Direct Transesterification of All Classes of Lipids in a One-Step Reaction

Abstract

Conventional techniques for the determination of fatty acid composition of lipids require solvent extraction, purification, hydrolysis, and derivatization procedures that are both lengthy and cumbersome. A 1-hr direct transesterification procedure carried out in methanol-benzene 4:1 with acetyl chloride circumvented all these steps and was applicable for analysis of both simple (triglycerides) and complex lipids (cholesteryl esters, phospholipids, and sphingomyelin). Recoveries (greater than 95%) of standards unaffected by the presence of 5% water and 200 mg of silica suggested that the technique could be used for the quantitative analysis of total fatty acids as well as of fatty acids in classes of lipids separated on silica from biological samples. When compared to the Folch procedure, the technique led to a 20.1% increase in total fatty acids for plasma, 3.9% for feces, 7.4% for bile, and 9.7% for rat liver. We therefore conclude that this one-step direct transesterification procedure is superior to currently used methods, not only because of its simplicity and speed, but also because of its added precision.

Full text

I
notes
on
methodology
Direct transesterification of
all
classes of
lipids
in
a one-step reaction
Guy Lepage' and Claude
C.
Roy
Pediatric Gastroenterology Unit, Centre de Recherche
Pe'diatriqua, Hdpital &-Justine, De'partement
de
Ediatrie, Universite'
de
Montre'al, Montrkal,
Qwbec,
Canada
Summary
Conventional techniques for the determination
of
fatty acid composition of lipids require solvent extraction, purifi-
cation, hydrolysis, and derivatization procedures that are both
lengthy and cumbersome.
A
1-hr
direct transesterification proce-
dure carried out in methanol-benzene 4:l with acetyl chloride
circumvented all these steps and was applicable for analysis of
both simple (triglycerides) and complex lipids (cholesteryl esters,
phospholipids; and sphingomyelin). Recoveries
(
>
95
%)
of
standards unaffected by the presence of
5%
water and 200 mg
of silica suggested that the technique could be used for the
quantitative analysis
of
total fatty acids as well as of fatty acids
in classes of lipids separated on silica from biological samples.
When compared to the Folch procedure, the technique led to a
20.1% increase in total fatty acids
for
plasma, 3.9% for feces,
7.4%
for bile, and 9.7% for rat liver.lWe therefore conclude
that this one-step direct transesterification procedure
is
superior
to currently used methods, not only because of
its
simplicity and
speed, but also because of
its
added precision.
-Lepage,
G.,
and
C.
C.
Roy.
Direct transesterification of all classes of lipids
in a one-step reaction.
J.
Lipid
Res.
1986.
27:
114-120.
Supplementary key
words
fatty acid triglyceride cholesteryl
ester phospholipid sphingomyelin gas-liquid chromatography
methylation
Fatty acid
(FA)
analysis of biological specimens
by
gas-
liquid chromatography
(GLC)
requires solvent extraction,
purification, and derivatization procedures that are both
lengthy and cumbersome (1-3). Attempts to bypass ex-
traction (4, 5) and purification steps (3,
6,
7)
have met
with varying degrees of success. This report proposes a
technique that circumvents most of the preparative steps
Abbreviations: GLC, gas-liquid chromatography;
TG,
triglyceride;
CE,
cholesteryl ester; PL, phospholipid; SP, sphingomyelin; FA, fatty
acid.
'Address
for
reprint requests:
Dr.
Guy Lepage, Pediatric Research
Center, HBpital Ste-Justine,
3175
Ste-Catherine Road, Montreal,
Quebec, Canada H3T 1'25.
and consists of a one-step reaction. It leads to more com-
plete recoveries of all classes of lipids which, during the
transesterification procedure, are freed from biological
specimens.
MATERIALS AND METHODS
Analytical grade solvents were redistilled in an all-glass
system. All glassware was rinsed with chloroform-metha-
no1
2:l
(v/v)
and dried under nitrogen. Acetyl chloride
(Fisher Scientific Ltd., Montreal, Quebec) was used with-
out further purification. Borosilicate glass tubes with
Teflon-lined screw-caps (100
x
13 mm) and magnetic stir-
ring bars (10
x
3
mm) were bought from Fisher Scientific
Ltd.,
Montreal, Quebec. Transesterification was carried
out in a Reacti-Therm heatinghtirring dry block which
controls temperature
f
0.5OC (Pierce Chemical Co.,
Rockford,
IL).
