US 3899397 A
An improved diazonium coupling procedure for L-aspartate: 2-oxoglutarate amino transferase, EC 18.104.22.168, or glutamic-oxalacetic transaminase (GOT) assay. The method for determining the glutamic-oxalacetic transaminase in biological fluids involves preparing a substrate of 1-aspartic acid, alpha -ketoglutaric acid, buffer, surfactant and water. The substrate has a pH between about 5 and about 8, preferably 7.5. A sample of biological fluid is admixed with substrate. The admixture is incubated. Then an aprotic solvent is added to the admixture to stop the reaction between the 1-aspartic acid and alpha -ketoglutaric acid which is catalyzed by the glutamic-oxalacetic transaminase. A diazonium dye and methanol is then added to the admixture. The activity level of the glutamic-oxalacetic transaminase by measuring the absorbance against a reagent-specimen blank is determined. The preferred diazonium dye is Diazo Red RC.
Description (OCR text may contain errors)
United States Patent [1 1 Morin et al.
[4 1 Aug. 12, 1975 GLUTAMIC OXALACETIC TRANSMINASE ASSAY METHOD  Inventors: Leo G. Morin; Jerome R. Prox, both of Miami, Fla.
 Assignee: Medico Electronic Inc., Indianapolis,
 Filed: July 5, 1973  Appl. No.: 376,333
 U.S. Cl 195/1035; 424/7  Int. Cl. Cl2k 1/04; C07g 7/02  Field of Search 195/1035  References Cited UNITED STATES PATENTS 3,069,330 l2/l962 Babson 195/1035 3,458,403 7/1969 Katsunuma 195/1035 Primary Examiner-A. Louis Monacell Assistant Examiner-Esther L. Massung Attorney, Agent, or FirmChristen & Sabol ABSTRACT An improved diazonium coupling procedure for L- aspanate: 2-oxoglutarate amino transferase, EC 2.6.l.l, or glutamic-oxalacetic transaminase (GOT) assay. The method for determining the glutamicoxalacetic transaminase in biological fluids involves preparing a substrate of l-aspartic acid,a-ketoglutaric acid, buffer, surfactant and water. The substrate has a pH between about 5 and about 8, preferably 7.5. A sample of biological fluid is admixed with substrate. The admixture is incubated. Then an aprotic solvent is added to the admixture to stop the reaction between the l-aspartic acid anda-ketoglutaric acid which is catalyzed by the glutamic-oxalacetic transaminase. A diazonium dye and methanol is then added to the admixture. The activity level of the glutamic-oxalacetic transaminase by measuring the absorbance against a reagent-specimen blank is determined. The preferred diazonium dye is Diazo Red RC.
9 Claims, 3 Drawing Figures GLUTAMIC OXALACETIC TRANSMINASE ASSAY METHOD BACKGROUND OF THE INVENTION 1. Field of This Invention This invention relates to an improved diazonium coupling procedure for glutamic oxalacetic transaminase assay. This invention also relates to a reagent for use in such an assay procedure.
2. Prior Art 7 The oxalacetate diazonium salt coupling method of Babson, A. L., Shapiro, P. 0., Williams, P. A. R., and Philips, G. E., The Use ofa Diazonium Salt for the Determination of Glutamic-Oxalacetic Transaminase in Serum, Clin. Chim. Aeta 7, 199 (1962), for L- aspartate: 2-oxoglutarate amino transferase determination represents a superior colorimetric approach to those utilizing dinitrophenylhydrazine, e.g., Reitman, S., and Frankel, S., A Colorimetric Method for the Determination of Serum Glutamic Oxalacetic and Glutamic Pyruvic Transaminases, Am. J. Clin. Pat/101. 28, 56 1957), and Cabaud, P., Leeper, R. and Wroblewski, F., Colorimetric Measurement of Serum Glutamic Oxalacetic Transaminase, Am. J. Clin, Pathol. 26, 1101 (1956). But a number of problems, well known to the art, exist with the Babson et a1. method. Numerous improvements have been made on the original procedure, including changes in the diazonium salt, see Morgenstern, S., Kaufman, J. H., and Klein, B., Automated Determination of Serum Glutamic Oxalacetic Transaminase,C1in. Chem. 13, 270 (1967), in the substrate concentration, see Sax, S. M., and Moore, J. J., Determination of Glutamic Oxalacetic Transaminase Activity by Coupling of Oxalacetate With Diazonium Salts, Clin. Chem. 13, 175 (1967), and by the introduction of color stabilizers, see Furuno, M., and Sheena, A., Adaption of Babsons Method for the Determination of Serum Glutamic Oxalacetic Transaminase in the Clinical Laboratory, Clin. Chem. ll, 23 (1965), and Amador, E., and Salvatore, A. C., Serum Glutamic Oxalacetic Transaminase Activity: Revised Manual and Automated Methods Using Diazonium Dyes, Am. J. Clin. Pat/ml. 55, 686 (1971 Despite these improvements, several problems still exist with the Babson et a1. procedure, namely, (1 the color development is continuous and requires the addition of a stopper or stabilizer at a precise time; (2) poor light sensitivity; and (3) precipitation occurs.
