NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
DETERMINATION OF FATS IN BIOLOGICAL MATERIAL.
357
Evidently the alcohol treatment has not completed the extraction as is often
believed to be the case. Consequent on this experiment a period of forty hours
ether extraction was adopted as sufficient. The neglected fraction was over 10
per cent. of the normal. No doubt the limits in these cases were set with a view
to bring the operations within practicable periods, on the assumption that
what was still left in the material was insignificant.
EXPERIMENTAL.
1. Extraction method.
The recovery of fats from any material without an appreciable change in the
nature of the compounds in which the fatty acids are present is altogether a dis-
tinct problem. The first common method of determination usually used is a con-
tinuous extraction of the dried material with a volatile solvent. For all practical
purposes, the changes incidental to the processes of drying and extraction are not
taken serious account of. In all the experiments reported here, the sample was
thoroughly dried and ground to pass through a 40-50 mesh sieve. The extrac-
tions with ethyl ether were all carried out in paper thimbles in Soxhlet tubes, with
2-5 grams of dried material in each. Where more fat was required for examina-
tion, it was collected from a series of such thimbles.
While ether is generally used for extraction, this extract is subsequently taken
up with petrol ether to give a determination of purer fats as opposed to other fatty
bodies such as waxes, resins or lipins. That the solvent power of petroleum
ether is not so specific, is shown clearly by the following figures :—
Ethyl ether and Petroleum ether. Comparative extractions with same quantity of
faeces.
Periods
6 hours
.
.
.
.
.
.
.
.
6
„
.
.
.
.
.
.
.
.
6
„
.
.
.
.
.
.
.
.
8
„
.
.
.
.
.
.
.
.
6
„
.
.
.
.
.
.
.
.
10
„
.
.
.
.
.
.
.
.
Total in over 40 hrs.
.
.
.
.
.
.
.
.
Ethyl ether
extract in
mgms.
125.5
20.0
9.0
14.0
2.5
5.5
176.5
Petrol ether
extract in
mgms.
109.0
22.0
15.0
19.0
5.0
4.0
174.0
358
THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [V IV
Given sufficient time petrol ether has extracted almost as much as ethyl ether.
But a great drawback to this extraction method is that, so far at least as
fodder and faeces containing a large bulk of interfering carbohydrates are con-
cerned, it has been found well nigh impossible to take the ether extraction to com-
pletion. The following illustrates the point :—
Progress of ether extraction with fodder and faeces, expressed as milligrams per 100
grams of dry material
Periods
1st 6 hours . . . . .
2nd
„
.
.
.
.
.
3rd
„
.
.
.
.
.
4th
„
.
.
.
.
.
5th
„
.
.
.
.
.
6th
„
.
.
.
.
.
7th
„
.
.
.
.
.
8th
„
.
.
.
.
.
Next 60 hours
.
.
.
.
Ragi straw
459.4
46.6
23.3
6.7
13.4
13.4
43.4
13.4
182.0
Groundnut
cake
9894.0
52.0
10.4
. .
. .
. .
10.4
10.4
156.0
Faeces
951.6
41.6
31.2
15.6
20.8
10.4
20.8
20.8
208.0
Showing the incompleteness of extraction
Official period 16 hrs. . .
Subsequent 100 hrs. . .
529.3
272.3
9956.4
176.8
1024.4
296.4
In discussing the results in the above table, it has to be pointed out that quite
a number of tests have been carried out to bear out these facts, only a typical one
being discussed here, and that at every stage of weighing, the ether extract was
DETERMINATION OF FATS IN BIOLOGICAL MATERIAL
359
evaporated to dryness only after filtration. These results were calculated to a
100 gram basis from the results of experiments with smaller quantities. Small
manipulative errors would thus appear magnified. Further the amount of extract
obtained during a period would depend upon the amount of ether that passed
through the material. A variation in the temperature of the water-bath would
cause a corresponding variation in the amount of the extract. These causes
partially explain the irregularities in the rate of extraction.