Fatty acid and fatty acid methyl ester standards (Ana-
labs, North Haven, CT; Terochem, Rexdale, Ontario;
Sigma, St. Louis,
MO;
and Mandel, Montreal, Quebec)
as well as cholesteryl ester
(CE),
phosphatidylcholine, and
chicken egg sphingomyelin
(SP)
standards (Sigma) were
certified to be
>
99%
pure. Unsaturated lipid standards
were bought packaged in ampoules under an inert gas to
prevent oxidation.
Biological specimens that were analyzed (Table 4) con-
sisted of plasma from a normal adult, fecal homogenate
from a human milk-fed premature infant, bile from an
adolescent with cerebrotendinous xanthomatosis, and rat
liver homogenate.
Recoveries
of
standards as a function
of
time,
the presence
of
silica, and added water
CE,
PL,
and SP standards were weighed and diluted
with methanol-benzene 4:l (v/v). After being mixed
thoroughly, the mixtures of three standards were placed
into individual reaction vials. These vials were submitted
to the direct transesterification procedure described below
for 1 hr, 4 hr,
or
16
hr.
To test whether silica could inter-
fere with the reaction,
200
mg of silica was added to the
mixture of standards. The liquid phase of this mixture
was brought to a total volume of
2
ml with methanol-
benzene 4:l (+) and transesterification was carried out
over periods of
1
hr to 16 hr. In order to check whether
biological samples could be processed without prior ex-
traction, the amount of water that could be added to
standards without interfering with the direct transesterifi-
cation procedure was tested in the following manner. One
hundred pl, 200 pl, or 300 pl of water was added to stan-
dards, the solution was then mixed for
1
min, and the
volumes were adjusted to
2
ml with methanol-benzene 4:l
(vh) for the transesterification reaction. The samples
114
Journal of
Lipid
Research
Volume 27, 1986
Notes
on
Methodology
by guest, on July 11, 2011www.jlr.orgDownloaded from

prepared in this manner contained
576,
lo%,
or
15%
of
water, respectively. From the molecular weights of
CE
and
PL
standards and of their constituent FA, the theoretical
100%
yield of fatty acid was calculated for each lipid stan-
dard. However, for the sphingomyelin solution, results
obtained after
16
hr of transesterification with and with-
out
5%
water were taken as
100%.
Direct transesterification method
One hundred
jd
of
either plasma or bile
or
100
mg of
either fecal or liver homogenate was precisely weighed in
glass tubes. As shown in
Fig.
1,
an internal standard
consisting of
50
pg to
300
pg of tridecanoic acid
(C13:0),
dissolved in
2
ml of methanol-benzene
4:l
(v/v) was pre-
cisely weighed and added to the biological samples. A
small magnetic stirring bar was added to each tube and,
while stirring,
200
pl
of acetyl chloride was slowly added
over a period of
1
min. Tubes were tightly closed with
Teflon-lined caps and subjected to methanolysis at
100°C
for
1
hr
(8).
Tubes were weighed before and after heating
as a check for leakage. After tubes had been cooled in
water,
5
ml
of
6% K2C03
solution was slowly added to
stop the reaction and neutralize the mixture. The tubes
were then shaken and centrifuged, and an aliquot of the
benzene upper phase was injected into the chromatograph.
Extraction
of
biological samples by.
the Folch method
(9,
10)
Biological specimens were processed as described earlier
(11).
After transesterification the pooled solvent extracts
were dried under a gentle stream of nitrogen at room
temperature. Residues were dissolved in 400
pl
of
hexane
containing
50-300
pg of methylated tridecanoic acid used
as an external standard. An aliquot was injected into the
chromatograph.
Add
0.2
ml
To
the aliquot,
add
a weighed
LJ
Weigh
0.1
ml
Aliquo
t
2
lal.
volume
of
CH3COC1
CH30H-C6H6(4:1)
with
Int. Std
Seal
Mix
U
Centrifuge
Add
5
ml
K2CO3 6%
Seal
&
Heat
At
100OC for
60
min
Injection
Phase
in
GLC
-
of
upper
Fig.
1.
Schematic diagram
of
the procedure
for
a biological sample.
Gas-liquid chromatography
FA were chromatographed as methyl esters on
a
30-m
fused silica column with an internal diameter of
0.32
mm
(Fig.
