A TITRATION

BY JAMES (From the Chemical Department Laboratory

METHOD

FOR BLOOD
AND PHOEBE

FAT.

L. STODDARD

E. DRURY. the

of Medicine, Harvard Medical School, and Hospital, Boston.) of the Massachusetts General

(Received for publication, July 2, 1929.) The basic idea of the method given in this paper is to isolate the fatty acids and titrate them. The blood is extracted with alcohol and ether, the extract saponified, the fatty acids separated, filtered, washed, dissolved in alcohol, and titrated with phenol blue as an indicator. In the development of the method, technical difficulties in the filtration and washing of the fatty acids were the chief obstacles. These were finally overcome by the technique of filtration through previously heated paper pulp mats in small Gooch crucibles, salt solution being used for washing. In a series of ten blood samples from normal fasting subjects the fatty acids averaged 294 mg. per 100 cc. All but one ranged between 260 and 333; the exception was 237. One other supposedly normal sample gave a figure of 193 mg. per 100 cc. It is believed that a titration method offers certain attractions in the way of simplicity of chemical procedure, and probably freedom from interfering factors in unusual blood samples. In 1925 Stewart and White published a method similar in general principle. The fatty acids, however, were not separated, washed, and titrated directly as in this method. The alcoholether extract of the blood was saponified with 5 cc. of 0.1 N NaOH, then an equivalent amount of 0.1 N HCl was added, the mixture boiled to get rid of COz, made up to volume, and an aliquot part The principal objections (one-tenth) titrated with 0.1 N NaOH. to this method are : (1) A very slight error in measuring either the NaOH used for saponification or the HCl used for neutralizing 1 Stewart, C. P., and White, A. C.,
Biochem. 741 J., 19,840 (1925).

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742

Titration

Method

for Blood Fat

makes a very large per cent error in the calculated blood fat. One of Stewart and Whites normal blood fat samples of 300 mg. per 100 cc. of blood would have 3 mg. per cc. or 0.3 mg. in the aliquot
TABLE I.

The materials used were Kahlbaums oleic and palmitic acids, and Baker and Adamsons C.P. stearic acid. Solutions were made in 95 per cent alcohol. The samples were saponified, etc., as in the method for blood, but of course the preliminary extraction with alcohol-ether mixture and filtration were omitted.
-7

Experiment No.

Ok&. 7n.w

Palmitic. ?nM

Stark.
_-

1:&a1 fatt)

acid.

Found. 7n.M

Error.

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Tndl

0.0646 0.0646 0.0646 0.0646 0.0646 0.0646 0.0323 0.0323 0.0969 0.0969 0.0626 0.0626 0.0969 0.0969 0.0969 0.0323 0.0323 0.0323 0.0323 0.0323

0.0195 0.0195 0.0195 0.0195 0.0195 0.0195 0.0097 0.0097 0.0388 0.0388 0.0194 0.0194 0 0 0 0.0101 0.0101 0.0101 0.0101 0.0101

0 0 0 0 0 0 0 0 0 0 0 0 0.030 0.030 0.030 0 0 0 0 0

mg. 0 0 0 0 10 10 10 10 0 0 0 0 10 10 10 9 9 9 9 9
-

7nM

per

cent

0.0841 0.0841 0.0841 0.0841 0.0841 0.0841 0.0420 0.0420 0.1357 0.1357 0.0820 0.0820 0.1270 0.1270 0.1270 0.0424 0.0424 0.0424 0.0424 0.0424

0.0838 0.0845 0.0850 0.0877 0.0838 0.0838 0.0430 0.0430 0.1290 0.131 0.0843 0.0808 0.1350 0.1320 0.1330 0.0422 0.0408 0.0420 0.0420 0.0407

-0.30 f0.50 +1.20 +4.40 -0.30 -0.30 +2.4 +2.4 -4.9 -3.8 +2.8 -.1.2 f6.3 +4.0 +4.7 -0.5 -3.8 -0.9 -0.9 -4.0

taken for final titration. This would take a titration of only 0.011 cc. of 0.1 N NaOH. An error of only 0.1 per cent in measuring either the NaOH used for saponification or the HCl used for neutralizing would make an error of 45 per cent in the blood fat.

J. L. Stoddard

and P. E. Drury

743

(2) In saponification the glass is attacked and sodium silicate formed, which interferes with the titration. (3) CO, will be absorbed during titration unless special precautions are taken. In the authors method the fatty acids are isolated and washed, and the only amounts of alkali which come into quantitative relations are those used in the direct titration of the fatty acids. Solution of the fatty acids in alcohol removes any silica which may have formed. CO2 is boiled off at a low pH, and special precautions are taken to avoid absorption during titration.
TABLE II.

