This invention relates to a new and improved reagent and a method for its use in the quantitative determination of aldopentoses aldohexoses, or their derivatives (e.g., glucose, arabinose, xylose, glucuronic acid, etc.) in a body fluid (e.g., whole blood, blood serum, blood plasma, urine, etc.).
The accurate determination of aldopentoses, aldohexoses and their derivatives in body fluids is of great importance in the diagnosis and control of certain body illnesses. For example, knowledge of the concentration of glucose in urine or blood is of great value to diabetics who must rely on such knowledge in controlling their sugar intake. Similarly, the concentrations of xylose or arabinose in urine may be indicative of pentosuria.
It is especially desirable that such determinations be easily performed with a minimum of manipulations to reduce human error and expense. It is also desirable that the results of such analyses be in close agreement with the results of standard test procedures. For example, it is desirable that a method of glucose analysis be available which yields results in close agreement with results obtainable with the widely employed "AutoAnalyzer" (Technicon, Incorporated) thereby permitting glucose determinations to be made in a hospital laboratory when the AutoAnalyzer is being employed for other purposes. The AutoAnalyzer analysis for glucose commonly employs a ferricyanide reaction. Manual ferricyanide glucose analyses unfortunately require many manipulations by skilled technicians and are time consuming and expensive. Such analyses are explained in R. J. Henry, Clinical Chemistry: Principles and Techniques, Harper & Row, New York, New York, 1965, pp. 625-627.
Analytical methods for glucose and related chemicals are known, and a number of such methods are explained in Henry, supra., Chapter 19. Such methods are characterized, however, by time-consuming preparations and numerous manipulative procedures.
A photometric procedure for the analysis of glucose which employed an o-toluidine/trichloroacetic acid reagent was reported by E. Hultman, Nature, 183; 108 (1959). In the Hultman procedure a sample of whole blood was deproteinized by addition of trichloroacetic acid and subsequent centrifugation. To the clear supernate was added o-toluidine in glacial acetic acid. A blue-green color developed in the resulting solution upon heating, and the optical density of the solution at 625 mμ was then measured and correlated with the glucose concentration of the blood sample. This procedure, together with certain variations which have been subsequently reported, are characterized by the requirement that protein be precipitated from the body fluid sample and removed by e.g., centrifugation or filtration. Protein appears to interfere with the optical density of the colored solution, and normally causes unduly high optical density readings. Attempts have been made to employ the Hultman procedure without first removing protein. Such methods have attempted to use constant correction factors to compensate for the resultant errors in optical density readings, but entirely satisfactory results have not been obtained.
It is an object of the present invention to provide a sensitive, simple and economical method for the quantitative determination of aldopentoses, aldohexoses or their derivatives in body fluids.
It is another object of the present invention to provide a novel analytical reagent useful in the method of the present invention.
It is a further object of the present invention to provide a highly sensitive reagent and method for the quantitative photometric determination of glucose in a body fluid.
Briefly, the novel analytical reagent of the present invention comprises a solution of
b. a dimethylsulfoxide-soluble aromatic amine capable of reacting with glucose in the presence of oxalic acid to form, with heating, a colored product;
c. a dimethylsulfoxide-soluble acid capable of promoting, with heating, a color-forming reaction between o-toluidine and glucose in dimethylsulfoxide.
The present invention further relates to an improvement in the method for the quantitative determination of a body chemical selected from the group consisting of aldopentoses, aldohexoses or their derivatives in a body fluid sample, comprising reacting the body chemical with an aromatic amine to form, with heating, a colored liquid having a optical density dependent upon the concentration of the carbohydrate in the body fluid. The improvement comprises carrying out the reaction in the presence of an acid capable of promoting the reaction in dimethylsulfoxide, and in the presence of sufficient dimethylsulfoxide to render the aromatic amine, the acid, and the body fluid sample soluble in the reagent.