2).
The column was wall-coated with
0.20
mm
SP-2330.
Analysis was performed on a Hewlett-Packard
5880
gas chromatograph equipped with a flame ionization
detector. Helium was used as carrier gas and nitrogen as
make-up gas. The split ratio was
17:l.
The injection port
temperature was
2OOOC
and the detector was
250OC.
The
column temperature was held at
8OoC
for
5
min and in
a step-wise fashion reached
a
plateau of
220OC.
The gas
chromatograph was calibrated using a standard mixture
of FA.
A
correction factor was applied to compensate for
the lower ionization detector response to unsaturated FA
relative to corresponding weights of saturated FA.
Calculations and validation
of
the method
In order
to
verify the purity and validate the exact
concentration of both internal and external standards,
they were alternately added to
a
known quantity of penta-
decanoic acid
(C15:O)
which invariably yielded the same
concentration on the chromatograph. Tridecanoic acid
proved to be a reliable standard for biological samples
since it invariably gave rise to a well-identified peak. It did
not interfere with the FA patterns of plasma, bile, feces,
and liver since they were shown to be free of endogenous
tridecanoic acid.
RESULTS
Table
1
shows that a 1-hr period of transesterification at
100°C
led to excellent recoveries
of
CE
standards. Silica
added to the standards neither interfered with the hy-
drolysis step nor with the esterification of the nascent FA.
Addition of
100
pl of water to the standards had no effect
on the completeness of the hydrolysis and methylation
reactions achieved by the transesterification procedure
carried out over
a
1-hr period. However, when the solu-
tions of
CE
standards were reconstituted with
10%
or
15%
of water there was impairment of the reactions.
CE
transesterification led to the appearance of three
very
polar peaks at the end of the chromatograms. Their sur-
face area was directly related to the degree of hydrolysis
that occurred in the absence or presence of water. After
injection of methylated cholesteryl formate standard,
it
was concluded that these three peaks originated from the
cleavage of cholesterol formate and FA. Attention should
be drawn to the fact that one peak coeluted with the
methyl ester of cerotic acid
(C26:O)
on the
SP-2330
fused
silica column.
Results with
PL
were similar
to
those obtained with
CE
in that transesterification was complete after
1
hr, and
Journal
of
Lipid
Research
Volume
27,
1986
NotCS
on
Methodology
115
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r-
----A-
Fig.
2.
milk-fed preterm neonate.
Gas-liquid chromatography profile of fatty acid methyl esters (650 pg-250 ng of each) obtained from the fecal homogenate
of
a human
silica did not seem to have an adverse effect on the reac-
tion
(Table
2).
However, in contrast to CE, neither
10%
nor
15%
water added to
PL
standards affected the per-
centage recovery of nascent methyl ester
FA
obtained after
1
hr.
In contrast to the results with solutions of CE and
PL
standards, hydrolysis of the
SP
solution was time-depen-
dent in the sense that a transesterification reaction over
a
4-hr period was advantageous
('Ifible
3).
Furthermore,
the presence
of
silica affected the rate of the reaction to a
significant extent. However, addition of
100
pl of water
(5%)
to
the
SP
solution transesterified over a 1-hr period,
even in the presence of silica, led to excellent recoveries
of
the nascent methyl ester
FA.
When the percentage of
water was increased to
10%
and
15%,
less satisfactory
results were obtained.
Table
4
compares individual fatty acids recovered from
biological samples processed with the classical procedure
TABLE
1.
The effect
of
time, silica and added water on direct
transesterification of cholesteryl ester standards"
NO
Added
HzO
Added
HzO
1
hr
with
Fatty
Acid
1
hr
4
hr
Silica
5%
10%
15%
8:O 100.3
100.2
99.7 98.0 89.6 63.5
12:o 99.6
99.8 99.6 97.4 70.8 34.9
14:O 100.1 99.8
99.9 97.2
64.5 27.3
16:O 100.3 100.4
100.1
97.6
59.7 24.7
16:l (n-7) 99.4 99.3 99.2
95.9 63.7 24.2
17:O 99.6
99.6 99.5 96.7 53.8 18.5
18:O 99.5 99.9
99.6 97.1
50.0
15.5
18:l (n-9) 98.8 99.2 98.8
95.6 54.1
19.4
18:2 (n-6) 99.2
99.6 99.2 95.7 58.2 22.5
18:3 (n-3) 98.0
98.5 98.3
94.6 61.3 25.5
20:4 (n-6) 95.7 96.9 95.2
92.4 53.4
20.9
"Values are expressed as
%
recovery and represent means of two
'One
hr
transesterification carried
out
with added
H20.
samples.
