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Fatty acid composition of pathological body fluids
188 SHORT
CLINICA
CHIMICA
ACTA
COMMUNICATIONS
Fatty acid composition of pathological body fluids It is only recently that the fatty acid content of biological fluids has become the object of detailed investigation. This has been made possible by the introduction of gas chromatography, which enables the investigator to study the fatty acid content in very small quantities of lipids. We used this method of research for the study of the fatty acid content in pleural and peritoneal effusions, either exudates or transudates, and in edema fluid. Up to now, only fragmentary investigation concerning chiefly the total lipid content or individual lipid fractions (particularly cholesterol) or lipoproteins has been made. MATERIALS
AND METHOD
IO samples of exudate of inflammatory or neoplastic nature, 16 of serous transudate and IO of edema fluid caused by heart failure were examined. These samples, together with venous blood, were taken in the morning from patients fasted for 12 h. The work on the blood serum and on the fluids (previously centrifuged to remove cells) was carried out simultaneously. Lipids were extracted from I ml of serum and ~--IO ml of fluid with a mixture of chloroform-methanol (z:I); another sample was treated in the same way with addition of carefully weighed heptadecanoic acid. Previous research carried out by us has shown that this procedure makes it possible to determine with satisfactory accuracy the total amount of fatty acids. On the lipid extract, transesterification was carried out with a mixture of methanol-benzene-sulphuric acid (IOO : 50 : 5) by boiling with a refrigerating system for I h. The methyl esters of the fatty acids thus obtained were purified on a silicic acid column and introduced into a Perkin-Elmer 226 gas-chromatograph with a flame ionization detector, using a capillary column (0.20” I.D. x IOO ft.) of butanediolsuccinate, and pure nitrogen as carrier gas. The temperature of the block was zoo” and of the column Igo’. The identification of the fatty acids on the chromatograms was made by comparing their I’r with the V, of pure samples run in the same conditions. The areas were calculated by triangulation.
RESULTS
The average fatty acid content of the fluids resulted as follows: 1.31 go/0o for exudates, 0.91 gO/00for transudates and 0.63 go/o0 for edema. With the method used 16 fatty acids were separated and identified. A few others, presumably unsaturated with 20-22 carbon atoms, were not identified with certainty because of the lack of standards; in any case, these are acids present only in minimal quantities both in the serum and in the other fluids (in the table they are grouped together). Clin. Chim.
Acta,
IO (1964)
188-191
4.25
0.91
jQ.70~IJ8)
(2.p8.24
traces (O-0.92)
0.14 jtr. -0.z9)
4,” (2.92-6.11)
traces (a-tr
)
I .46 (0.~~~Z.10)
1.03 (a.64-1,4oj
a.63 (0.48 -o.Bz) traces (o-ir.) I.64 (r.os-z.4ri
traces jn-tr.) 0.30 (wo-0.3:) tlzes (tr. -0.42)
0.2g (a-0.q
23,39 (zr.za-26.90)
24.m (I9.69-28.60)
4.09 (3.43-5.73)
(3-32-6494
6.01 (1.35-8.46)
tracts (ii”. -0.1
traces jtr. - 0.30)
0.28 (tr. - 3.73)
5.00
j)
traces (O-2 ,osj
traces (tr* -0.47)
p’
7.5: (b-7WPf
15.553,331
31.31 i27w%To)
32.46 13~.38-33.50)
13.35 (20.~wz5.05)
P5-95-91.3q
20.70
traces
(o--o.clS) traces +.43j
0.25 (tr. -c.61) il.31 k-54-7&) 31.98 (z&21)-41.42) 20.86 (‘5.4YW4)
traces (0 a.gq
traces (o-0.83)
traces
traces (“-0 42)
(n-0
I.64 !LOj-I.@
sg.12 (23 3 j-Zj.62)
53)
2.07 @. - 5.07)
5.94
5.15 (q-%68)
(3.82-8.00) cl.63
traces [o-a .8 I)
The highest quantities are those of olefc, palmitic, linoleic, palmitoleic, stearic and arxchidic acids; the myristic and cicosalrienoic acid content is low (about: I-Z% on the average) and the amount of the remainingacids (someof which were
absent in sctne cases)is even
less.
