a colorimetric method for the …removes interfering substances. the procedure is carried out in the...

A COLORIMETRIC METHOD FOR THE DETERMINATION OF ALLANTOIN*

BY HARDY W. LARSON

(From the Biochemical Laboratory of the Metropolitan Life Insurance Company, New York)

(Received for publication, November 4, 1931)

INTRODUCTION

Since the publication, in 1908, of Wiechowski’s (1) method for the determination of allantoin, various attempts have been made to simplify the method and to increase its accuracy. These attempts have not met with much success.

The Wiechowski method depends upon the precipitation of allantoin by a mercuric acetate reagent after the urine has been freed from interfering substances by a very complicated and time- consuming procedure. The mercury-allantoin precipitate obtained is decomposed by hydrogen sulfide, and the allantoin recrystallized or a Kjeldahl nitrogen determination made on the precipitate. Handovsky (2), in 1914, introduced the use of a known amount of standard mercuric acetate-sodium acetate reagent. In his modification, the mercury-allantoin compound is filtered off and the mercury remaining in the filtrate is titrated against standard ammonium thiocyanate. This involves a very difficult end-point. Unfortunately, small errors in this titration make considerable differences in allantoin values.

In more recent literature (3-6), methods have appeared which are based upon the hydrolysis (by acid, alkali, or enzyme) of allantoin. Certain products of hydrolysis, such as urea, hydan- toin, glyoxylic acid, and oxalic acid, are determined, and from these the amount of allantoin is estimated. These methods are based upon the hypothesis that the hydrolysis of allantoin pro- ceeds in a clear cut, orderly manner, and that the so called end-

* Presented at the meeting of the American Chemical Society at Buffalo, September, 1931.

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Determination of Allantoin

products of hydrolysis represent quantitative values. There is wide-spread disagreement as to the exact course and mechanism of these reactions, as well as to the products involved. Until proof is established concerning the true character of these allantoin hydrolyses, such methods are open to considerable doubt.

All of the published methods for allantoin determination require from 6 to 24 hours to perform. Many of them are probably in- accurate, at best giving not more than approximate values for allantoin. Because of the importance of animal purine metab- olism, the need for a more accurate and rapid method has become urgent. The present investigation was undertaken in the en- deavor to provide such a method.

Folin and Svedberg (7), in working with various copper reagents for carbohydrate determination, developed an ammoniacal copper reagent which is practically unaffected by urinary sugar, but which is reduced by nitrogenous compounds such as creatine, creatinine, and allantoin. It is the use of this reagent which makes the following calorimetric method possible.

Calorimetric Determination of Allantoin

General Outline

5 cc. of animal urine are treated with an excess of 30 per cent phosphotungstic acid, followed by an excess of saturated basic lead acetate solution, and 5 per cent sulfuric acid. This treatment removes interfering substances. The procedure is carried out in the same 50 cc. centrifuge tube. After the addition of each reagent, the tube is gently rotated to insure proper mixing, and the mixture is centrifuged until perfectly clear. 2 cc. of this liquid are pipetted into a Folin-Wu sugar tube together with 2 cc. of Folin ammoniacal copper regent which is reduced by allantoin. This is then heated in a boiling water bath for 10 minutes, cooled, and 2 cc. of acid molybdate reagent added. The color obtained is read against a 1 mg. allantoin standard. Recoveries of allantoin added to rat urine range from 90 to 100 per cent. Values obtained calorimetrically check with those obtained by use of the mercury-allantoin reagent on the same solution. 2 hours are required for the complete determination, as against the 10 or 12 hours required by the Wiechowski-Handovsky method now in general use.


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H. W. Larson

Reagents Required

Phosphotungstic Acid-The phosphotungstic acid solution is made up in the centrifuge tube directly before using, as the solution seems to deteriorate rapidly on standing. Eight different lots of C.P. phosphotungstic acid obtained from four leading manufacturers varied greatly in their precipitating power, and gave inconsistent results. It is therefore necessary to prepare pure phospho-24-tungstic acid.