But the really important fact is that, though with small quantities of sub-
stances during one or two periods the extract was small, a sufficiently prolonged
period of 60 hours has yielded appreciable quantities. This shows clearly that
the extraction has not ceased. Nevertheless, about 50 per cent more lipoids from
ragi straw and about 30 per cent more from faeces have been recovered than would
have been the case if the extraction had been confined to the 16 hours recommended
by the Official method [Methods of analysis, A. O. A. C, 1931]. Further work
has shown that the extraction might still be continued with profit. But such a
lengthy extraction ceases to have any value as a practicable routine laboratory
method. That the later extraction, little though the extract be, is not complete
even with such a highly proteid fatty substance as groundnut cake is also impor-
tant. Allusions to such a drawback in the case of animal tissues such as kidneys
and liver are also to be found [Jowett and Lawson, 1931].
Viewed in the light of the results obtained in this paper with ragi straw, it
is clear that if about 50% of fatty bodies already obtained are still left over in a
material, resisting the action of solvents, it would appreciably vitiate the results
of a quantitative analysis of fractions, such as, the sterols and other aliphatic.
alcohols, the chlorophyll derivatives, the phospho and galacto-lipins and the
fatty acids.
The value of the results, qualitative or quantitative, obtained'with such a
method is further minimised when, as is made clear by the following figures, the
subsequent extracts are richer in fatty acids than the first fractions :—
Nature of the extract obtained at later stages
Period
1st 10 hours . . .
Next 34 hours . . .
Acid value
4.1
8.1
Saponifi-
cation
value
13.2
119.2
Fatty acid
per cent.
29.6
40.5
Unsaponi.
fiablo
matter
per cent.
65.0
30.5
360 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [V, IV
An examination of the saponification values incidentally obtained in other
experiments also indicates that the resistant fractions consist, at least in part, of
fatty acids. The results are given below :—
Saponification values of the extract in stages
Period
1st 6 hours
.
.
.
.
Next 42 hours
.
.
.
.
Further 60 hours
.
.
.
.
Ragi straw
148
164
468
Groundnut
cake
149
366
701
Faeces
113
315
561
It is evident from these further facts that the nature of the later extracts
differs considerably from that of the first portions. Such a difference would
materially alter the meaning of the figures obtained by such incomplete extractions.
The seriousness of the error in the method will be more pronounced in its appli-
cation to nutritional studies, where corresponding excreta have to be extracted
with the same solvent in a similar manner. In the absence of a really quantitative
method of determination of fats, there is no knowing to what extent the solvent
attacks the feed and to what other extent the excreta and what specific substances
are left over. The ether extraction therefore appears to be inadequate for the
purpose.
2. Hydrolysis method.
The other method in common use is a thorough hydrolysis of the raw material
with strong alkali followed by a wet extraction with ether. As a matter of fact,
the efficiency of extraction methods has been decided by comparison with
hydrolysis figures. Improvements in this line are generally aimed at perfecting
the hydrolysis so as to break down the material for complete exposure to the
solvent. This same method has been used for the determination of total lipoids
in faeces, etc. [Sharpe, 1913, Gephant and Csonka, 1914, Sperry and Bloor,
1924, Leaths and Raper, 1925]. To avoid the considerable difficulties experienced
in the filtration after saponification and the permanent emulsions with ether, the
following modified method was adopted.
The substance, weighed out into a conical flask, is hydrolysed in aqueous 20%
alkali for 24 hours over a water bath. The mass is then neutralized and rendered
decidedly acid to litmus with H 2 SO 4 in a distillation flask. The volatile acids, thus
liberated, are steam-distilled and titrated against standard alkali. The mother
liquid is rendered alkaline and re-transferred to a porcelain evaporating basin.
When the mass is nearly dry, it is acidified completely with dilute HCl and dried
thoroughly over a water bath, being powdered at the same time. Now it is
transferred to a thimble and extracted in a Soxhlet. This gives the total lipoids
with the fatty acids freed from their bases. Apart from the ease of the operations
and a better extraction, this result includes all the water-soluble fatty acids,
portions of which are likely to be lost in the usual wet method.
DETERMINATION OF FATS IN BIOLOGICAL MATERIAL
361
The completion of the hydrolysis was put to the test by rehydrolysing the
residue after extraction and repeating the whole process. Not only did this yield
a comparable quantity of non-volatile fatty acids but also an appreciable titration
for steam-distilled acids. The accompanying table gives the relevant figures from
three independent tests with ragi straw and with faeces. The figures obtained on
hydrolysis of a sample of faeces already ether extracted for 54 hours are of extra
importance.
