NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
280 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [II, III
One pair of black spots are present in the Graber's organ of the larva of the
first instar. With every moult there is an addition of another pair of black spots.
In summer the larval stage lasts for about five to seven weeks.
The full-grown larvae pupate in the soil close to and a little drier than the
larval habitat. The pupal period lasts for about seven to nine days.
The life-history may be summed up as: egg, four days; larva, five to seven
weeks or if hibernation takes place about eight months; and pupa about eight
days.
Broods.
Three broods in the year are possible if food is plenty and climatic conditions
are favourable. The adults of the first brood appear about the month of April,
another brood of flies appears in about July to August and a third appears in about
October. As starvation or extreme cold causes retarded growth in the larvae the
broods are likely to overlap. The largest number of flies are present in the July
to August brood.
REFERENCES.
1. Cameron, A. E. (1926). Bionomics of the Tabanidae (Diptera) of the Canadian Prairies. Bull.
Ent. Res. 17, 25.
2. Cameron, A. E (1930). Oviposition of Haemaopota pluvialis, Linno. Nature 126, 001-602.
3. Flotchor, T. Brainbrigge (1921). Report of the Imperial Entomologist. Sci. Rept. Agric. Res.
Inst., Pusa, 1920-21, 46.
4. Pattern, W. S., and Cragg, F. W. (1913). Text-book of Med, Ent., 297.
5. Patton, W. S., and Evans, A. M. (1929). Insects, ticks, mites and venomous animals of medical
and veterinary importance. Part I. Medical 52.
EXPLANATION OF PLATE XXIII.
Haematopota roralis Fb.
1. Egg mass × 3.
2. Single egg × 20.
3. Second instar larva × 20.
4. Full-grown larva × 4.
5. Pupa × 4.
6. Female fly × 4.
7. Side view of head of female fly × 6.
8. Male fly × 4.
9. Side view of head of male fly × 6.
PLATE XXIII.
THE QUANTITATIVE PRODUCTION OF BENZOIC ACID AND
PHENOLS IN THE ANIMAL SYSTEM
BY
F. J. WARTH, M.Sc,
AND
N. C. DAS GUPTA, B.Sc.
(From the Animal Nutrition Section, Bangalore.)
(Received for publication on the 4th June 1932.)
The substances to be considered originate from the food; they are residues
which the body is unable to oxidise and are poisonous.
It has long been known that these substances are not excreted in the free state
but are first detoxicated by conversion to more complex compounds, benzoic acid
being converted to a large extent into hippuric acid, and phenols into ethereal
phenolic sulphates. Thus the elimination of benzoic acid and phenols is liable to
make demands on the body nitrogen and sulphur. On this account alone the
production of these substances is of importance. But they have great significance
in other respects also. For example, work done in this section has shown that
hippuric acid is a potent factor in the acid-base balance of cattle urine [ Warth
and Krishna Ayyar, 1930 ]. Then again, as will be shown in the present paper, the
quantity of hippuric acid formed from certain types of ration is significantly high,
amounting at times to as much as 4.5 per cent. of the total food digested.
Very little work has been done on the determination of the quantitative produc-
tion of these substances. The only recent determinations are those carried out by
Brigl and Pfahler [1929]. These authors were able to show that benzoic acid
production from hay rations was higher than from straw rations. Our experiments
described in the sequel establish the following main facts:—
1. The production of benzoic acid is quantitative. Tests carried out with
several animals on the same fodder give uniform results.
2. The production of benzoic acid depends upon the nature of the fodder.
Certain fodders give high values, others low values. Data have been
obtained for a series of typical foodstuffs.
3. The. production of phenols is loss regular, but in a general way, the amount
of phenol produced is typical of the fodder.
( 281 )
282 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [II, III
PROCEDURE.
