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NOTES ON THE EXTRACTION AND SAPONIFICATION OF LIPIDS FROM BLOOD AND BLOOD SERUM
BY EVELYN B. MAN AND EDWIN F. GILDEA
(From the Department of Psychiatry and Mental Hygiene, Yale University School oj Medicine, New Haven)
(Received for publication, July 12, 1937)
Recently Boyd (5, 6) and Stewart and Hendry (19, 20) have published data on the extraction and saponification of lipids from whole blood, serum, and red blood cells. The following experi- ments were performed to test the modifications of the Stoddard and Drury titrimetric method for the determination of fatty acids (12), the gravimetric procedure for cholesterol (14), and for the colorimctric estimation of lipoid phosphorus (11, 14) which have been used by us.
EXPERIMENTAL
Ten cxpcrimcnts in each of which two samples have been hy- drolyzed with sodium hydroxide and two with potassium hydrox- ide have been performed. Blood serum was used in seven of the ten experiments; in two a commercial lecithin was employed; in one the lipid material was a lecithin preparation from the liver of a guinea pig which had just been killed.
In Table I are the results of eighteen experiments in which six different samples of blood serum and twelve of whole blood were extracted by different methods and were analyzed for titrated fatty acids and lipoid phosphorus. Cholesterol determinations were also made, but the values are omitted from Table I because the variations were not significant. All extractions were made in duplicate and the average of the two results is given in Table I. Before the measurement of each 2 cc. aliquot the blood was mixed to insure even distribution of cells and plasma. All the aliquots of.blood were pipetted as rapidly as possible and were in alcohol and ether within 20 minutes from the measurement of the first to the measurement of the last sample. The alcohol and ether
77
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TABL
E I
Seru
m
and
Bloo
d Li
pid
Valu
es
afte
r Ex
tracti
on
by
Dise
rent
M
ethod
s
Expe
rimen
t No
.
Titra
ted
fatty
ac
ids
Lipo
id
phos
phor
us
Dev
iatio
n fro
m
Man
an
d G
ildea
M
inim
um.
_ .
. .
Max
imum
. .
. .
. Av
erag
e . .
. .
.
- -
m.-e
q.
12.8
5 27
.53
9.39
11
.13
11.1
0
Tn.-q
.
12.5
5
?I%.-e
q. m
.-cq.
??
a.-e
q.
12.7
7 26
.97
, 10
.73
9.28
10
.51
11.2
6
%ir
10.4
1 25
.10
11.6
7 9.
83
8.01
8.
21
11.2
5 8.
56
10.1
2 10
.16
‘;& %
: 10
.41
25.7
2 7.
90
7.82
8.
34
10.1
0
-0.3
0 -0
.94*
0
-0.3
9 +0
.44
+O.lS
* +0
.04
+0.6
2 +0
.10
-0.3
6*
+0.0
2 +0
.07
Expe
rimen
ts
on
seru
m
-
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7 8 9 10
11
12
13
14
15
16
17
18
Devia
tion
from
Ma
n an
d Gi
ldea
Mini
mum
. .
. M
axim
um.
Aver
age
.
18.3
8 12
.55
10.8
5 9.
87
9.82
11
.39
10.2
5 8.
90
10.8
3 13
.14
13.3
0 10
.33
18.1
4 15
.96
13.0
2 12
.11
10.7
7 8.
48
10.1
8 8.
80
9.46
9.
81
11.5
4 10
.77
9.89
7.
60
8.75
7.
12
18.8
5 11
.60
8.79
8.
76
9.19
10
.36
8.43
8.
32
14.3
8 12
.95
9.73
9.
20
8.69
11
.20
9.09
8.
39
11.7
5 10
.75
12.0
3 9.
35
12.0
5
15.3
5 15
.00
13.2
0 12
.91
9.68
8.
42
12.0
5 11
.32
10.8
1 10
.20
11.6
0 12
.00
9.78
9.
