Microdetermination of Fatty Acids in Serum ELIZABETH K.4ISER
AND
B. M. KAGAN, Michael Reese Hospital, Chicago, 111. Ten milliliters of distilled water are added to the residue, and the beaker with its contents is warmed slightly to form a water emulsion. The fatty acids are precipitated from the emulsion by concentrated hydrochloric acid, which is added hopwise unt,il a p H of approximately 2 is reached. Thymol blue may be used as an indicator. The beaker is kept overnight in a refrigerator. The fatty acids are filtered with suction throu h a sintered glass crucible of fine porosity (a Gooch-type crucigble, low form with fritted disk of fine porosity. capacity 15 ml., disk diameter 20 mm.). Any residue in the beaker is washed carefully into the crucible with 5% Sodium chloride solutio^, w h i d has been freshly boiled and cooled uickb in a bath containing ice water. The precipitate in %e crucible is washed with the sodium chloride solution. This washing is continued until about 10 ml. of the wash water give a definite pink color in the presence of phenolphthalein when 1 drop of freshly prepared 0.01 N sodium
~ I O X Gthe methods used for the determination fatty L 4 acids in blood serum, the acidimetric titration are the nlost u-idely used. The bases of these methods were deof
veloped by St'ewart and White ( 2 ) in England and by Stoddard and Dmry (3) in this country. The Stoddard and Dmry method wad later modified by Man and Gildea ( 1 ) . In determining blood constituents it is always advantageous t o us(' the smallest possible amounts for each test. It is even more important t o use tests that can be performed with small samples from infants and children. For when the blood is t o be this reason the acidimetric titration method described below was developed for the determination of fatty acids in small amounts of serum. The general principle of this method is similar t o that of Man and Gildf?& The changes in the Procedures, however, make it possible not only to perform the determination with greater ease and more rapidity but to do this with serum samples of only 0.2 ml. or less instead of 2.0 ml. as previously required. The major differences are in the t.echnique by which the precipitate is filtered and the solvents are evaporated, and the manner in which the greater part of the reaction is carried out in a single vessel. Elimination of the paper pulp mat and the Underwriter condrnerr make this method much simplw and more rapid. ____~ ~ _ _ _ _ _ ~ ~
Table I .
Sample,
Resulta, SIe./1000 M I .
1
0.2
10.3 10.2 10.7 11.3
111.
0.4 2
E
3
0 2
1
6
_ _ ~
___________
Difference, Me. 0.1
Table 11. Reproducibility Sample.
Results
KO.
1
MI. 0.2
Me./lM)O h l . 10.5 10.0
2
0.2
0.2
10.7 10.7
0
0
3
0.2
0.2
11.5 11.5
0
0
4
0.2 0.2
I1 3 11.3
0
0
0.2 0.2
17.5 17.0
0.5
2.9
0.2 0.2
42.3
1 .o
2.3
-__
Difference, 7' 0 9 1.6
11.5 11.2
0.3
?.8
0.3
2.7
0 2 0.4
11.3 11.6 17.3 17.2
0.1
0.6
0.2 0 4
48.0 42.7
0.3
0.7
0 4 0.2 0 4
0.2
1
6
0.4
-
Sample
Effect of \-ariation in I'oliime of Sample
Sampl~ NO.
4
h y ~ ~ ~ f ~ t ~ ~crucible ~ f ' $with & the fatty acids is placed in the beaker in which the fatty acids had been precipitated. Fifteen
.
43.3
__
Difference, Me. 0.5
Ditierenoe,
% 4.9
-____
~
Table 111. Recovery of Fattj Acids Added to Serum Samples Serum Sample A
Me./ 1000 ml. 10.3
B
11.5
_______
The p?ncipIe of the method is as follom. Blood serum is refluxed with a mixture of 3 to 1 alcohol-ether. The precipitate is filtered and the a t r a t e is saponified with alkali. The solvents are evaporated and the remaining soaps are taken up in distilled xater. The fatty acids are precipitated with concentrated hydrochloric acid, filtered, x-ashed, dissolved in alcohol, and titrated with alkali.
Solution _ _ _ _Stearic _ _ _hcid ~__ 'I1. added to 0.2 ml. _ ivle./1000 _ _MI. _
%rum 0.2 0.4 0 8 1.6 O ?