Check

Determinations
-7

on Various
Average. Maximun deviation from average.

Normal
1 L

and Pathological
-Y-

Blood
Average.

Samples.

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Sai%?e
% iY
1 362 360 364 376 372 379 370 360 378 367 242 237 288 274

S%?
26 ET
6 7 8 270 270 538 543 209 209 1180 1225 498 510 229 247

--

per cent

270 540 209 1203 504 238

0 0.6 0 2.0 1.2 3.3

362

0.6

374

1.4

9 10

368 239 281

2.8 11 1.2 2.5

4 5

Much work was done on known mixtures of oleic, palmitic, and stearic acids. Not more than 4 parts of oleic to 1 of palmitic or stearic can be filtered and washed quantitatively. These pure fatty acid mixtures are much more difficult to deal wit,h than those derived from blood, partly because cholesterol is present in the blood extracts at the time of filtration, which makes a harder precipitate. In a series of various pathological blood samples none has been found that offers as much difficulty in filtration as the artificial mixtures which, nevertheless, give accurate results.

744

Titration

Method

for Blood Fat

Addition of cholesterol to the artificial mixtures had no effect on the results. Tables I to III give the results of test analyses with the method.
TABLE III.

Additions

of Fatty

Acid

to Blood.

A mixture of approximately 3 parts of oleic acid to 1 part of palmitic was used, dissolved in 3 parts of 95 per cent alcohol to 1 part of ether. The blood sample was run into the alcohol-ether mixture used for extraction, and then a measured amount of fatty acid mixture was added. This procedure was adopted to avoid precipitation of proteins by a direct addition of the alcohol-ether solution to blood. Figures are in mg. of fatty acid per 100 cc. of blood.
-

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Experiment No.
_-

Blood I mnple.

Average.
_-

a! d I

L I

Blood

with added fatty acid. Found.

Average.

ERW.

i :alculated _
510.6 867.6

per

cent

398.6

384.6 384.6 384.6 290 391.6

495.2 498.3

496.7

-2.8 +2.1 -l-o.03 -0.4 -1.2 -1.8 $3.4

887.6 886.0 1220.9 1231.8 406 409 756 758 1104 1107 394 406 403 760 759 770 1103 1133

886.8 1226.3 407.5 757 1105.5 401

1225.6 409 766 1124

265 272

387.5 268.5 744.5

763

+2.5

1101.5
-

1118

+1.7

J. L. Stoddard

and P. E. Drury

745

Procedure. Extraction and SaponiJication.--5 cc. of whole blood or of plasma or serum are measured into a 100 cc. flask containing about 75 cc. of a mixture of 3 parts of 95 per cent alcohol and 1 part of ethyl ether (redistilled). The blood or plasma is made to enter in a slow stream of drops and the liquid in the flask is kept rotating rapidly to prevent the formation of large aggregates of precipitate. At once, or after waiting until a convenient time, the flask is immersed in boiling water with frequent and strong rotation (to prevent superheating) until the liquid begins to boil, then cooled to room temperature, made up to volume, mixed by pouring back and forth three times into a dry 300 to 500 cc. flask or beaker with stirring, and filtered through a fat-free filter paper. Evaporate 75 cc. of the filtrate in a 100 cc. beaker (put in 50 cc. at first). Add a few grains of coarse sand (previously boiled with acid, and then washed, dried, and extracted with ether) to prevent bumping. Place on an electric stove at low heat, or on the cover of a steam bath, or elsewhere, so that it does not boil perceptibly, and allow to evaporate until the ether is practically gone; then place in a gentle current of steam, and continue evaporation until a volume of about 30 cc. is reached. Then add 0.1 cc. of saturated NaOH,2 mix, add a few grains more of sand, cover with a watch-glass, and boil gently for 20 to 30 minutes (to saponify). Then remove the cover-glass, drop in a small piece of litmus paper, make acid with 30 per cent HCI; then run back to alkalinity with 10 per cent NaOH (in order to avoid an excess of alkali on evaporating to dryness). Evaporate to dryness (in order to get rid of all alcohol). Add 15 cc. of water, heat on the steam bath, and stir to dissolve the soaps. While hot, add a drop of thymol blue indicator and make acid until a faint pink with 30 per cent HCI. Set the beaker for 10 minutes in cold water, then swirl almost continuously for 5 minutes to produce a better separat,ion of the fatty acids. The filters, which should have been prepared beforehand, are made as follows: Use a Gooch crucible, smallest size (top 28 mm.,
2 Make a saturated solution of C.P. sodium hydroxide settle. Make in Nonsol glass if possible, if not, in Pyrex, cool during solution. sticks; keeping allow to the flask

