NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
S. C. RAY 235
reagent is not only reduced by allantoin but also by other constituents of
urine such as sugar, creatine and creatinine when they are present in more than
certain minimal amounts.
The following method for the determination of allantoin has been evolved
in view of the limitations and short-comings of the earlier methods. The
method is in reality a simple and workable combination of the Larson and
Handovsky-Wiechowski techniques.
Outline of the proposed method
The urine is subjected to clarification from interfering substances on the
same principle as that used by Larson. The clear liquid is then carefully made
just pink to phenolphthalein with an alkali solution. The allantoin is precipi-
tated from this medium by adding a known excess of allantoin reagent contain-
ing mercuric acetate. The precipitate is filtered off and the excess mercuric
ion in the filtrate is titrated against a standard thiocyanate solution, with ferric
alum as indicator. The ferric indicator used in this method gives a sharp end-
point. The estimation of allantoin in six samples can be completed in less
than 1½ hours.
The reagents
(1) Phospho-tungstic acid:—It is essential that the phosphotungstic
acid should be pure phospho-24-tungstic acid.
(2) Saturated basic lead acetate :—To 500 ml. of hot water in a suitable
pyrex vessel, add enough basic lead acetate (A. R.) to allow of a permanent
solid phase remaining after a thorough shaking. Cool to room temperature
and then filter.
(3) 5 per cent sulphuric acid.
(4) N/40 ammonium thiocyanate solution.
(5) N/40 silver nitrate solution.
(6) Ferric indicator:—Dissolve 10 gm. of ferric ammonium sulphate in
100 ml. water, add 100 ml. concentrated nitric acid (sp. gr. 1.42). Boil the
mixture to drive off the nitrous fumes. Cool.
(7) Allantoin reagent.—Dissolve 5 gm. mercuric acetate (A. R.) in 500
ml. of water. The solution should be very clear. It should be filtered before
use and kept in a dark bottle. The strength of the mercuric ion should also
be periodically checked.
The method
To a 25 ml. test-tube containing 10 ml. of 10 per cent acetic acid, add 10
ml. of the filtered urine. Shake and mix thoroughly.
Weigh out approximately 1-2 gm. phosphotungstic acid in a 50 ml. cen-
trifuge tube. Add 10 ml. acid treated urine, rotate the tube to ensure thorough
mixing and allow it to stand for 15 minutes. Centrifuge for five minutes, then
add 5 ml. saturated basic lead acetate, rotate slowly and then centrifuge for
five minutes. Add 5 ml. of 5 per cent sulphuric acid, rotate the tube as before
and centrifuge again for five minutes. Pipette out 10 ml. of the clear super-
natant liquid—which is equivalent to 2.5 ml. of original urine—into a 50 ml.
volumetric flask. First carefully neutralize with 2 Neaustic soda, then just
236
Allantoin in the Urine of Farm Animals
acidify with N/10 sulphuric acid and finally just pink to phenolphthalein with
N/10 caustic soda. Rinse the sides of the flask with a little distilled water
and then add 10 ml. allantoin reagent. Stopper the flask, shake the contents
and make up to the volume with distilled water. Mix thoroughly by gently
inverting the flask several times. Leave the flask in a dark place for 20 minutes
to allow the precipitate to settle. Filter into a dry Erlenmeyer flask. Take
duplicates of 10 ml. filtrate in glazed porcelain basins, add 2 ml. ferric indicator
and titrate with N/40 ammonium thiocyanate solution from a 5 ml. micro-
burette. During the titration, stir the contents in the basin constantly with a
glass rod. The end-point is indicated at the moment when the solution turns
to a chocolate-brown colour.
Calculation : 10 ml. allantoin reagent is equivalent to 25.08 ml. N/40
ammonium thiocyanate solution, so that 25.08—(titration volume × 5) ×
1.09 × 40 = mg. allantoin in 100 ml. urine.
1 ml. N/40 ammonium thiocyanate solution is equivalent to 1.09 mg.
allantoin.