Calcd. 1.5.8
li.8
0 4
0.8 1 6
c
11.3
D
10.7
E
12.3
F
11.3
EXPERIMENTAL
The blood sample is centrifuged within 1.5 hours after being drawn. The lipides are extracted from the serum sample by refluxing in a 3 to 1 alcohol-ether mixture. To 7 ml. of alcohol-ether mixture in a 125-mI. ground-glass Erlenmeyer flask, 0.2 ml. of serum is added dropwise while the flask is agitated. After refluxing for an hour, the mixture is filtered into a lipless 200-1111. Berzelius beaker, 90 mm. long and 30 mm. wide. The residue is washed three times, each time with 7 ml. of alcohol-ether mixture. The remainder of the procedure is conducted in this same beaker. The whole filtrate with the t,hree washings in the special beaker is saponified with 0.1 ml. of 50% aqueous potassium hydroxide solution. The beaker is covered with a watch glass and placed in a special rack (made $ the authors' shop, to hold 12 beakers) in a water bath a t about ( 0 " C2. for 1.5 hours. The watch glass is then removed and the solvents are completely evaporated in a water bath a t about 90' C. Even the last traces of the alcohol can be evaporated in this manner.
0.2 0.4 0.8 1.6 0.2 0.4 0.8 1.6
17.1
22.1
Found 16.0 15 8 15.8 15.6 15.0 15.4 15.1 15.0 17.0 17.2 16 7 16.8 21.4 21.4 22.0 21.4
1879
15.3
% 101.3 100.0 100.0 98.3 94.9 97.5 95.6 94.9 99.4 100.6 97.6 98.3 96.7 96.7 99.5 96.7
Linoleic Acid Solution 0.2 24.3 24.8 0.4 24.3 0.8 23.8 1.6 24.8
102.1 100.0 97.9 102.1
0.2 0.4 0.8 1.6
100.8 100.0 102,s 97.7
24.3
24.5 24.3 25.0 23.4
Palmitic and Oleic Acid Solutions" 0.2b 25.2 24.3 0.4 25.6 0.8 24.5 H 12.5 0.1 25.2 24.3 0.2 25.3 0.4 24.2 0.8 24.9 1.6 24.0 a Palmitic, 25.5 me./1000 ml; oleic, 24.9 me./l000 ml. b M1. of each added. Q
~ Recovery,
96.2 101.4 97.2 96.2 100.4 95.6 98.6 95.0
ANALYTICAL CHEMISTRY
1880 riiilliliters ot 95% alcohol are added. The beaker is covered with a wat,ch glass and the alcohol is allomd to boil slowly for 15 minutes. The beaker is agitated a few times. The watch glass is removed and the alcoholic solution is allowed to boil for a f e x seconds to boil off‘ the carhon dioxide. The solution is titrated with freshly prepared 0.01 A‘ sotliuiii hytlroxide in a nijcrohuret graduated in 0.02 nil. HE S U LTS
The results ol)tainrd ivith 0.2- and 0.4-nil. serum samples are shown in Table 1. The difference is less than 5%. When the f:itt,y acid conwiltrat ion3 :irc? high, as in cert.ain diseases, 0.1 1111. gives similar results. Table I1 shom that the difference is less than 5% for duplicat,ea of different sera when 0.2-ml. samples are used. Such reproduciI,ility support^ the validity of single fatty acid deterniiri:itionp ith this method. The recoveries of aaturttted and unsaturated fatty acids added t o serum samples of known fatt,g acid content are shown in Table 111. The acids were added t,o the serum samples a t the beginning of the determination. Therefore, the samples went through each s t t y of the entire proc.edurrA. DIscussIox
I t was found that using aliquots of the filtrate of the serum for t,he fatty acid determination was unnecessary and the entire filtrat,e could be used. The filtrate is allowed to pass directly into the Berzelius beaker and is saponified in the same beaker together wit.h the three washings. In this way possible errors in making the filtrate up to volume and measuring out aliquots are Avoided. By using the whole sample for the dcterniination of fatty acids, the loss of sample is diminished. The serum extract is saponified in a water bath rather than on a hot plate. The last traced of alcohol can be evaporated in t,hc water bath. I n contrast t’o previous methods, bhis eliminates the need for a hot plate arid :t drying oven.