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746

Titration

Method

for Blood Fat

Set the crucible in a rubber washer bottom 18 mm. in diameter). which fits over the top of a small funnel. Push the stem of the funnel through the hole in a rubber stopper and set the stopper in a suction flask (500 cc. capcity). Have ready a paper pulp emulsion, made by shaking up a piece of soft filter paper (such as Schleicher and Schiill black ribbon, No. 589) in 300 to 400 cc. of distilled water. Shake this emulsion vigorously and immediately pour some into the crucible while there is a strong suction on. Repeat until a layer about 1 mm. thick is formed. Tamp the layer down carefully all over with the end of a glass rod. Allow the larger masses of filter pulp fibres in the emulsion to settle out, and pour on successive amounts of the thin upper suspension of isolated shreds, keeping a strong suction on and tamping down occasionally, until the filter is dense enough to offer a definite resistance to the suction. Take the crucible out of the rubber washer and dry in an air oven at 110 for 15 minutes. Take out and allow to cool before using. Set up a row of the funnels which fit the crucible washers. Place a crucible in its washer in a funnel. Place under the funnel a test-tube, and pour into the crucible some of the fatty acid suspension. If the filtrate is not perfectly clear, put it through the crucible again. If the filtration does not start in a few minutes, change the crucible to the funnel in the filter flask and start the suction very gently, with a test-tube under the funnel. After filtration has started, continue without suction. After the fatty acid suspension is filtered and has drained, wash with 4 cc. of 5 per cent NaCl solution, which has been neutralized to methyl red. Use a pipette and run the salt solution down the walls of the beaker all around, then, tipping the beaker, use a fine bent glass rod to rinse the side of the beaker more thoroughly with the solution, then pour this rinsing into the Gooch crucible, rinsing its side with the aid of the rod. Wash until the filtrate from one washing takes not over 0.05 cc. of 0.02 N NaOH to neutralize it to phenolphthalein. Usually this is true of the third washing. Put the crucible back on the suction flask with a non-protein nitrogen tube (cut off to a convenient height and calibrated at 1 cc. intervals from 10 to 15 cc.) under the funnel. Wash down the walls of the beaker with 5 cc. of 95 per cent alcohol, heat to

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J. L. Stoddard and P. E. Drury

747

boiling, and pour into the crucible. With the glass rod quickly loosen up any fatty acid fragments on the wall of the crucible. Allow to run nearly out, then put on a moderate suction. Rinse out the beaker and crucible twice more with 3 cc. of alcohol each time, heating it to boiling. Then wash off the outside of the crucible and the funnel. Titration.-Add a few grains of sand, boil the filtrate for 1 minute, cool in a beaker of water, note the volume of alcohol, add 3 drops of 0.3 per cent phenol blue in 50 per cent alcohol, titrate with 0.02 N NaOH to a pure blue which stays practically unchanged with no yellow tinge while stirring it for 2 minutes with a stopper in the mouth of the tube to avoid absorption of CO,. For a blank boil 10 cc. of alcohol and titrate. Calculate the correction necessary for the amount of alcohol present before titration. Add a correction amounting to 0.005 cc. for each cc. of NaOH used in titration (a simple correction for the volume of solution). Subtract the total correction from the titration. Multiply by the normality factor, thus getting the number of millimols of fatty acid. Multiply by
cc. filtrate 100 evaporated X 100 cc. blood used =

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millimols of fatty acid per 100 cc. of blood. To translate into terms of weight (not a very significant figure) multiply by an average factor for the fatty acids as they usually occur in blood = 277.2. The molecular weights are so nearly alike that a considerable variation in the proportions will not affect the calculated weight more than about 2 per cent. Note that cholesterol is present while the fatty acids are titrated. The cholesterol does not affect the titration. Determination of Cholesterol.-Transfer the titrated fatty acids to a 100 cc. beaker, make acid with 3 N HCl, add a few grains of sand to prevent bumping, and evaporate to dryness on the steam bath. Add 10 cc. of chloroform, stir to dissolve, allow to stand 10 minutes, filter through a paper pulp filter in a Gooch crucible into a 20 CC. volumetric flask. Extract twice more with 5 cc. of chloroform for 5 minutes each time. Make the flask up to volume with chloroform, mix by pouring back and forth three times into a flask, then

748

Titration

Method for Blood Fat

take 2 cc. for cholesterol determination according to Bloors method by the Liebermann-Burchard reaction. Note that the fatty acids are present while the cholesterol is being determined. It was found that they give no color reaction and do not interfere with the color reaction given by cholesterol. This paper is the first in a series on fat metabolism, Dr. Chester M. Jones and the authors. projected by

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CORREClIO?S. On page 741, Vol. lxssiv, line 9, read thymol blue for No. 2, November, phenol blue. 1929, line 4, and psge 747,

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