1980
ANALYTICAL CHEMISTRY
daylight compared to darkness-also showed no effect. ,4 precision within 2% was obtained when the conditions prescribed for color development iTere adhered to strictly. Nature of Colored Compound. Little has been learned about the nature of the color reaction. The literature is nearly devoid of mention of color formation between halogens and vitamins D or related sterols. Although the reaction of Tortelli and Jaff6 ( I f ) may be related in principle to the reaction disclosed here, the techniques used are different, and the resulting colors are dissimilar. I blue-green color observed by Green ( 5 ) between his iodine trichloride reagent and p-carotene may be related also. Green postulated a mesomeric change in the carotene moleculd under the influence of IC&, C1-, or Cia- similar to that produced by antimony trichloride on carotenoids and vitamin D. Although no direct evidence for a mechanism was observed in the authors’ experiments, it is suggested that a loose union occurs between iodine and the unsaturated center of the D vitamins. More specifically, because traces of bromine increased both €he intensity of color and the speed with which maximum color was attained, it appears that iodide ion may be the active form which unites with the vitamin to form the colored product. As the enhancing agents did not change qualitatively the spectral ab-
sorption properties of the colored vitamin D complex, their function may be to promote the reaction by increasing the concentration of the iodide ion. LITERATURE CITED (1)
Brockmann, H., and Chen, Y . H., 2. p h y s i o l . Chem., 241, 129 (1936).
(2) Campbell, J. 8., ASAL. CHEM.,20, 766 (1948). (3) DeWitt, J. B., and Sullivan, XI. K., 1x0, EX. CHEW,A N ~ L . ED.,18, 117 (1946). (4) Ewing, D. T., Powell, 11.J., Brown, R. A. and Emmett, A. D., (5)
ANAL.CHEY.,20, 317 (1948). Green, J., Biochem. J . , 49, 36 (1951).
(6) Mueller, 8.. J . Am. Chem. SOC.,71, 924 (1949). (7) Sield, C. H., Russel, W. C., and Zimmerli. b.,J . Biol. Chem., 136, 73 (1940). (8) Pirlot, G., Anal. Chim. Acta, 2, 744 (1948). (9) Raoul, Y . , and hleunier, P., Compt. rend., 209, 546 (1939). (10) Sobel, A . E., Mayer, A. M., and Kramer, B.. IND.ENG.C m x , Asvlr.. ED.,1 7 , 160 (1945). (11) Tortelli, M., and Jaff6, E., Ann. chim. a p p l . . 2, 80 (1914).
RECEIVED for review August 2 6 , 1963. Accepted September 6, 1965. Journal Paper No. 713 of the Purdue Agricultural Experiment Station, Lafayette, Ind.
Determination of Urea in Blood and Urine with Diacetyl Monoxime HAROLD L. ROSENTHAL Division of Biochemistry, Clinical and Pathological Laboratories, Rochester General Hospital, Rochester, N. Y. The condensation of urea with acid diacetyl monoxime (Fearon reaction) with concomitant oxidation by arsenic acid has been extensively studied in an effort to improve reproducibility and the linearity of response of the reaction. The concentration of mineral acid and oxidizing arsenic acid was found to be critical. By performing the reaction in 3.8N hydrochloric acid and 0.08V arsenic acid maximum color is produced which conforms to Beer’s law at urea concentrations up to 60 y per 10-ml. reaction volume. Dilution of the reaction mixture results in a deviation from Beer’s law, and the urea response curve no longer passes through the origin. By the study, a rapid and accurate method for the determination of urea in blood and urine has been developed. Comparative studies with existing methods and recovery studies have shown the suitability of the procedure. Analysis of a sample in duplicate requires less than 1 hour.