In its preferred embodiment, the method of the present invention may be carried out as follows:
a. a measured sample of the body fluid (e.g., blood serum) which contains a carbohydrate (e.g., glucose) is dissolved in a measured quantity of a reagent containing;
1. an aromatic amine (e.g., o-toluidine) which is capable, with heating, of reacting with glucose in the presence of oxalic acid to form a colored reaction product;
2. an acid (e.g., oxalic acid) which is capable of promoting, with heating, a color forming reaction between o-toluidine and glucose in dimethylsulfoxide; and
3. dimethylsulfoxide in sufficient quantity to render the aromatic amine, the acid, and the body fluid sample soluble in the reagent.
b. The resulting solution is heated (e.g., by placing a glass tube containing the solution into boiling water) for a short period (e.g., 4 to 10 minutes) and is then cooled to room temperature. During heating, the reaction between the carbohydrate and the aromatic amine produces a colored product which colors the solution.
c. The optical density of the colored solution is measured at a wavelength which is characteristic of the absorption of the colored product.
d. The measured optical density is correlated with the concentration of the carbohydrate in the body fluid by known means.
Acids which may be employed in the present invention are characterized by their ability to promote a color forming reaction between glucose and o-toluidine in dimethylsulfoxide with heating. Usefulness of a candidate acid in the present invention may be determined as follows: To each of a number of test tubes, each containing 100 ml. of dimethylsulfoxide, is added from 0.01 to 0.25 moles of the candidate acid. Thereafter, to each tube is sequentially added 4.0 ml. of o-toluidine and 0.5 ml. of an aqueous glucose solution having a concentration of 100 milligrams of glucose per 100 ml. of solution. After brief agitation, each tube is heated in boiling water for 6 minutes. Coloration of the solution in any of the tubes indicates that the candidate acid may be employed in the present invention. Examples of such acids include oxalic acid, phosphoric acid, sulfuric acid, phthalic acid and hydrochloric acid. Oxalic and phosphoric acids are especially preferred because the colored solutions which are obtainable through the use of such acids in the dimethylsulfoxide reaction medium unexplainably exhibit much more intense colors than do the colored solutions of Hultman. Such intense coloration appears to depend primarily upon the selection of oxalic or phosphoric acid as the acid component rather than upon the selection of a specific aromatic amine. The intense coloration obtainable by the use of oxalic acid greatly increases the sensitivity of the system and is believed to be primarily responsible for the unusual capability of the present invention to determine the concentration of such carbohydrates as, for example, arabinose, xylose and glucuronic acid in body fluids. Although the present invention may employ other acids in the determination of these carbohydrates, oxalic acid is greatly preferred.
Aromatic amines which may be employed in the present invention are characterized by their capacity to form, with heating, a colored product with glucose in the presence of oxalic acid and dimethylsulfoxide. To test the usefulness of a candidate aromatic amine, 4 ml. of the amine are added to 100 ml. of dimethylsulfoxide containing 6.0 g. of oxalic acid in a test tube. To the resulting solution is added 0.5 ml. of an aqueous glucose solution having a concentration of 100 gm. of glucose per 100 ml. of solution. The tube is briefly agitated and is heated in boiling water for 6 minutes. The appearance of a visually colored solution indicates that the candidate aromatic amine may be employed in the present invention. Examples of such aromatic amines include o-toluidine, 2,6-dimethyl aniline, and 2,5-dimethyl aniline (as reported by Hultman, supra.) and such other amines as p-bromoaniline, 3,5-dimethyl aniline and 2,4-dimethyl aniline.
The reagent of the present invention will tolerate the inclusion therein of numerous other components. For example, quantities of methanol, ethanol and isopropanol may be added to the reagent without significant adverse effect. The reagent will tolerate the addition of various other amines and acids, such as formic acid. Certain compounds, such as acetone, tend to interfere with the color forming reaction and hence should be avoided. To be avoided also are those compounds (e.g., trichloroacetic acid) which tend to precipitate protein from body fluids such as blood serum, since the necessary addition thereto of sufficient dimethylsulfoxide to counteract such precipitants may unduly dilute the color of the solution.