116
Journal
of
Lipid
Rerearch
Volume
27,
1986
Nota
on
Methodolqy
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TABLE
2.
The effect
of
time, silica and added water on direct
transesterification
of
phospholipid standards'
~~
Fattv Acid
12:o
14:O
16:O
17:O
18:O
18:l
(n-9)
18:2
(n-6)
20:o
~ ~~
No
Added
Hz0
1
hr
with
1
hr
4
hr Silica
100.6 100.1
100.1
99.1
99.4 97.9
98.7 99.8
96.8
98.5 99.8
96.6
98.0 99.6 96.2
98.4 99.8
96.0
99.1 100.5 96.7
96.4 98.1
94.5
Added
HzOb
5%
100.6
98.3
97.8
97.7
97.2
97.6
98.2
95.9
10%
99.5
97.7
96.5
96.1
95.4
96.5
96.7
94.2
-
15%
98.4
96.0
95.5
95.3
94.9
95.1
96.0
93.2
"Values are expressed as
%
recovery and represent means
of
two
'One hr transesterification carried out with added
H20.
samples.
of Folch, Lees, and Sloane Stanley (9) and the direct
transesterification method. Aliquots of plasma, feces, bile,
and liver were added with internal standard dissolved in
methanol-benzene
4:l
(v/v) to achieve a final water con-
centration of less than 5%. The comparative FA content
was higher with the direct transesterification technique
for all specimens. The percentage increase of total FA
recovered was
20.1%
for plasma, 3.9% for feces,
7.4%
for
bile, and 9.7%
for
liver.
The stability of the methyl ester FA in the benzene
upper phase was excellent.
A
weekly injection of the same
sample stored at 4OC gave identical results after 3 months.
Losses of unsaturated FA by oxidative processes, during
the direct transesterification reaction or following the
3-month period of storage were never observed.
DISCUSSION
As we pointed out recently for triglycerides
(ll),
this
one-step reaction is rapid and reproducible. During the
initial phase of our study we tried to transesterify CE, PL,
and
SP
standards with
100
pl
of acetyl chloride using the
same technique as with FA and
TG
standards. However,
poor results were obtained especially for SP which under-
went only
*
55% hydrolysis. This was not surprising in
view
of
the observations of MacGee and Williams
(12)
that sphingolipids are extremely resistant to alkaline treat-
ment. Sphingolipid fatty acids exist as amides rather than
esters, and sphingolipids undergo acid hydrolysis
or
acid
methanolysis very slowly. After testing up to 350 pl of
acetyl chloride,
200
pl was found to be sufficient to hy-
drolyze and methylate all lipid classes.
To
our surprise
it
was noted that transesterification of SP was helped by
the presence of 5% water. This may very well be due to
the fact that SP are amphipathic (13). From our experi-
ence with the measurement of FA content of
SP
in both
standards and biological specimens, it may be concluded
that the technique described is reliable without extraction
and requires only
1
hr as opposed to 1.5 to
2
hr for the
hydrolytic phase alone, once extraction of SP from tissues
has been carried out
(12,
14).
Because recoveries
of
linoleic, homo-gamma linolenic,
and arachidonic acids are temperature-dependent, we
tried working at
a
lower temperature as recommended by
Haan, Van Der Heide, and Wolthers (3). This failed as
only 65% hydrolysis took place when the reaction was
carried out at 7OoC for
2
hr. Moreover, the ratio of poly-
unsaturatedhaturated FA did not improve over that ob-
tained at 100OC. It was then concluded that loss of the
polyunsaturated FA at high temperatures is associated
with saponification procedures rather than acid hydrolysis.
Different proportions of methanol-benzene were also
studied.
It
appeared that larger amounts of benzene did
not help the transesterification. In fact, decreasing the
percentage of benzene from
20%
to
10%
led to recoveries
of only
20%
for palmitoleic and arachidonic, 26% for
linoleic, and 30% for oleic.