T
H E need for a direct, simple, and accurate method for determining urea in blood and biological fluids has resulted in the development of a variety of direct and indirect procedures. The indirect methods, which are the most widely used, depend on the hydrolysis of urea with the enzyme, urease, to form ammonia. The liberated ammonia is usually determined by direct nesslerization, by aeration and nesslerization, or by aeration and titration ( 3 , 7 , 9, If). The methods for the direct determination of urea depend upon the condensation of urea with a-isonitrosopropiophenone (1) or diacetyl derivatives (2, 5, f2, f3) in the presence of strong acid solutions. The reaction with a-isonitrosopropiophenone requires special precautions because of the long heating time required for the production of color, and because of the photosensitivity of the color formed. The reaction between diacetyl monoxime and urea [Fearon reaction (a)] to yield a yellow color appears to offer distinct advantages for the determination of urea. However, the various published modifications generally suffer from the fact that the c31or formed does not obey Beer’s law and, at low concentrations,
is not proportional to the concentration of urea. Preliminary studies using the Kawerau (8) modification indicated that both the concentration of hydrochloric acid and the concentration of arsenic acid greatly affected the production of the color. A more complete study of the conditions for the reaction was undertaken with the concomitant development of a reproducible and accurate procedure for the determination of urea in blood and biological fluids. REAGENTS
Urea Stock Standard, 1 mg. of urea per ml. Dissolve 100 mg. of dried, reagent grade urea in 100 ml. of water, adding a few drops of chloroform as preservative. The solution is stable for a t least 4 months when refrigerated. Urea Working Standard, 0.05 mg. of urea per ml. Dilute 5 ml. of stock standard to 100 ml. with water. Add a few drops of chloroform as preservative. Diacetyl Monoxime, 2.5% in 5y0 acetic acid. Dissolve 2.5 grams of diacetyl monoxime in 100 ml. of 5% acetic acid. The solution is stable at room temperature for at least 6 months. The appearance of a slight yellow color does not interfere. Arsenic Acid, saturated stock solution. Suspend 50 grams of arsenic pentoxide (Baker’s Analyzed reagent) in 1000 ml. of concentrated hydrochloric acid, and allow uspension to stand with occasional mixing one or more d a v . For use, decant the clear yellow supernatant or filter through sintered glass. The solution is standardized as follows. Dilute 2 ml. of stock solution with 50 ml. of 4aVhydrochloric acid in a 250-ml. Erlenmeyer flask. Add 2 grams of iodate-free potassium iodide and 15 ml. of carbon tetrachloride. Mix, and after 10 to 30 minutes titrate the liberated iodine with 0.1N sodium thiosulfate using the disappearance of purple iodine color from the carbon tetrachloride as the end point. This solution is approximately 0.9 to 1 . O X with respect to quinquevalent arsenic ion. Arsenic Acid Working Solution. Dilute an appropriate amount of saturated stock solution with concentrated hydrochloric acid to yield a final solution which is 0.26 to 0 27*V with respect to quinquevalent arsenic. EXPERIMENTAL
The yellow color formed between diacetyl monoxime and urea has an absorption maximum at 480 to 485 mH, as measured with a Beckman DU spectrophotometer. The Klett-Summerson filter
V O L U M E 2 7 , NO. 1 2 , D E C E M B E R 1 9 5 5
the yellow reaction product was determined by varying the concentration of the oxidizing acid in the reaction mixture. All other components of the reaction mixture were maintained at optimal concentrations as described under procedure, and the concentrations of arsenic acid and urea are expressed in terms of the final 1 0 4 . reaction mixture. For absorbance measurements all reaction mixtures were diluted to a volume of 20 ml.
4-
m
9 -
The optimum concentration of arsenic acid for the production of color is critical over a narrow range of concentrations and varies with the concentration of urea as shown in Figure 2. For all urea concentrations tested, maximum color formation is obtained with 0.08S quinquevalent arsenic. ilt greater concentrations of arsenic acid, color formation is inhibited at some, but not all, concentrations of urea. Since some color is produced in the presence of optimal hydrogen ion concentration in the absence of arsenic acid, the data are presented on a relative basis for comparative purpose.