Attention is drawn to US. Pat. No. 3,069,330, inventor Arthur L. Babson.
BROAD DESCRIPTION OF THIS INVENTION The rate of the reaction of the L-aspartic acid with a-ketoglutaric acid catalyzed by the enzyme glutamicoxalacetic transaminase present in an unknown body fluid, and thus the concentration of this enzyme in the body fluid, may be determined by measuring the depth of color developed by the coupling of a Diazonium salt with the oxalacetic acid formed by the reaction.
The rate of this reaction is directly proportional to the concentration of such enzyme in the reaction medium. It is known that this transaminase enzyme is released within the body in certain types of cell destruction and for this reason the measurement of the enzyme concentration is a valuable aid in the diagnosis of diseases where such cell destruction takes place as, for example, myocardial infraction, intrahepatic lymphoma or carcinoma, hepatitis, cirrhosis and the like. This enzyme is released into body fluids, such as the blood, spinal fluid and the like.
This invention involves a method for determining the amount of glutamic-oxalacetic transaminase (enzyme) in biological fluids. Broadly, the process includes (i) preparing a substrate of l-aspartic acid, a-ketoglutaric acid and water, the substrate having a pH between about 5 and about 8. A sample of biological fluid is ad mixed with the substrate. The admixture is incubated. An aprotic solvent is added to the admixture to stop the reaction between the l-aspartic acid and a-ketoglutaric acid which is catalyzed by the glutamic-oxalacetic transaminase. A diazonium dye and methanol, is then added to the admixture, It is essential that methanol be used. The activity level of the glutamic-oxalacetic transaminase by measuring the absorbance against a reagent-specimen blank is determined.
Preferably the substrate has a pH of 75. Preferably the substrate contains a buffer. A preferred substrate also contains dibasic potassium phosphate, tetrasodium ethylene-diaminetetraacetate, polyvinylpyrrolidone and Triton X-100. The preferred aprotic solvent is di methylformamide. The preferred diazonium dye is diazo-2-amino-4-chloro-anisole. In the most preferred embodiment, the aprotic solvent is dimethylformamide, and the substrate has a pH of 7.5.
This invention also includes the various reagents used herein.
This invention eliminates several difficulties with the known diazonium coupling procedures. The use of dimethylformamide greatly enhances the rate of color development and, together with methanol and Diazo Red RC, results in complete and stable color development within 5 minutes. Further, the use of dimethylformamide reduces remarkably the turbidity and precipitation problems. Dimethylformamide inhibits the enzyme activity and allows for the preparation of specimen blanks by the addition of serum to a completed reagent blank. The use of methanol virtually eliminates the problem oflight sensitivity and helps to stabilize the color, eliminating the necessity to add a color inhibitor at a precise time. There has been an improvement of linearity extending to 360 I.U. Diazo Red RC is comparable to Fast Red PDC in specificity and sensitivity, but is considerably less sensitive to pH changes, a distinct advantage. Further, Diazo Red RC is a more economical dye to use.
The method of this invention provides a rapid, accurate and simple procedure for measuring the concentration of glutamicoxalacetic transaminase in blood serum or other body fluids. The method provides accuracy and simplicity not attainable by methods heretofore known. The diazonium salt forms a colored coupling product only with oxalacetic acid and not with any other ingredient present in the incubated test mixture. The coupling product has a visible color so that usage of a complex ultraviolet spectrophotometer is not required, and simple comparison of the color developed with a standard can be used.
DETAILED DESCRIPTION OF THIS INVENTION The substrates of this invention, when buffered, are stable for at least one month when refrigerated. The substrates of this invention contain at least l-aspartic acid, a-ketoglutaric acid, and water and have a pH between about 5 and about 8.