12
12.1
5 11
.10
10.2
3 13
.32
Boyd
. M
ay,
1036
10.1
4 12
.96
Nitro
gen
10.4
0 12
.50
12.3
7 12
.36
13.0
6 13
.15
6 hr
s. he
at ing
in
air
13.0
3 13
.10
10.7
6 10
.39
10.7
7 13
.49
13.5
6 12
.06
13.0
6
-0.6
0 -2
.65t
-1
.16
-2.0
6f
-0.4
3 +0
.47
-0.0
1t
-0.1
9 +0
.47t
+1
.00
-0.0
4 -1
.42t
-0
.77
-0.9
6t
+0.2
6
Expe
rimen
ts on
wh
ole
blood
* In
Ex
perim
ent
3 th
e ca
rbon
dio
xide
value
wa
s us
ed
in
plac
e of
th
e Ma
n an
d Gi
ldea.
t
Expe
rimen
ts 7
to
14
inclu
sive.
Bwd.
M
ay.
1936
11.8
5 12
.27
13.3
3 12
.62
-0.9
5t
+0.6
2t
-0.2
3t
-
8.63
11
.62
9.62
11
.90
9.15
11
.75
12.5
0
-1.1
3 -0
.13
-0.4
1
14.3
1
8.96
11
.81
11.0
0 11
.80
9.68
11
.15
Nitro
gen
12.5
0
i hm
,. he
at-
ing
m a
ir
12.7
5
+o.w
-0
.9Ot
-0.0
9t
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Blood Lipids
used in all extractions was a mixture of 3 parts of alcohol, redis- tilled after being refluxed with potassium hydroxide as detailed by MacArdle (lo), and 1 part of ether, redistilled wit,h an eight bulb pear column as described by King (9). These methods of distillation are the same as previously described by Man and Gildea (12). In every instance the ether was distilled within 28 hours of the time when it was used. The fat-free filter paper, either Schleicher and Schiill No. 589 or Whatman No. 43 (12), was reextracted for 4 to 6 hours with boiling redistilled alcohol. In all extraction methods the lipid extract was not diluted to volume until after filtration, washing of the filter paper and pre- cipitate, and cooling of the extract to room temperature.
In Column 1 of Table I are given the results of the Man and Gildea extraction method which has been detailed previously (12). This consisted of the slow addition with shaking of 2 cc. of blood or serum to 25 cc. of alcohol-ether. This mixture was then refluxed for 1 hour at the lowest heat on a hot-plate insulated with four wire gauzes. The temperature in the refluxing flask rises slowly during the process of refluxing, but in one test experiment 90 minutes after the beginning of extraction the temperature regis- tered 66” on a thermometer the bulb of which was in the boiling mixture. After 60 minutes of refluxing the lipid extract was filtered into a 50 cc. volumetric flask and the protein precipitate and filter paper were then washed with five to seven small por- tions of cold alcohol-ether until the volume of the extract was almost 50 cc.
In Column 2 of Table I are the results of the “CO2 extraction” method which was identical to the process in the Man and Gildea extraction except that during the hour of refluxing a stream of carbon dioxide was admitted through the cork in the top of the Erlenmeyer flask. Carbon dioxide was added to prevent oxida- tion of fatty acids and an increase in carboxyl groups, after the suggestion of Stewart and Hendry (19).
In Table I, Column 5, is one experiment in which nitrogen was substituted for carbon dioxide during the hour of refluxing. In that same column is another single experiment called “6 hours heating in air.” 2 cc. of blood were precipitated in 50 cc. of alcohol-ether. The alcohol-ether was then refluxed for 6 hours, a prolongation of the Man and Gildea technique without the use of either nitrogen or carbon dioxide.
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E. B. Man and E. F. Gildea 81
The “Boyd water hemolysis” extraction followed the method for ext,raction of red blood cells, recommended by this author in August,, 1936 (6). 2 cc. of blood were hemolyzed with 2 cc. of distilled water and 50 to 60 cc. of alcohol and ether were then added with ronsta,nt agitabion. The flasks were then shaken for 3 minutes before filtration. The prccipitatc and filter paper were washed with seven or more portions of alcohol and ether although Dr. Boyd used only one washing in some of his experiments ((6) P. 39).
