New Color Reaction for Vitamins D W. 1. LYNESS’ and F. W. QUACKENBUSH Ind.
Department of Biochemistry, Purdue University, Lafayette,
Vitamins Dz and D3 were found to react with an i o d i n e ethylene dichloride reagent to produce a strong yellow color which showed maximal absorption at 450 mp. The intensity of the color was enhanced by mercuric p-chlorobenzoate and certain other compounds. The reagent showed high specificity for the vitamins D, no other compound having been found to give rise to a similar color. Various sterols produced no color; vitamin A produced a weak violet color which showed some absorption at 450 mp. While the reagent showed greater sensitivity as well as greater specificity than the glycerol dichlorohydrin reagent, it was less sensitive than the antimony trichloride reagent. I t is easy to prepare and its use is not complicated by the formation of films or corrosive volatiles.
ethylene dichloride was shaken in a separatory funnel with two 50-ml. portions of concentrated sulfuric acid. After removal of the hypophase the ethylene dichloride was washed with distilled water and with 2% sodium bicarbonate solution, dried over anhydrous sodium sulfate, and allowed to percolate through a 20 X 200 mm. column of silica gel (28- to 200-mesh, Davison Chemical Corp.). Mercuric p-chlorobenzoate. A concentrated aqueous solution of sodium p-chlorobenzoate was added to a saturated aqueous solution of mercuric chloride. The white precipitate was filtered and washed with water. Iodine-ethylene dichloride reagent (subsequently referred to as “the reagent”). To a flask of ethylene dichloride were added 80 p.p.m. of mercuric p-chlorobenzoate and sufficient iodine to produce an absorbance at 500 mp of 0.182 3t 0.002 (absorbance a t 450 mp of 0.076 =!= 0.002). This solution was approximately 0.20mM with iodine. A Beckman ;\lode1 DU spectrophotometer with matched 1-cm. cells was used for all absorbance measurements. An incandescent lamp served as the source of illumination, and the slit width was varied t o accord with the region of the spectrum being examined.
T
WO interrelated problems have thus far impeded the development of a satisfactory chemical or physicochemical method of assay for the vitamins D. First, the vitamins have proved to be extremely difficult to separate quantitatively from other closely related compounds. Secondly, known reactions have lacked specificity or sensitivity, or both, for use nith the vitamin D concentrates from most natural oils and synthetic irradiation mixtures. Of the several methods of assay which have been proposed, none has come into common use. Direct spectrophotometry in the ultraviolet ( 4 ) and infrared (8) regions lacks specificity; measurement of colors derived from the vitamins has found wider acceptance. Trio of these color reactions, one with antimony trichloride developed originally by Brockmann and Chen ( I ) , and a second Kith glycerol dichlorohydrin advanced by Sobel, Mayer, and Kramer (IO),have continuously stimulated interest. Improvements on the original antimony trichloride procedure which have been proposed by Raoul and Meunier ( 9 ) , Nield, Russel, and Zimmerli (71, and others have produced a method which has good sensitivity but lacks the desired specificity. A modification of the Sobel procedure which increased the sensitivity of this reagent by a factor of 20 was introduced by Campbell ( d ) ; however, sensitivity is still one of its limitations. In the present work a new color reaction has been discovered which is highly specific for vitamins D and which, it is hoped, may give rise to a procedure with fewer shortcomings than those which are currently available. This paper describes responses of pure vitamins and related sterols to the reagent.
EXPERIMENTAL
Development and Measurement of Colored Vitamin D Complex. When a 0.lOmM solution of vitamin D1 or D3 in ethylene dichloride was added to an equal volume of the reagent, the pink color of the reagent was superseded by a yellow color which developed in the solution. This colored vitamin D complex showed a sharp absorption maximum at 450 mp (Figure l ) , reached its maximal intensity in about 7 minutes, then faded slowly. Different samples of crystalline vitamins Dn and D, showed essentially identical behavior.