The method is applicable to all urines containing 75-300 mg. allantoin
per 100 ml. urine . If the urine sample happens to be acid, it is not necessary
to acidify it before mixing. When ammonia in the urine sample is very high,
the amount of phosphotungstic acid required for treatment will probably not
be less than 2 gm. To ensure the complete removal of interfering materials
at different stages, it is advisable to test the supernatant liquid after centrifu-
gation with a small drop of the precipitating reagent. The heavy precipitate
encountered during the clarification process does not remove allantoin from
the medium when the strength of the latter ranges from 75 to 300 mg. The
concentration of chloride ion present in the final clarified urine does not affect
the allantoin reagent.
EXPERIMENTAL
The accuracy of the method has been tested out in several experiments
one of which is described below :—
Allantoin in a sample of cow's urine was estimated in triplicate according
to the following plan :
(1) As undiluted urine.
As diluted urine in the proportion of 1 vol. of urine : 1 vol. of water.
As diluted urine in the proportion of 1 vol. of urine : 2 vols. of water.
(2) A known solution of allantoin in water—200 mg. per 100 ml. was
prepared and allantoin in the solution was estimated in triplicate according
to the following plan:
As pure solution.
As a mixture containing 7 vols. of allantoin solution and 3 vols. of cow's
urine taken in (1).
As a mixture containing 3 vols. of allantoin solution and 7 vols. of cow's
urine.
The results of the analysis are shown in Table I.
S. C. RAY 237
TABLE I
Specimen
A. Cow's urine . . .
B. Diluted cow's urine (1:1)
C. Diluted cow's urine (1:2)
D. Allantoin solution . .
E. Allantoin solution + cow's
urine (7:3)
F. Allnatoin solution + cow's
urine (3:7)
Proposed method (1)
mg. allan-
toin in 100
ml.
214
208
210
105
104
104
50.8
54.1
51.7
198
204
204
200
200
202
204
206
206
The mean
value esti-
mated
211
104
52.2
202
201
205
Percentage
deviation
from the
mean
+1.5
—1.0
—0.5
+0.5
Nil
Nil
—2.7
+3.6
—1.0
—1.0
+1.0
+1.0
—0.5
—0.5
+0.5
—0.5
+0.5
+0.5
Larson's method (2)
mg. allan¬
toin in 100
ml.
248
244
240
108
112
107
51.4
53.7
53.7
202
200
200
206
204
204
220
216
220
The mean
value esti-
mated
244
109
52.9
201
205
219
Percentage
deviation
from the
mean
+ 1.6
Nil
—1.6
—1.0
+2.1
—1.8
—2.8
+1.5
+1.5
+0.5
Nil
Nil
+0.5
—0.5
—0.5
+0.5
—1.4
+ 0.5
It will be seen from the percentage deviation from the mean value that
in most cases the error in estimation by the proposed method is within ±1.5.
When the concentration of allantoin is very low, as in the case of (C), the
margin of error is slightly increased. But since a case such as (C) can always
be avoided by taking a bigger aliquot of the sample for allantoin estimation,
this point need not be considered seriously. The recoveries of added allantoin
of known concentration in the mixtures (E) and (F) and also in the pure solu-
tion (D) are quite satisfactory.
The proposed method, based on the titrimetric principle, has an advantage
over Larson's colorimetric technique in that the accuracy of Larson's method
is limited by other factors which will be seen from the following experimental
evidence.
The samples (A), (B), (C), (D), (E) and (F) were simultaneously analysed
for allantoin by Larson's method.
The results set out in Table 1 show that while the triplicate analyses of
the same sample fairly agree with one another, the mean values obtained of
the different samples compare satisfactorily with those by the proposed
method only when the urine sample is considerably diluted, viz. the samples
C. and E. or when allantoin is estimated from a pure solution, viz. the sample
D. Folin's ammoniacal copper reagent used in the estimation of allantoin by
Larson, is known to be acted upon by other constituents of urine, such as
creatine, creatinine, sugar, uric acid, etc. The higher values obtained by
Larson's method may therefore be due to constituents other than allantoin
present in the urine. That this is so, can be shown from the data presented
in Table II.