The column labeled “Boyd, May, 1936” refers to the extraction methods recommended by Dr. Boyd at this time (5). For serum or plasma, in order to extract all the lipids, he adopted a dilution in which 3 cc. of serum or plasma were dropped slowly with shaking into 80 cc. of alcohol-ether. For blood he advised a dilution of 1: 30 or 1: 35. In all experiments on blood 2 cc. were precipitated in 75 cc. of alcohol-ether. Boyd stipulated that the extracts be heated for 5 minutes with frequent shaking to prevent superheat- ing and after cooling, filtration, and washing, that the final volume should be 100 cc.
In the Bloor extraction 2 cc. of blood were added drop by drop with shaking to 26 cc. of alcohol-ether. The flasks were then immersed in boiling water and were shaken continuously to pre- vent superheating. After the alcohol-ether mixture appeared to boil, the flasks were kept immersed in the hot water for 1 minute. In Column 4 of Table I called “Bloor water cooling” are given the results on the lipid extracts when the containing flasks immediately after removal from the hot water were cooled in cold water and the extracts were filtered immediately after cooling (1, 2). In contrast,, Column 5 called “Bloor air cooling” contains the results on the extracts when the flasks after immersion in hot water were not chilled in cold water but were allowed to cool spontaneously to room temperature (3, 4). This air cooling required 40 to 60 minutes, a length of time almost as great as in the Man and Gildea refluxing method. In both of these Bloor methods the extract was filtered into a volumetric flask, the precipitate and filter paper were washed five to seven times, and the extract was made up to volume subsequent to the processes of filtration and washing.
The method of extraction devised by Man and Gildea was designed to be applicable to both serum and whole blood, so that
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Blood Lipids
when lipids were to be analyzed simultaneously in both serum and whole blood every step in the technique would be com- parable. Since it is generally assumed that whole blood is more difficult to extract than serum or plasma, only six experiments on serum, but twelve on whole blood, were conducted to reinvestigate extraction methods.
At the end of the six experiments on serum and the twelve experiments on blood are given the average deviations of the yields of each extraction method compared with the yields of the Man and Gildea technique. A negative sign indicates that the yield was lower than by the Man and Gildea method. Average deviations have been calculated after consideration of positive and negative signs. This means that, if the differences in yields by different extraction methods were only governed by the laws of chance, variations should cancel each other and the average deviation would approach zero. The minimum and maximum values represent the extreme differences from the Man and Gildea yields. In comparing the yields of lipids obtained by different methods one criterion of a significant variation is that obtained by a statistical analysis of the duplicates of 100 consecutive deter- minations of serum lipids. It was found that, given the average of a pair of duplicate fatty acid determinations, the average of two other duplicates should agree within ho.36 milli-equivalent of fatty acid, or given one duplicate, the other can be estimated within ho.49 milli-equivalent. Given the average of a pair of duplicate lipoid phosphorus determinations, the average of another pair of duplicates should agree within f0.21 mg. per cent of lipoid phosphorus.
Results
Sodium Hydroxide Compared with Potassium Hydroxide in Saponification
In the saponification experiments there was much overlapping of the four fatty acid values obtained after hydrolysis of two sam- ples with sodium and two with potassium hydroxide. This over- lapping prohibits drawing any distinction between these two saponifying agents. Although in seven of the ten experiments the average of the two potassium hydroxide duplicates was higher than the average of the two sodium hydroxide duplicates, in three sodium hydroxide gave higher yields of fatty acids than potassium
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E. B. Man and E. I?. Gildea
hydroxide. With the exception of the crude lecithin all the lipids saponified in these experiments were not dried or exposed to the oxidizing influence of air between extraction or purification and saponification. It is not known whether the differences between these two saponifying agents may be minimized when the phos- phatides are saponified immediately after extraction or puri- fication.
E$ect of Rejihxing on Completeness of Extraction
The data in Table I show that for whole blood refluxing for 1 hour increases the yield of fatty acids by alcohol and ether extraction. This increase is apparent in samples which have been protected from oxidation by carbon dioxide and therefore is not dependent on decomposition of the fats or upon an increase in the number of carboxyl groups. Because the differences between the serum fatty acid values obtained by the Man and Gildea and the Bloor water cooling methods are not uniform, it would seem that some sera are more difficult to extract than others and that the hour of refluxing is advisable for those sera which are difhcult to extract.
