Spectrophotometric Determination of Uric Acid and Some Redeterminations of Its S o h bility DANIEL R. NORTON', MARY A. PLUNKETTZ, and FRANCIS A. RICHARDS Woods Hole Oceanographic Institution, Woods Hole, Mass. Beckman DU spectrophotometer and pH measurements with a Beckman Model G p H meter. A Model N Leeds and Xorthrup Electrochernograph was used to record the polarographic data. Chemicals. Except where otherwise specified, inorganic chemicals are C.P. or reagent grade, while organic chemicals are Eastman White Label grade. Disodium dihydrogen ethylenediamine tetraacetate dihydrate (Versene), Hach Chemical Co. Sodium tungstate dihydrate, C.P. (Folin) Uric Acid, C.P. Reagent Solutions. Sodium cyanide, 10% solution. Disodium dihydrogen ethylenediamine tetraacetate dihydrate (Versene), 4% solution. Arsenophosphotungstic Acid Solution (Color Reagent). The solution was prepared according to the method of Benedict and Franke (3'). One hundred grams of sodium tungstate dihydrate were dissolved in 600 ml. of distilled water. Fifty grams of arsenic pentoxide, 25 ml. of 85% phosphoric acid, and 20 nil. of concentrated hydrochloric acid were added to the solution. After the solution had been brought to a boil, 20 ml. of bromine water were added and the boiling was continued for 2 hours After cooling, the solution was diluted to 600 ml. (in the original procedure described by Benedict and Franke the solution was diluted to 1 liter). The p H of the clear yellow reagent was 1.48 and it was stable for a period exceeding 2 months. According to Benedict and Franke the solution should be practically colorless. In the Folin procedure using phosphotungstic acid reagent, the yellow color is attributed to phospho-24-molybdic acid and smaller blanks can be obtained by removing molybdenum ( 7 ) . Uric Acid Standard Solutions. The uric acid primary standard solution was prepared according t o a modification of the method of Benedict and Hitchcock ( 4 ) . Nine grams of sodium monohydrogen phosphate dodecahydrate, 1 gram of sodium dihydrogen phosphate monohydrate, and 0.2000 gram of uric acid were dissolved in 500 ml. of distilled water a t 50" C. After cooling, the solution was diluted to 1 liter. By using sterile equipment, adding several milliliters of chloroform, and keeping the solution refrigerated, the primary standard was kept for 1 month without decomposition. The p H of the standard solution was 7.20. Although the original procedure calls for the addition qf acetic acid to the primary standard solution, it was omitted in thls work as it was found to lower the p H to 5.62, thereby causing the precipitation of uric acid. A secondary standard solution was prepared immediately prior to use by diluting a 25-ml. aliquot of the primary standard solution to 500 nil. One milliliter of this solution contains 10y of uric
The present study was initiated in order to develop a rapid and accurate method for the determination of uric acid in fresh, brackish, and sea water. It was found that the spectrophotometric determination of uric acid based upon its reaction with arsenophosphotungstic acid reagent in the presence of cyanide ion meets this objecthe. The absorbancy of the blue coinplex was measured at 890 mp. Slight variations from Beer's law were generally found. The results show the effects of pH, reaction time, concentration of reagents, and temperature upon color development and precipitate formation. Disodium dihydrogen ethy-lenediamine tetraacetate (Versene) was used as a buffering and complexing agent. The results are significant in that they give the absorption spectrum of the blue complex and the effects of variables upon its absorbancy. Studies were made with the method to determine the stability of reagents and standard solutions and to determine the rate of bacterial decomposition of uric acid. Measurements of the solubility of uric acid are reported.
