A METHOD FOR THE RAPID DISTILLATION OF THE LOWER VOLATILE FATTY ACIDS FROM STOOLS.

BY W. H. OLMSTED, C. W. DUDEN, W. &‘I. WHITAKER, AND

R. F. PARKER.

(From the Department of Internal Medicine, Washington University School of Medicine, St. Louis.)

(Received for publication, August 10, 1929.)

Methods for the accurate identification of the volatile fatty acids of the stools have long been known. Ludwig Brieger (1) in 1878 published a method, the principles of which are still in use; namely, the distillation of the acids from an acid mixture and the purification and identification of their metallic salts. Schmidt and Strasburger (2) in their well known work used the same principles. Edelstein and von Csonka (3) in 1912 and Bahrdt and Edelstein (4) in 1911 distilled stools under a partial vacuum, claim- ing by this technique to obtain the acids in a fairly pure state. They identified the acids by the fractionation of their silver salts. The latter method is undoubtedly very accurate but time- consuming. Hoppe-Seyler (5) after extracting the stool with alcohol, neutralized the alcoholic extract with sodium carbonate, evaporated it to dryness, took up the residue in water, acidified, and then distilled it. Hecht (6) distilled the acidified stools in steam. Cecchini (7) distilled stools directly but redistilled the first portion of distillate. These later methods are simple but open to the objection that acid-volatile products of protein and carbo- hydrate digestion would contaminate the distillate. The appear- ance of these products would be accentuated by boiling stools in the presence of strong mineral acid.

The principle we have used is the precipitation of the mass of organic material with mercuric chloride and then steam distillation of the filtrate. Before the filtrate is distilled, however, it is saturated with magnesium sulfate in order, first, to increase the rapidity of distillation and thus shorten the time-consuming

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116 Rapid Distillation of Fatty Acids

process of collecting large volumes of distillate, and, second, to obtain the maximum effect of the salting out process on the distillation rate. The distillation rate of a fatty acid will vary if it is in solution with varying amounts of any salt. Since stool filtrates do contain salts varying in concentration with each speci- men, it is necessary to control this factor if the distillation rate is to be used for the identification of the acid.

Method.

Stools mixed with an equal amount of 10 per cent alkali are not only effectively deodorized but their emulsification is very greatly facilitated. If more than one stool is to be collected, a sufficient amount of alkali may be placed in a commode and the stool passed directly into the alkali. Before precipitation the alkaline stool mixture is passed through a wire sieve, such as used by the housewife. There are certain definite objections to the collection of stools in alkali. A slow hydrolysis of nitrogenous material and sugar takes place, so that after a period of a week or more there is a definite increase in organic volatile acids. In a month’s time we have found an increase of 20 per cent in the volatile acids. These processes are so slow, however, that one has from 48 to 72 hours in which to work.

100 gm. of alkaline stool (50 gm. of stool) are made acid to litmus with 50 per cent sulfuric acid and ‘diluted with 100 cc. of water. 200 cc. of 10 per cent mercuric chloride in 1.5 per cent hydrochloric acid are added. Mercury is precipitated with a thick suspension of calcium hydroxide. The latter is added until the reaction is strongly alkaline to litmus. The volume is made up to 500 cc. A Buchner funnel fitted with a piece of muslin makes rapid filtration of this bulky material possible. The first filtrate is very cloudy but the muslin is soon filled with the course precipi- tate and the filtrate becomes clear. Centrifugation in 100 cc. centrifuge tubes is also a satisfactory method. Mercuric chloride precipitation of stools yields a filtrate that is clear but of amber color, giving a faint biuret reaction. 95 per cent of the stool nitrogen as determined by Kjeldahl is removed. Of late we have been experimenting with mercuric sulfate precipitation of stools and find the filtrate to be water-clear. At this time we have no data as to the recovery of added volatile acid with the latter

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Olmsted, Duden, Whitaker, and Parker 117

method of precipitation. Excess mercury is removed by hydrogen sulfide and blown off in the usual way. No loss of volatile fatty acid occurs from running an air current at high speed through acid solutions of such concentration as we are dealing with. To 100 cc. of mercury-free filtrate, representing from 5 to 10 gm. of stool, are added 2 cc. of 50 per cent sulfuric acid and 70 gm. of magnesium sulfate. Distillation is carried out as described in the preceding paper.

Blanks were run on materials including the alkali used for collection. The blanks on each of six fractions of distillate

TABLE I.

