A NEW METHOD FOR THE DETERMINATION OF FAT

AND FATTY ACIDS IN FECES.

BY OTTO FOLIN AND A. H. WENTWORTH.

(From the Biochewkcal Laboratory of Harvard Medical School.)

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(Received for publication, March 31, 1910.)

An adequate knowledge of fat metabolism is particularly im-

portant as a prerequisite for a rational treatment of the numerous
cases of digestive disorders occurring among infants and young
children. Without reasonably reliable methods for determining
what can properly be called fat in the stools such knowledge
is scarcely obtainable. In the course of several years’ study in
this field involving many fat metabolism experiments, one of US
(Wentworth) has gradually come to see that the methods in
use for the determination of the different fat constituents in
feces are not satisfactory.1 We believe that the method de-
scribed in this paper, simple as it is, meets the needs of the situa-
tion.
In working out this method we have had two important con;
siderations in mind. First, that everything which can be taken
out by means of organic solvents from dried stools is not fat;
secondly, that it is practically impossible to determine separately
the free fatty acids and the soaps.
That a solvent like chloroform will take out substances which
are not fat or fatty acids is well known, but it appears to be less
well known to what extent the substances so extracted interfere
with the determination. After extracting dried stools with anhy-
drous ether for twenty hours and then for another twenty hours
with ether acidified with hydrochloric acid gas, we have found
that chloroform will still take out relatively large quantities of

‘This statement refers only to the extraction methods ordinarily in use.

With the recent method of Kumagawa and Suto (Biochemische Zeitschrift, ix,
p. 337, 1908) we have had no experience.
422 Determination of Fat and Fatty Acids

a mixture possessing an intense fecal odor. Since fat and fatty

acids are readily soluble in all the ordinary organic solvents,
and since there are other constituents in feces which are soluble
in some such solvents, it would appear more or less self-evident
that the solvent to be used should be selected with reference to
how little it will take out rather than with reference to
how much. We conclude from our experience that the use of
chloroform is not permissible, for even with the subsequent treat-
ment of the chloroform extract with petroleum ether it is not
possible to separate the fat and fatty acids from the impurities.

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For special reasons which will be given later we use anhydrous
ether for our extractions and then purify the extract by treatment
with low boiling petroleum ether (ligroin).
With reference to the separate determination of the soaps and
fatty acids there is this to be said. The ordinary fatty acids are
extremely weak acids and the extent to which they will combine
with bases to give soaps in such a complex mixture as the stools
is not very important. Numerous different factors of no special
bearing on fat metabolism will materially effect the equilibrium
between the soaps and fatty acids. The water, the hydrogen
sulphide, the carbon dioxide, the calcium and magnesium, to say
nothing of the heat and ammonia formation during the drying
of the stools, all affect the soaps. The proportion of free fatty
acid extracted by means of neutral organic solvents from the
dried stools bears, therefore, no definite relationship to the pro-
portion as it existed at the time of defecation or at any time pre-
vious to the defecation.
In view of these considerations we set ourselves the task of
determining (a) the total fat including neutral fat and total fatty
acid; (b) the total fatty acid including the fatty acids of the soap.
The neutral fat is obtained by the difference between the two
results.
Having found that anhydrous ether acidified with hydrochloric
acid gas readily takes out the fatty acids from soaps, we now ac-
complish the determination by means of a single extraction.
And having discovered a method by means of which even the
higher fatty acids can be titrated as sharply as the strongest
mineral acids, the determination involves but one weighing and
one titration.
 Otto Folin and A. H. Wentworth 423
Our titration method represents, we believe, a new departure
in alkalimetry and is not only interesting from a theoretical
standpoint but practically should become applicable to a large
number of organic acids which hitherto it has not been possible to
determine by titration.
The method is based on the use of sodium alcoholate as the
alkali and certain organic solvents such as chloroform, carbon
tetrachloride, benzol or toluol as solvent for the acid which is
to be titrated. Sodium alcoholate and certain organic solvents
have of course been used before in titrations but so far as we know,

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none of the above mentioned solvents have been used. The or-
ganic solvents which have been employed, alcohol, ether or ace-
tone, are almost useless. It is, in fact, largely because of the as-
tounding differences in the quality of the end-point of the titra-
tions obtained by means of these two sets of solvents that we
have concluded that the availability of the first named set has
until now remained undiscovered.
Organic solvents are held to be much less desirable than water.
The ordinary oxygen-containing organic solvents act like weak
acids with an indicator like phenolphtalein, consequently a con-
siderable excess of alkali is used up and the end-point obtained
is extremely uncertain. These phenomena are explained on the
basis of the associating effects of the organic solvents as against
the dissociating effects of water. With the above-mentioned
oxygen-free solvents, no such “associating” results are obtained.
Further the addition of alcohol, ether or acetone to these solvents
is as disastrous to the end-point of the titration as it is in the case
of water.
These phenomena are being further investigated in this labora-
tory with reference to the different organic solvents and indica-
tors and their applicability to the titration of a number of or-
ganic acids, hence it is unnecessary to discuss the matter any
further in this paper. Here we wish only to emphasize the em-
pirical finding that on titrating a higher fatty acid like stearic
acid in hot solutions of benzol or carbon tetrachloride, theore-
tical figures are obtained and that the end-point, a deep purple,
is obtained with the addition of the last o .o 5 cc. of the decinormal
sodium alcoholate solution.
The sodium alcoholate solution is prepared by dissolving about
424 Determination of Fat and Fatty Acids