The determinations were all carried out on urine samples collected in the course
of digestion trials, the food consumption and digestion being determined over a
period of 12 days and the urine collected quantitatively for six consecutive days
during the digestion experiments. The average excretion of benzoic acid and
phenols was ascertained in these samples. The determination of benzoic acid was
carried out by our method described in the Bicchemical Journal [Warth and Das
Gupta, 1928]. The volatile phenol was determined by a modified bromine titration
method which has not yet been described.
EXPERIMENTAL RESULTS.
1. The quantitative production of benzoic acid.—In several series of experiments
three animals were fed the same fodder. It was found that all the animals produced
the same amount of benzoic acid per 100 grms. of food consumed. This is a proof
that the production is quantitative. The accompanying Table gives typical data
obtained in experiments with three fodders.
TABLE I.
Benzoic acid excretion per 100 grms.of fodder consumed.
Fodders
Ragi Straw
.
.
.
.{
Jowar Straw .
.
.
. {
Bolaram hay .
.
.
{
Animals
R
V
A
L
N
K
N
K
A
Per cent. bonzoic acid excretion from
fodders
0.755
0.785
0.764
1.093
1.035
1.029
1.517
1.374
1.486
PRODUCTION OF BENZOIC ACID AND PHENOLS IN THE ANIMAL SYSTEM.
283
It is evident from these figures that ragi straw consistently yields low values
while jowar yields high values.
2. The quantitative production of volatile phenols (para-cresol).—The evidence
on this point is given in Table II in which the benzoic acid and para-cresol content
of typical urines are shown.
TABLE II.
The ratio between benzoic acid and para-cresol excreted.
Fodders
Ragi Straw ...{
Jowar hay
...{
Tornagallu
hay...
{
Animals
H
J
L
R
K
A
Benzoic acid
per 100 c.c.
urine
1.058
0.677
1.107
1.079
1.127
2.281
Para-cresol
per 100 c.c.
urine
0.062
0.035
0.102
0.096
0.132
0.268
Ratio
B: P.
1: .059
1 : .052
1 : .087
1: .089
1: .117
1 : .118
It will be observed that for any given foodstuff the ratio benzoic acid : para¬
cresol is fairly constant. As the benzoic acid has already been shown to be pro-
duced quantitatively, the para-cresol production is also quantitative. The ratio
which may range between 1: 0.11 and 1: 0.05 for different fodders is fairly constant
for each fodder.
The figures show that the para-cresol formed does not amount to more than
one-tenth of the benzoic acid while it may fall as low as one-twentieth in some
exceptional fodders. The following is a full table of B: P ratio obtained with
typical fodders.
284 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ II, III
TABLE III.
Benzoic acid and para-cresol production from typical coarse fodders.
Fodders
Ragi Straw
......
Ditto
Ditto
....
Ditto
Jowar fresh (young) .
Ditto
(prime) ,
Ditto
(ripe)
.
Jowar hay (young)
.....
Jowar silage Do.
.....
Bolaram hay
......
Ditto
Millet straw
Aurangabad hay
.....
Tornagallu hay
......
Benzoic acid
excretion per
100 fodder
0.82
0.83
0.86
0.83
1.36
1.14
1.21
1.46
1.44
1.06
1.05
1.14
1.22
1.09
Para-cresol
excretion per
100 fodder
0.042
0.047
0.042
0.037
0.143
0.125
0.080
0.131
0.226
0.016
0.020
0.042
0.010
0.072
Ratio
B: P.
1: .051
1: .057
1: .049
1 : .045
1: .105
1 : .110
1: .066
1 : .090
1 : .157
1: .015
1: .019
1: .037
1: .008
1: .066
3. Other phenolic substances.—These have been estimated indirectly by deter-
mining the total ethereal sulphates and deducting from this figure the amount
equivalent to the para-cresol. Such determinations show that, in most cases, para-
cresol accounts for almost the entire amount of total phenols. In a few cases other
phenolic substances may amount to one-tenth of the para-cresol. In one excep-
tional case other phenols were equal to half the para-cresol. Summing up these
results we find the distribution of the aromatic compounds to be as follows:—
Bonzoic acid
.......