238
Allantoin in the Urine of Farm Animals
TABLE II
The proposed method vs. Larson's method
Specimens
B . . .
C . . .
E . . .
F . . .
Proposed method
mg. allantoin in
100 ml.
Calculated
(1)
106
52.7
203
208
Estimated
(2)
104
52.2
201
205
percentage
difference
between
(1) and
(2)
1.9
1.0
1.0
1.5
Larson's method
mg. allantoin in
100 ml.
Calculated
(1)
122
61.0
213
231
Estimated
(2)
109
52.9
205
219
percentage
difference
between
(1) and
(2)
12.0
15.2
3.9
5.5
The allantoin content in samples B, C, E and F has been calculated from
the original values of allantoin estimated in the urine and the pure allantoin
solution by the respective methods. When these calculated values are com-
pared with the values actually estimated, by the respective methods, it
will be seen that the maximum percentage difference between the calculated
and the estimated value is only 1.9 in the case of the proposed method, while
the same is so wide as 15.2 in the case of Larson's method. The lower
values actually estimated in these samples by the latter method are due to
partial elimination, by dilution with water or pure allantoin solution, of
constituents other than allantoin, which could react with the ammoniacal
copper reagent. In fact, it was possible to demonstrate that such consti-
tuents could be creatine and preformed creatinine in cow's urine, which
contributed from + 1 to + 28 per cent positive error, according to the
quantity present under different physiological and nutritional conditions.
Excretion of allantoin in the urine of ruminants
It has already been pointed out that there are few published reports on
the quantitative excretion of allantoin by farm animals. Hunter and Givens
[1914] have given figures for allantoin and other purine bodies (uric acid and
purine base) in the urine of cows, goats and sheep which are now generally
accepted. The figures for allantoin obtained by these workers were based
upon the method proposed by Handovsky [1914] with slight modifications.
In order to compare the values of allantoin given by Hunter and Givens with
those obtainable by the present method, a number of urine samples were analy-
sed for allantoin. The average value of all estimations for each animal are
shown in Table III.
S. C. RAY 239
TABLE III
Daily excretion of allantoin in the urine of cows, sheep and goats
Cow 1 . . .
2 . . .
3
.
.
.
Goat 1 . . .
2 . . .
3 . . .
Sheep 1
.
.
.
2 . . .
3 . . .
Allantoin
gm.
21.36
14.49
16.17
0.89
0.89
1.76
2.72
2.55
2.89
Allantoin
N.
gm.
7.45
5.07
5.66
0.31
0.31
0.62
0.95
0.89
1.01
Total-N.
gm.
66.30
50.92
56.55
4.84
4.50
6.18
9.06
8.47
9.86
Allantoin.
N.
as percen-
tage of
Total-N.
11.2
10.0
10.0
6.4
7.0
10.0
10.5
10.5
10.2
Total.
purin-N.
9.24
6.08
6.38
0.38
0.38
0.79
1.22
1.16
1.32
Allantoin.
N. as
percen-
tage of
total
purin-N.
82.2
83.4
88.6
81.6
81.6
78.5
78.0
76.7
76.5
Remarks
The figures are
averages of 18
determinations
The figures are
averages of 33
determinations
The figures are
averages of 14
determinations
The animals chosen for the above experiment were all apparently healthy
and under normal conditions of feeding and management. The cows and
goat No. 3 were in milk. It will be seen that, whereas Hunter and Givens
found a markedly different distribution of allantoin (given as a percentage of
total purin-N) for cows, goats and sheep, in the present work the difference in
distribution between the three types of ruminants is slight. This may or may
not be significant.
Allantoin-N as percentage of
total purin-N.
Cow
H.&G.
92
P. W.
85
Goat
H.&G.
81
P.W.
80
Sheep
H.&G.
64
P.W.
78
H. & G.—Hunter and Givens
P. W.—Present work
It may also be seen from Table III that the percentage contribution of
nitrogen by allantoin towards total urinary nitrogen is on an average about
10.4,7.8 and 10.4 per cent respectively for cows, goats and sheep.