No
change was observed in the
recoveries of saturated FA. The use of
400
pl of benzene
present in the
2
ml of benzene-methanol
(4:l)
supplied
a
sufficiently concentrated upper phase extractant after
transesterification to allow direct injection into the chro-
matographic unit, circumventing thereby the need for a
time-consuming and ineffiicient evaporation step.
In view of the consistent recoveries (96-100%) of the
three internal standards used in the report recently pub-
lished
(ll),
we elected to use only one standard, namely
tridecanoic acid, which efficiently monitors the complete-
ness of the extraction of methyl ester FA into the benzene
phase after transesterification. Pentadecanoic (15) and
heptadecanoic acids (16) have also been used but trideca-
noic acid was selected because it was the only one totally
absent from biological samples studied.
A
further advan-
tage is that it forms a well-individualized peak in a rela-
tively uncrowded part of the fatty acid methyl ester
chromatographic run.
The addition
of
5 ml of potassium bicarbonate should
be done slowly because
of
the quick liberation of COn
TABLE
3.
The effect
of
time, silica and added water on direct
transesterification
of
sphingomyelin standard'
No
Added
H20
Added
HzO
1
hr
5%
with
with
Fatty
Acid
1
hr
4
hr
Silica
5%
10%
15%
Silica
14:O 90.5
94.6 87.8
97.3 89.2 91.9 98.6
16:O 92.7
97.8
85.4 97.9
91.2
92.7 98.1
18:O 94.1
98.4 87.5
99.2 88.8 94.6 95.7
18:l
(n-9)
92.9
100.0 92.9
103.6 89.3 85.7 100.0
"Values are expressed as
%
recovery and represent means
of
two
'One hr transesterification in the presence
of
water with or without
samples.
silica.
Journal
of
Lipid
Research
Volume
27,
1986
Noh
on
Mtthodology
117
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TABLE
4.
Comparative fatty acid content of biological specimens using the classical technique
of
Folch et al. and the direct transesterification method“
Plasma Feces Bile Liver
Folch et al. Direct Folch et
al.
Direct Folch et al. Direct Folch et al. Direct
Fatty Acid Extraction Transesterification’ Extraction Transesterification Extraction Transesterification Extraction Transesterification
12:o
14:O
14:l (n-5)
15:O
16:O
16:l (n-7)
17:O
18:O
18:l (n-9)
18:2 (n-6)
18:3 (n-3)
20:o
20:l (n-9)
20:2 (n-6)
20:3 (n-6)
20:4 (n-6)
22:o
22:l (n-9)
22:4 (n-6)
22:5 (n-6)
22:6 (n-3)
24:O
24:l (n-9)
26:O
444
f
1.7 522
f
2.5
25
f
0.1 31
i
0.5
158
f
0.6 186
f
0.1
374
f
11.5 444
i
11.9
831
i
4.1 968
f
3.8
33
f
0.4 41
f
0.5
165
i
0.8 201
f
1.1
trace 20
f
0.2
trace 25
i
0.4
mg/72
hr
Ps/ml
32
*
0.3 40
f
0.3
4
*
0.1
4
f
0.1
148
f
1.1 160
i
0.5 59
f
1.8 66
i
0.5
19
f
0.2
20
f
0.1 44
f
0.9 48
f
0.2
993
f
7.7
1035
f
7.5 3064
f
10.5 3259
f
18.6
53
*
0.4 56
f
0.4 226
i
1.7 243
f
2.2
28
f
0.3
29
f
0.2 26
f.
0.2
28
f
0.1
709
f
7.2 733
i
5.8
317
f
1.0 338
i
2.4
1390
f
15.0
1432
f
10.4 1087
i
2.7
1153
f
9.8
307
i
4.4 313
i
2.8 1897
f
6.2 2046
f
15.4
14
i
0.2 15
f
0.1 40
i
0.2 43
f
0.3
14
f
0.2
15
f
0.1
23
f
0.5 24
i
0.2
20
i
0.5 20
f
0.1
13
i
0.2 13
f
0.1 198
f
0.9 217
f
1.9
17
i
0.2 19
f
0.2 720
f
3.5 788
i
6.1
7
f
0.1
8
i
0.1
4
f
0.1
4
f
0.1
6
f
0.1
6
i
0.1
5
f
0.1
6
f
0.2 104
f
0.4 118
f
0.9
8
+
0.1 10
f
0.1
6
*
0.2 6
i
0.1
12
f
1.2 15
f
0.5 74
i
3.3 89
i
4.3
trace 15
f
0.1
1522
f
11.5 1668
f
10.2
38
i
0.3 41
f
0.3
45
i
0.5 49
i
0.4
2059
f
18.1 2216
f
15.3
541
i
7.5 576
i
4.6
1601
f
14.7 1718
-t
12.5
102
i
1.8 112
f
0.8
1878
f
19.3 2042
f
15.4
trace 22
i
0.4
21
f
0.2 23
i
0.2
95
i
1.0 105
f
1.0
883
i
9.8 970
f
7.9
42
f.
