March 15, 1935
ANALYTICAL EDITION
LITERATURE CITED Barnes, R. B., and Matosi, F., 2. Physik, 76, 24 (1932). Beattie, J. A., Rev. Sci. Instruments, 2, 458 (1931). Beattie, J. A., and Jacobus, D. D., J . Phus. Chem., 34, 1254 (1930).
Hull, A. W., Gen. Elec. Rev., 32, 213, 390 (1929). Lange, B., and Voos, E., 2. tech. Physik, 15, 323 (1934). Moser, H., Ibid., 13, 383 (1932). Newton, R. H., and Furnas, C. C., Chem. Met. En*,, 39, 455 (1932).
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(8) Roebuck, J. R., Proc. Am. Acad. Arts Sci., 60, 537 (1925). (9) Smith, L. B., Mech. Eno., 48, 153 (1926). (10) Southard, J. C., and Andress, D. H., J . Franklin Inst., 207, 324 (1929). (11) Workman, J. E., Phys. Rev., 37, 1706 (1931). (12) Zabel, R. M., and Hancox, R. R., Rev. Sci. Instruments, 5, 28 (1934). RECEIVIDD December 28, 1934. Contribution No. 118 from the Research Laboratory of Organic Chemistry, Massachusetts Institute of Technology
Determination of Uric Acid in the -Mixed Excrement of Birds JAMESC. FRITZ,Bureau of Animal Industry, U. S. Department of Agriculture, Beltsville, Md.
A
N ACCURATE analysis of the feces is essential for the calculation of digestion coefficients. It is difficult to
obtain the necessary data in the case of birds because the urine and the feces are mixed in the cloaca and excreted together; a method for differentiating the urinary constituents from the fecal constituents present in the mixed excrement is necessary if one is to obtain data for measuring digestibility. In general, the methods used to study digestion in poultry are based on either of two principles. The first is to alter the bird by surgical means so that the urine and the feces are voided separately. The second is to make a chemical estimation of the constituents excreted only in the urine or only in the feces. The protein digestion and metabolism of birds can be studied if we are able to measure the amounts of nitrogen excreted in the forms of uric acid and ammonia, which are the chief end products of nitrogen metabolism in birds. Uric acid is excreted only in the urine. Some ammonia is excreted in the feces, and Katayama (6) introduced a factor t o correct for the small amount which is normally present. Not all the urinary nitrogen is present in the forms of uric acid and ammonia (5-6). Therefore, it is necessary to apply a correction factor to the sum of the uric acid nitrogen and the ammonia nitrogen to obtain the total urinary nitrogen. The various formulas developed to calculate the partition of the excrement nitrogen can be expressed in the following general forms: 1. Fecal nitrogen = excrement nitrogen
4-ammonia nitrogen)
- k (uric acid nitrogen
k is a constant to correct for ammonia nitrogen in the feces and for the urinary nitrogen not present in the forms of uric acid and ammonia. 2. Urinary nitrogen = excrement nitrogen
- fecal nitrogen
Working with Katayama’s data, Titus (11) found that the following modification of Equation 1 gave less variable results than the original form: Fecal nitrogen = 0.9784excrement nitrogen - 1.0253 uric acid nitrogen - 1.2641 excrement ammonia nitrogen - 0.0554 \
\
I n order to use these formulas one needs an accurate method
kor the determination of uric acid in the mixed excrement of fhwls. Several methods, based upon different principles, are reported in the literature. Considerable difficulty was experienced by Brown (W), Bartlett ( I ) , Katayama (4, St. John (7), and other workers who attempted to use the methods previously recorded in the literature. All the methods
give good results when tested on pure uric acid, but are not so rapid and accurate when used on excrement samples. There is a real need for an accurate and convenient method for the determination of uric acid in excrement. The writer has subjected a number of these methods to critical tests with a view to selecting or developing an accurate and rapid technic for the determination of uric acid in the mixed excrement of fowls. The following methods have been studied :I Brown’s isolation and piperidine titration (2). Brown’s method, modified to recover the uric acid gravimetrically instead of titrating it. Katayama’s method for separating the uric acid in the form of ammonium urate, converting it back to the acid form, and then determining nitrogen by the Kjeldahl method (6). Katayama’s method modified for gravimetric recovery of the isolated uric acid. Kionka’s piperidine titration (6). St. John’s method for isolating uric acid (7). Suauki’s method for measuring the amount of ammonia required to convert the uric acid into ammonium urate (IO). Woodman’smethod for isolating uric acid (1.9). The writer’s differential extraction method, which is based on the assumption that acidified water extracts the same nitrogenous constituents, with the exception of uric acid, as does piperidine.