2.3 grams of metallic sodium in about I liter of absolute alcohol

It is easily and quickly prepared and can be standardized in the
usual manner against decinormal hydrochloric acid. The coating
of oxide which always covers sodium sticks should of course be
scraped off or cut away before the sodium is weighed.
The ethereal hydrochloric acid solution used for the extraction
should be decinormal or a little stronger. It is conveniently pre-
pared by dropping concentrated sulphuric acid on about IO
grams of powdered sodium chloride and leading the gas into about I
liter of ether. Cold ether holds hydrochloric acid fairly well

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and that is why we use this solvent by preference for the extrac-
tions. The ether must be of the absolute sodium dried variety
as both water and alcohol must be carefully excluded. The hy-
drochloric acid content of the ether is determined by titration
(in the presence of water) and is then diluted to the desired con-
centration by the addition of more anhydrous ether.
The determination is carried out as follows: The thoroughly
dried stool is pulverized and sifted through a 40 mesh sieve.
All should go through the sieve. The whole is then sifted through
once more in order to insure thorough mixing of the sample.
The thorough powdering of the stools is an important detail,
for without it it is well nigh impossible to obtain complete extrac-
tion.
One gram of the powder is then weighed out, wrapped up in
a piece of fat-free filter paper and the whole transferred to a fat-
free filter paper “thimble.” This is inserted in the extraction
apparatus which is then attached to a 250 cc. Erlenmeyer flask
containing about I 50 cc. of the ethereal hydrochloric acid solution.
The boiling of the ether should then be kept up for about 20 hours.
After disconnecting the flask the ether is distilled off. With it
goes the hydrochloric acid, provided that no alcohol or water is
present. (Any traces of HCl which may remain are removed
during the ligroin treatment). When practically all the ether
has been thus removed about 50 cc. of low boiling petroleum ether
is added and the flask is set aside over night. The petroleumether
should have a boiling point of 30 to 60’ C., i.e., when distilled
all should go over below 60’.
The following day the petroleum ether solution is filtered
through a small plug of absorbent cotton inserted in the stem of
 Otto Folin and A. H. Wentworth 425
a suitable funnel or ‘I adapter.” The filtrate and washings are
collected in a weighed, tall, IOO cc. beaker. The solvent is boiled
off, the residue is dried at about 95’ C. for five hours, cooled and
weighed. This gives the total weight of the neutral fats and the
fatty acids.
The fatty residue is then dissolved in 50 cc. of benzol, one or two
drops of a 0.5 per cent alcoholic solution of phenolphtalein is
added, and the mixture is heated until the boiling point is nearly
reached. It is then immediately titrated with the standardized
sodium alcoholate solution. The titration should be continued

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until the maximum color of the indicator is obtained. The sub-
sequent more or less rapid fading away of the color does not indi-
cate that the true endpoint was not reached. This fading seems
to be due mainly to the fact that on cooling the soap which is
formed is transformed into basic soap, thereby setting free alittle
of the acid.
Each cubic centimeter of decinormal alkali solution used cor-
responds to 28.4 mgm. of stearic acid and all our results thus far
obtained indicate that the fatty acid in stools consists mainly of
stearic acid.
The following results obtained on the stools of normal adults
were obtained by the method described above:

PER CENT 1 2
______
Total fat.. . .. . . . .. 13.5 16.0
Neutral fat. .. . . . . . . . . 6.8 6.6
Fatty acid.. .. . 6.7 9.4

While the total fat varies between 13 and 30 per cent in round
numbers, the neutral fat varies only between 5 and o per cent.
In other words the larger the amount of total fat the feces con-
tains, the more of that fat is present as fatty acids (including the
soaps).
In the case of an “atrophied” infant the results obtained are
even more striking, for here the total “fat” seems to consist al-
most entirely of fatty acids and soaps.
The following figures were obtained: On modified cow’s milk,
total fat 54.5 per cent, fatty acid 54.6 per cent. On mother’s
milk, total fat 28.8 per cent, fatty acids 25.8 per cent.
426 Determination of Fat and Fatty Acids

It may be thought that these high fatty acid values are due
to the splitting effect of the acid ether, but such is not the case.
By special experiments we have satisfied ourselves that neutral
fats are not saponified by being boiled with decinormal solu-
tions of hydrochloric acid in anhydrous ether. Neither can the
figures be explained on the basis of lower fatty acids for the ex-
tracts melted at about 53” C., a figure easily accounted for on the
basis of a small admixture of oleic acid.

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 A NEW METHOD FOR THE
DETERMINATION OF FAT AND FATTY
ACIDS IN FECES
Otto Folin and A. H. Wentworth
J. Biol. Chem. 1910, 7:421-426.

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