THE OXIDATION OF DIXANTHYDRYL UREA, A MICRO METHOD FOR DETERMINING UREA.

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1 THE OXIDATION OF DIXANTHYDRYL UREA, A MICRO METHOD FOR DETERMINING UREA. BY JAMES MURRAY LUCK. (From the Department of Chemistry, Stanford University, California.) (Received for publication, June 13, 1928.) The method described by Kiech and Luck (1) for the estimation of urea suffers from three undesirable features. Like many gravimetric methods it requires special apparatus and the expenditure of much time. A fairly large amount of urea, somewhat more than 0. mg., is also needed. This quantity is contained in about 20 cc. of the tungstic acid filtrate from normal blood and tissues and only too often it was found inconvenient to spare so large a quantity. We were therefore driven to exercise further economies in time and material. In the course of his extensive description of xanthydrol, Fosse (2) pointed out that this substance is readily oxidized to xanthone. From this it occurred to us to investigate the action of oxidizing agents upon dixanthydryl urea in the hope of developing a quantitative volumetric method for the estimation of urea. A few preliminary experiments showed that dixanthydryl urea dissolved in strong acids to give fluorescent solutions of a brilliant canaryyellow color, which on oxidation with potassium permanganate, became colorless. As the end-point could be determined quite readily under the optimum conditions, we were enabled to utilize these facts as the basis of a quantitative method. Before describing the procedure, it would be well to point out that oxidation appears to proceed far beyond the formation of xanthone. Method. 2 to cc. of the tungstic acid filtrate (1, 3) contained in a 1 cc. centrifuge tube are diluted to cc. with water. (For normal We have been employing Eastman s preparation of xanthydrol. We have not succeeded in obtaining complete solution of the substance in methyl alcohol, although Fosse appears to have done so. The solution 211

2 212 Determination of Urea blood use cc. of filtrate.) cc. of glacial acetic acid and 0. cc. of 10 per cent xanthydrol in methyl alcohol2 are added. The contents must be intimately mixed. After 1 hour the tube is centrifuged, the supernatant fluid is decanted off, and the precipitate of dixanthydryl urea washed once with cc. of a saturated solution of dixanthydryl urea in methyl alcohol. This serves to remove the excess xanthydrol. The tube is again centrifuged and the fluid removed by decantation. The excess of alcohol which clings to the precipitate and the wall of the tube is removed by drying. A few minutes at 100 is sufficient for this purpose. 8 cc. of cold 1:l sulfuric acid (equal volumes of water and concentrated acid) are now added.3 The residue dissolves slowly to form a yellow solution which is washed with 32 cc. of water into an evaporating basin of white porcelain, 10 cm. in diameter. The contents are heated to 70-7 and titrated at this temperature with 0.0 N potassium permanganate contained in a cc. burette. It is advisable to carry out the titration in a bright, white light having at hand a second porcelain basin containing 40 cc. of water. The latter is of great assistance as a standard and is used for purposes of comparison as the endpoint is neared. It should not be artificially illuminated. If the basin containing the solution of dixanthydryl urea be removed from beneath the burette and placed beside the standard, or between two such standards after each addition of permanganate the end-point may readily be determined. The fluid must be titrated until the last tint of yellow has disappeared. It is necessary to maintain an acid concentration of 10 per cent (by volume). Since the latter changes by dilution during the titration, it is advisable to add, when near the end-point, an addi- used in this work was prepared by suspending 10 gm. of xanthydrol in 90 gm. of pure methyl alcohol, shaking at intervals during 3 days, and filtering. 2 Since this method is applicable to the estimation of dixanthytlryl urea however prepared, it is apparent that the use of other protein precipitants or conditions of precipitations are permissible,-provided only that the subsequent formation of dixanthydryl urea be quantitative and productive of a pure, crystalline ureide. 3 Complete solution of the dixanthydryl ureais essential. It is advisable to pulverize the precipitate by grinding it against the bottom of the centrifuge tube with a stirring rod before adding the sulfuric acid.