Para-cresol
Other phenolic substances.....
Per cent.
87—93
5—10
1
4. Benzoic acid production from typical coarse fodders.—The accompanying Table,
IV, shows characteristic results obtained from numerous tests with each of the
fodders and the figures may be accepted as representative average values.
PRODUCTION OF BENZOIC ACID AND PHENOLS IN THE ANIMAL SYSTEM
285
TABLE IV.
Benzoic acid production from typical coarse fodders.
Fodders
Ragi Straw
......
Paddy Straw
.....
Millet Straw .
Wheat Straw
.....
Meerut Farm Hay .
Tornagallu Hay
Aurangabad Hay .
Oat Hay
Rhodes grass Hay .
Spe ar grass Hay
Bolaram Hay (Prime)
Guinea grass .
Dhub grass
Jowar (Prime)
.
„ (Ripe)
Jowar Hay (Prima)
„ „ (Ripe) .
Jowar Silage .
Benzoic acid excreted
for 100 fodder
fed
0.780
0.813
0.904
0.930
1.023
1.115
1.179
1.208
1.212
1.263
1.459
1.065
1.144
1.172
1.191
1.507
1.397
1.407
The range of values may be noted in the first place. Of the fodders examined
ragi straw gives the lowest value and jowar the highest. The highest value is about
double the lowest.
It is instructive to consider the highest value which corresponds to about 2.22
grms, of hippuric acid from 100 grms, of fodder. The digestibility being about
50 per cent., this means that for every 100 grms, of food digested 4.44 grms, of
hippuric acid are excreted in the urine. This is a notable quantity.
286 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [II, III
5. Benzoic acid and para-cresol production from concentrates.—Data on this
point were obtained during experiments to determine the digestibility of concen-
trates. This was done by carrying out three tests with small, medium and large
rations of concentrates, respectively, the coarse fodder remaining constant through-
out. The benzoic acid and para-cresol production was determined at each stage,
and was found to increase with the concentrate ration. The increase is evidently
due to benzoic acid and para-cresol produced from the extra concentrate. In this
way the figures in the accompanying Table were obtained.
TABLE V.
Benzoic acid and para-cresol production from concentrates.
Concentrates
Ground-nut cake .
Rape cake .
Til cake
Linseed cake
Cotton seed cake .
Per cent.
Benzoic acid
excretion from
concentrate
2.077
1.525
1.000
1.225
0.836
Per cent.
para-cresol
excretion from
concentrate
0.752
—
—
0.212
—
It will be noticed that weight for weight ground-nut cake actually produces
more benzoic acid than any coarse fodder. Til cake and cotton-seed cake give
low values.
It should be pointed out that the figures given in previous tables for coarse
fodders have been duly corrected for the benzoic acid and para-cresol derived from
the concentrates fed together with the fodders.
6. Subsidiary observations on factors influencing benzoic acid production.
(a) Influence of stage of maturity on benzoic acid production. It should be
mentioned that the samples of each of these crops used in these tests were
obtained from the same plot on an area of uniform growth and therefore any
differences are due to the stage of maturity only. Results are shown in Table VI.
PRODUCTION OF BENZOIC ACID AND PHENOLS IN THE ANIMAL SYSTEM
287
TABLE VI.
Influence of stage of maturity on benzoic acid production.
Jowar Hay (1928)...
Do
(1930)
Bolaram hay
Rhodes grass hay
Per cent. benzoic acid excreted.
Young
1.347
1.417
1.033
1.150
Prime
1 .541
1.472
1.459
1.212
Ripe
1.353
1.440
0.882
1.153
Without laying great emphasis on the figures there is an indication that the
prime cut yields the highest amount of benzoic acid.
(b) Effect of curing.—In the accompanying Table results are shown for identical
material (a) fed fresh, (b) converted into hay, (c) converted into silage. It appears
that the benzoic acid is increased by both these treatments.