Usually there is a greater yield of lipoid phosphorus after reflux- ing blood for 1 hour with alcohol and ether than after heating the alcohol, ether, and blood mixture for 1 to 5 minutes. Comparison of the values for lipoid phosphorus in the extracts which have been refluxed in air and carbon dioxide shows that in nine of the eleven experiments on whole blood higher yields of lipoid phosphorus were obtained in those samples which were refluxed in the presence of carbon dioxide. In the two experiments on blood serum the differences were less than 0.05 mg. per cent. In the one experi- ment, when whole blood was refluxed for 1 hour in air, in the presence of carbon dioxide and of nitrogen the three values agreed within 0.14 mg. per cent. The above results suggest that, when the somewhat unstable phosphatides in whole blood are protected by an inert gas, higher quantities of phospholipid may be obtained. Since the method of refluxing in air gave higher results than those without refluxing, the yields in the carbon dioxide-relluxed samples are appreciably larger than in the non-refluxed samples.
Statistical analysis of 100 consecutive samples of serum cho- lesterol showed that,. given the average of two duplicates, the average of another pair can be estimated within a probable error
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Blood Lipids
of f7.69 mg. per cent. It has been concluded that any of the above methods is equally good for the extraction of total choles- terol, because in a comparison of cholesterol yields of all different methods the mean value, regardless of sign, of the 98 differences was only 5.96 mg. per cent. Page, Kirk, Lewis, Thompson, and Van Slyke in two experiments on plasma found no significant differences between the amounts of total and free cholesterol in extracts made by the Bloor and the Man refluxing methods (18).
DISCUSSION
The saponification experiments showed no significant dif- ference between hydrolysis with potassium and sodium hydroxide. These results do not substantiate the saponification experiments originally published by Man and Gildea (12). Stewart and Hendry (19) have very justly crit,icized these original findings of ours.
In our original calculations (12) it was concluded that only 82 per cent of the fatty acids of phosphatides were saponified by our method, because we had not been able to recover more than approximately 80 per cent of the theoretical fatty acids saponified from a purified sample of lecithin. The proportion of fatty acids to be ascribed to phosphatides in blood or serum still remains obscure, because these phosphatides are not homogeneous but are composed of a variety of compounds. In view of this uncer- tainty it seems better to us not to attempt to correct titrated fatty acids for phosphatide fatty acids as we did previously or to try to calculate non-phosphatide fatty acids. Now the fatty acids are calculated directly from the titer and are called “Titrated fatty acids.” The last sentence before the summary in our original paper has, therefore, no significance. “The value ob- tained in Equation 1 + 18.0 per cent phospholipid fatty acids is considered to be equivalent to the total fatty acid content of the serum, as far as can be judged at the present time” (12). This step in the calculations has affected the fatty acid values by about 10 per cent or less in a number of our published papers. For- tunately, the omission of this step in calculation does not alter the relative values or conclusions reported in previous studies.’
1 To change to “Titrated fatty acids ” “Total fatty acids” in the follow- ing papers already published (7,8,13,14,17) multiply the lipoid phosphorus by 2/31 and subtract 18 per cent of this value from the “Total fatty acids.”
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E. B. Man and E. F. Gildea
It has been shown above that higher yields of fatty acids and of phosphatides are obtained from whole blood and to a lesser degree from serum when the alcohol-ether mixture is refluxed for 1 hour than when the Bloor or the Boyd red blood cell extraction method is used.
Boyd (5) questioned the refluxing modification because it required more time and apparatus. In his method the extracts were heated 5 minutes and, “When cool, the extracts were filtered” ((5) p. 225). It was found in our laboratory that 40 to 60 minutes were necessary for the extracts to reach room temperature when they were allowed to cool spontaneously. This air cooling method was the one apparently recommended by Bloor in 1922 and 1928 (3, 4) and must be contrasted with the quicker water cooling methods of 1914 and 1915 (1, 2). There is no appreciable dif- ference in time between refluxing the alcohol-ether mixture for 1 hour as recommended by Man and Gildea and between heating the extract for 5 minutes and then letting it cool 40 to 60 minutes before filtration.