Showing the unsuitability of alkali hydrolysis. Ether extractives expressed as
milligrams fatty bodies per 100 grams of dry material
No. of
test
I
II
III
Nature of test
Alkali hydrolysis for
24 hours.
Rehydrolysis of resi-
due from above.
Alkali hydrolysis for
24 hours.
Rehydrolysis of resi-
due from above.
Alkali hydrolysis for
24 hours.
Rehydrolysis of resi-
due from above.
Analysis
Volatile acids . .
Non-volatile acids .
Total .
Volatile acids . .
Non-volatile acids .
Total .
Volatile acids . .
Non-volatile acids .
Total .
Volatile acids . .
Non-volatile acids .
Total .
Volatile acids . .
Non-volatile acids .
Total .
Volatile acids . .
Non-volatile acids .
Total .
Ragi
straw
32
468
500
. .
199
..
31
600
631
15
621
636
31
517
548
13
669
682
Faeces
19
1,193
1,212
10
377
387
35
1,314
1,349
10
1,240
1,250
32
1,286
1,318
11
1,619
1,630
Faeces
ether
extracted
for 54 hrs.
14
636
650
12
282
294
I
362 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [V, IV
That the extracts obtained by ether with every subsequent hydrolysis are
fatty substances has also been tested fairly well. The following figures detail the
neutralization or ester values of the fatty acids obtained on different occasions :—
Showing the fatty acid nature of the extract obtained on hydrolysis of material
Neutralization value of part of the acids obtained
Ragi straw
.
.
.
.
.
.
.
.
Do
.
.
.
.
.
.
.
.
Do
.
.
.
.
.
.
.
.
Faeces . . . . . . . .
Do
.
.
.
.
.
.
.
.
Do . . . . . . . .
I. Hydrolysis
Saponification
value
159
133
154
130
197
173
II. Hydrolysis
Saponification
value
401
368
391
288
252
287
The fact that the acids obtained on rehydrolysis always show a higher saponi-
fication value indicates that they belong to groups with lower molecular weights.
In two instances iodine values were also determined on the mixed acids
obtained by extracting one sample each of rehydrolysed ragi straw and. faeces :—
Nature of the fatty acids obtained on rehydrolysis
Part of fatty acids extracted from material after
rehydrolysis.
Ragi straw.
Iodine value
49
Faeces.
Iodine value
70
They gave the iodine numbers 49 and 70 respectively, indicating that the
extracts contain fatty acids with unsaturated linkages also. It is rather strange
that, under the conditions of this work, where special precautions for avoiding
oxygen absorption were not taken, acids present in the material should remain
unsaturated after innumerable operations so conducive to their oxidation. Indi-
cations therefore, are not wanting that here is a case of formation of fatty acids
from the carbohydrates present in the material.
DETERMINATION OF FATS IN BIOLOGICAL MATERIAL
363
CONCLUSION
These observations suggest that the alkali hydrolysis method does not offer
a suitable means for a quantitative determination of fats. One has, therefore, of
necessity to resort to extraction methods alone for a determination of lipoids in
plant products. Ether alone or with alcohol has been found unable to do it.
Other solvents have been tried by a few but not with much thoroughness.
[Maxwell, 1891, Jowett & Lawson, 1931, Walker, 1933]. Under the circumstances,
it was thought advisable to make a study of the composition of the ether extrac-
tives at successive stages, so that the information then available on the nature of
the persistent fractions may help in deciding upon suitable and ready solvents
for such fractions. Work is proceeding along these and similar lines.
SUMMARY
The discrepancy in accepting figures obtained by the usual extraction method
is pointed out. The difficulty of extraction, the incompleteness of the estimation,
the differing nature of the subsequent extracts and hence the inadequacy of the
method are all supported by experimental results.
The alkali hydrolysis method is improved in technique by the substitution
of dry extraction for a wet one, making the process easier and surer.
The destructive nature of alkali hydrolysis is revealed. The results obtained
suggest the formation of fatty acids from carbohydrates during the hydrolysis.
Other tests indicate the presence, in the acids formed, of groups with lower mole-
cular weights and unsaturated linkages also.
It is concluded that a satisfactory method has yet to be evolved by means of
extraction only.
The author gratefully thanks Dr. F. J. Warth for his inspiration and valuable
guidance throughout.
REFERENCES
Allodi, A., and Palomba, G. (1931). Chem. Zentr. II, 603.
Chibnall and Channon (1927). Bio-chem. Jour., 21, 225.