TABLE VII.
Effect of curing on benzoic acid production.
—
Jowar fresh 1928
Do.
1930
......
Do. Hay 1928
Do.
1930
Do.
Silage 1928
Per cent. benzoic acid excreted
Young
1.254
1.337
1.347
1.417
1.685
Prime
1.180
1.164
1.541
1.472
1.407
Ripe
1.180
1.202
1.353
1.440
1.409
288 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [II, III
SUMMARY.
The production of benzoic acid and phenols from typical foodstuffs has been
determined.
It has been shown that for any given fodder the production is constant per
100 grms, fodder consumed.
A number of fodders examined gave values ranging from 0.78 to 1.51 grms,
benzoic acid per 100 grms, fodder consumed.
For each fodder there is a constant ratio between the benzoic acid and para.
cresol produced.
REFERENCES.
Warth, F. J., and Ayyar, N. K. (1930). Biochem, J., 24, 1505.
Brigl, P., and Pfahler, A., (1029). Tierernahrung 1, 30-6.
Warth, F. J., and Das Gupta, N. C. (1928). Biochem, J., 22, 621.
THE CONJUGATION OF BENZOIC ACID BY INDIAN CATTLE
BY
N. C. DAS GUPTA, B.Sc.
(From the Animal Nutrition Section, Bangalore,)
(Received for publication on the 4th June 1932.)
Two modes of conjugation of benzoic acid are known, namely, the conjugation
with glycuronic acid to form benzoyl glycuronic acid and with glycine to form
benzoyl glycine, commonly known as hippuric acid.
For the first conjugation the body has to provide glucose, for the second type,
glycine, a nitrogenous substance, and for this a demand is made upon the body
nitrogen. It has to be emphasised that the nature of the demand is very different
for the two types of conjugation.
That the conjugation is essential for the safety of the animal is proved by the
fact that whenever benzoic acid is ingested it is excreted almost entirely in the
conjugated (detoxicated) form. Only when very large doses of the poison are
given, is a considerable proportion excreted in the unconjugated form.
The literature indicates that the form of detoxication depends to a considerable
extent on the species of animal tested. Quick [ 1926 ] showed that the dog produced
glycuronic more readily than hippuric acid. Certainly, when large doses of benzoic
acid were fed the glycuronic conjugation became by far the most important
mechanism for detoxication. Csonka [ 1924 ] found that in the pig both glycuronic
and hippuric acid conj ugation take place ; the glycuronic ranging from 9 to 45 per
cent. of the whole. Ringer [ 1911-12 ] showed that with the goat the hippuric
acid conjugation is important.
Regarding cattle it is a well known fact that these animals produce a large
amount of hippuric acid. The literature offers no evidence to show that hippuric
acid is the only form of detoxication with this species. Experiments described in
the sequel prove definitely that with Indian cattle there is only one form of
detoxication, namely, the hippuric acid form.
Hewitt [1929] has carried out extensive experiments on the detoxication
of benzoic acid by cattle. He did not determine hippuric acid but assumed
that this conjugation occurred and directed his attention to the extent of the
conjugation. He found that some free benzoic acid is invariably excreted. That
is to say, the conjugation is not perfect.
The present author's results do not agree with this conclusion. Figures will be
given in the sequel to show that with the animals employed conjugation was always
perfect, the benzoic acid being eliminated entirely in the form of hippuric acid
even under conditions of nitrogen hunger.
( 289 )
H
290 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [II, III
EXPERIMENTAL RESULTS.
The results will be considered under two separate heads, namely, (1)
presence of free benzoic acid in urine and (2) the form of conjugation of benzoic
acid.
1. The presence of free benzoic acid in urine.—For the determination of free
benzoic acid in urine, the method of Warth and Das Gupta [1928] was employed.