3 J. M. Luck 213 t,ional amount of the 1: 1 sulfuric acid equal to one-fourth of the volume of permanganate employed. Not more than 3 cc. of permanganate should be employed in the titration. Somewhat beyond this point the relationship between the amounts of permanganate and urea ceases to be linear. Below this upper limit 1 cc. of 0.0 N KMn04 = mg. of urea. For the estimation of urea in urine, dilute 1 cc. of urine in a volumetric flask to 100 cc. Transfer 1 cc. of the diluted urine to a 1 cc. centrifuge tube, add 4 cc. of water, cc. of glacial acetic acid, and 0. cc. of the methyl alcohol solution of xanthydrol. Continue with the procedure given above. EXPERIMENTAL. Preparation of Dixanthydryl Urea. To 40 cc. of 0.20 per cent urea were added 140 cc. of glacial acetic acid and 20 cc. of 10 per cent xanthydrol in methyl alcohol. The latter was added in cc. portions at minute intervals. 1 hour and 2 minutes after the last addition, the mixture was centrifuged and the crystalline precipitate washed with 100 cc. of ethyl alcohol and -again centrifuged. The residue was washed onto a suction filter with 0 cc. of ethyl alcohol and dried at 60. The product was employed in the first few experiments and for preparation of the saturated solution of dixanthydryl urea in methyl alcohol. Optimum Concentration of Sulfuric Acid mg. of dixanthydryl urea were dissolved in 100 cc. of 1: 1 sulfuric acid. 3 cc. portions were diluted with water and sulfuric acid to volumes of 40 cc. and sulfuric acid concentrations of to 0 per cent. They were titrated at with 0.09 N potassium permanganate. It was found that the titration value is dependent upon the concentration of sulfuric acid and is indeed a linear function of the concentration between and 30 per cent. For future work it was decided to employ an acid concentration of 10 per cent. The color given by the dixanthydryl urea in per cent sulfuric acid was relat,ively faint. It is also obvious that a

4 214 Determination of Urea maximum depth of color with a maximum titration value is desirable. Nevertheless it was not permissible to use very high concentrations of sulfuric acid. In an acid concentration of 0 per cent the fluid became brownish during titration, the end-point was very obscure and, indeed, charring appeared to have proceeded. In order to minimize the possibility of such a happening, the relatively low acid concentration of 10 per cent was selected. This was used in all later work. Only on completion of the experiments described in this paper was it discovered that in view of the approximately linear relationship between acid concentration and cc. of permanganate the use of at least 20 per cent sulfuric acid would probably have been permissible. As the titrations have proceeded satisfactorily under the conditions described we have not seen fit to attempt this change, although the delicacy of the method might be thereby enhanced. Volume of Sulfuric Acid. Although the quantity of permanganate required for the oxidation of a given amount of dixanthydryl urea is a function of the concentration of sulfuric acid it is independent of the volume. Five portions of dixanthydryl urea solution, each of 3 CC., were each diluted with 12 cc. of water. Increasing amounts of 10 per cent sulfuric acid (1 cc., 2 cc., 2 cc., 40 cc., 40 cc.) were added. The solutions were then titrated at 70-7 with 0.09 N K(Mn04. The following titration values were obtained: 1.34 cc., 1.29 cc., 1.32 cc., 1.28 cc., and 1.33 cc. respectively. The experiment was repeated with 4 cc. portions of dixanthydryl urea solution to which were added 16 cc. of water and to 40 CC. of 10 per cent sulfuric acid. Titrations were made with N KMn04 at Fourteen samples were run. All values fall between 2.0 and 2.19 cc. of potassium permanganate. Oxidation of Varying Quantities of Dixanthydryl Urea mg. of dixanthydryl urea were dissolved in 1: 1 sulfuric acid and made up to a volume of 200 cc. Ten portions of increasing size (2 cc. to 8 cc.) were diluted with water and sulfuric acid to final volumes of 40 cc. and acid concentration of 10 per cent. Titrations were made at It was found that up to 2 mg. of dixanthydryl urea the quantity