Comparison of Total Acid Appearing in Each Fraction of Distillate When Acids Are Distilled from (A) 100 Cc. Watery Solution of Acids Plus

70 Gm. of Magnesium Sulfate; (B) 100 Cc. Blank Filtrate Plus Acids Plus 70 Gm. of Magnesium Sulfate.

100 cc. distilled, Formic acid, 10 co. 0.1 N. 1 Acetic acid, 10 cc. 0.1 N. 1 Butyric acid, 10 cc. 0.1 N.

Fr%? A ,I.

B B A

cc. cc. cc. cc.

1 3.0 3.3 6.2 6.6 2 2.2 2.3 2.3 2.16 3 1.6 1.6 0.9 0.7 4 1.0 1.0 0.3 0.2 5 0.6 0.55 0.1 0.1 6 0.45 0.46 0.0 0.05

A B

cc. cc.

9.2 8.8 0.6 0.7 0.0 0.2

0.08 0.03 0.02

(0.24, 0.13, 0.06, 0.06, 0.06, 0.06 cc.) totaled 0.6 cc. of 0.1 N alkali. These blanks are not large enough to interfere with the accuracy of determining rates of distillation.

The purpose of distilling from a solution of magnesium sulfate was to minimize the effects of salt concentrations in stool filtrates. To test out this point, blank filtrates were prepared, starting with 100 cc. of 10 per cent alkali and to this filtrate was added formic, acetic, or butyric acid. Table I shows that the rates of distillation under the conditions of our method are comparable to the rates of distillation of pure acids distilled from a saturated solution of magnesium sulfate.

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118 Rapid Distillation of Fatty Acids

Recovery of Lower Volatile Acids Added to Stools.

Table II shows the recovery of added volatile acid to stools. The accuracy of the method appears to be ZIZ 5 per cent. A well known method for the determination of formic acid depends upon the reduction of mercuric chloride to the mercurous salt. The recovery of added formic acid in this method is due to the fact that mercuric chloride is reduced by formic acid very slowly and with long continued heating. We are convinced that mercuric

TABLE II.

Recovery of Volatile Fatty Acids Added to Stools.

0.1 N acid dietilled from 20 gm. stool.

A

0.1 N acid added to 20 gm. stool.

B

-

_

0.1 N acid recovered.

A+B C

Acid recovered. C-A B

cc. cc. cc. per cent

13.73 4.0 Formic. 17.01 82 13.73 8.0 “ 20.38 83 11.78 4.0 “ 15.31 88 10.68 4.0 “ 14.30 90

7.88 2.0 Acetic. 9.97 104 7.88 4.0 “ 11.94 101 7.88 8.0 “ 16.19 104

10.74 4.0 “ 15.23 112 10.74 8.0 “ 18.28 94 13.70 4.0 (‘ 17.90 105

7.88 8.0 Butyric. 15.93 100.5 10.74 8.0 “ 18.77 100

13.70 4.0 “ 17.57 97 8.88 4.0 “ 13.02 103

11.80 4.0 Propionic. 15.78 99

-

-

Expected recovery.

per cent 85 88 85 85 99 99 99 99 99 99 98 98 98 98 98

chloride precipitation does not result in the reduction of mercuric chloride by formic acid in the stool.

Quantity of Lower Volatile Acids Found in Stools.

The distillation rate of a mixture of two known volatile fatty acids will be in proportion to the quantity of the acids present. This fact was first pointed out by Duclaux. Gillespie and Walters (8) have shown that it is possible to calculate the amount of acids from distillation rates even when there are three or more

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Olmsted, Duden, Whitaker, and Parker 119

in the mixture. Therefore the distillation rates obtained from mixtures of volatile fatty acids in the stool will demonstrate the quantity of the individual acids present provided their identity is known.

The literature on volatile fatty acids of stools furnishes sufficient data on this point. Rubner (9) in 1883 reported 79 per cent of butyric and 20 per cent of acetic acid. Nencki (10) in 1891 found that in material obtained from an iliac fistula only acetic acid was present. Brieger’s results in 1878 show that three-fourths of the acid was acetic, and that there were definite amounts of butyric acid and traces of the higher volatile fatty acids. Schmidt and Strasburger reported that butyric was the chief acid with much less of acetic and traces of formic acid present. Von Csonka and Edelstein found acetic to be the principal acid, with butyric and traces of formic acids in the mixture. Bahrdt and McLean (11) give data on stools of infants in which acetic acid predominated; lesser amounts of butyric. Fischer (12) found acetic and butyric acids; Cecchini, also. Hecht, working with infant’s stools found that acetic acid predominated with butyric acid in lesser amounts and formic acid present at times and absent at others. The find- ings of von Csonka and Edelstein, Bahrdt and McLean, and of Fischer are to be emphasized because they identified and purified the acids by the silver salt method. In general we can say that in human stools there appear acetic, butyric, and traces of formic acids and that in only rare instances have the higher acids been found.