The quantity of dimethylsulfoxide required in the reagent of the present invention will vary according to the nature of the body fluid tested, the aromatic amine employed, the colored product produced, etc. It is required only that sufficient dimethylsulfoxide be present to render the aromatic amine, the acid, and the body fluid sample soluble in the reagent to form, upon heating, a colored solution (i.e., a colored fluid having no visually discernable cloudiness).
The relative quantities of acid and aromatic amine which may be employed in the present invention may vary widely. The optimum ratio of acid to amine depends upon the acid and amine chosen, and can be determined by simple experimentation. Normally the quantity of aromatic amine employed is in excess of that theoretically required to react with all of the carbohydrate in the body fluid sample, although lesser quantities of amine may be used. Acid is normally added in sufficient quantity so as to provide maximum coloration of the solution formed upon heating. In the most preferred embodiment of the present invention which employs oxalic acid, o-toluidine and dimethylsulfoxide, acceptable results have been obtained using a ratio by weight of oxalic acid/o-toluidine of from 1/1 to 2/1.
A weight ratio of oxalic acid/o-toluidine of from 1/1 to 3/2, however, is preferred.
The reaction mechanism of the present invention is not completely understood. It is believed, however, that several competing reactions occur, as represented by the following schematic diagrams wherein the carbohydrate, aromatic amine and acid are exemplified by glucose, o-toluidine and oxalic acid respectively. ##SPC1##
Reaction I (color-forming) is believed to be an equilibrium reaction which is far more rapid than reaction II. As the equilibrium of reaction I is shifted to the right, more colored product is formed. It appears that the equilibrium is shifted to the right by the use of oxalic or phosphoric acids in the present invention, since, as noted above, the use of such acids give rise to intensely colored solutions.
Heating of the combined body fluid-reagent may be easily accomplished by means of a boiling water bath, for example, although other methods of heating may be employed. The time and temperature required to cause formation of the colored solution will vary according to the contents of the solution. The temperature should be sufficiently high to permit a rapid color-forming reaction. The use of excessive temperatures or long periods of heating, however, may give rise to decomposition of the dimethylsulfoxide. In one experiment, for example, an odor believed to be dimethylsulfide was evolved from a solution of blood serum, oxalic acid, o-toluidine and dimethylsulfoxide upon prolonged heating of the solution in a boiling water bath. Temperatures of from 80° to 100° C. may be successfully employed, although 100° C. (the temperature of boiling water) is preferred. Heating by means of a boiling water bath for a period of 4 to 10 minutes is usually sufficient to develop full color in the solution.
In the method of the present invention, it has been found that if the aromatic amine is added to a heated solution of dimethylsulfoxide and body fluid prior to addition thereto of the acid, little color formation occurs. If, however, the acid is added prior to or concurrently with the aromatic amine, good color formation is obtained, the reason for which is not entirely understood.
Determinations could not easily be made by the Hultman procedure, supra., for such carbohydrates as xylose or arabinose, and could not be made at all for glucuronic acid. Arabinose and xylose normally are found in urine rather than in blood, and present procedures for their analysis are cumbersome and time consuming (see Henry, Clinical Chemistry: Principles and Techniques, supra., pp. 662-664). In the method of the present invention, the optical density of colored fluids produced from these carbohydrates is measured at wavelengths identifiable with such carbohydrates. Using the oxalic acid/dimethylsulfoxide/o-toluidine reagent, for example, the colored product from glucuronic acid exhibits distinctive absorption at 530 mμ, and the colored products of arabinose and xylose exhibit distinctive absorption peaks at 455 mμ. Simultaneous reduction of the data may be accomplished, for example, by the method of Swann, Adams and Weil as reported in Analytical Chemistry, Vol. 27, No. 10, pp. 1604-1606 (Oct., 1965). The use of oxalic or phosphoric acid in the reagent is especially useful in the determination of such carbohydrates, as explained above.