8w 3 -
z4 P
2-
I
c
I
0
I I ' I I 1 2 3 4 NORMALITY OF ACID
I 5
I 6
Figure 1. Effect of acid concentration on color f o r m a t i o n at various concent r a t i o n s of urea 0 10 y A 30 y 0 45
y
of urea per tube of urea per tube of urea per tube
0 GO y of urea per tube 90 y of urea per tube
t 1.0
1981
Effect of Diacetyl Monoxime Concentration on Color Production. The concentration of diacetyl monoxime necessary for the production of maximum color was determined by varying the amount of the reagent in the reaction mixture. Maximum color production is obtained at a concentration of 0.25% diacet!l monoxime. At lower concentrations of reagent, color production falls off rapidly, and no color is produced in the absence of the reagent. At greater concentrations of diacetyl monoxime, color production is depressed. Effect of Heat on Color Production. Color formation increases rapidly with heating in a boiling water bath for the first 25 minutes, in which time approximately 90% of the color is formed. Since continued heating for an additional 35 minutes increases the amount of color by only lo%, a 30-minute heating period 15 as selected as the optimal time for color formation. The final color is somewhat photolabile and fades at a rate of approximately 5% per hour under fluorescent lighting in this laboratory.
v 0
I 0.05
I aio
NORMALITY
OF
I
I
020
0.30
ARSENIC
+5
Figure 2. Effect of arsenic acid concentration on color f o r m a t i o n a t various concentrations of urea Numerals on curres indicate micrograms of urea per reaction tube
KO.47 was, therefore, selected for use with the Klett-Summerson photometer. Effect of Acid Concentration on Color Production. The concentration of hydrochloric acid necessary for maximum production of the yelloR- reaction product was determined by varying the concentration of hydrochloric acid in the reaction mixture. All other Components of the reaction mixture were maintained at optimal concentration. as described under procedure. The concentrations of hydrochloric acid and urea are expressed in terms of the final 10-ml. reaction mixture. For absorbance measurements all reaction mixtures were diluted to a volume of 20 ml. The optimum acid concentration (Figure 1) shifted from 3.6N for 10 y of urea to 4 . 0 s for 60 y of urea and to 4.5iV for 90 y of urea. An average value of 3.KV acid was selected to cover the working range of concentration from 10 to 60 y of urea. High concentrations of acid inhibit color formation to a great degree, and little, if any, color is produced when the acid concentration approaches 10N. The data of Figure 1 were obtained at optimal arsenic acid concentrations. Because the arsenic solution alone is sufficiently acid to produce some color, the data are presented on a comparative basis. Effect of Arsenic Acid Concentration on Color Production. The concentration of arsenic acid for the maximum formation of
Effect of Urea Concentration on Color Formation. K i t h the information available for the optimal conditions of the condensation reaction, standard curves were prepared with graded quantities of urea. The reaction mixture, in a final volume of 10 ml , was read photometrically a t 470 mp, using a Klett-Summerson photometer. As shown in Figure 3, the color formed under these conditions (curve A ) obeys Beer's law for concentrations of urea up to 60 y per tube, and the curve passes through the origin. At concentrations above 60 y per tube, the curve rapidly decreases in slope. When the final reaction mixture is diluted to 20 ml. (curve B ) , the curve is linear up to a concentration of 80 y per tube, but no longer passes through the origin. On further dilution t o 40 ml. (curve C) the curve remains linear for concentrations up to 160 y per tube, but does not go through the origin. Each of the curves, however, is reproducible from day to day and the choice of standard curves for use depends on the sensitivity desired and on the concentration of urea to be measured. PROCEDURE