Table III shows the titration figures of each 100 cc. fraction of distillate and Chart 1 the percentage of total acids distilled that appear in each fraction. In this and the succeeding paper we have distilled the filtrates from 150 stools. The percentage of acid appearing in the first 100 cc. of distillate has always ranged between 68 and 77 per cent. At the end of 200 cc., 85 to 91 per cent has appeared; at the end of 300 cc., 92 to 96 per cent; at the end of 400 cc., 95 to 97 per cent; in the fifth and sixth fractions there is but a trace of acid. On comparison of these findings with the rates of distillation obtained with pure acids (Chart l), it would appear that in the first two fractions the rate is due to the rapid distillation of butyric and acetic acids, whereas in the latter fractions the rate decreases, probably because of the presence of formic acid in small amounts.

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Olmsted, Duden, Whitaker, and Parker 121

In Table IV of the preceding paper it was shown that all of the butyric and acetic acids are distilled over in 400 cc. of distillate and that in the fifth fraction 6 per cent of formic acid remains. The amounts of acid found in the fifth fraction of distillate should represent formic only and since they represent 6 per cent of the total formic acid present, it is a simple matter to calculate this amount. We have done this and have found that 20 to 25 per cent of the total volatile acids is formic acid. This amount in the light of the results recorded in the literature

TABLE III.

Amount of Acid Appearing in Each of Six 100 Cc. Fractions in Normal Stools.

The results are expressed in cc. of 0.1 N acid.

Subject. raction 1.

Fr. 8.14 PI. 10.27 01. 6.25 EV. 8.69 St. 8.72 Gr. 13.50 Ko. 13.87 Ad. 4.22 Ku. 5.82 Ai. 9.92 McC. 12.17 Ma. 5.27

- -

‘raction 2.

FWC- tion 3.

FGXC- tion 4.

FKN- tion 5.

FK%C- tion 6. Total.

1.65 0.53 0.22 0.10 0.04 10.68 2.22 0.74 0.30 0.12 0.06 13.71 1.64 0.64 0.27 0.11 0.04 8.95 2.17 0.82 0.36 0.17 0.06 12.27 2.14 0.76 0.28 0.15 0.06 12.11 2.95 1.03 0.52 0.23 0.11 18.34 2.12 0.93 0.35 0.17 0.12 17.56 0.81 0.32 0.11 0.08 0.02 5.56 1.18 0.47 0.22 0.14 0.10 7.93 2.30 0.94 0.42 0.18 0.06 13.82 2.17 0.72 0.29 0.19 0.14 15.68 0.97 0.28 0.09 0.04 0.03 6.68

*s? listilled

gm. cc.

6.2 145 9.5 240 2.9 115 4.1 193 7.0 172

20.0 269 4.1 143 4.0 141 4.4 210 9.8 158 4.1 312

10.0 138

I

Total 0.1 N

mid for 24 hrs.

seems too high. To check the amounts of formic acid in stools by another method, we determined it by the reduction of mercuric to mercurous salt. 600 cc. of distillate from a normal stool were concentrated after neutralization to a small volume and the method as employed by Shaffer and Friedemann (13) was used. Blank determinations were always run and duplicate determina- tions checked. Table IV shows that 6 to 15 per cent of the total volatile acid was in the form of formic acid, an average of 10 per cent. If we accept this figure as accurate, then one must conclude that in distilling these stool filtrates there appears in the distillate

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122 Rapid Distillation of Fatty Acids

a volatile acid, or acids, not of the fatty series. Every fact seems to point to these acids being present in very small amounts.

TABLE IV.

Determination of Formic Acid by Mercury Reduction Method. -T-

Stool No.

H68 G66 A62

gi B74 T82 E86 A88 K92

-- Total acids.

cc. 0.1 N

4.59 13.03 11.11 11.42 8.76 6.83

10.47 9.04 4.50 7.63

-

_-

-

Formic acid. P er cent of total acids.

cc. 0.1 N

0.43 1.12 1.35 0.74 1.13 0.56 1.59 1.u) 0.51 0.91

9 9

12 6

13 8

15 13 11 12

TABLE V.

Amounts of Acetic Acid and of Butyric Acid, on the Basis That 10 Per Cent of Formic Acid Is Present in Stools.