T
H E various methods for the determination of uric acid were studied critically by Hutchinson ( I f ? ) , who recommended preliminary separation of uric acid and its determination with arsenophosphotungstic acid or lithium arsenotungstate reagent. The method for the determination of uric acid based upon its reaction with arsenophosphotungstic acid in the presence of cyanide ion to give a blue color was developed by Benedict and coworkers (1-3). The use of cyanide ion to increase the sensitivity of the method was first reported by Benedict and Hitchcock ( 4 ) in the case of phosphotungstic acid reagent. It was later shown to have the same effect with arsenotungstic acid reagent ( 1 7 ) and with arsenophosphotungstic acid reagent ( 1 ) . While there are no references to the structure of the bluecolored substance, it is probably related to tungsten blue or similar compounds. Sidgqick (19) ascribes the blue color formed on gentle reduction of tungstates to products containing tungsten(V) and (VI). Sidgwick also refers to a complex cyanide of tungsten(V) which cannot be further oxidized by strong oxidants. The stabilization of tungsten(V) by formation of a complex cyanide would shift the redox potential in the direction to increase the relative strength of tungsten(V1) as an oxidant toward uric acid. The resultant shift in the equilibrium would favor the formation of tungsten blue or related colored compounds. While the method of Benedict and coworkers gives reliable rcsults when close attention is given to a prescribed procedure, slight deviations from the procedure may cause significant errors. In modifying this method for the determination of uric acid in natural waters, studies were made of the effects of pH, reaction time, concentration of reagents, and temperature upon color development and precipitate formation. Disodium dihydrogen ethylenediamine tetraacetate (Versene) was used to control the pH of the reaction mixture and to complev alkaline earth ions in the natural water samples.
..oo
METHOD
[I .W
800
500
WAVE
Apparatus. Absorbancy measurements were made with a
700
eo0
900
,ow
LENGTH (MILLIMICRONS)
Figure 1. Absorption Spectra
1 Present address, Department of the Interior, U. S. Geological Survey, Washington 25, D. C. * Present address, Vassar College, Poughkeepsie, N. Y.
1. Abaorption spectrum before 1 hour; lOOy uric acid per 50 ml. 2.
454
Abaorption spectrum a t 24 hours; 407 uric acid per 50 ml.
V O L U M E 2 6 , NO. 3, M A R C H 1 9 5 4
455 Table 11.
Effect of pH on Color Development and Precipitate Formationa (Reagent concentrations constant)
pH
Absorbancy a t 890 mp
6.40 8.02 8.20 8.20 8.32 9.00
0.105 0.215 0.236 0.236 0.232
Ppt. formed Conditions. Small amounts of NaOH or hydrochloric acid were added to flasks containin 12.0 ml. of Versene and 5.00 ml. of dilute uric acid standard. After ajdition of 2.50 ml. of color reagent and 4.00 ml. sodium cyanide, the reaction mixtures were diluted t o 50 ml. and allowed to stand for 25 minutes before pH and absorbanries were measured. T h e sodium cyanide solution was 1 month old.
Table 111. Absorbancies at 890 mg as a Function of Uric Acid Concentration and Reaction Time L-ric .4cid, I
t.0
.
I
.
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7 s
TOO
.
,
.
I
*w
004
W A V E LENDTW ( Y I L L I Y I C I O N S )
Figure 2. C u r v e No. 1 2 3 4 5
Effect of Time on -4bsorption Spectrum M i n u t e s Elapsed after Addition of Color R e a g e n t 920 m u 690 m u 36 41 49 60 61 ._
72 95 132
6
Table I.
Effect of pH on Color Development and Precipitate Formationa
- aCN, 1oQ21.