SUMMARY
A rapid semi-micro method of estimating allantoin in the urine of farm
animals has been described. The allantoin content in the urine of cows, goats
and sheep has been estimated by the proposed method and compared with
existing figures.
240
Allantoin in the Urine of Farm Animals
ACKNOWLEDGEMENT
The major portion of the experimental work described in this paper was
carried out in the physiological laboratory, Hannah Dairy Research Institute,
Scotland. The author is grateful to Dr N. C. Wright, Director of the Institute
for giving him facilities for carrying out this work.
REFERENCES
Allen, F. W. and Cerecedo, L. R. (1931). J. Biol. Chem. 93, 293
Champagne, M. and Murot, G. (1931). Bull. Soc. Chem. Biol. 13, 86
Christman, A. A. (1926). J. Biol. Chem. 70, 173
Fosse, R., Brunel, A. and de Greave, P. (1929). C. R. Acad. Sci. 188, 1418 & 1632
Fosse, R., Brunel, A. and Thomas, P. E. (1931). C. R. Acad. Sci. 192, 1615
Handovsky, Z. (1914). Z. Physiol. Chem. 90, 211
Hunter, A., Givens, M. H., and Guion, C. M. (1914). J. Biol. Chem. 18, 387
Hunter, A. and Givens, M. H. (1914). J. Biol. Chem. 18, 403
Larson, H. W. (1932). J. Biol. Chem. 94, 727
Plimmer, R. H. A., and Skelton, T. (1914). Biochem. J. 8, 70 and 641
Terroine, E.F. (1933). Le metabolisme de L'azote (Paris), presses Universitaires de france,
(Paris)
Wiechowski, W. (1908). as cited in Practical Physiological Chemistry by Hawk,8th edn.
544
PHOSPHATASE IN FAECES
BY
S. C. RAY, M.Sc., PH.D., A.I.C.
Animal Nutrition Section, Imperial Veterinary Research Institute, Izatnagar
(Received for publication on 22 September 1941)
IT appears to be an accepted fact that the enzyme phosphatase is
present in an active form in the intestinal mucosa of animals and Cayla
[1935] is of the opinion that bile favours both the secretion and activation of
this enzyme, which subsequently plays an important rôle in the absorption of
calcium and phosphorus from the middle and lower intestinal regions. Accord-
ing to Verzár [1936], the enzymes secreted by the intestine for digestive pur-
poses are not ordinarily re-absorbed but pass out in the faeces. It seems pro-
bable, therefore, that a study of the activity of the enzyme phosphatase in
faeces may give interesting information on the problem of calcium and phos-
phorus absorption from the intestine. In a recent publication Duckworth
and Godden [1941] have shown that in rats large increases in faecal phosphatase
excretion take place when the fibre intake is raised. From the results obtained,
the authors have expressed the view that studies of faecal phosphatase excre-
tion may be valuable aids in investigations of secretory activities of the intes-
tinal tract.
In order to study the subject, an investigation was carried out to determine
the faecal phosphatase of normal animals under different physiological condi-
tions. In this preliminary paper, a method of determining the activity of
faecal phosphatase is described and some data are given, showing the phos-
phatase content in goats faeces under normal condition and the effect on its
excretion during prolonged fast.
Method of determining the activity of faecal phosphatase
The faeces of a 24-hour collection was thoroughly mixed and a sample—
(an aliquot of the total excretion) was taken. The sampled faeces was passed
through a mincer. Depending upon the amount available, 5 to 50 gm. of the
' minced ' faeces was weighed out, transferred to a mortar and worked up into
a thin suspension with small known amounts of distilled water. The suspen-
sion was transferred to an Erlenmeyer flask. The residue in the mortar was
then washed out completely with a known amount of water and added to the
contents of the flask. Finally, enough water was added to make the faecal
suspension up to one per cent. A few drops of toluene were added and the
suspension thoroughly shaken and left to stand in a refrigerator for 24 hours.