While it is true that the refluxing of the alcohol-ether mixture does require condensers and some heating device, this initial expense for apparatus is offset in Boyd’s modification by the ex- pense of large amounts of solvent. To obtain the greatest extrac- tion of lipids from serum when the samples were not refluxed he recommended the use of at least twice as much solvent (5) as Man and Gildea had found necessary. He has discussed in detail the dilutions recommended by Bloor at different times. In addi- tion to the expense of these quantities of alcohol and ether the large volume in which the lipids are subsequently dissolved has its disadvantages. More time is required for the removal of the solvent from the lipids. In the case of the phosphatides which have been shown to be unstable and easily broken down (11) the additional heating and time for removal of the solvents may easily result in decomposition and a marked loss of phospholipids.
To change “Non-phospholipid fatty acids” in the following papers (13, 15-17) to “Titrated fatty acids” multiply the lipoid phosphorus by 2/31 and add 82 per cent of this value to the non-phospholipid fatty acids. In these calculations it was assumed that one combining weight of phosphorus, 31, unites with 2 of fatty acid in each gm. molecule of serum phosphatide, and that only 82 per cent of the phosphatide fatty acids was actually saponified and titrated.
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86 Blood Lipids
In his dilution experiments on serum he found that, “Phospholipid and free cholesterol were extracted almost as readily with the smaller volumes as with the larger, but with smaller volumes the extraction of neutral fat and cholesterol esters was much more incomplete” ((5) p. 225). But in the experiments on whole blood he stated that, “The phospholipid and free cholesterol only of these extracts were analyzed” ((5) p. 226). Since these two fat fractions were more easily extractable from serum, their use as criteria for the efficiency of extraction from whole blood can be criticized.
Boyd (5, 6) and Stewart and Hendry (19) have questioned in the Man and Gildea modification of the Stoddard and Drury titrimetric method the refluxing process, because it may cause an increase in titratable substances in the extract. Stewart and Hendry found in samples of blood refluxed 5 hours, five times as long as the samples are refluxed in the Man and Gildea modifica- tion, that there was an increase of 40 per cent in the titer of fatty acids. They do not state the temperature of their heating appa- ratus. They found that if the blood was refluxed in a %ontinuous current of purified hydrogen” this increase in fatty acids did not occur. For this reason in fifteen of our eighteen experiments refluxing was conducted in the presence of air and also in carbon dioxide. In seven of the fifteen experiments higher titers were obtained in the carbon dioxide samples and in eight experiments higher yields in the air specimens. In one experiment in which nitrogen was used instead of carbon dioxide and in another single experiment, when the alcohol-ether blood mixture was refluxed for 6 hours, the fatty acids all agreed within 0.43 milli-equivalent. These results show that the high yields of whole blood fatty acid obtained after refluxing for 1 hour, as recommended by Man and Gildea, represent a greater extraction of fatty acids and are not caused by oxidative decomposition of the fatty acid chains and an augmentation of carboxyl groups.
Boyd (6) has presented evidence to show that heating alcohol- ether extracts of red blood cells extracts colored material which contaminates the lipid material. “The colored material was found to be soluble in alcohol, ether, acetone, petroleum ether, methyl alcohol, chloroform, and dilute sodium hydroxide solution; it was insoluble in water and dilute hydrochloric or sulfuric acid” (p. 41).
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E. R. Man and E. F. Gildea 87
The fact that this substance is soluble in these lipid solvents makes it difficult to judge whether such a colored substance should be classed as a contaminant of lipids or as a lipid itself. However, the differences in Table I of 0.01 to 2.65 milli-equivalents in the fatty acids of whole blood by the Man and Gildea refluxing process and the Boyd hemolysis extraction method without heat do not approach the differences in fatty acid found by Boyd in extracts free from or contaminated with this colored substance. He found the neutral fat three times as high after 1 hour’s heating as without heat ((5) p. 228). Boyd states that, “When the colored mat,erial was dissolved in 0.1 N NaOH and this extract.ed with petroleum ether, the color remained in the aqueous alkaline medium and did not pass visibly into the petroleum ether” ((6) p. 41). This insolubility in petroleum ether is no proof that the “con- taminating substance” is non-lipid in nature, because petroleum ether does not extract quantitatively all lipids from an aqueous alkaline solution. Boyd himself has prescntcd evidence to shop that extraction of red blood cells without heating gave values fol total fatty acids below 240 mg. per cent, while there were 275 mg. per cent of fatty acids in the samples which had been both heated 15 to 60 minutes and treated for the removal of the colored “con- taminating substance” ((6) Fig. 2). If a short period of heating can result in 13 per cent higher yields of fatty acid, this increase in determined fatty acids after 60 minutes of heating cannot be attributed entirely to contaminating substances. With the pres- ent definition of lipids, it seems to the authors that the additional titration value for fatty acids in the Man and Gildea refluxed samples above that in samples not heated or heated for a short time may represent a more complete extraction of lipid material from whole blood.