5 J. M. Luck 21 of potassium permanganate required for the oxidation is a linear function of the amount of ureide present. All values for the urea equivalent (in mg.) of I.0 cc. of 0.0 N potassium permanganate fell between and The average was Application of Method to Solutions of Urea. An aqueous solution of urea containing 100 mg. of urea per liter was prepared. Portions of the solutions contained in small Urea solution. cc Estimation N KMnOa. cc TABLE I. f Urea. Urea found. Urea calculated. m mfl centrifuge tubes were made up to cc. with water. The urea was precipitated as dixanthydryl urea by the addition of cc. of glacial acetic acid and 0. cc. of xanthydrol solution. The washed precipitates were estimated by oxidation with potassium permanganate, according to the procedure already described. The results are presented in Table I.

6 216 Determination of Urea Estimation of Urea in Blood. Six portions of rabbit blood, each of 2 cc., were treated as follows: To the first was added 14 cc. of water, to the second 13 cc. of water and 1 cc. of urea solution (0.2 mg. per cc.), to the third 12 cc. of water and 2 cc. of urea, to the fourth 11 cc. of water and 3 cc. of urea, to the fifth 10 cc. of water and 4 cc. of urea, and to the sixth 8 cc. of water and 6 cc. of urea. Each received 2 cc. of sodium tungstate (10 per cent) and 2 cc. of sulfuric acid (0.66 N). Of the six filtrates which were then obtained, 2 and 3 cc. portions of each were treated with water, glacial acetic acid, and xanthydrol according to the routine procedure. The experiment was repeated upon the blood of another rabbit. The results of both experiments are summarized in Table II. Found. ml TABLE Recovery of Urea from Blood. Urea in 2 cc. of blood. II. Experiment 1. Experiment 2. _I Calculated. Found. Calculated. I WJ m mg Each value represents the average of two determinations, one of which was obtained from 2 cc. of filtrate, and the other from 3 cc. Estimation of Urea in Animal Tissue. An adult female rat was killed by stunning and deeply incising the thorax. The blood was collected over potassium oxalate and the liver and muscles of one hind limb excised and frozen with liquid air. The powdered tissues, 4 gm. samples, were treated with tungstic acid as described in a preceding paper (1). Urea estimations were made upon portions of each filtrate by the volumetric method described in this paper. These results, together with those from a second rat, are presented in Table III.

7 J. M. Luck 217 Use of Tanret s Reagent as a Protein Precipitant. Kiech and Luck (1) reported that the application of their method to muscle gave impure, yellowish precipitates of the ureide. In the course of the experiments which have been summarized in Table III, trouble was again experienced with the muscle preparations. The unwashed dixanthydryl urea obtained after the first centrifugation was somewhat discolored. Practically all of the colored impurity appeared to dissolve in the methyl alcohol for the washed product was quite white. Nevertheless when the attempt to dissolve the dried precipitates in 1:l sulfuric acid was made, there appeared to be a small portion of the muscle samples which resisted solution. TABLE Urea Content of Rat Blood, Liver, and Muscle. Rat 1. -~ Rat 2. cc. cc. Blood I Liver Muscle I III. 0.0 N KMnO4. Urea in 100 gm. of tissue. Rat 1. Rat Rat 1. Rat 2. cc. cc. mg. ml It seemed advisable therefore to check the tungstate procedure by the use of another protein precipitant. For this purpose we employed Tanret s reagent4 as modified by Fosse, Robyn, and Franpois (4). Duplicate 4 gm. samples of frozen, powdered liver and muscle were weighed out. Two rats had to be employed to supply enough liver tissue, the latter being intimately mixed before weighing. The first of each pair of tissue samples was treated with 28 cc. of ice-cold water, 4 cc. of ice-cold sodium tungstate (10 per cent), and 4 cc. of ice-cold sulfuric acid (0.66 N). The second sample of each pair received 32 cc. of water, and 4 CC. of Tanret s 4 Tanret s reagent consists of: mercuric chloride 2.71 gm., potassium iodide 7.20 gm., and glacial acetic acid 66.6 cc.; diluted with water to 100 cc.