241
S. C. RAY 243
balance trial, equilibrium of the intake and outgo of nitrogen, calcium and
phosphorus. Four healthy goats were selected for the experiment, of which :—
Goat 1 was a non-lactating adult animal.
Goat 2 was a non-lactating growing animal.
Goat 3 was lactating adult animal.
Goat 4 was lactating adult animal.
The two lactating animals had kidded just before the experiment and at the
commencement of the experiment, they were at the height of lactation. The
average production performance of goat 4, during the eight-weeks of the experi-
ment was superior to that of goat 3. The average daily milk yield of goat 3
was 320 ml., and that of goat 4, 540 ml.
The ration, which consisted of dried green grass and crushed oats, was
the same for all the four animals. After conducting a preliminary feeding trial
lasting for two to three weeks, the following ration card was drawn up :—
—
Dried grass
.
.
.
.
.
Crushed oats
.
.
.
.
.
Goat 1
400 gm.
120 gm.
Goat 2
350 gm.
120 gm.
Goat 3
750 gm.
220 gm.
Goat 4
750 gm.
220 gm.
Before the period of collection of the excreta for analytical purposes, the
animals were fed the scheduled ration for three to four weeks. This prelimin-
ary feeding period was considered sufficient to ensure the maximum effect the
feed would have on the metabolism of the animals. At the end of the period,
the animals were put in the metabolism cages. As soon as they had become
accustomed to the new environment, a daily collection of faeces was made for
two weeks for the determination of its phosphatase content. The animals were
weighed just before and immediately after the period during which the col-
lection of faeces was made. The average weights were as follows :—
Goat 1 . . . .
Goat 2 . . . .
Goat 3 . . . .
Goat 4 . . . .
29.95 kg.
21.20 kg.
36.70 kg.
35.75 kg.
The daily record of fresh faeces voided, the dry matter in the faeces and
the units of faecal phosphatase excreted are shown in Table I.
F
244
Phosphatase in Faeces
TABLE I
Faecal phosphatase activity under normal condition
Number of days
1 . . . .
2 . . . .
3 . . . .
4 . . . .
5 . . . .
6 . . . .
7 . . . .
8 . . . .
9 . . . .
10 . . . .
11 . . . .
12 . . . .
13 . . . .
14 . . . .
Average
.
.
.
Goat 1
Fresh
faeces
(gm.)
378
360
355
422
378
357
351
404
425
382
408
373
411
364
383
Dry
matter
(gm.)
152.4
152.0
169.4
186.3
166.1
163.6
150.2
168.2
177.4
170.0
169.4
158.3
180.8
160.1
166.0
Total
units of
phos-
phatase
3,290
3,334
3,528
3,803
3,630
3,544
3,308
3,682
3,764
3,781
3,668
3,584
3,817
3,704
3,603
Goat 2
Fresh
faeces
(gm.)
270
223
270
378
326
379
395
320
344
278
284
291
320
306
313
Dry
matter
(gm.)
118.0
108.1
115.5
139.8
130.9
141.7
145.1
137.4
134.7
121.4
144.7
148.1
140.4
132.7
132.6
Total
units of
phos-
phatase
3,000
2,746
3,012
3,504
3,540
3,608
3,780
3,304
3,312
3,087
3,814
3,807
3,774
3,208
3,392
Goat 3
Fresh
faeces
(gm.)
750
580
600
725
550
625
570
629
601
684
667
694
523
688
635
Dry
matter
(gm.)
305.8
243.6
257.2
360.0
223.1
304.0
285.2
282.4
252.8
344.4
341.2
338.8
204.7
348.2
299.3
Total
units of
phos¬
phatase
6,100
5,608
5,881
7,003
5,434
6,504
6,434
6,139
6,012
6,777
6,814
6,794
5,283
6,888
6,262
Goat 4
Fresh
faeces
(gm.)
660
600
655
625
525
601
605
618
652
554
581
572
608
622
605
Dry
matter
(gm.)