SUMMARY
In ten experiments in which lipid extracts were saponified simul- taneously with sodium and potassium hydroxides no significant differences in fatty acids were found.
Eighteen experiments were conducted in which serum or blood was extracted by the Bloor method, by the Boyd modifications, and by refluxing for 1 hour in the presence of air and carbon dioxide. Data have been presented to prove that the Man and
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Blood Lipids
Gildea refluxing process extracts more material which by present definition is classed as lipid in nature than the Boyd modifications of the Bloor extraction method.
When whole blood was used, the refluxing process gave yields of fatty acid 0.01 to 2.65 milli-equivalents higher than did the other methods except for one analysis. Serum gave slightly larger amounts of fatty acid after the refluxing process, the average difference being 0.36 milli-equivalent. Samples refluxed in air and in carbon dioxide yielded identical amounts of fatty acids.
Phosphatides of whole blood were highest in the aliquots re- fluxed for 1 hour in the presence of carbon dioxide and exceeded the samples refluxed in air by an average deviation of 0.26 mg. per cent of lipoid phosphorus. The air refluxing method gave higher yields than the other extraction methods by average varia- tions of 0.09 to 0.41 mg. per cent of lipoid phosphorus.
Cholesterol determinations in all extracts agreed satisfactorily.
BIBLIOGRAPHY
1. Bloor, W. It., J. Biol. Chem., 17,377 (1914j. 2. Bloor, W. R., J. Biol. Chem., 22, 133 (1915). 3. Bloor, W. R., J. Biol. Chem., 77, 53 (1928). 4. Bloor, W. R., Pelkan, K. F., and Allen, D. M., J. Biol. Chem., 62, 191
(1922). 5. Boyd, E. M., J. Biol. Chem., 114, 223 (1936). 6. Boyd, E. M., J. Biol. Chem., 116,37 (1936). 7. Gildea, E. F., Kahn, E., and Man, E. B., Am. J. Psychiat., 92, 1247
(1936). 8. Gildea, E. F., and Man, E. B., J. Clin. Znv., 16, 295 (1936). 9. King, H. J., J. Chem. Sot., 738 (1929).
10. MacArdle, D. W., The use of solvents in synthetic organic chemistry, New York (1925).
11. Man, E. B., J. Bid. Chem., 117, 183 (1937). 12. Man, E. B., and Gildea, E. F., J. Biol. Chem., 99,43 (193233). 13. Man, E. B., and Gildea, E. F., J. Clin. Znv., 16, 203 (1936). 14. Man, E. B., and Peters, J. P., J. Biol. Chem., 101, 685 (1933). 15. Peters, J. P., and Man, E. B., J. Biol. Chem., 107,23 (1934). 16. Ptlan, E. B., and Peters, J. P., J. Clin. Inv., 13, 237 (1934). 17. Man, E. B., and Peters, J. P., J. Clin. Znv., 14, 579 (1935). 18. Page, I. H., Kirk, E., Lewis, W. H., Jr., Thompson, W. R., and Van
Slyke, D. D., J. Biol. Chem., 111, 613 (1935). 19. Stewart, C. P., and Hendry, E. B., Biochem. J., 29, 1677 (1935). 20. Stewart, C. P., and Hendry, E. B., Biochem. J., 29, 1683 (1935).
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Evelyn B. Man and Edwin F. GildeaBLOOD AND BLOOD SERUM
SAPONIFICATION OF LIPIDS FROM NOTES ON THE EXTRACTION AND
1937, 122:77-88.J. Biol. Chem.
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