In this process kerosene oil, which has been shown to be a more specific solvent
than toluene or chloroform, is used for the extraction. This solvent has been
shown to give accurate results with small quantities of benzoic acid in aqueous
solution. With urine it gives lower values than toluene or chloroform. It may
be contended that kerosene is not effective for the extraction out of urine. To
test this possibility, a sample of urine was exhaustively extracted with chloro-
form. The acid bodies were taken up with NaOH and the kerosene method
applied to this solution. Results are as follows :—
Total benzoic acid in 100 c.c. urine
....
Free benzoic acid in 100 c.c. urine
....
Free benzoic acid after CHCl 3 extraction
. 2.433 grms.
. 0.0063 „
. 0.0099 „
The figures show that kerosene extraction by the method already described is
perfectly effective with urine.
The liberation of free benzoic acid by fermentation.—It is well known that the
entire amount of conjugated benzoic acid in urine is completely liberated by fermen-
tation in three or four days. In tests for free benzoic acid, fermentation must be
rigidly avoided. The following tests show that some free benzoic acid is produced
within a few hours even in the presence of preservatives as toluene and formol.
Total benzoic acid in 100 c.c. urine
Free benzoic acid in 100 c.c. fresh urine .
1.838 grms.
0.0115 „
(0.65 per cent. of total.)
After 4 hours
—
Grms, free benzoic acid in 100 c.c. urine .
Free benzoic acid as per cent. of total .
No
preservative
0.0333
1.81
5 c.c.
toluene per
100 c.c.
urine
0.0333
1.81
5 c.c. of
formol per
100 c.c.
urine
0 0214
1.17
Kept in the
refrigerator
0.0166
0.90
CONJUGATION OF BENZOIC ACID BY INDIAN CATTLE
291
After 24 hours
Grms. free benzoic acid in 100 c.c. urine .
Free benzoic acid as per cent. of total
0.774
42.1
0.184
10.0
0.079
4.3
0.041
2.2
The conclusion is that tests for free benzoic acid have no significance unless
absolutely fresh urine is employed.
In the succeeding tests the urine was examined as soon as it was voided. A
few typical results are given below :
A. With normal concentrate ration
—
Free benzoic acid per 100 c.c. urine .
Average total benzoic acid per 100 c.c. urine
Mysore
bullocks
0.002
1.100
Ongole
heifers
0.002
1.500
Scindhi
heifers
0.001
1.500
Half bred
Scindhi
Ayreshirs
nil
1.500
B. With low concentrate ration
Average total benzoic acid
per 100 c.c. urine
1.
2.
2.000 grms.
2.000 „
Free benzoic acid per
100 c.c. urine
0.004 grms.
0.002 „
Nitrogen
balance
—2.07 grms.
—2.68 „
2. The form of conjugation.—To determine the form of conjugation the sub-
stance must be extracted out of the urine. This is done as follows. The urine
is saturated with. NaCl, acidified, stirred and allowed to stand. Almost all the
hippuric acid separates out. It is filtered and washed with acidified saturated
NaCl. The filtrate and washings are combined and extracted thoroughly with
H 2
292 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [II, III
ether. The ether is evaporated and the residue added to the precipitate of
hippuric acid. The following are typical results from this procedure:—
Total in urine
Total separated
Amount remaining in urine
.......
1
2
c.c. N/7
17.88
17.90
nil
17.94
17.80
0.14
The figures show that practically all benzoic acid compounds have been ex-
tracted. Occasionally there appears to be a very small unextracted remainder in
the urine which cannot be examined. To this small extent the information is
necessarily incomplete.
Before attempting an examination of the material extracted from urine, tests
were carried out with pure hippuric acid by Quick's [1926] method. This did not
yield quantitative results until the time of hydrolysis had been increased from
half-an-hour to an hour and a half For urine it was found necessary to
hydrolyse for four hours. It was also found advisable to add a small amount of
lime after removal of the acid and before final concentration. Quick's method was
employed, with these two modifications, for the estimation of hippuric acid.