The preparation of the pure acid is essentially according to Wu (8) and is as follows: Dissolve 100 gm. of Na2W04.2Hz0 in about 100 cc. of water with the aid of heat. Add 10 cc. of 85 per cent H3P04 and then 80 cc. of concentrated HCl. Cool. After 4 hours or more, filter on a Buchner funnel and suck as dry as possible. Redissolve the precipitate in 120 cc. of HzO, pour the solution into a liter separatory funnel, add about 90 cc. of ether, and then add 40 cc. of concentrated HCl. Shake. After standing a few minutes, there should be three layers of liquid. The lowest layer contains nearly all .the complex acid. If there are only two layers, more ether must be added and the mixture shaken again. Transfer the lowest layer to another separatory funnel, add about 120 cc. of water, and shake vigorously; then add 50 cc. of ether and finally 50 cc. of concentrated HCl. After standing, the lowest layer, which should be perfectly clear, is transferred to a crystallizing dish. Add 30 cc. of Hz0 and 1 drop of liquid bromine and evaporate on a steam bath. The solution should be greenish in color. If the slightest trace of dust or organic matter is present, a pinkish color develops; and 1 or 2 drops more of liquid bromine must be added to oxidize this foreign material.

Evaporate on the steam bath until crystals begin to form on the surface. Let stand overnight. The crystals obtained are sucked as dry as possible on a large Buchner funnel. After air-drying for 1 week, powder the crystals and keep in an amber glass con- tainer. This phosphotungstic acid should dissolve instantly to give a perfectly clear, practically colorless solution.

Basic Lead Acetate Solution (9)-Dissolve 180 gm. of lead acetate in about 700 cc. of distilled water, with boiling. Add 110 gm. of lead oxide (litharge) and boil for 3 hour with occasional stirring. Cool, filter, and add sufficient distilled water to the filtrate to make the weight 1 kilo.


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730 Determination of Allantoin

Folin Ammoniacal Copper Solution (7)-Dissolve 100 gm. of ammonium sulfate in about 400 cc. of water and filter into a volumetric liter flask. 100 cc. of 10 per cent sodium hydroxide are then added, 12 gm. of sodium tartrate, and finally a solution of 5 gm. of copper sulfate. Dilute to volume and mix. This reagent will not give a blank for months, if kept in the dark in well filled, tightly stoppered, amber glass bottles. The bottles should be of small volume.

5 Per Cent Sulfuric Acid (by Weight). Folin Acid Molybdate Reagent (IOj-Prepare a stock solution

of 30 per cent brominated sodium molybdate as follows: Dissolve 300 gm. of sodium molybdate in water and make up to 1 liter. The solution is slightly turbid. Add 2 or 3 drops of liquid bromine and let stand overnight. Transfer 500 cc. of the clear super- natant liquid to a liter flask and add, with stirring, 225 cc. of 85 per cent phosphoric acid. Then add 150 cc. of cool sulfuric acid (25 volumes per cent). The bromine which is liberated is removed by aeration. Add 75 cc. of 99 per cent acetic acid, mix, and dilute to 1 liter.

Allantoin Standard-Dissolve 100 mg. of allantoin in about 50 cc. of water with the aid of heat, but do not allow to boil. Cool, transfer to a 100 cc. volumetric flask, and dilute to volume. Cover with toluene. Such a standard will keep for about 2 weeks at room temperature without deterioration. Without toluene, a loss of 1 per cent of the allantoin is noted after standing 1 week. Givens (11) reports a loss of 1.7 per cent after 90 days.

Procedure

Transfer 1.5 gm. of phosphotungstic acid to a 50 cc. centrifuge tube and add 5 cc. of water. Rotate gently to insure solution; then add 5 cc. of animal urine. Centrifuge immediately, place the tube in a refrigerator for 3 hour, then centrifuge again until perfectly clear. The addition of a crystal of phosphotungstic acid should not cause further precipitation. Add 5 cc. of basic lead acetate solution which precipitates the excess phosphotungstic acid as well as the remaining interfering substances. Centrifuge the mixture, then treat with 5 cc. of 5 per cent sulfuric acid to remove the excess lead, and then centrifuge until perfectly clear. Pipette 2 cc. of the resulting water-clear liquid into a Folin-Wu


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H. W. Larson 731

sugar tube, neutralize with 5 per cent sodium hydroxide, and then add 2 cc. of Folin ammoniacal copper reagent. Place the tubes in a rapidly boiling water bath for 10 minutes, cool, then add 2 cc. of acid molybdate reagent. Dilute the tubes to volume and read against a 1 mg. allantoin standard.