Gephant, J. C., and Csonka, T. A. (1914). Jour. Biol. Chem., X, 19, 522.
Jonas, K. B., Wochbl (1931). Papier-Fabr., 61, 25A, 97 to 98.
Jowett and Lawson (1931). Bio-chem. Jour., 25, 1981 to 82.
Leathes and Raper (1925). Monographs on Bio-chem., The Fats, page 59.
Long, J. H. (1906). Jour. Amer. Chem. Soc., 28, 704.
Maxwell, W. (1891). Amer. Chem. Jour., 13, 13.
Methods of analysis (1931). A. O. A. C., 3rd Edn., pages 170 and 279.
Otto Folin and Wentworth (1909). Jour. Biol. Chem., 7, 421.
Seshan, P. A. (1932). Ind. Jour. Vet. Sci. & Anim. Husb., 2, 4, 333 to 337.
Sharpe, J. S. (1913). Jour. Biol. Chem., 7, 96.
Sperry and Bloor (1924). Jour. Biol. Chem., 40, 522.
Tomula, E. S., and Takala, E. (1932). Acta Chemica Fenica., 5, 1 to 4.
Waksman, S. A., and Tenney, F. G. (1927). Soil Sci., 24, 275 to 283.
Walker, L. S. (1933). Jour. A. O. A. C, 16, 190 to 191.
ACUTE AMPHISTOMIASIS OF CATTLE IN ASSAM:
A PRELIMINARY REPORT
BY
P. G. PANDE, M.Sc., M.R.C.V.S.,
Veterinary Investigation Officer, Assam.
(Received for publication on 10th April 1935.)
INTRODUCTION.
In October 1934, reports of cattle mortality from certain villages of
the Kamrup district were received by the Civil Veterinary Department of the
province, in which it was stated that about 60 per cent of the infected animals had
died of the disease. It was considered expedient, therefore, that the outbreak
should be investigated, and the cause of the disease ascertained, if possible.
During the course of the investigation which commenced in November, 1934,
about fifteen villages were visited, and about forty animals were examined post-
mortem. Over one thousand animals were examined clinically, where faeces
and blood examination for helminthic ova and protozoan parasites respectively
had yielded negative results. Samples of dung from affected animals, however,
upon a closer examination with the naked eye were found to contain minute
pinkish bodies which, when pressed between two slides and examined under the
microscope, were discovered to be the immature amphistomes. Postmortem
evidence regarding the pathogenic effects of these forms of the parasite was obtain-
ed from forty animals which were known to have voided immature amphistomes
in their dung. On autopsy these forms were found attached to the mucous coat
of the pylorus, and further back to the mucosa of the duodenum and the ileum.
Although during past years considerable attention has been given by workers
in this country to diseases caused by the helminths, it is a truism to say that
amphistomes have enjoyed the reputation of being harmless parasites for a long
time. It is due to this that no record is available to show that these parasites
which normally inhabit the rumen and reticulum of cattle are able to produce
a disease amongst livestock culminating in a severe economic loss. Losses in
sheep due to immature forms of Cotylophoron cotylophorum are known in South
Africa where the condition has to some extent been investigated.
The writer has been informed by the veterinary assistant surgeon in whose
jurisdiction the investigation was made that losses from similar outbreaks in
the past in his jurisdiction were attributed to Fasciola, but evidence has been
brought forward in the present communication to indicate that an exaggerated
importance has been hitherto given to the question of fascioliasis of cattle in
Assam. During postmortem examination of animals in the present outbreak
one was struck with the rarity of this class of parasite in the liver, though upon
clinical examination one would not have entertained any doubt in diagnosing the
condition as fascioliasis.
( 364 )
ACUTE AMPHISTOMIASIS OF CATTLE
365
Another outbreak with a similar mortality report was investigated in Cachar
district in February, 1935, where also immature amphistomes were found to be
the cause of the trouble. Before the investigation in this district was made
by the writer in February, 1935, he was informed by the veterinary assistant
surgeon that the condition had been persisting in those localities for the preceding
five months, and besides a few immature flukes in the liver and Mecistocirrus
digitatus in the abomasum, to which the cause of the outbreak was assigned, no
other form of parasite known to be pathogenic to cattle was discovered. It
is to be noted, however, that from 10 cases on which an autopsy was performed
the writer was able to recover a large number of immature amphistomes attached
to the mucosa of the small intestines in the Cachar outbreak. It is doubtful,
therefore, whether during the earlier investigations a complete and careful post-
mortem examination of carcases was made.