The present invention may be better understood by reference to the following examples, which are presented for illustrative purposes only and which should not be construed as limiting the scope of the invention.
A reagent solution was prepared by combining 100 ml. of dimethylsulfoxide, 6.0 grams of oxalic acid (anhydrous) and 4.0 ml. of o-toluidine. Using this reagent, the concentration of glucose was determined in 50 different blood plasma samples. The results were statistically compared to AutoAnalyzer results, to results obtained using the Hultman procedure, supra. [as reported by K. M. Dubowski, Clin. Chem. 8:215 (1962)] and to results obtained from a modification of the Hultman procedure.
A. Method of Present Invention
The concentration of glucose in each of the 50 plasma samples was determined as follows:
To 4.0 ml. of the above-prepared reagent in a glass tube were added 20 microliters of plasma. The tube was capped and placed in boiling water for 6 minutes. After cooling with cold water for 5 minutes, the optical density of the resulting bluish-green solution was measured at 630 mμ using a Coleman Jr. II spectrophotometer (Perkin-Elmer Corporation) which had been calibrated at zero on the reagent alone. In identical fashion, the optical densities of a series of aqueous 0.2 percent benzoic acid to glucose solutions of known concentration were measured, and a standard curve of glucose concentration versus optical density was graphically prepared therefrom. The standard curve was then employed to relate the concentration of glucose in each of the plasma samples to the optical density of the colored solution prepared therefrom.
B. Hultman Procedure
The concentration of glucose in each of the 50 plasma samples was determined by the method set forth in Dubowski, supra. Briefly, this procedure involves precipitating the protein from 20 microliters of blood plasma with 1.8 ml. of 3.0 percent trichloroacetic acid, filtering out the precipitate, combining 1.0 ml. of the filtrate with 3.0 ml. of 6 percent v/v o-toluidine/glacial acetic in a glass tube, immersing the tube for 10 minutes in a 100° C. fluid bath, cooling the tubes, and measuring the optical densities of the solutions at 630 mμ. Glucose concentration values were then derived as in "A" above.
C. Modified Hultman Procedure
The concentration of glucose in each of the 50 plasma samples was measured by the Hultman procedure described in "B" above, except that the step of precipitating protein from the plasma was omitted.
The concentration of glucose in the 50 plasma samples was measured by an AutoAnalyzer (Technicon Incorporated) which employed the ferricyanide reaction.
The concentrations of glucose were reported in milligrams of glucose per 100 ml. of plasma.
The results obtained by each of procedures A, B and C were separately plotted as ordinates against the AutoAnalyzer results as abscissas on rectangular coordinate paper. Straight lines were drawn through the data points by the method of least squares, each line relating the AutoAnalyzer results with the results respectively obtained by one of the above procedures A, B and C. The equation for each line was derived in the form y=mx+b wherein y represents values of glucose concentration according to procedures A, B and C above, x represents values of glucose concentration according to the AutoAnalyzer procedure, m is the slope of the straight line, and b represents the value of glucose concentration at the point of interception of the straight line with the y axis. Perfect agreement between results from procedure A and the AutoAnalyzer, for example, would give rise to a line wherein m=1.0 and wherein b=0 (i.e., y=x). As the values of m and b approach 1.0 and zero, respectively, the agreement between the results of the compared methods increases. A "correlation coefficient" Dixon, W. J. and Massey, F. J., Introduction to Statistical Analysis, 2nd Ed., McGraw-Hill, New York, 1957, Chapter 11) was computed for each comparison. As the value of the correlation coefficient approaches 1.0, the statistical probability that the compared values are identical increases. Table I lists the values for m and b for the comparisons of procedures A, B and C respectively with procedure D, together with correlation coefficients. --------------------------------------------------------------------------- TABLE
I Correlation Comparison m b Coefficient __________________________________________________________________________ A vs. D 1.028 -2.305 0.9912 B vs. D 0.953 +3.980 0.9870 C vs. D 0.842 -3.715 0.9891 __________________________________________________________________________
the correlation of procedure (present invention) with procedure D was significantly greater than the correlation of procedure C with procedure D, even though protein was not removed in either of procedures A or C.