DIFFERENTIAL EXTRACTION METHOD Weigh out two equal samples of the excrement. Two grams make a convenient quantity to handle. To each sample add 20 cc. of a hydrochloric acid solution (5 parts of the concentrated reagent and 95 parts of water), and let the samples stand overnight. This treatment converts urates into free uric acid. Filter, and wash each residue with about 25 cc. of cold water. Transfer the residues, plus the filter papers, t o their respective original beakers. To one of the residues add sufficient piperidine to make the mixture distinctly alkaline to phenolphthalein. Bring the volume to approximately 25 cc. with distilled water. To the other residue add a similar quantity of 0.1 N hydrochloric acid. Digest both mixtures in a water bath at 60’ C. for 1 hour. Filter each mixture through a layer of Celite on a filter cloth (the filter cloth recommended by the Association of Official Agricultural Chemists for use in the determination of crude fiber is satisfactory), and wash the residues with equal quantities of cold wash water. Continue washing until the wash water from the piperidine-extracted material is free from an alkaline reaction. Transfer the residue (and Celite) from the piperidine extraction to a Kjeldahl flask for a total nitrogen determination, A . Transfer the residue (and Celite) from the water extraction t o a second Kjeldahl flask for a total nitrogen determination, B. B - A = uric acid nitrogen Uric acid nitrogen X 3 = urio acid 1 Since this work was completed, another method for determining fecal nitrogen has been reported in the literature. Stotz (8, 9) has described a method for oxidizing uric acid by nitric acid, and washing out the resulting soluble products. After applying suitable corrections, the remaining nitrogen can be considered as fecal nitrogen.
INDUSTRIAL AND ENGINEERING CHEMISTRY
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PLANOF TESTS Each method was tried first on known amounts of chemically pure uric acid, and if uniform and accurate results were obtained, the method was then applied to a standard sample of droppings. This standard sample consisted of a large quantity of droppings from adult Rhode Island Red cocks, dried, well mixed, and finely ground to provide a uniform sample on which t o test the methods. If the results justified further study, known amounts of pure uric acid were added to weighed samples of the excrement and the mixture was analyzed for uric acid. The percentage of the added uric acid which was recovered was considered to be an indication of the accuracy of the method. A summary of the results of the tests is shown in Table I. The highest and the lowest values obtained on the standard sample of excrement are given to indicate the variation in the results obtained with each method.
Vol. 7 , No. 2
erably lower results than any, but one, of the other methods tested. This is contrary to what one might expect from the relative ability of the methods to determine uric acid, alone or when added to excrement. Several experiments were made in the hope that they would offer some explanation for this discrepancy. A synthetic sample was prepared to contain a known amount of uric acid. Besides the uric acid this sample contained urea, sodium chloride, ammonium chloride, sucrose, and gelatin. The differential extraction method gave a more accurate measure of the amount of uric acid present in this synthetic sample than did any other of these methods. When the xanthoproteic test is made on the material extracted by solvents used in the methods of Brown, Katayama, St. John, and Woodman, a positive reaction is noted. Pure uric acid does not give this color reaction. Therefore, these methods must extract nitrogenous substances other than uric
OF METHODS FOR DETERMINING URICACID IN MIXEDEXCREMENT OF BIRDS TABLEI, SUMMARY OF TESTS
METHOD
RECOVERY OF c. P. URICACID No. of deths. Av.
URICACIDI N STANDARD SAMPLE OF EXCREMENT Range AV.
No. of detns.