The substrate employed need only contain cz-ketoglutaric acid and L-aspartic acid as the active components and the latter may be added either as the pure L-form or as the racemic mixture, D,L-aspartic acid. The amounts present are not critical, with the exception that a substantial excess of each should be present.
The substrate preferably contains a nonionic surfactant. The preferred nonionic surfactant is Triton X- 100. Examples of other useful nonionic surfactants are the various polyoxyethylene sorbitan esters of fatty acids available under the tradename TWEEN and the nonylphenyl-polyethylene glycol esters available under the tradename BRIJ. From 0.01 to percent ofa nonionic surfactant can be used.
The preferred substrate also contains a-ketoglutaric acid, tetrasodium ethylene-diaminetetraacetate, and polyvinyl-pyrrolidone.
The pH of the substrate should be between about 5 and about 8, but most preferably is 7.5. The pH can be achieved by adding a base, such as. potassium hydroxide (preferred), calcium hydroxide, lithium hydroxide, sodium hydroxide, potassium carbonate, calcium carbonate, ammonium hydroxide, lead hydroxide, zinc hydroxide, barium hydroxide, barium carbonate, lithium carbonate, ammonium carbonate, borax, lime, ammonium bicarbonate, magnesia, sodium bicarbonate, ferrous hydroxide, hydrazine, hydroxylamine, lithium car bonate, and sodium carbonate. Preferably a buffer is added to the substrate before the pH is adjusted to the desired level. The buffer serves the function of keeping the pH at the desired level during the assay. Examples of useful buffers are dibasic potassium phosphate (preferred), monobasic dipotassium phosphate, trisodium phosphate, sodium barbital and tris(hydroxymethyl) aminomethane.
As used herein, the term biological fluids includes blood, blood serum. spinal fluid, and the like. Blood serum, which is the liquid fraction obtained by centrifuging clotted whole blood, is the most commonly used biological fluid.
The time and temperature of the incubation can be varied with good results resulting, but preferably the incubation is conducted at 30C. for minutes. In general the incubation should be for a controlled time of 5 to 30 minutes at a constant temperature within the range of about C. to about 40C. to permit the desired reaction to proceed whereby the oxalacetic acid is liberated. With low temperatures, relatively longer times are required, while with higher temperatures, more rapid reaction is obtained. For reproducible results, of course, uniform incubation conditions should be maintained for every determination.
Some enhancement of the color development has been found for all of the aprotic solvents which have a high dielectric constant, but unexpectedly it has been found that dimethylformamide gives excellent enhancement of the color development. The rate of diazonium coupling is greatly enhanced by the presence of dimethylformamide (see FIG. 1). This is true for diazonium dyes such as diazotate 6-benzoyl-amino-4- methoxy'5-toluidine (Fast Violet B) and diazotate N-butyl-4-methoxymetanilamide (Fast Red PDC or Fast Ponceau L) as well as Diazo Red RC. Another effect of the dimethylformamide on all three dyes is to increase the light sensitivity by at least six-fold. In balancing enhanced color development against increased photosensitivity, a level of 20 to percent dimethylformamide was selected as optium (same for the other aprotic solvents), although 5 to 50 percent can readily be used.
Monograph of the National Bureau of Standards defines dipolar aprotic solvents. Aprotic solvents are almost devoid of acidic or basic properties, for example, aprotic solvents do not interact strongly with acidic solutes, such as carboxylic acids, phenols and mineral acids, or with basic solutes, such as amines and derivatives of quanidine or pyridine. They are comparatively inert in character. Aprotic solvents are not masking or leveling solvents, such as water. Indifferent solvents are other names for aprotic solvents. In general, aprotic solvents are organic compounds.
There are generally two classes of aprotic solvents, but this invention uses those aprotic solvents which are termed dipolar aprotic solvents having a high dielectric constant (e), i.e., ranges from about 20 to about 50. The preferred aprotic solvent is dimethylformamide. Examples of other aprotic solvents in this class are acetone, acetonitrile, nitrobenzene, nitromethane, methyl sulfoxide, benzonitrile, nitromethane, dimethylacetamide, sulfolane (tetramethylenesulfone), N-methyl formamide, formamide, N-methyl propionamide, dimethyl sulfoxide, diethylformamide, diethylacetamide, diethylsulfoxide, dipropylformamide, dipropylacetamide, dipropylsulfoxide, dimethylpropiamide, dimethylbutyramide, and mixtures thereof.