Color of Minutes before Absorbancy Soln. pH Pptn. a t 890 mu 2.50 Yellow .5 . 4 1 00 0.035 2.50 Bluish-green 6 3 2 00 0.130 2.50 4 00 Blue 8.1 0.310 2 50 6.00 Blue 8 7 30 2.50 8 00 Blue 9.1 10 ... 0.50 4 00 Blue 9.0 50 ... 1 .oo 4.00 Blue 8.9 50 ... 1.60 4.00 Blue 8.6 50 . . 2.00 4.00 Rlue 8.4 50 2.50 .. 4.00 Blue 8.1 o:iio a Conditions. 12 ml. of Versene and 5.00 nil. of dilute uric acid standard were added t o flasks containing the volumes of reagents indicated. After diluting t o 50.0 ml. a n d allowing t o stand 50 minutes, pH a n d absorbanries were measured. T h e sodium cyanide solution was freshly prepared.
MI.
.~ .
~~~
~~~
~
RI1. 0 20 40 60
so
100
Reaction Time, \fin. 34
36
41
46
51
56
61
0.031 0,091 0.171 0.258
0.032 0.092 0.172 0.258 0.355 0 443
0.034 0.095 0.174 0.261 0.354 0 445
0 036 0.097 0.176 0.261 0.353 0 442
0.038 0.099 0.176 0.260 0.349 0 441
0.039 0.097 0.173
0.039
. .
...
0.355 0.448
.. .
... ...
...
71 .-
82 108 143
(Reagent concentrations varied) Color Reagent,
-,/SO
~
acid. It is important to use freshly prepared secondary standards as uric acid-utilizing bacteria may be present and t,heir action in decomposing uric acid is rapid. Procedure. An aliquot of the nat'ural water sample, or standard solution, containing between 20 and 120y of uric acid is transferred to a 50-ml. volumetric flask and diluted to 30.0 ml. Twelve milliliters of 47, Versene solution are added, 2.50 ml. of arsenophosphotungstic acid solution and 4.00 ml. of 10% sodium cyanide solution are introduced from burets, and the solution is mixed. (The usual precautions should be taken in handling and disposing of the poisonous solution?.) After standing the required length of time, the solution is made up to volume, mixed thoroughly, and the absorbancy measured in 1-em. cells a t 890 m p against a reference cell containing distilled water. % . blank and a standard should be determined Ji-ith each set of samples. It is recommended that a calibration curve be determined frequently and that the uric acid concentration be read directly from the curve, The time required for the absorbancy to reach a maximum depends largely upon the age of the cyanide solution and this must be determined experimentally. If a turbidity develops in the solution prior to reading the absorbanry, it, is necessary to change the ratio of the volume of the color reagent to the volume of the cyanide solution until optimum conditions are obtained. It was determined experimentally that the salt cont'ent of natural water sampler has no effect upon color development. DISCUSSION
Absorption Spectra. The absorption spectrum of the blue complex formed by the reaction of uric acid with arsenophospho-
tungstic acid reagent in the presence of cyanide ion changes with time. Curve 1 of Figure 1 shows the absorption spectrum of the hlue complex early in it. development. It has a high abqorbancy between 430 and 960 mp with a plateau between 730 and 900 mp. When observed for longer periods of time the absorbancyat 890 mp is practically constant, while ahqorbancies in the range 680 to 870 nip continue to increase aq illuqtrated in Figure 2. If the reaction iq allowed t o continue 24 hours, a maximum is found a t 700 mp as shown in curve 2 of Figure I. Since the abqorbancy a t 890 mp reaches a maximum in a comparatively short time, the absorbancy a t this wave length is uqed to determine the concentration of uric acid in the reaction mixture. The absorption spectrum of the reagent blank shows the same characteristics as described above. Effect of pH. By changing the amount and ratio of arsenophosphotungstic acid and sodium cyanide solutions it was possible to develop the color in reaction mixtures between p H 5.4 and 9.1. The results in Table I show that absorbancy increases with pH. The absorption spectrum of the light yellow solution at p H 5.4 has the same characteristics as the blue solution at p H 8.1, with the exception that the relative absorbancies below 550 m p are much greater in the former case. The blur solution a t pH 8.1 !vas prrfectly clear arid gave no Tyndall beam. At p H 8.4 and above a n hite cry4alline precipitate formed. It n as qoluble in strong baqe, precipitated from strong acid, and gave a characteristic test for tungstate ion Since the formation of the precipitate limits the pH range for this method, further experiments were made to determine the effect of pH upon color development. Keeping the concentrations of the reagents constant, the pH mas changed by adding varying amounts of hydrochloric acid and sodium hrdroude. The results of this qtudy reported in Table I1 show concluqivelg that pH iq the important factor in controlling color development and precipitate formation. The increase of sensitivity with p H may be attributed to the increase of cyanide ion concentration. Reaction Time. The time required for the absorbancy t o reach a maximum varies between IO and 60 niinutes, depending upon the conditions eniploj ed, and inust be determined experimentally. The absorbancy is then constant for a period of 5 to 20 minutes before it begins to decrease. The time required for maximum color development depends to some extent upon the concentration of uric acid. As shoxn in Table I11 the absorbancy a t 890 mp reaches a maximum more rapidly n-ith increasing concentration of uric acid. This may account for the observed deviations from Beer's law.