%
%
%
50 96.8 47 10.60-12.32 Brown 38 9.37-13.16 Brown with gravimetric recovery 18 99.1 21 96.5 20 7.57- 9.57 Katayama *. ... Katayama with gravimetric recovery 26 97.7 21 97.2 Analysis impossible-heavy Kionka 21 94.7 9 11.43ffi-14.18a St. John 23 99.9 40 10.14-14.48 Susuki 54 96.5 42 9.97-13.30 Woodman 20 99.1 26 8.10-10.18 Differential extraction 0 This is not the standard sample which was used in the determinations by the other methods.
For the recovery of uric acid in the presence of excrement, two equal samples of the excrement were taken. To one of these a known amount of pure uric acid was added. Then the total uric acid was determined in each sample, and the difference in the results was the portion of the added uric acid which was recovered. DISCUSSION OF METHODS Numerous objections, either theoretical or practical, may be raised about each of the methods tested. The writer has found that all efficient solvents of uric acid have a destructive action upon the acid itself, but this can be minimized by leaving the uric acid in solution for as short a time as possible. The extreme difficulty in filtering the alkaline solution, presumably because of the colloidal material present in the excrement, complicates the rapid removal from the solvent. The most successful method for speeding the filtration was to freeze the alkaline uric acid solution with the suspended insoluble matter, and then to filter with suction while the material melted. Suzuki’s method and the differential extraction method are free from errors attributable to the destruction of uric acid by alkaline solvents, because they do not depend upon isolating and measuring the uric acid as such. Titration of uric acid with either piperidine or potassium permanganate was considered impractical, because in all cases the end points were indistinct. Heavy pigmentation of the solution interfered with the colors that indicated the end points of the titrations. The most accurate measurements of pure uric acid, either alone or when added to excrement samples, were obtained with Suzuki’s method and with the differential extraction method. The average values for the per cent of uric acid present in the standard sample of excrement varied with the different methods. The differential extraction method gave consid-
RECOVERY OF QDDED C. P. URIC ACIDI N PRESENCE OF EXCREMENT No. of detns. Av.
11.65 11.32 8.88
...
% 20 59.6 19 70.4 Inconsistent and low
pigmentation 13.0Z0 11.73 11.76 8.89
..
..
18 22 2 74
50.2 92.9 14.5 93.8
acid. This phenomenon may account for the high values obtained with the isolation methods. After considering the results of these tests, the writer is inclined to place more confidence in the lower values obtained on the standard sample. The differential extraction method is very simple, and presents no difficulties in manipulation. For this reason the writer believes that it offers some distinct advantages over any other method which has been proposed. SUMMARY AND CONCLUSIONS Kine different procedures for the determination of uric acid in the mixed excrement of birds have been tested. None of the methods reported in the literature yielded satisfactory results. A differential extraction method proposed by the writer is recommended because of its simplicity and accuracy. Two equal samples of excrement are extracted, one by piperidine and the other by acidified water. The difference in unextracted nitrogen is considered to be uric acid nitrogen. ACKNOWLEDGMENT The author wishes to thank Harry W. Titus of the Bureau of Animal Industry for valuable suggestions made in connection with this study. LITERATURE CITED (1) B a r t l e t t , J. M., M a i n e Agr. E x p t . Sta., Bull. 184 (1910). (2) Brown, E. R., Bur. Animal Ind., Bull. 56 (1904). (3) Coulson, E. J., a n d Hughes, J. S., Poultry Sci., 10, 53 (1930). (4) Davis, R. E., J . Biol. Chem., 74,509 (1927). ( 5 ) K a t a y a m a , T., Bull. Imp. Agr. Expt. Sta. Japan, 3, No. 1 (1924). (6) Kionka, H., Arch. intern. pharmacodynarnie, 7, 55 (1900). (7) St. John, J. L., a n d J o h n s o n , O., J . Biol. Chem., 92,41 (1931). (8) Stotz, H., Arch. Tierernuhrzing Tierzucht, 7, 29 (1931). (9) Ibid., 9,426 (1933). (10) Suzuki, J., a n d Nishizaki, A., J . Agr. Chern. SOC.Japan, 7, 507 (1931). (11) T i t u s , H. W., Poultry Sci., 7, 145 (1928). (12) W o o d m a n , H. E., J . Agr. Eci., 14,413 (1924). RECEIVED October 10, 1934.