Remarks

The above method was designed primarily for use with rat urines, but has been used successfully with other urines. The method depends upon a proper balancing of reagents involved. The amount of phosphotungstic acid used above is in slight excess for normal urines. The lead is sufficient to precipitate the excess phosphotungstic acid and certain reducing materials, and the 5 cc. of sulfuric acid completely remove the excess lead. However, when certain urines, e.g. from a high protein diet, are used, the amount of phosphotungstic acid is not in excess. When such a urine is encountered, simply add more solid phosphotungstic acid until no further precipitation occurs (usually, 0.2 gm. will suffice). To insure complete removal of lead, in such a case, after treatment with the 5 cc. of sulfuric acid an additional cc. should be added to make sure that there is no further formation of lead sulfate. It is only in rare instances that urines are encountered which require the above variations in procedure. Of course, initial dilution of such a urine or the use of a smaller quantity entirely circumvents the difficulty. When a urine contains large amounts of allantoin, as little as 1 cc. may be used. On the other hand, where a urine is low in allantoin, 10 cc. should be employed instead of 5. In such a case, the solid phosphotungstic acid may be added directly to the urine, instead of first dissolving it in water.

It has been claimed that certain amino acid-phosphotungstate precipitates dissolve when allowed to remain in excess phosphotungstic acid for any length of time. In order to minimize this possibility, the precipitated urine is centrifuged before it is placed in the refrigerator. The use of the refrigerator is not a necessary part of the method. It merely cuts the time of complete phosphotungstate precipitation to a minimum.

Sometimes particles of lead sulfate remain floating on the surface of the liquid or adhere to the walls of the tube and it is diffi-


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cult to pipette the liquid without contamination. To avoid this difficulty, the liquid is transferred to another tube by decantation, and centrifuged for a minute or two.

Comparison of the Wiechowski-Handovsky and Calorimetric Methods

The Handovsky modification of the Wiechowski procedure for allantoin determination is the most generally accepted method. For this reason, it was chosen as a basis for comparison with the calorimetric method. During the past 2 years, the writer has had occasion to run a great number of these Wiechowski determinations. The enormous, heavy precipitates encountered have been centrifuged off, instead of being filtered. It was felt that this would increase the accuracy of the method, and this was the only deviation from the regular Wiechowski procedure. In spite of every precaution, it was impossible to obtain consistent results by the Wiechowski-Handovsky method, even for duplicate determinations. There was wide discrepancy between the results obtained by this method and those by the calorimetric method. In general, the higher results were obtained by the latter.

In Table I are shown results obtained by the Wiechowski- Handovsky and calorimetric methods. The calorimetric values vary from those of Wiechowski from -24.0 per cent to +31.7 per cent.

Recovery of Added Allantoin

Allantoin added to rat urines may be recovered satisfactorily by the calorimetric method. Consistent recoveries of from 90 to 100 per cent are obtained, the average for twenty-five recoveries being 97.2 per cent. If the recovery of added allantoin is taken as a criterion of the accuracy of the method, the calorimetric method is vastly superior to the Wiechowski-Handovsky procedure. Recoveries by the latter method range from 20 to 87 per cent, the majority being in the neighborhood of 70 per cent. It is reasonable to assume that the method which gives the higher percentage recovery is the more nearly accurate one.

In Table II are given the recoveries of added allantoin by the calorimetric method and the low recoveries obtained by the Wie- chowski-Handovsky method from the same urines.

In Table III are given allantoin recoveries for urines containing


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H. W. Larson 733

TABLE I

Comparison of Allantoin Values Obtained by Two Methods on the Same Urine

Rat urine No.

1 2 3 4 5 6 7 a 9

10 11 12

U&hod

mo.

183.8 206.0 252.4 212.0 220.0 147.2 52.4

mo.

283.9 301.2 349.4 308.8 310.0 240.8 144.8

mo. per cent Calorimetric Rat Urine 9 100 100.0

“ “ 10 100 95.2 “ “ 11 100 97.0 “ “ 12 109 96.8 “ “ 13 100 90.0

Dog urine 100 93.6 Monkey urine 100 92.4

- . Wiechowski- Rat Urine 9 60 79.3

Handovsky (( “ lo* 60 29.1 “ “ 11 165 65.8 “ “ 12 100 86.7 “ “ 13 165 76.4

Dog urine 100 87.3 Monkey urine 100 84.4

- * This urine required a great excess of silver nitrate which was removed

with difficulty.

241.8 83.6

204.7 168.5 166.6 143.8 78.4

289.4 101.1 313.3 255.2 292.8 231.1 162.8

Allantoin per co.

mo.

3.36 3.45 2.40 2.93 2.70 3.10 2.58 1.77 2.41 2.04 1.69 1.67

-

Calorimetric method

mo.