456 Table IV.
Effect of Concentration of Reactants on Color Development5
.,
ANALYTICAL CHEMISTRY ,
,
I
Volume during Color Development, rlbsorbancy 311. a t 890 mp 22 0.475 27 0,454 32 0.400 47 0.988 Conditions. Varying amounts of distilled water were added t o flasks containing 12.0 ml. of Versene a n d 4.00 ml. of dilute uric acid standard. T w o milliliters of color reagent a n d 4.00 ml. of sodium cyanide were added a n d the color developed a t t h e volumes indicated. After 45 minutes the solutions were diluted t o 50.0 ml. a n d the absorbancies measured. 0
'
'
0
' 20
TIME
,370
I
I
I
1
Figure 4.
J
ee
40
OF
8"
100
110
1.a
STORAGE LhUURSl
Decomposition of Uric Acid in a Brackish Water Sample Sample stored at 3' C.
Table V. Redistilled Water,
MI. 100 80
6n .. 40 20 0
I
,110 0
I
I
I
I
2
S
10
AGE O P C Y A N I D E SOLUTION ( D A Y S )
Figure 3.
Effect of Age of Cyanide Solution
AbsorbancieR mcasured at 890 m p i reaction time, 50 minutes: 50y uric acid per 50 ml.
Forsham et al. (8) used sodium polyanethole sulfonate to stabilize the colored complex in a modification of the KernStiansky method ( 1 4 ) employing Folin reagent ( 7 ) and it is possible that it would be beneficial in this method. Concentration of Reactants. The results in Table I V illustrate that absorbancy is proportional to the concentration of reactants during color development. Therefore, it is important to adjust t,he volume of solution prior to the addition of reagents. I n the adopted procedure the sample, or standard, is adjusted to 30.0 ml. before the addition of reagents and the volume during color development is 48.5 ml. Temperature. From 5' to 33' C. temperature had no effect on absorbancy while between 33" and 50' C. the absorbancy increased 9%. Decomposition of Sodium Cyanide Solutions. Ricca and D' Amore (18) concluded that the decomposition of sodium cyanide solutions was caused by hydrolysis of cyanide ion to hydrocyanic acid, sodium formate, and ammonia. Benedict used urea to stabilize sodium cyanide solutions ( 1 ) . Benedict and Behre ( 2 ) added 2 ml. of concentrated ammonium hydroxide to each liter of sodium cyanide solution and reported that it improved during the first 2 to 3 weeks after its preparation but that it should not be used after 6 to 7 weeks. blargolin and Buchteyev (16) studied the stability of sodium cyanide solution with reference to its use in the Benedict procedure and stressed the importance of constructing calibration curves at frequent intervals. Brown ( 5 ) studied the stability of cyanide solutions using phosphotungstate color reagent. The results of the present investigation shown in Figure 3 give further evidence for the rapid decomposition of this reagent. To avoid the high initial rate of decomposition of freshly prepared sodium cyanide solutions, they were allowed to stand for 2 weeks before use. The solutions were discarded when their sensitivities became too low.