Procedure A was repeated 20 times for one sample of blood serum to determine the reproducibility of the procedure. A standard deviation of 0.84 mg./100 ml. was obtained.
If the oxalic acid of Example I A is replaced with H3 PO4, H2 SO4, phthalic acid or HCl, solutions exhibiting the following colors are obtained (Table II). --------------------------------------------------------------------------- TABLE
II Acid & Quantity Other Reactants Color Obtained __________________________________________________________________________ 1.0 M H3 PO4 5% (v/v) o-toluidine blue in dimethylsulfoxide (+ glucose) 0.18 M H2 SO4 4% (v/v) o-toluidine red in dimethylsulfoxide (+ glucose) 10% w/v phthalic 4% (v/v) o-toluidine yellow-green acid in dimethylsulfoxide (+ glucose) 0.36 M HCl 4% (v/v) o-toluidine red-yellow in dimethylsulfoxide (+ glucose) __________________________________________________________________________
The o-toluidine employed in Example I A was replaced with other aromatic amines to provide solutions having the colors set forth in TABLE III. --------------------------------------------------------------------------- TABLE III
Aromatic Amine Colored Product Obtained __________________________________________________________________________ 2,5-dimethylaniline blue-green 2,6-dimethylaniline blue-green p-bromoaniline deep yellow 3,5-dimethylaniline yellow 2,4-dimethylaniline yellow __________________________________________________________________________
The procedure of Example I A was duplicated on identical samples of blood serum containing glucose, except that various solvents were substituted for portions of the dimethylsulfoxide. Results are presented in Table IV. --------------------------------------------------------------------------- TABLE IV
Solvent (ratio by volume) Solution Characteristics __________________________________________________________________________ dimethylsulfoxide/methanol (2/3) clear, blue-green dimethylsulfoxide/ethanol (2/3) clear, blue-green dimethylsulfoxide/isopropanol (2/3) clear, blue-green dimethylsulfoxide/acetone (3/1) no reaction observed dimethylsulfoxide/dimethylacetamide (1/9) green, cloudy __________________________________________________________________________
The relative sensitivities of the method of the present invention, the method of Hultman, and a modification of the Hultman procedure, were compared as follows:
To 4.0 ml. of each of the six reagent solutions listed below was added 50 microliters of an aqueous glucose solution containing 102 mg. of glucose per 100 ml. of solution. Each reagent solution was then heated for 6 minutes in boiling water and cooled. The optical density of each solution (less the optical density of the reagent blank) was measured in a 1 cm. cuvette at 630 mμ in a Hitachi Model 124 Spectrophotometer (Perkin-Elmer Corporation). The results, which are reported below show the increase in optical density obtained with the method and reagent of the present invention (reagents A-D), as compared to the method of Hultman and a modification thereof (reagents E and F).
reagent A: dimethylsulfoxide 50 ml. o-toluidine 2 g. oxalic acid 3 g. Optical density at 630 mμ 0.210
Reagent B: dimethylsulfoxide 50 ml. o-toluidine 2.5 g. oxalic acid 5.0 g. Optical density at 630 mμ 0.450
Reagent C: dimethylsulfoxide 50 ml. o-toluidine 2 ml. phosphoric acid 4.16 g. Optical density at 630 mμ 0.250
Reagent D: dimethylsulfoxide 50 ml. o-toluidine 2 ml. phosphoric acid 6.37 g. Optical density at 630 mμ 0.228
Reagent E: acetic acid 50 ml. o-toluidine 3.0 ml. Optical density at 630 mμ 0.165
Reagent F: 6% aqueous o-toluidine 50 ml. 3% aqueous trichloroacetic acid 1 ml. Optical density at 630 mμ 0.125