Total acids appearing in first 100 cc, fraction. Acetic acid. I Butyric acid.

per cent per cent per cent

68 60 30 69 56 34 70 51 39 71 48 42 72 44 46 73 41 49 74 38 52 75 35 55 76 30 60 77 27 63 78 25 65

Table V shows the percentage of acetic and butyric acids appearing in the stools for the rates of distillation that have been observed. The average distillation rate is 72 per cent in the first 100 cc. fraction, which indicates equal amounts of acetic and butyric acids.

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Olmsted, Duden, Whitaker, and Parker 123

/c7 Subiect P

/

/-

Before hddinq acid- MI;~inq Bcc.Pormic

Calcuiat edrate*--X

Fractions of Dist Slate

100cc. 200 300 400 500

CHART 2.

60

70

Before addinq acid - Aft ey adding 4ccFormic

Y Subject D

Acid.- Calculated rate -

60 _

1oocc. ZOO 300 400 500 Fractions of Distillate

CHART 3.

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124 Rapid Distillation of Fatty Acids

If one adds to a stool a known amount of, say, formic acid in sufficient quantity to change definitely its rate of distillation, one should be able to predict the new rate of distillation. We have first calculated the acids present in a stool by the use of Table V, then added a known amount of formic or butyric acid to the same stool, and calculated the rate of distillation of this new mixture of acids by Table I presented in the preceding paper. The actual observed rate of distillation was compared with the calculated.

100.

50 I

Calculated xate X-X

100ec. 200 300 400 500 Fractions of Distalate

CHART 4.

The amounts of natural stool acids determined varied in the specimen from 9 to 14 cc., and the amounts of acid added, from 4 to 8 cc. Charts 2 to 6 show the rates of distillation before and after addition of a known amount of acid, as well as the calculated rates. The rather close agreement of the observed and calculated rates gives further evidence that our deductions as regards formic acid are correct, and that the amounts of non-fatty volatile acids are so small as to be negligible.

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Olmsted, Duden, Whitaker, and Parker 125

100 CC. 200 300 400 500 Fractions of Distillate

CHART 5.

100

Acid o----o Calculated rate x-x

60 1oocc. 200 300 400 500

Fractions of Distillate CHART 6.

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Rapid Distillation of Fa,tty Acids

CONCLUSIONS.

1. A method for the rapid distillation of the volatile fatty acids from stools is presented.

2. Volatile acids added to stoolsmay be recovered. 3. Evidence is presented that acetic and butyric acids appear in

amounts varying from 30 to 60 per cent each of the total volatile fatty acids in normal adult stools.

4. Formic acid appears in very small amounts, averaging 10 per cent of the total volatile fatty acid content.

BIBLIOGRAPHY.

1. Brieger, L., J. prakt. Chem., 17-18,124 (1878). 2. Schmidt, A., and Strasburger, J., Die Faces des Menschen im normalen

und krankhaften Zustande mit besonderer Berticksichtigung der klinischen Untersuchungsmethoden, Berlin, 3rd edition (1910).

3. Edelstein, F., andvon Csonka, F., Biochem. Z., 42,372 (1912). 4. Bahrdt, H., Edelstein, F., Langstein, L., and Welde, E., 2. Kinderheilk.,

1, 139 (1911). 5. Hoppe-Seyler, cited by Cammidge, P. J., Feces of adults and children,

New York, 222 (1914). 6. Hecht, A. F., Mtinch. med. Woch., 67,63 (1910). 7. Cecchini, A., Arch. pat. e clin. med., 2,361 (1923). 8. Gillespie, L., and Walters, E., J. Am. Chem. Sot., 39, 2027 (1917). 9. Rubner, cited by Schmidt, A., and Strasburger, J., (2) p. 189.

10. Macfadyen, M., Nencki, M., and Sieber, N., Arch. exp. Path. u. Pharmakol., 28,311 (1891).

11. Bahrdt, H., andMcLean, S., 2. Kinderheilk., 11,143 (1914). 12. Fischer, H., 2. exp. Path. u. Therap., 14,179 (1913). 13. Shaffer, P. A., and Friedemann, T. E., J. Biol. Chem., 61,585 (1924).

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and R. F. ParkerW. H. Olmsted, C. W. Duden, W. M. Whitaker

STOOLSVOLATILE FATTY ACIDS FROMDISTILLATION OF THE LOWER

A METHOD FOR THE RAPID

1929, 85:115-126.J. Biol. Chem. 

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