300.7
277.8
312.6
295.6
236.3
294.4
272.4
284.2
313.8
248.7
244.9
259.4
260.7
278.4
277.1
Total
units of
phos-
phatase
6,788
6,501
6,706
6,589
6,108
6,914
6,762
6,624
7,002
6,388
6,334
6,582
6,517
6,811
6,616
S.
C.
RAY
245
TABLE II
faecal phosphatase activity during prolonged fast
Number of days
1 . . . .
2 . . . .
3 . . . .
4 . . . .
5 . . . .
6 . . . .
7 . . . .
8 . . . .
9 . . . .
10 . . . .
11 . . . .
12 . . . .
Goat 1
Fresh
faeces
(gm.)
202
162
84
77
...
Nil.
23
Nil
71
Nil
...
...
Dry
matter
(gm.)
82.9
84.2
47.9
46.2
...
Nil
11.5
Nil
16.3
Nil
...
...
Total
units of
phos-
phatase
1,802
1,944
603
319
...
Nil
62
Nil
101
Nil
...
...
Goat 2
Fresh
faeces
(gm.)
277
100
73
85
...
70
Nil
Nil
35
Nil
...
...
Dry
matter
(gm.)
110.8
44.0
32.1
52.1
...
32.2
Nil
Nil
22.7
Nil
...
...
Total
units of
phos-
phatase
3,062
1,108
485
511
...
196
Nil
Nil
194
Nil
...
...
Goat 3
Fresh
faeces
(gm.)
720
...
270
...
60
80
50
27
28
26
33
30
Dry
matter
(gm.)
493.2
...
170.8
...
36.6
41.6
25.5
17.6
17.4
16.6
17.5
17.4
Total
units of
phos-
phatase
7,111
...
1,888
...
212
289
143
121
83
76
114
42
Goat 4
Fresh
faeces
(gm.)
600
...
184
...
30
45
40
74
48
22
39
31
Dry
matter
(gm.)
268.4
...
106.7
...
20.4
27.9
24.8
37.6
32.6
13.2
23.4
16.4
Total
units of
phos-
phatase
7,042
...
1,847
...
261
178
192
198
127
81
104
76
F 2
246
Phosphatase in Faeces
Experiment 2.—Faecal phosphatase excretion during prolonged fast
Immediately the previous experiment was concluded the animals were
starved. The non-lactating animals were starved for 10 days and the lactat-
ing animals for 12 days. During this period, water was supplied ad lib. With
the exception of goat 1, which died on the first day of re-alimentation, the
animals stood the fast well and no untoward symptoms appeared during the
subsequent re-alimentation. The lactating goats continued to secrete milk,
though in very small quantities, up to the last day of the fast. The voiding
of faeces never stopped at any stage of the fast; in the case of goats 1 and 2,
however, it was somewhat irregular.
Faecal phosphatase determination was made every time the faeces were
collected. The amount of fresh faeces voided, the dry matter therein and the
total faecal phosphatase excreted are shown in Table II.
DISCUSSION
In spite of the fact that the number of animals used in experiment 1 was
limited and that they were not of uniform type, the results of the experiment
have revealed several interesting points for consideration.
During normal metabolism, a significant amount of the enzyme phos-
phatase is excreted in the faeces of goats. The source of this enzyme cannot
be ascribed to food, because firstly, the enzyme within the ingested food ma-
terial cannot escape digestion by the gastric juice and secondly, even if the
food phosphatases happen to escape digestion, it is an established fact that
phosphatases of plant origin cannot act in an alkaline medium such as that
used in this experiment.
Table III shows the phosphatase units excreted :—
(a) Per gram of dry food ingested.
(b) Per gram of dry faeces voided.
(c) Per kilogram of body weight.
TABLE III
Phosphatase units excreted
Animals
Goat 1
.
.
.
.
.
.
.
.
Goat 2
.
.
.
.
.
.
.
.
Goat 3
.
.
.
.
.
.
.
.
Goat4
.
.
.
.
.
.
.
.
Per
gm. of
food
6.9
7.2
6.5
6.8
Per
gm. of
faeces
21.7
25.5
21.0
23.9
Per kgm.
of body
weight
120
160
170
185