To obtain the data for the absorption curvea (Figure l),a series of test tubes, each containing 3.0 ml. of reagent, was prepared, and to each were added 3.0 ml. of a 0.10 mM solution of the vitamin, but at successive intervals. After several of the tubes had been used to establish the 450 mp reading a t the time of maximal color development, each of the remaining tubes was used to obtain a point at a different wave length, the reading being taken in each case as quickly as possible after the maximum reading at 450 mp had been reached. Yo attempt was made to correct the absorption curves for light absorbed by free iodine in the
Table I. Reactivity of Reagent with Vitamins and Sterols
REAGENTS AND APPARATUS
Vitamin D2 (crystalline, 40,000,000 U. S. P. units per gram), Sterwin Chemicals, Inc. Vitamin D3 (Crystalline, 40,000,000 U. S. P. units per gram), Sterwin Chemicals, Inc. Ergosterol, Eastman Kodak Co. Recrystallized tn-ice from ethyl acetate and once from ethylene dichloride before use; melting point 165-166’ C. 7-Dehydrocholesterol, E. I. du Pont de Nemours & Co., Inc. Cholesterol, Eastman Kodak Co. Recrystallized from ethyl acetate before use; melting point, 147-148’ C. Stigmasterol (melting point, 170-171 C.), The Glidden Co. Sitosterols (purified mixture, melting point, 137’ to 140’ C.), The Glidden Co. Lumisterol. Prepared by saponification of the 3,s-dinitrobenzoate, which was obtained from Smith-Sew York, Inc. Vitamin A (crystalline alcohol), Distillation Products, Inc. Iodine (c.P.resublimed), J. T. Baker Chemical Co. Ethylene dichloride. A 750-ml. quantity of a technical grade of I
Present address, The Procter & Gamble Co., Cincinnati 31, Ohio.
1978
(Readings taken a t 7 minutes) Concentration, Millimole/ Liter Compound i’ione (blank) 0:h Vitamjn Dz 0.0: Vitamin Dz 0.10 Vitamin A 0.50 Lumisterol 0.50 Ergosterol 0.50 7-Dehydrocholesterol 0.50 Cholesterol 0.50 Stigmasterol 0.50 Sitosterol
Absorbance, 450 m p 0.038 1.060 1.060 0.130 0.039 0.039 0.033 0.037 0.037 0.037
Table 11. Constancy of Color Production of Reagent with 0.05mM Vitamin D2in Presence of Related Sterols Sterols Present None Lumisterol Cholesterol Ergosterol Cholesterol and Ergosterol
(Readings taken a t 7 minutes) Concentration, Millimole/ Liter
0:i: 0.25 0.25 0.25 0.25
Absorbance, 450 mp 1.040 1.045 1.045 1.035 1.040
V O L U M E 2 7 , NO. 1 2 , D E C E M B E R 1 9 5 5
0.00
WAVE LENGTH, rnk
1979 Factors Affecting Intensity of Color. When the mercuric p chlorobenzoate was omitted from the reagent, color development was qualitatively the same but quantitatively about 10 to l5Y0 as intense. An optimal concentration of the mercury salt was observed, above which increments in concentration caused a decrease rather than an increase in absorbance. Similar effects were observed with mercuric benzoate (Table IV) and other mercuric salts; however, mercuric p-chlorobenzoate was the most effective of the several mercuric salts tested (Table V). Other compounds which produced a similar, but weaker, effect included ferric, ceric, and ammonium benzoates, and oommercial lots of ethylene chlorohydrin, trichloroacetic acid, and iodobenzene. Benzaldehyde produced as much color as the mercuric salts but the amounts required varied from 0.06 to 1.5% depending on the source of the material. Apparently an impurity was responsible for the effect. KO definite relation was observed between the various compounds and their activity as color enhancers. The time required for maximum color development varied nith the different compounds and their concentrations. Interference with the color reaction was observed with some commercial samples of ethylene dichloride. The interfering substances were not removed completely by