contains considerable quantities of interfering earbonyl compounds, apparently derived from ingested foods, which can be removed by Lloyd's reagent. For every 10 ml. of the acidified urine add 0.75 g. of Lloyd's reagent; shake vigorously and filter immediately. Tissue suspensions, as used in in vitro studies of intermediary metabolic reactions, may be treated directly with TCA solution, which precipitates the proteins and stops the reactions. Tissues from living animals are frozen in liquid nitrogen; they are crushed 9 or ground; the cold samples are weighed and added to measured volumes of cold TCA solution. Weighed, fresh plant tissues are ground in measured volumes of cold TCA solution in the Waring blendor. The tissue suspensions are cleared by eentrifugation. General Procedure. Transfer 3.0 ml. of sample extract to each of two 18 X 200-ram. test tubes. For each series of not more than ten tubes, prepare three reagent blanks containing 3.0 ml. of TCA or acetic acid solution as in the sample extract. Incubate all tubes 10 minutes in a bath or pan of water maintained at 25 ___ 2 °. At accurately timed intervals, t j. B. Graeser, J. E. Ginsberg, and T. E. Friedemann, J. Biol. Chem. 104, 149 (1934).
[66]
DETERMII~IATION
OF a - K E T O ACIDS
417
say 30 seconds, add in turn 1.0 ml. of hydrazine solution to each tube. Continue incubation at 25 ° for the exact time indicated below, at which time add the exact volume of appropriate solvent from a buret to each tube at the timed intervals. Mix immediately by aerating the contents for 2 minutes with a rapid stream of air or nitrogen through a Wright capillary pipet in each tube (10-ram. o.d. glass tubing; upper portion 4 inches long; lower portion 8 inches long, drawn to fine capillary with square-cut tip; both ends flamed). Remove the capillary pipets. Centrifugate at low speed to facilitate separation of phases. Ethyl benzene and ethyl acetate separate readily without centrifugation. Attach rubber bulbs to capillary pipets, and remove the aqueous reaction phase. Give a violent circular motion to the tube to dislodge any remaining solution. (If considered necessary, add 1 ml. of water, mix contents by circular motion of tube, and centrifugate.) Remove the remaining aqueous phase. Add 6.0 ml. of Na2C03 solution from a pipet. Remove the rubber bulbs from the capillary pipets, and again mix the contents by rapid aeration for 2 minutes. Remove the capillary pipets; centrifugate if necessary. Insert rapidly a 5-ml. pipet through the solvent layer, and gently blow out any solvent which may have entered. Transfer 5.0 ml. of the ioa:er carbonate extract to a colorimeter tube, held in the bath or pan of water at 25 °. At definite intervals, say 30 seconds, add 5.0 ml. of NaOH solution in turn to each colorimeter tube. A red to orange-red color is produced. At the end of 5 minutes, at the same time intervals between each tube, determine the color density, after setting the galvanometer reading to 100 by means of the reagent blanks. Use a photoelectric colorimeter provided with a filter having maximal transmittance at 420 rag; if a spectrophotometer is used, set the instrument at wavelength 435 mg. METHOD 1. Determination of pyruvic acid and other m onocarboxylic keto acids. Proceed as above. Incubate the sample extract and hydrszine solution for exactly 5 minutes; then add 3.0 ml. of ethyl benzene. METHOD 2. Determination of a-ketoglutaric acid and other dicarboxylic keto acids. Proceed as above. Incubate for exactly 25 minutes; then add 8.0 ml. of benzyl alcohol. METHOD 3. Determination of total keto acids. Proceed as above. Incubate the sample extract and hydrazine solution for exactly 25 minutes; then add 8.0 ml. of ethyl acetate. Calculations
The procedure for calculating the. individual acids is illustrated by the following data and formulas based on analysis of standard solutions, using the Evelyn colorimeter with filter 420. Thus, the following respective calculated color densities per micromole of pyruvic and ~-ketoglutaric acids
418
CITRIC ACID CYCLE
[6~]
were observed: by method 1, 2.037 and 0.149; by method 2, 0.181 and 1.862; by method 3, 2.119 and 1.919. If D1, D2, and Da represent the observed densities by methods 1, 2, and 3, and P and G represent micromoles of pyruvic and a-ketoglutaric acids in 3 ml. of the sample extract taken for analysis, then D1 = 2.037P ~ 0.149(7 D2 = 0.181P ~- 1.862G D3 = 2.119P -{- 1.919G By using any two of the methods and by solving the two respective equations for P and G, the individual acids may be evaluated. However, the most accurate results are obtained with methods 1 and 2 which provide sharp separation of the hydrazones.
Concentrations in Blood and Urine The methods have yielded the following results. The blood from ten moderately active subjects contained 0.86 to 1.63 rag. of pyruvic acid per 100 ml., with an average of 1.204 mg.; it contained 0.10 to 0.32 mg. of ~-ketoglutaric acid, with an average of 0.19 mg. The 24-hour urine contains an average of 20 __+ 8 mg. of pyruvic acid and an average of 33 _ 15 mg. of a-ketoglutaric acid.
[67] Preparation and Assay of Oxalacetic Acid B y SAMUEL P. BESSMAN
Preparation Reactions
2 Na diethyloxalacetate ~- H~SO~ --* 2 Diethyloxalacetate W Na2S04 Diethyloxaiacetate W 2 HOH ~ Oxalacetic acid ~ 2 ethanol Method. 1-8 One hundred grams (0.48 mole) of sodium diethyloxalacetate is dissolved in 500 ml. of 10-8 M disodium ethylenediaminetetraacetate (Na2-EDTA). When solution is complete, the mixture is placed in a liter separatory funnel and concentrated sulfuric acid is added dropwise until acid to congo red paper. Diethyloxalacetate separates as a heavy oil. Sufficient ether is added with shaking to bring all the oil into
1L. S. Simon, Compt. rend. 137, 855 (1903). 2p. p. Cohen, J. Biol. Chem. 136, 565 (1940). 3L. O. Krampitz and C. H. Werkman, Bioehem. J. 35, 595 (1941).