Dimethylformamide is preferably admixed with water to form a diluent solution; such diluent solution is stable almost indefinitely, e.g., at least 6 months when refrigerated.
Any diazonium dye, such as, 6-benzoyl-amino-4- methoxy-S-toluidine and N-butyl-4- methoxymelamilamide, can be used in conjunction with the use of the aprotic solvent and methanol, with an increase of color enhancement and color development control. But only when diazo-2-amino-4 chloroanisole or Diazo Red RC is used is there an unexpected very large increase in color enhancement and color development control. The preferred diazonium dye is diazo-2-amino-4-chloro-anisole. When the diazo- 2-amino-4-chloro-anisole is admixed in anhydrous methanol for use as a reagent, the reagent is stable for 1 working day.
In re color development, Diazo Red RC was found to be comparably sensitive and specific for oxalacetic as Fast Red PDC, yet considerably less sensitive to pH changes (see FIG. 3). In this procedure, the final color development pH is preferably 7.5. The optimum level of Diazo Red RC was selected as 0.816 mg. per assay, although other levels can readily be used, but normally at least 0.4 mg. per assay should be used (herein assay equals 0.1 ml. of serum). In other words, an excess of diazonium dye should be used.
When methanol is admixed with the diazonium dye, methanol was found to virtually eliminate light sensitivity under test conditions. When methanol was used in conjunction with dimethylformamide, stabilized color development was achieved with diazonium dyes, such as, Fast Red PDC and Diazo Red RC (see FIG. 2). It was found that relatively small and uncritical quantities of methanol were required, 5 to 15 percent being optimum, but 1 to 40 percent could be effectively used.
Experiments have shown that other alcohols, such as, isopropanol, butanol and ethanol, do not work in the manner which methanol has been found to work. The
action of methanol appears to be unique and critical.
Any suitable colorimeter or spectrophotometer can be used to measure the absorbance. Examples of useful colorimeters are: Coleman, Model 44; Perkin-Elmer, Model 124; the colorimeter disclosed in US. patent application Ser. No. 224,457, applicants: Raymond W. Kiess and Peter H. Stewart, filed: Feb. 8, 1972 assignee: Kiess Instruments, Inc., 8768 S. W. 131st Street, Miami, F1a., 33156; and the direct reading colorimeter disclosed in US. Pat. No. 3,561,878, inventor: R. W. Kiess. Also, the absorbance can be measured by visual comparison with a standardized color chart.
The absorbance is measured at 470 nm (point of maximum absorbance). The absorbance is measured against a reagentspecimen blank, which is prepared by adding the serum to a complete reagent blank.
The color is essentially developed in about 5 minutes after the diazonium dye or salt is added. The diazonium dye forms a colored coupling product only with oxalacetic acid and not with any other ingredient present in the incubated test mixture. The coupling product has a visible color.
FIG. 1 demonstrates the effect of dimethylformamide (DMF) on the rate of diazonium coupling with oxalacetate. In FIG. 1: PDC Fast Red PDC, pH 4.2; RC Diazo Red RC, pH 7.5; B Fast Violet B, pH 7.5. A black dot before the symbol indicates the presence of 25 percent of dimethylformamide. The oxalacetate level in all instances was 0.8p.mol/3.3 ml.
FIG. 2 demonstrates the effect of 7 percent methanol and 25 percent dimethylformamide (DMF) on the rate of diazonium coupling with oxalacetate and on color stability. In FIG. 2: PDC Fast Red PDC, pH 4.2; RC Diazo Red RC, pH 7.5; B Fast Violet B, pH 7.5. In all instances the oxalacetate level was 0.8}1-11101/33 m1., except that it was 0.4,u.mol for lower RC curve.
FIG. 3 demonstrates the effect of pH on standard curves of diazonium coupling with oxalacetate. Except for the dye and pH, all of the parameters are as used in Example l. Concerning FIG. 3:
Fast Red PDC, p Fast Red PDC. p Diazo Red RC. p Diazo Red RC. p
EXAMPLE 1 A buffered substrate is prepared by dissolving 17.4 gm. of anhydrous dibasic potassium phosphate, 17.3 gm. of l-aspartic acid. 2.9 gm. of a-ketoglutaric acid, 0.8 gm. of tetrasodium ethylene-diaminetetraacetate, 8 gm. of polyvinylpyrrolidone (Plasdone C, G.A.F., New York. N. Y. 10020), and 1 ml. of Triton X-100 (Rohm and Haas, Philadelphia, Pa. 19105) in 900 ml. of distilled water. The pH of the substrate was adjusted to 7.4 with dropwise addition of 1N KOH, and the volume was brought to 1 liter. This solution was stable for at least 1 month when refrigerated.