fractional distillation but were eliminated by treatment xith sulfuric acid and silica gel by the procedure outlined above. Ethylene dichloride samples from five different manufacturers, when so treated, had excellent stability and gave uniform results in the color reaction. Other chlorinated solvents which were tested included carbon tetrachloride, chloroform, ethylidene chloride, the trichloroethanes, and 1,1J2,2-tetrachloroethane.While varying amounts of yellow color were observed in these solvents, the strong color with the characteristic sharp absorption peak vias observed only in ethylene dichloride. Temperature differences between 20" and 35" C. were shown to have no effect on the reaction. Light conditions-i.e., diffuse
Figure 1. Spectral absorption of ( A ) iodine-ethylene dichloride reagent, ( B ) reagent with 0.05 mM vitamin DP, and (C) reagent with 0.05 mM vitamin Da Reaction time 7 minutes
Table 111. Comparison of Absorptivitiesof Vitamins D and Certain Sterols in Three Color Reactions
reagent. However, the reagent before reaction, but in the same dilution, showed an absorbance of only 0.038 a t 450 mp (Figure 1). Lower concentrations of iodine in the reagent did not produce maximal absorbance (450 mp) of the vitamin D complex. Specificity of Color Reaction. Six different sterols tested at 0.05 and 0.5mN concentrations showed no apparent reaction with the reagent (Table I ) and produced no interference when present with vitamins D (Table 11). However, vitamin rl (0.20mM solution) produced a medium blue color which changed after approximately 1minute to medium violet. The solution shoved a broad absorption band with a maximum at 555 mp and some absorption at 450 mp. In admiuture with vitamins D, vitamin -4interfered to give low values: Vitamin D P (0.10mM) n i t h vitamin -4(0.10mM) produced an absorbance of 0.990, Removal of the vitamin A would appear necessary for use of the reaction with natural materials such as fish liver oils. The chromatographic method of DeWitt and Sullivan ( 3 ) has been used satisfactorily for this purpose. Polar compounds such as alcohol and acetone u. hen present in large quantities reacted n ith the reagent to produce high absorption in the near ultraviolet n ith some overlapping into the 400to 450-mp region. Hox-ever, no interference from such compounds was encountered when standard separation procedures were used prior to color development. In specificity for vitamins D, the new reaction compared favorably with well-known reactions (Table 111). However, the sensitivity attained in the study was below that of the improved (Nield) antimony trichloride reagent.
Compound Vitamin D2 Vitamin Ds Ergosterol 7-Dehydrocholesterol Cholesterol a
Absorptivity a t Wave Length of Maximum Absorption Glycerol Iodine Antimony dichlororeagent trichloride5 hydrin b 530 1800 360 545 1850 370 0 0 5 14 0 0 8 0 3 0 0 25 0
Improved reagent containing acetyl chloride; from d a t a of Mueller ( 6 ) .
b From data of Campbell ( 8 ) .
Table IV. Effect of RIercuric Benzoate Additions on Intensity of Color Developed with 0.05mM Vitamin DI Mercuric Benzoate Added, P.P.M. A-one
Maximum Absorbance, 450 mp 0 132
n - ~_ _ 4" n
6.1 . .
11.5 12.7 13.5 14.3 19.0
0 0 0 0 0
915
970 990
970 900
No color formation
38.0
Table V. Effect of Various Mercuric Salts on Intensity of Color Developed with 0.05 mM Vitamin Da hlercuric Salt Mercuric acetate Mercuric trichloroacetate Mercuric benzoate Mercuric p-chlorobenzoate Mercuric p-aminobenzoate
Optimal Concn., P.P. 11,
-
11 14 40
11
Maximum Absorbance, 450 mp 0 600 0 990 0 990 1 060 0 890
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., ANAL.CHEY.,20, 317 (1948). (5) 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