1.3 76
f
0.5
15
i
0.1
27
f
0.5
56
f
3.3
77
f
2.8
Total 2051
f 15.0 2463
i
20.1 3810
f
37.9 3957
f
28.4 7856
f
21.6 8436
i
55.4 8871
f
97.0 9730
f
64.9
“Mean
f
SE
of
five samples
from
the same specimens.
’See text
for
details.
bubbling the mixture. Addition of this strong base is
necessary to bring the pH back to neutral. This is an im-
portant step in the procedure because, after transesterifi-
cation with
200
pl of acetyl chloride, the pH of the solu-
tion is less than 1 and injection of this acidic benzene
supernatant can break down the very thin stationary
phase coating the capillary column which is thereby
rendered useless. Rogiers (17) and Christie (18) have
reported the same problem.
The experiments carried out with silica were important
in validating the usefulness of the method for the analysis
of various lipid classes after their separation on silica.
Following the thin-layer chromatographic separation of
lipid classes on silica of Bitman, Wood, and Ruth (19),
bands can be scraped from the plate and added directly
to the
2
ml of transesterification mixture with excellent
recoveries. In fact, we found that up to
200
mg of silica
can be used without concern that it will in any way inter-
fere with the direct transesterification method.
In an attempt to improve recoveries of methyl ester FA
migrating in the benzene upper phase after transesterifi-
cation, solvents such as pentane, hexane, or isooctane
were added but were not beneficial. Chromatographic
profiles on
30
M
SP-2330
capillary columns were remark-
ably good. Since the glassware was thoroughly cleaned
and redistilled solvents were used, no artefact peaks were
seen on blank runs and solvent peaks were narrow with-
out trailing. Furthermore, baseline resolution was ob-
tained with all fatty acids including the polyunsaturated
fatty acids which on some columns are reported to coelute
(20).
After processing hundreds of standard mix-
tures and specimens and injecting them into the gas
chromatograph, no alteration
of
the chromatographic
column has
so
far been observed.
The comparative FA content
of
biological specimens
was higher with the direct transesterification technique
than that obtained using the classical technique of Folch
et al.
(9).
The percentage increases of total FA in plasma,
feces, bile, and liver were
20.196,
3.996, 7.496, and
9.796,
respectively. The physicochemical properties of classes of
lipids and their varying distributions in biological speci-
mens tested most likely explain the fact that the degree of
discrepancy between the two methods is not the same for
all samples.
The Folch extraction takes advantage of the low solu-
bility of lipids and of their preference for water-immiscible
organic solvents, conferred by the long side chains of FA.
Since membrane and plasma lipids are normally associ-
ated with proteins, Folch et al.
(9)
used chloroform-
methanol
2:l
(./.). Because of its water solubility and
118
Journal
of
Lipid
Research
Volume
27,
1986
Notes
on
Meihodology
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hydrogen-bonding ability it induces splitting of the lipid-
protein complex and even in some cases denatures the
protein.
There was little advantage
(+
3.9%)
in the direct trans-
esterification method over the Folch extraction in the FA
recovery from feces. This is likely due to the fact that
84.5%
of the lipids were in the form of free fatty acids and
the rest was almost entirely made up of
TG
that are easily
extracted from
a
water solution with a nonpolar solvent
such as the one used by Folch et al.
(9).
Fasting plasma
lipids are mostly made up of CE and PL. As water is
strongly hydrogen bonded to protein and to polar lipids
in lipoproteins, covalently bound lipids must be subjected
to a hydrolysis procedure before they can be extracted
completely with organic solvents of any polarity
(21).
We
feel that these bound lipids likely account for the higher
discrepancy
(+
20.1%)
found between the two methods
for plasma. In terms of physical properties, PL “bridge the
gap” between the completely water-insoluble neutral
lipids and molecules that form true aqueous solutions.