On the basis of the above information, the procedure finally adopted is as follows. Place 1 ml. of 1 to 10 protein-free filtrate of blood in the bottom of a test tube and dilute with approximately 4 ml. of water. A blank solution containing distilled water and a standard solution containing 0.05 mg. of urea are incorporated in each series of tests. To each tube are added 3 ml. of dilute arsenic acid solution and 1 ml. of 2.5Q/, diacetyl monoxime solution. The volume is adjusted to 10 ml., and the solution is mixed by gentle shaking. The tubes are capped with small beakers and placed in a boiling water bath for 30 minutes. The level of the boiling water bath must a t all times be above the level of the reaction mixture. At the end of 30 minutes the tubes are removed and are placed in tap water ( 5 " to 15" C.) for 3 minutes. Absorbance is determined immediately with a Klett-Summerson photometer adjusted to zero with the blank. The concentration of urea of the unknown sample may then be determined by reference to a standard curve, or may be calculated over that portion of the standard curve which is linear. If desired, the blank, standard, and unknown solutions may be diluted with water to an appropriate volume before absorbance measurements are made. For the determination of urea in urine or other fluids containing no protein, the samples are diluted with water to a suitable con-
ANALYTICAL CHEMISTRY
1982 centration and incorporated in the test. If proteinaceous material is present this may be removed by suitable methods before analysis. RESULTS
Because the Fearon reaction is not specific for urea, but reacts with other substances containing the ureido group such as citrulline (5-ureidonorvaline), substituted ureas, and carbamyl amino acids, three samples of blood and three samples of urine were treated with an excess amount of urease by the Gentzkow procedure (6) as modified in this laboratory. Following precipitation of the proteins with tungstic acid, the filtrates were analyzed for residual urea using curve B , Figure 3. It was found that urease destroyed all of the chromogens in blood and 94 to 96% of the chromogens in urine. It would appear, therefore, that the proposed procedure primarily measures urea in this type of material. The concentration of urea in blood and urine was determined by the diacetyl procedure and compared with the direct nesslerization modification of Gentekow (6). As can be seen from Table I, the two methods are in good agreement for blood samples containing essentially normal amounts of urea. At high concentrations of urea, the diacetyl method yields values up to 8% higher than the urease method. It is probable that the diacetyl monoxime procedure for uremic blood samples yields more nearly correct values. Since many of the uremic samples were obtained from persons undergoing drug therapy, it is felt that some substances may inhibit the action of urease enzyme. This factor, however, requires further study. With urine samples, good agreement between the two methods was obtained. Recovery experiments were performed by adding known amounts of urea to samples of blood and urine. The average recoveries and standard deviations are shown in Table 11. Individual recoveries ranging from 94 t o 103% in blood and 92 to 110% in urine were obtained. DISCUSSION
The preceding experiments indicate that the condensation of diacetyl monoxime with urea to yield a yellow color is suitable for the estimation of urea in blood and biological fluids. The pro-
Comparison of Diacetyl Monoxime and Direct Nesslerization Procedures
Table I. Sample 1 2 3 4 5 6
69
10 11 12