Results with pure hippuric acid may be quoted here.
Hippuric acid weighed out.
„
„ estimated as glycine .
.
.
.
.
.
.
0.1495 grms.
0.1503 „
Similar determinations with material extracted from urine gave following
results. Total benzoic acid was determined by the method of Warth and Das
Gupta [1928].
A.—With normal concentrate ration.
Total benzoic acid per 100 c.c. urine
....
Hippuric acid estimated as glycine
....
1
1.25
1.24
2
1.32
1.26
3
2.18
2.08
4
1.30
1.29
B.—With low concentrate ration.
Total benzoic acid in 100 c.c. urine.
....
Hippuric acid estimated as
glycine.
....
1
0.713
0.718
2
2.115
2.081
3
1.113
1.087
CONJUGATION OF BENZOIC ACID BY INDIAN CATTLE
293
(The above animals were on low concentrate for about eight months prior to the
experiment.)
These figures show that almost the entire amount of benzoic acid present in
the urine is accounted for, and that the glycine obtained is equivalent to the
benzoic acid. There can be no doubt from these figures that the benzoic acid exists
entirely as benzoyl glycine or hippuric acid, and that this is the only form of
conjugation which occurs with Indian cattle.
SUMMARY.
Absolutely fresh urine should be used for the estimation of free benzoic acid
in urine.
The conjugation of benzoic acid with Indian cattle is always perfect, the
benzoic acid being eliminated entirely in the form of hippuric acid.
Acknowledgment.
My thanks are due to Mr. F. J. Warth for his helpful guidance during the
course of this work and in the preparation of this paper.
REFERENCES.
Quick, A. J. (1926). J. Biol. Chem., 67, 477.
Csonka, F. (1924). J. Biol. Chem., 60, 545.
Ringer, A. I. (1911-12). J. Biol. Chem., 10, 327.
Hewitt, E. A. (1929). Cornell Vet., 19, 318.
Warth, F. J., and Das Gupta, N. C. (1928). J. Biochem., 22, 621.
AN EXPERIMENT TO DETERMINE THE EFFECT OF HIPPURIC
ACID EXCRETION ON THE NITROGEN BALANCE
BY
F. J. WARTH, M.Sc.,
AND
N. C. DAS GUPTA, B.Sc.
(From the Animal Nutrition Section, Bangalore.)
(Received for publication on the 4th June, 1932.)
As far as we are aware it has not been definitely established whether hippuric
acid excretion does or does not influence the protein metabolism. The experiments
of McCollum and Hoagland [1913-14] indicated that the addition of benzoic acid,
in amounts equivalent to 40 per cent. of the urinary endogenous protein nitrogen,
could be removed in the form of hippuric acid without influencing the amount of
protein metabolism. On the other hand, Magnus Levy [1907], Ringer [1911-12] and
Delprat and Whipple [1921] have found increased protein metabolism. Our results
agree with these workers in showing that hippuric acid excretion (corresponding
to 25 per cent. of the digested nitrogen) exerted a small but definite influence on
the protein metabolism.
Previous work by us has shown that hippuric acid production is dependent
upon the fodder. Certain fodders produce large amounts, others produce small
amounts. It seemed to be a question of practical importance to determine whether
such variations have an appreciable effect upon the nitrogen balance. The experi-
ment consisted of a long period feeding test with the two fodders, ragi straw and
Bolarum hay, of which the former is known to yield less hippuric acid than the
latter. Four bullocks were maintained on each ration. At definite intervals
digestion experiments and nitrogen balance determinations were carried out, the
protein (ground-nut cake) in the ration being increased or decreased from time to
time to adjust the balance. The average total nitrogen content of the food stuffs
used was as follows :—
Ragi straw
Bolarum hay
.
.
Ground-nut cake
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
0.589 per cent.
0.362 per cent.
8.312 per cent.
It has to be noted at the outset that the ragi straw contained distinctly more
nitrogen than the hay.
( 294 )