So far, this disease has been investigated only in the districts of Kamrup
and Cachar, and although no investigations have personally been made in other
parts of the province, it is suspected that it may occur as an epizootic in other
parts of the province.
SYMPTOMATOLOGY.
The disease is characterised in its initial stage by an oedematous infiltration
in the subcutis of the intermandibular space. Slight thermal reaction has been
observed during this stage of the disease and is atrributed mainly to inflammatory
processes in the alimentary canal. Later, a pronounced degree of anorexia sets
in; the animal goes off colour and the suspicion of disease becomes confirmed.
The oedema subsides naturally in about a week, but on cauterization (a practice
followed by the people) with a hot iron, it begins to disappear within twenty-four
hours, and develops again on the third day of treatment. The oedema has
also been noticed in the subcutis of the prepuce of bullocks and this follows the
same course. After this phase of the disease, the animal shows diarrhoea, the
consistency of the faeces becoming softer day by day until it assumes a fluid
character. No blood has been observed in recently passed diarrhoeic fluid, but
stringy mucous flakes have been observed and when these were examined under
the microscope they were found studded with numerous young amphistomes in
various stages of development. The diarrhoeic fluid is dark in colour and gives
an offensive odour. Although the appetite returns at this stage, emaciation
rapidly sets in, eyes become sunken, and the perineal region at the root of the
tail sinks considerably. The animal continues like this for about a week or more.
Finally it lies down, defaecates in the recumbent position, and shows a hurried
respiration. The pulse becomes feeble and frequent, and the animal succumbs
within a period varying from fifteen to twenty days.
Samples of urine collected from recent cases as well as from cases after death
showed no abnormality whatsoever. The mucous membrane of external orifices
365 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY. [V, IV
was found considerably anaemic. Examination of blood smears from about
a dozen clinical cases showed varying degrees of anaemic changes in the red blood
cells, and eosinophiles on differential count numbered as high as 29 % on an
average.
EPIZOOTIOLOGY
Outbreaks of acute amphistomiasis are known to have a seasonal occur-
rence, and the appearance of the disease is to be suspected immediately after the
monsoon which is about the end of September. It has further been observed
that acute amphistomiasis alternates with rinderpest, which it immediately
follows in the month of October and continues as late as the end of February,
which is about the time when reports regarding outbreaks of rinderpest are in-
variably received. Rinderpest in its turn is known to continue as late as Sep¬
tember after which time acute amphistomiasis makes its appearance. In the
district of Kamrup, where an investigation on acute amphistomiasis was made
in November and December, the local people call those diseases in which diarrhoea
is a. marked symptom as " Sera ", and they asserted that since March, 1934 Sera
had been responsible for causing death in cattle of their villages. Upon a scruti-
nised inquiry from well-informed people, however, it appeared that from March
to September, though the disease was characterised by diarrhoea and known as
Sera, it was also associated with mouth lesions, and the writer therefore con-
cluded that it was rinderpest which took its toll for those seven months. The
cause of the mortality in October, November and December, when the outbreak
was investigated, was found to be due to infection with immature amphistomes,
and it is to be remarked that the outbreak started immediately after the rinder-
pest had subsided. The outbreak at Cachar was investigated in February at
three tea estates, and on perusal of a mortality chart compiled by the medical
officers of these estates, it appeared that the outbreak in these localities continued
from October to February.
On the results of clinical investigation and information rendered by the
veterinary assistant surgeons of Kamrup and Cachar—two widely separated
districts, one in the Assam Valley and the other in the Surma Valley—it is to
be presumed that acute amphistomiasis is a disease of great economic importance
in Assam.
During the early part of the outbreak, i.e., in October, 1934, a few goats and
buffaloes were also reported to die of the same disease. This information has
been received from five villages where goats and buffaloes were kept. Although
no clinical cases in these classes of ruminants were brought up for investigation
in November and December, it is not unlikely that the outbreak prevailed in these
animals also, since they are kept under the same environment as cattle and harbour
the same parasites naturally. Sufficient reliance can be placed on the statement
given by the people who were much affected by the loss of their stock, and thus
were only too familiar with the nature of the disease.