Blood, Mg./lOO M1. Diacetyl Nessler's Diff., % ' -6.6 99.0 106.0 -4.4 13.5 12.9 -0.8 20.8 20.6 4-3.8 25.8 26.8 34.7 -0.6 34.5 21.4 -1.9 21 . o -0.3 34.3 34.2 2.6 7.4 I .6 4-1.7 88.5 90.0 7-7.1 90.0 96.9 t7.1 44.6 48.0 -7.8 55.3 60.0
Urine, Mg./RIl. Diacetyl Nessler's Diff., Yo 15.0 15.6 -3.8 25.1 +4.2 26.2 14.5 13.9 +2.8 25.2 26.2 +3.8 20.4 4-6.4 21.8 14.0 l5,O -6.6 12.9 +4.4 13.5 36.3 f4.0 37.8 f2.2 31.2 30.5 0.0 7.5 7.5 14.5 14.8 +2.0
Table IT. Recovery of Urea Added to Blood and Urine as Determined by Diacetyl Monoxime Procedure Urea Urea Added, Recovered, Mg./lOO M1. % 97 3.9 5 100 i 4 . 5 10 100 32. 3 . 7 20 101 i 3 . 3 40 2.5 102 f 6 . 5 UrineC 5.0 100 f 3 . 9 10.0 103 i 4 . 4 103 f 4 . 8 20.0 a Each sample analyzed in duplicate or triplicate. b Blood samples contained 17.9 t o 45.4 mg. of urea per 100 ml. 0 Urine samples contained 4.0 to 34.5 m g . of urea per ml. Sample Bloodb
No. of, Detn. 7 8 10 7 5
s
i
*
0
100
50
UREA,
y
I50
PER TUBE
Figure 3. Effect of urea concentration on color formation
cedure is relatively simple, a t least five times more sensitive than existing enzymatic procedures, and i6 more satisfactory for rapid, accurate estimations of urea. Although the procedure is not specific for urea and some color is given by other compounds containing the ureido grouping ( I O ) , only urea yields a yellow color with an absorption maximum at 480 t o 485 mp. Citrulline and other carbamyl amino acids react to form compounds with absorption maxima in the vicinity of 550 mp. Moreover, Koritz and Cohen ( I O ) have shown that only citrulline, carbamyl-glycine, and substituted ureas have chromogen equivalents greater than urea. 111 other substances tested by these authors have chromogenic equivalents less than 25% of urea Citrulline does not interfere in our procedure, as the concentration in biological fluids is apparently too low to be measured except under unusual physiological conditions. For highly uremic blood samples, further dilution of the filtrates with water prior to incorporation in the test may be necessary. Dilution of the final colored reaction mixture t o lesser absorbance is not recommended unless the standard solution is also diluted, because the absorbance is not reduced proportionately by dilution. When the yellow reaction product is treated with alkali the color is markedly reduced a t reactions more alkaline than p H 2. The effect of alkali and the deviation from Beer's law on dilution indicates that the colored reaction product map be in the form of a dissociable complex molecule. L'arious types of deproteinating agents such as trichloroacetic acid, tungstic acid, or cadmium sulfate-sodium hydroxide may be used satisfactorily. When present a t high concentrations, tungstic acid may, however, yield a cloudy reaction mixture. As much as 5 ml. of tungstic acid-blood filtrate may be used satisfactorily, but greater quantities of filtrate develop a precipitate. LITERATURE CITED
Archibald, R. XI., J . Biol. Chem.. 157, 507 (1945). (2) Barker, S. B., Zbid., 152, 453 (1944). (3) Day, H. G., Bernstorf, E., and Hill, R. T., ANAL.CHEM.,21, 1290 (1949). (4) Fearon, W. R., Biochem. J . ( L o n d o n ) , 33, 902 (1939). (5) Friedman, H. S.,ANAL.CHEM.,25, 662 (1953). (6) Gentzkow, C. J., J . B i d . Chem., 143, 531 (1942). (7) Hughes, J., and Saifer, 9., J . Lab. Clin. M e d . , 27, 391 (1941). (8) Kawerau, E., Sci. Proc. R o g . D u b l i n Soc., 42, 63 (1946). (9) Kibrick, -4.C., and Skupp, S.,Proc. SOC.Esptl. Biol. Mcd., 73, 432 (1950). (IO) Koritz, S. B., and Cohen, P. P., J . Bid. Chem., 209, 145 (1954). 111) Loonev. J. M.. Ibid.. 88. 189 (1930). i12j Natelsk, S., Scott, M. L., and Beffa, C., Am. J . CZin. Pathol., 21, 275 (1951). (13) Ormsby, A. A., J . Bid. Chem., 146, 595 (1942). (1)
RECEIVED for review February 11, 1955. Accepted August 18. 1955. Presented in part before Division of Biological Chemistry, .4CS, S e w Pork, N. Y . , September 1954.