8 218 Determination of Urea reagent. Of the four f&rates which were then obtained, 3 cc. and cc. portions of each were treated with glacial acetic acid and xanthydrol. The rest of the procedure was that described in the first port,ion of this paper. This experiment was performed a second and third time. In a fourth experiment a pregnant female rat (18th day of gestation) was employed and analyses were made upon the fetus as well. This animal had received urea by subcutaneous injection 2 minutes before. The results are summarized in Table IV. TABLE Tanret s Reagent as Protein Precipitant. Tissue. Protein precipitant. Liver... I I Muscle I Whole fetus I# Tungstic acid. Tanret s reagent. Tungstic acid. I Tanret s reagent. I I IV. - - V OlUUll 3 of 6 l1trat.e, - used. I.- _- CC urea content&eer 100 gm. of Experi. I nent 1 ml mg mg In Experiment 4, the animal received urea by subcutaneous injection 2 minutes before being killed. CXpEIioent 4. mv From these it may be concluded that tungstic acid and Tanret s reagent are equally satisfactory for the precipitation of the proteins of the liver and fetus. Identical urea values are obtained. When applied to muscle, however, tungstic acid permits the passage into the filtrate of some product which contaminates the dixanthydryl urea ultimately obtained, interferes with its solution in 1: 1 sulfuric acid, and leads to low titration values. Believing that it might be possible to precipitate the interfering substance by adding Tanret s reagent t,o tungstic acid filtrates of muscle, we made several experiments with that end in view. The addition of 0.1 to 0.3 cc. of Tanret s reagent gave no precipi-

9 J. M. Luck 219 tate. It was found that the subsequent addition of glacial acetic acid and xanthydrol gave precipitates of dixanthydryl urea which were less contaminated, and which dissolved more readily in 1: 1 sulfuric acid, but complete solution of the precipitates was not obtained. The permanganate titration values were still inclined to be lower that those which followed the use of Tanret s reagent only. Were it not for the need of estimating substances other than urea in the protein-free tissue extracts we would be inclined to use Tanret s reagent as the protein precipitant. We have however been mindful of the value of using a reagent which would be of general utility in tissue analysis. Thus we have found Tanret s reagent quite unsuitable if the estimation of amino acids be desired. It is nevertheless necessary to use this reagent as the protein precipitant in estimating the urea content of muscle. The presence of glycogen in the tissue extracts does not interfere in the determination of urea by this method. SUMMARY. Dixanthydryl urea dissolves in sulfuric acid to give fluorescent yellow solutions which are rendered colorless by oxidation with potassium permanganate. These facts have been employed in the development of a volumetric method for the estimation of urea. 0.1 mg. may be estimated with an experimental error of about per cent. Alan Archer McCray and Frank Hilton Smith assisted in part of this work, BIBLIOGRAPHY. 1. Kiech, V. C., andluck, J. M., J. Biol. Chem., 1928, Ixxvii, Fosse, M. R., Ann. chim., 1916, vi, Folin, O., andwu, H., J. Biol. Chem., 1919, xxxviii, Fosse, M. R., Robyn, A., and Franpois, F., Compt. rend. Acad., 1914, clix, 367. Addendum.-Since this work was submitted for publication, a paper by F. Beattie appeared (Biochem. J., 1928, xxii, 711) in which dixanthydryl urea is estimated calorimetrically by comparison of the yellow solution, here described, against a standard.

10 THE OXIDATION OF DIXANTHYDRYL UREA, A MICRO METHOD FOR DETERMINING UREA James Murray Luck J. Biol. Chem. 1928, 79: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#ref-list-1

A COLORIMETRIC METHOD FOR THE DETERMINATION OF INORGANIC PHOSPHATE IN BLOOD SERUM.

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