The amphipathic character of PL makes them more easily
extractable than CE
(13).
PL are the major lipid constitu-
ents of bile and liver
(22).
Bolton et al.
(23)
encountered
serious problems in the lipid analysis of bile after Folch
extraction. They proposed that biliary PL were subject to
some error because water-soluble PL such as lysolecithin
were removed by aqueous washes
(23).
A
7.4%
discrepancy
for bile between the two methods therefore seems reason-
able. Lipids bound to membranes should be taken into
account for the higher
(9.7%)
difference found for liver
homogenate. From the above discussion, it may be con-
cluded that the more complete recovery of FA from com-
plex lipids is due to the fact that they were directly and
completely freed from biological specimens during the
transesterification procedure.
The determination of FA composition is conventionally
done by GLC. It requires solvent extraction, purification,
hydrolysis, and derivatization procedures that are both
lengthy and cumbersome
(1-3).
The proposed method
provides a total FA profile after a one-step reaction with-
out losses and in less time than that required for a gravi-
metric determination
(24).
Furthermore, the FA pattern
of various lipid classes may be easily obtained following
TLC and direct transesterification of the silica scrapings
since silica does not interfere with the reaction.
Hundreds of biological specimens have now been run
in duplicate by the direct transesterification method. The
coefficient of variation for the different FA varied from
0.4
to
2.0%
for several samples that were processed at least
five times each. The proposed technique gave better re-
sults than the
3%
to
11.8%
coefficient of variation re-
ported
by
MacGee and Williams
(12).
Although simple
and rapid, it is also more precise than currently used tech-
niques because it bypasses extraction and purification
steps and also calls for the addition of an internal stan-
dard at the beginning. However, good reproducibility can
only
be
expected if both the specimen to be analyzed and
the internal standard are carefully weighed.
In conclusion, the direct transesterification technique
described recently for FA and
TG
(11)
has been modified
and is now applicable to both simple and complex lipids.
Because of its simplicity, speed, and added precision, it
has proved to be
very
useful in our hands and should at-
tract the attention of lipidologists.
I
This study was supported by Grant MT 4433 of the Medical
Research Council of Canada and by the Canadian Cystic Fi-
brosis Foundation.
Manucnipt
received
12
Jub
1985.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Thomas, L.
H.,
P. R. Jones,
J.
A.
Winter, and
H.
Smith.
1981. Hydrogenated oils and fats: the presence of chemically-
modified fatty acids in human adipose tissue.
Am.
J.
Clin.
Wolff,
J.
A.,
S.
Margolis, K. Bujdoso-Wolff, E. Matusik,
and W. C. MacLean,
J.
1984. Plasma and red blood cell
fatty acid composition in children with protein-calorie
malnutrition.
Pcdiatr.
Rcs.
18:
162-167.
Haan, G. J.,
S.
Van Der Heide, and
B.
G. Wolthers. 1979.
Analysis of fatty acids from human lipids by gas chroma-
tography.
J.
Chmmatogz
162:
261-271.
MacGee, J., and
K.
G. Allen. 1974. Preparation of methyl
esters from the saponifiable fatty acids in small biological
specimens
for
gas-liquid chromatographic analysis.
J.
Shimasaki,
H.,
F. C. Phillips, and
0.
S.
Privett. 1977.
Direct transesterification of lipids in mammalian tissue for
fatty acid analysis via dehydration with
2,2
-dimethoxypro-
pane.
J.
Lipid
Rcs.
18:
540-543.
Gerhardt, K.
O.,
and C. W. Gehrke. 1977. Rapid micro-
determination of fatty acids in biological materials by gas-
liquid chromatography.
J.
Chmmatogz
143:
335-344.
Penttila,
I.,
A. Huhtikangas,
J.
Herranen,
S.
Eskelinen,
and
0.
Moilanen. 1984. Simultaneous measurement of free
and esterified fatty acids by gas chromatography from
normal and type
IV
hyperlipoproteinaemic sera.
Ann. Clin.
Lillington,
J.
M.,
D. J. H. Trafford, and
H.
L.
J.
Makin.
1981. A rapid and simple method for the esterification of
fatty acids and steroid carboxylic acids prior to gas-liquid
chromatography.
Clin. Chim.