3.18 3.38 2.73 2.90 2.51 2.62 2.66 2.05 1.83 2.52 2.12 2.20

TABLE II

Recovery of Allantoin Added to Urine

-

1 I

?ercentage dii%rence

-5.3 -2.0

+13.7 +1.0 -7.5

-15.5 f3.1

+15.8 -24.0 +23.5 +25.4 +31.7

Specimen Allantoin

per loo cc. urine

Total allan toin found per loo cc.

urine

Allantoin recovered


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varying amounts of allantoin, as well as other reducing substances. Recoveries range from 90 to 102 per cent, and tend to show that allantoin recoveries by the calorimetric method are independent of the kind of animal urine used, as well as of the concentration of reducing substances. The recovery is as good for rabbit urine containing only a small amount of allantoin as it is for rat urines which contain large amounts of allantoin.

TABLE III

Recovery of Allantoin Added to Urine Containing Varying Amounts of Allantoin. Calorimetric Method

Urine Allantoin per cc.

Dog, normal.. . . . . . . . . . “ diabetic (1.7 per cent sugar) “ fasting (1.45 tine). ...........

Dog, fasting ...... Rabbit ............

‘I ............ Monkey. ..........

‘I ........... Rat*. ..............

‘( t .............. ‘( $. .............

:i ‘L

.......

.......

....... ....... ....... ....... ....... ....... .......

crea- ...... ...... ...... ...... ...... ...... ...... ...... ......

no. mo.

1.47 260.4 2.06 206.0

per cent

93.6 98.4

4.66 326.2 100.0 2.19 481.7 98.3 0.39 128.7 102.0 0.53 136.2 94.1 0.81 181.8 90.8 0.78 184.2 90.2 7.65 66.3 93.6 6.92 73.8 102.0 8.57 97.1 96.2

rota1 allantoin excreted per

24 hrs. Recovery of

dded allantoin

* The diet contained 72.5 per cent dried liver residue and 2.5 per cent nucleic acid.

t The diet contained 70.0 per cent dried liver residue and 5.0 per cent nucleic acid.

$ The diet contained 65.0 per cent dried liver residue and 10.0 per cent nucleic acid.

Creatine and sugar, while not completely removed by the calorimetric procedure, are present in such small amounts that their reduction may be considered negligible. A 0.1 per cent creatine solution gives a faint blue coloration with the copper reagent, whereas a 0.1 per cent creatinine solution gives a stronger color than a 0.1 per cent allantoin solution.

It is realized that higher allantoin recoveries do not constitute final proof of the accuracy of the calorimetric method. Inasmuch


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H. W. Larson 735

as this method gives, in general, higher allantoin values than the Wiechowski method, it was felt that proof must be given that reduction of the copper reagent comes from allantoin only, and not from any other substances which might be present.

In order to furnish this proof, the following experiments were devised, in which the allantoin was determined on the same filtrate, not only by copper reduction, but also by mercury precipitation. Allantoin may be quantitatively recovered from its solutions by precipitating the mercuric acetate-sodium acetate reagent. After filtering off the mercury-allantoin precipitate, the excess mercury remaining in the filtrate is titrated with standard ammonium thiocyanate and the allantoin calculated from this titration. In order to apply this Handovsky determination to the final liquid obtained by the calorimetric procedure, it is first necessary to remove the chlorides. Chlorides interfere with the ammonium thiocyanate titration, and must be removed by treatment with silver nitrate, and the excess silver precipitated by hydrogen sulfide.

The procedure devised for this purpose is identical with the regular calorimetric procedure, save that three times the quantity of urine and various reagents are used, so that the final volume is 60 cc. instead of 20. A 100 cc. centrifuge tube is used instead of the 50 cc. size. The chlorides are removed from the 60 cc. volume by treatment with 15 cc. of 0.1 per cent silver nitrate solution. The precipitate is centrifuged off; the liquid is transferred to a beaker, and the excess silver removed by hydrogen sulfide. After filtering off the precipitated sulfides and aerating to remove any hydrogen sulfide, the filtrate is ready for the dual determination.

2 or 3 cc. of this filtrate are used for the calorimetric determination. For the Handovsky determination, 50 cc. of the filtrate are pipetted into a 100 cc. volumetric flask, carefully neutralized, and 40 cc. of the standard mercuric acetate-sodium acetate reagent added, and the contents of the flask diluted to volume. From this point, the regular Handovsky procedure is followed.

In Table IV are shown the results obtained by the two methods. The agreement is quite close and within the limits of error of the ammonium thiocyanate titration. Thus, by two entirely different means, the same allantoin values are obtained, showing that


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in the calorimetric procedure the urine is freed from all reducing material other than allantoin.