Solubility of Uric Acid at 30.2' C.
Sea. Water (Chlorinity 20.21 1\11, 0 20 40 60 80 100
O/OO),
PH
5.21 5.68 5 04
S,io
6.20 6.29
Solubility, Mg./100 MI. 5.07 11.3 18 1 24.2 28.4 34.6
Destruction of Uric Acid by Bacteria. The method described in this paper was used to study the distribution and bacterial decomposition of uric acid in fresh, brackish, and Rea water samples taken in the neighborhood of poultry farms. The bacterial decomposition of uric acid occurs under both aerobic and anaerobic conditions and can be brought about by a number of different organisms (6, 11, 16). According to Karlsson and Barker (13) Clostridium m i d i urici decomposes uric acid anaerobically to give ammonia, acetic acid, and carbon dioxide while allantoin and urea, which are products of the aerobic decomposition of uric acid, are neither formed nor decomposed. The present studies indicate that uric acid-utilizing bacteria are ubiquitous and that the rate of bacterial deromposition of uric acid is rapid. The concentration of a uric acid solution contaminated with bacteria decreased from 1.4 to 0.7 mg. per liter within 6 hours a t room temperature. The decomposition of uric acid in a brackish water sample taken from the neighborhood of a poultry farm and stored a t 3 " C. was measured. The data presented in Figure 4 show that decomposition was appreciable even a t this temperature. Since destruction of uric acid by bacteria can be rapid, it is
URIC ACID I Y I C R O O R A Y 8 PER 50
Figure 5.
mll
Calibration Curve
V O L U M E 2 6 , NO. 3, M A R C H 1 9 5 4 advisable to use sterilized equipment when feasible and to analyze the natural water samples as soon as possible after their collection Accuracy, Precision, and Sensitivity. The studies reported i n this paper show the effects of pH, reaction time, concentration of reagents, and other variables on the accuracy, precision, and sensitivity of the method. In order to achieve accuracy it is necessary to construct calibration curves frequently. Even under optimum conditions absorbancy ie seldom exactly proportional to concentration. Figure 5 is a calibration curve using the recommended procedure. Absorbancies were read 50 minutes after the addition of reagents and the reagent blank was 0.066 absorbancy units. In this case, using 2-week-old cyanide solution, Beer's law was closely followed. However, the age of cyanide solution has an effect on the shape of the calibration cuive. With fresh cyanide solutions Beer's law v,-as followed a t concentrations greater than 40y per 50 ml but a t lower concentrations the absorptivities vere low. On the other hand, the absorptivities a t these lower concentrations were high when cyanide solutions 3 weeks and older were used. The precision of the method was determined by running blanks, standards, and samples in triplicate. The average deviations were less than 2%. It is possible to measure quantities as small In a 20-ml. R S 2-/ of uric acid in a total reaction volume of 50 ml. aliquot of a natural water sample the method is capable of measuring uric acid concentrations as low as 0 . 1 per ~ milliliter. Interferences. Although the color reagent is not specific for uric acid in the presence of certain easily oxidizable substances, i t appears to be highly selective in its action. For example, allantoin, barbituric acid, guanine, hydantoin, urea, and xanthine did not react with the color reagent a t concentrations of l O O r per 50 ml. of reaction volume. Alloxan interfered a t this concentration, 1OO-y giving an absorbancy which was equivalent to 203 of uric acid. A polarographic study was made to determine whether alloxan was a stable bacterial decomposition product of uric acid. Solutions of uric acid in natural waters were examined polarographically before, during, and after bacterial decomposition had taken place and no alloxan was detected. The apparent absence of interfering substances in natural waters makes it possible to upe the method for the determination of uric acid directly. However, to be absolutely certain that interferences are not present it would be necessary to use the procedure on an aliquot of the .ample after the uric acid was selectively oxidized by the enzyme uricase.