Acta.
111:
91-98.
Folch,
J.,
M. Lees, and G. H. Sloane Stanley. 1957.
A
simple method for the isolation and purification of total
lipids from animal tissues.
J.
Biol. Ch.
226:
497-509.
Klein, R.
A.,
D. Halliday, and P. G. Pittet. 1980. The use
of 13-methyl-tetradecanoic acid
as
an indicator of adipose
tissue turnover.
Lipidr.
15:
572-579.
Lepage, G., and C. C. Roy. 1984. Improved recovery of
fatty acid through direct transesterification without prior
extraction
or
purification.
J.
Lipid
Res.
25:
1391-1396.
MacGee,
J.,
and M. G. Williams.
1981. Preparation of
sphingolipid fatty acid methyl esters for determination by
gas-liquid chromatography.
J.
Chmmtop.
205:
281-288.
NUX
34:
877-886.
Chmmtogr:
100:
35-42.
Res.
16:
13-17.
Journal
of
Lipid
Research
Volume
27,
1986
Notes
on
Methodoloo
119
by guest, on July 11, 2011www.jlr.orgDownloaded from

13.
Gurr, M.
I.,
and A.
T.
James.
1975.
Lipid Biochemistry: an
Introduction. 2nd ed. Chapman and Hall, London.
138.
14.
Morrison,
W.
R.,
and
L.
M.
Smith.
1964.
Preparation of
fatty acid methyl esters and dimethylacetals from lipids
with boron fluoride-methanol.
J.
Lipid
Rex.
5:
600-608.
15.
Ohlrogge,
J.
B., E.
A.
Emken, and
R.
M.
Gulley.
1981.
Human tissue lipids: occurrence of fatty acid isomers from
dietary hydrogenated oils.
J.
Lipid
Res.
22:
955-960.
16.
Goetz,
N.,
G. Kaba, and
P.
Bort.
1982.
Capillary gas
chromatographic analysis of human skin surface lipids after
microsampling on ground-glass platelets.
J.
Chromutogr
17.
Rogiers, V.
1977.
The application of an improved gas-
liquid chromatographic method for the determination of
the long chain nonesterified fatty acid pattern of blood
plasma in children.
Clin. Chim.
Acta.
78:
227-233.
18.
Christie, W.
W.
1982.
A
simple procedure for rapid trans-
methylation of glycerolipids and cholesteryl esters.
J.
Lipid
19.
Bitman,
J.,
D.
L.
Wood, and
J.
M.
Ruth.
1981.
Two-stage,
one-dimensional thin-layer chromatographic method for
separation of lipid classes.
J.
Liq. Chromutogr
4:
1007-1021.
233:
19-28.
Res.
23:
1072-1075.
20.
Putnam,
J.
C.,
S.
E.
Carlson, and P. W. De Voe.
1982.
The
effect of variations in dietary fatty acids on the fatty acid
composition of erythrocyte phosphatidylcholine and phos-
phatidylethanolamine in human infants.
Am.
J.
Clin.
Nutr
Nelson, G. J.
1975.
Isolation and purification of lipids from
animal tissues.
In
Analysis of Lipids and Lipoproteins.
E. G. Perkins, editor. The American Oil Chemists’ Society,
Champaign,
IL.
1-22.
22.
Yunker,
R.
L.,
A.
S.
Hassan, and M. T.
R.
Subbiah.
1981.
Simultaneous quantitation of biliary cholesterol, bile acids,
and phospholipids (as fatty acids), by gas-liquid chromatog-
raphy, with campesterol as internal standard.
Clin. Chem.
23.
Bolton, C. H.,
T.
S.
Low-Beer, E. W. Pomare, A. C. B.
Wicks, J. Yeates, and K. W. Heaton.
1978.
A simplified
procedure for the analysis of cholesterol, phospholipids and
bile salts in human bile.
Clin. Chim.
Acta.
83:
177-181.
24.
Esteves,
W.,
L.
A.
S.
Soares, L. Hartman, and
A.
S.
Pereira.
1982.
Determination of lipids, including medium-
chain fatty acids, in human feces.
Clin. Chem.
28:
603-605.
36:
106-114.
21.
27:
1779-1780.
120
Journal
of
Lipid
Research
Volume
27, 1986
Nota
on
Methodology
by guest, on July 11, 2011www.jlr.orgDownloaded from