When allantoin was added to the urine, recoveries were practically identical for the two determinations on the same filtrate. It is interesting to note here, that the percentage recoveries obtained are 10 to 15 per cent lower than by the regular colorimetric procedure. Inasmuch as the silver nitrate-hydrogen sul-

TABLE IV

Comparison of Allantoin Values Obtained by the Two Methods on Same Filtrate. Filtrate from 16 Cc. of Rat Urine. Calorimetric Procedure

Urine

Rat 100. ......................... “ lOl.......................... “ 102 .......................... “ 103. ......................... “ 104 .......................... “ 105 .......................... “ 106. ......................... “ 107. ......................... “ 108. ......................... “ 109. ......................... “ 110 .......................... “ ill..........................

Dog 1.. .......................... IL 2. .......................... “ 3 ............................ “ 4. ...........................

-

- -

-

Added allantoin

mg.

15

15

15

15

-

f

-

-

Calorimetric zleterminstion

mQ. mg.

38.6 37.4 33.2 31.8 49.7 50.5 38.0 37.8 55.0 50.7 26.6 26.3 40.7 37.9 25.5 25.3 30.6 31.3 44.4 44.5 23.9 24.8 32.5* 33.7* 72.4 70.9 30.5 31.3 20.3 21.5 33.6 34.6

Wiechowski- Handovsky

Hg precipita- ion,, NH&XX

titration

* The filtrate was obtained by the Wiechowski procedure.

fide treatment is the only deviation from this procedure, it suggesm one of the sources of loss of allantoin in the Wiechowski-Handov- sky determination. It is not the purpose of this paper to point out the various sources of error in the Wiechowski-Handovsky method, but from the writer’s experience with this method, the loss of allantoin due to adsorpt,ion on the tremendous bulky precipitates of lead-silver sulfides is considerable (see Table II, Urine 10).


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H. W. Larson 737

Allantoin in Human Urine

By using a modification of his method for animal urine, Wie- chowski (12) found minute amounts of allantoin present in human urine, 5 to 12 mg. per liter. (At such a dilution it is almost impossible to precipitate allantoin from pure solution by the mercuric acetate reagent.)

If human urine contains allantoin in such small amounts, it would appear that a better yield would result by employing a modification of the colorimet.ric procedure for ridding urine of interfering sub&ances. By this modified procedure 25 mg. of allantoin were obtained from a 24 hour specimen of 1205 cc. Specimens of mixed human urines yield 25 and 30 mg. of allantoin per liter. These figures agree closely with those recently published by Fosse (13), using the spectrophotometric method.

The mercury-allantoin precipitates obtained were decomposed with hydrogen sulfide, and the small amount of allantoin recrystallized twice. The melting point determined was 236-237”. Wie- chowski reports a melting point of 232” for his recrystallized prod- uct. Watt (14) gives 235” as the melting point of pure allantoin. The crystals obtained, while few in number, were of characteristic structure. A solution of these crystals readily reduced the Folin ammoniacal copper reagent.

SUMMARY

A rapid and accurate method for the determination of allant,oin is described.

BIBLIOGRAPHY

1. Wiechowski, W., Be&r. them. Physiol. u. Path., 11, 109 (1908). 2. Handovsky, H., Z. phlysiol. Chem., 90, 211 (1914). 3. Christman, A. A., J. Biol. Chem., 70, 173 (1926). 4. Fosse, R., Brunei, A., and de Graeve, P., Compt. rend. Ad., 188, 1418,

1632 (1929). 5. Allen, F. W., and Cerecedo, L. R., J. Biol. Chem., 93, 293 (1931). 6. Fosse, R., Brunel, A., and Thomas, P. E., Compt. rend. Acad., 192, 1615

(1931). 7. Folin, O., and Svedberg, A., J. Biol. Chem., 70, 415 (1926). 8. Wu, H., J. Biol. Chem., 43, 197 (1920). 9. Hawk, P. B., and Bergeim, O., Practical physiological chemistry, Phil-

adelphia, 10th edition, 1888 (1931). 10. Folin, O., J. Biol. Chem., 82, 88 (1929).


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11. Givens, M. H., J. Biol. Chem., 18, 417 (1914). 12. Wiechowski, W., Biochem. Z., 19, 378 (1909). 13. Fosse, R., Brunei, A., and Thomas, P. E., Corn@. rend. Bcad., 192,

1619 (1931). 14. Watt, H. E., Pharm. J., 99, 283 (1917).


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Hardy W. LarsonDETERMINATION OF ALLANTOIN

A COLORIMETRIC METHOD FOR THE

1932, 94:727-738.J. Biol. Chem.

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