457
mechanical shaker for 2 hours a t 30.2' C. The p H of the unfiltered solutions was then determined. The samples were filtered through No. 42 Whatman filter paper and an aliquot was taken for the determination of uric acid. The results are shown in Table V. The solubility of uric acid in distilled water is 5.1 mg. per 100 ml. a t 30.2' C. and its saturated solution has a p H of 5.21. The solubility of uric acid in sea water having a chlorinity of 20.21 grams per kilogram and an original p H of 8.29 is 35 mg. per 100 ml. a t 30.2" C. and its saturated solution has a pH of 6.29. Intermediate values of uric acid solubility in sea water of varying dilutions are reported. The results are in general agreement with those of other investigators. Gudzent (9) gives a value for the solubility of uric acid of 6.49 mg. per 100 ml. of distilled a a t e r a t 37" C. while His and Paul (IO)report a value of 2.54 mg. per 100 ml. of distilled a a t e r a t 18" C. In the same papers Gudzent reported an ionization constant for uric acid of 2.3 X a t 37' C. while His and Paul reported a value of 1.5 X l o w 5a t 18" C. From these results it can be calculated that a substantial increase of solubility should occur a t pH 5.6 and this relationship was found in the experiments reported. The large differences in the solubility of uric acid in natural water samples are therefore attributed to the pH of the solutions. ACKNOWLEDGMENT
The authors xish to thank Alfred C. Redfield, Ralph F. Vaccaro, and Sathaniel Corwin for their assistance in this study. LITERATURE CITED
Benedict, S.R., J . Biol. Chem., 51, 187 (1922). Benedict, S.R.,and Behre, J. A,, Ibid., 92,161 (1931). Benedict, S. R.,and Franke, E., Ibid., 52, 387 (1922). Benedict, S.R.,and Hitchcock, E. H., Ibid., 20, 619 (1915). Browi, H., Ibid., 158, 601 (1945). Ecker, E. E.,and Morris, J. L., J . Infectious Diseases, 35, 479 (1924).
Folin, O., J . Biol. Chem., 101, 111 (1933). Forsham, P.H.,Thorn, G. W., Pruntr, F. T. G., and Hills, A. G., J . Clin. Endocrinol., 8,15 (1948). Gudrent, F., Z. physiol. Chem., 56, 150 (1908). His, IF7.,and Paul, T., Ibid., 31, 65 (1900). Hutchinson, G. E.,B u l l . Am. M u s e u m N u t . Hist., 96,76 (1950). Hutchinson, J . C.D., Biochem. J., 35, 81 (1941). Karlsson, J. L., and Barker, A. A., J . Biol. Chem., 178, 891 (1949).
Kern, A.,and Stransky, E., Biochem. Z., 290,419 (1937). RIargolin, L. T., and Buchteyev, S. F., Zhur. Eksptl. Biol. i Med., 11,110(1929).
Morris, J. L., and Ecker, E. E., J . Infectious Diseases, 34, 592 (1924).
SOLUBILITY MEASUREMENTS
The solubility of uric acid in distilled water and in sea water of varying dilutions was determined. One hundred milliliters of the water and 0.5 gram of powdered uric acid were added to glassstoppered Erlenmeyer flasks. The mixtures were placed on a
J., and lIacLeod, A. G., J . Biol. Chem., 50, 55 (1922). Ricca, B., and D'Amore, G., Gatz. chim. ital., 79,308 (1949). Sidgwick, N. V., "The Chemical Elements and Their Compounds," Volume 11, pp. 1047-52,Oxford, Clarendon Press lforris,
1950. RECEIVED for review August
13, 1953. Accepted December 16, 1953. tribution S o . 661 from t h e Woods Hole Oceanographic Institution.
Con-