A DMF diluent solution was prepared by bringing 250 m1. of dimethylformamide (DMF) up to 1 liter with distilled water. This diluent solution was stable almost indefinitely (at least 6 months when refrigerated).
A Diazo Red RC reagent was prepared by dissolving 40.8 mg. of Diazo Red RC (Sigma Chemical Co., St. Louis, Mo. 63118) in 10 ml. of anhydrous methanol,
and stored in an amber vial. This reagent solution was suitable (stable) for only one working day.
To a series of tubes, 0.5 ml. of the buffered substrate was added. The tubes were warmed to a constant 30C., 0.1 m1. of serum added to each and mixed in; and the tubes were incubated at 30C. for 15 minutes. The reaction was stopped by adding and mixing in 2.5 ml. of the DMF diluent solution. Then, 0.2 ml. of Diazo Red RC reagent was added and mixed. After 5 minutes, the activity from the absorbance read at 470 nm against a reagent blank or specimen blank (i.e., serum added to complete reagent blank) was determined.
Standardization was made with Versatol E (General Diagnostics, Morris Plains, N. J. 07950) and dilutions thereof, reassayed kinetically (according to the method of Amador, E., and Wacker, W. E. C., Enzymatic Methods Used in Diagnosis", Methods of Biochemical Analysis (Vol. 13). Ed. by Glick, D., Interscience Publishers, Inc., New York, 1965, pp. 265-356), and correlated to an oxalacetate standard curve to yield results in International Units. With the several lots of Versatol E'"' used, it was found that the given TransAc"" assay values were within 1 1% of applicants assay; consequently, except for the most exacting work, the given assay values could cautiously be used for standardization.
EXAMPLE 2 The precision of applicants method was tested with 40 replicate assays each of Versatol E"' diluted to 200 I.U., I.U., and 30 I.U. The means i 1 standard de viation were 200 i 6 I.U., 100 i 5 I.Uv and 30 i 2 I.U. Day-to-day precision was tested in the same manner 5 times over a 7 month period (total of 200 determinations) and the variation coefficient was 3.9%.
Sixty samples from a general hospital population were assayed by this procedure on the DMA 16/P (Kiess Instruments, Inc., Miami. Fla. 33156), and by the Morgenstern et a1. procedure on the SMA 12/60 (Technicon Corporation, Tarrytown, N. Y. 10591 Correlation was good, the regression equation being y =O.98 1.63 with a coefficient of determination (r of 0.96. There was no significant difference between the means of 89.3 for this procedure and 83.2 for the autoanalyzer by t test pair analysis (0.50 p 0.40).
- What is claimed is:
1. A method for determining the glutamic-oxalacetic transaminase in biological fluids which comprises: (a) preparing a substrate of l-aspartic acid, a-ketoglutaric acid and water, said substrate having a pH between about 5 and about 8; (b) admixing a sample of biological fluid and said substrate; (0) incubating admixture (b); (cl) admixing a dipolar aprotic solvent and methanol with admixture (b) to stop the reaction between the l-aspartic acid and a-ketoglutaric acid which is catalyzed by the glutamic-oxalacetic transaminase; (e) admixing a diazonium dye with admixture (d); and (f) determining the activity level of the glutamic-oxalacetic transaminase by measuring the absorbance against a reagent-specimen blank.
2. A method as described in claim 1 wherein said substrate has a pH of 7.5.
3. A method as described in claim 1 wherein said substrate contains a buffer.
4. A method as described in claim 3 wherein said substrate also contains dibasic potassium phosphate, tetra- 7 8 sodium ethylene-diaminetetraacetate, polyvinylaprotic solvent is dimethylformamide. pyrrolidone and Triton X-lOO. 8. A method as described in claim 7 wherein the pH 5. A method as described in claim 1 wherein said is 7.5. aprotic solvent is dimethylformamide. 9. A reagent for use in determinations of glutamic- 6. A method as described in claim 1 wherein said dia- 5 oxalacetic transaminase in biological fluids which conzonium dye is diazo-2-amino-4-chloro-anisole. sists of diazo-2-amino-4-chloro-anisole and methanol.
7. A method as described in claim 6 wherein said