The filt’rat’ionof the fatty acid precipitate with suction through a sintered-glass crucible of fine porosity results in a clear filtratts. This e1imin:ttes the use of a paper pulp mat and makrs the filtrxtioii of the fatstyacids niuch Piinp1i.r and faster. The sodium chloride solut imi is boiled before btaing used for
washing the fatty acid residue iri order to eliminate the diesolved carbon dioxide. This increaees thtl rtxliak)ility of tht. test for ~rlmxiceof acids in the filtrate. In previous methods, extrdution of fatty scids from the Goorh crucible’s paper pulp mat riecessitated the use of an Underwriter c,ondenscr. This is a time-consuming and laborious procedure. In the new procedure the sintered-glass crucible with the fatty acida on it i8 placed in the beaker in which t,he fatty acids had previously been precipitated. This eliminates the use of an Underwriter condenser. The fatty acidP are extrac-t,ed and titrated in the same beaker. Because of the small amounts of fatty arids i n the samples, 0.01 N instead of 0.02 N sotliuiii hydroxide solution is uwd iu the tit~~tion. CONCLUSIONS
X relatively simple and rapid method for the determination of the fatty acid concentration* in 0.2 ml. of blood serum has been developed and its accurwy and reproducihilit,y have been demonstriit’ed to be within 5%. LITERATURE CITED
(1) Man, E. B., and Gildeil, E: F.,J . Btol. C ‘ h e n t . . 99, 43 (1932). (2) Stewart, C. P., and White. A. C., Biochem. J . , 19, 840 (1925). (3) Stoddard, J. L., and Drnry, P. E., J . Bid. Chem., 84, 741 (1929). RECEIVED h‘overnber 10, 1950. Presented before the Division of Biological Chemistry a t the 118th Meeting of the AMZRICAN CHEMICAL SOCIETY, Chicago, Ill. From the Kunstadter Laboratories for Pediatric Research, Medical Research Institute, and the Garah Morrb Hospital for Children, Michael Reese Hospital, Chicago, Ill. Aided by grants from the National Yitairiin Foundation and the Chicago League for Nephritic Children.
Determination of Available Oxygen and Total Manganese in Manganese Oxides KOBEHT F. STALZEK
AND
W. C. VOSBURGII, Duke Uniaersity, Dirrhum, iV. C.
TOR the determination of the composition of a nianganese oxide in terms of the evaluation of L in the formula MiiO,.nH@, both available oxygen and total manganese must be deter-
E
mined. The arsenious oxidr method described by Kolthoff and Sandell ( 1 ) has been used in this laboratory for the former and the method of Lingane and Karplus (2) for the latter. Occasionally it has been desirable or necessary to make both determinations on a single sample. This was tried, the available oxygen being determined first and then the total manganese, with a correction for the manganese introduced in the first titration, Comparison with the results obtained when separate Jamplea were used for the two determinations showed that the single-sample method gave results 2 t o 5 parts in a thousand too low. To find the source of the error aliquot portions of a manganese (11) sulfate solution were titrated by the method of Lingane and Karplus with and without addition of the substances suspected of causing the interference. Arsenate in the quantity present after determination of the available oxygen in manganese dioxide did not interfere. When the iodate catalyst needed for the titration of the arsenite was present the results were usually low by 3 to 5 parts in a thousand. Osmium tetroxide caused more error than iodate. The mechanism of the interference was thought to be the catalysis of oxidation of the divalent manganese by air in presence of the pyrophosphate. Accordingly, addition of the pyrophosphate and titration in an atmosphere of nitrogen were tried and led to satisfactory results.
With this change the analysi.’ of mangane8e oxides with a single sample gave satisfactory agreement with analysis with separate samples for the two determinations. For example, a precipitated manganese dioxide gave 56.47 and 56.46% manganese by the Lingane and Karplus method and 56.35 and 56.40% when available oxygen and manganese were determined on the same samples. The small difference may have been the result of the uRe of unpurified tank nitrogen. LITERATURE CITED
(1) Kolthoff, I. M.,and Sandell, E. B., “Textbook of Quantitative Inorganic Analysis,” revised ed., p. 605, New York, Macmillan
Co., 1943. (2)
Lingane, J. J., and Karplus, R., IND.ENQ.CHEM.,ANAL. ED., 18, 191 (1946).
REChIVED
July 3. 1951
Correction In the paper on “Design and Interpretation of Interlaboratory Studies of Test Methods” [ANAL.CHEM.,23, 1574 (1951)] the definition for degrees of freedom given in Table I11 should read: “The number of deviations minus the number of constants determined from the experiment and used to fix the points from which the deviations are measured.” GRANTWERNIMONT