each d the three types cf fluidexamincd -exudates,trausndatcs and cdcma -the cases show differences as regards both the total fatty acid content and the percentage of individual acids. These differences, however, do no; depend on the site In
SHORT
190
COMMUNICATIONS
SERUM
I
EXUDATE
I
A-
l”
Fig. I. Chromatograms of a sample of edema, of transudate and of exudate. Above each chromatogram is that of the corresponding serum. In the sample of exudate and its corresponding serum, heptadecanoic acid has been added.
of origin of the fluid (pleura, peritoneum, subcutis) and have no relationship with the length of the fatty acid chain nor with their saturation degree. For the sake of we have shown in Table I the mean and &pme v&es .- hrevitv. _‘_.__,, ..- __-._ -.__.... ___ obtained
in each group of subjects
both in the blood serum and in the fluid.
In Fig. I are represented the chromatograms of a sample case from each of the three groups. The comparative examination of the blood serum and fluids shows on the whale a very similar aspect in the fatty acid composition. In individual cases, however, some Clin. Chim. Acta, IO (1964) I88--1gI
SHORT COMMUNICATIONS -L
5
4
5
6
7
191 1(
mu
E
t
Ill
ml
111
art
M
M
M
In
In
Ill
in In Jn
In
M -
In
In_
ICL
Fig. 2. Percentual values of myristic, palmitic, palmitoleic, stearic, oleic, linoleic, eicosatrienoic and arachidic acids in the 16 patients with transudates. The shaded column represents the value in serum, the blank column that in exudate.
differences in the fatty acid content and in the percentages of single fatty acids are to be found. This is seen clearly in Fig. 2, which represents graphically the percentage values of the main fatty acids in the different transudates and their corresponding sera. Institute of Clinical Medicine, University of Rome (Italy) *
C. PIETROPAOLO
L. PISANO G. CALI’
E. FIORENTINO Received March Isth, 1964 * Director: Prof. L.
CONDORELLI.
C&n. Chim.
Acta,
10 (1964)
r&-r91
CLINICA
CHIMICA
ACTA
COMMUNICATIONS
Fatty acid composition of pathological body fluids It is only recently that the fatty acid content of biological fluids has become the object of detailed investigation. This has been made possible by the introduction of gas chromatography, which enables the investigator to study the fatty acid content in very small quantities of lipids. We used this method of research for the study of the fatty acid content in pleural and peritoneal effusions, either exudates or transudates, and in edema fluid. Up to now, only fragmentary investigation concerning chiefly the total lipid content or individual lipid fractions (particularly cholesterol) or lipoproteins has been made. MATERIALS
AND METHOD
IO samples of exudate of inflammatory or neoplastic nature, 16 of serous transudate and IO of edema fluid caused by heart failure were examined. These samples, together with venous blood, were taken in the morning from patients fasted for 12 h. The work on the blood serum and on the fluids (previously centrifuged to remove cells) was carried out simultaneously. Lipids were extracted from I ml of serum and ~--IO ml of fluid with a mixture of chloroform-methanol (z:I); another sample was treated in the same way with addition of carefully weighed heptadecanoic acid. Previous research carried out by us has shown that this procedure makes it possible to determine with satisfactory accuracy the total amount of fatty acids. On the lipid extract, transesterification was carried out with a mixture of methanol-benzene-sulphuric acid (IOO : 50 : 5) by boiling with a refrigerating system for I h. The methyl esters of the fatty acids thus obtained were purified on a silicic acid column and introduced into a Perkin-Elmer 226 gas-chromatograph with a flame ionization detector, using a capillary column (0.20” I.D. x IOO ft.) of butanediolsuccinate, and pure nitrogen as carrier gas. The temperature of the block was zoo” and of the column Igo’. The identification of the fatty acids on the chromatograms was made by comparing their I’r with the V, of pure samples run in the same conditions. The areas were calculated by triangulation.
RESULTS
The average fatty acid content of the fluids resulted as follows: 1.31 go/0o for exudates, 0.91 gO/00for transudates and 0.63 go/o0 for edema. With the method used 16 fatty acids were separated and identified. A few others, presumably unsaturated with 20-22 carbon atoms, were not identified with certainty because of the lack of standards; in any case, these are acids present only in minimal quantities both in the serum and in the other fluids (in the table they are grouped together). Clin. Chim.
Acta,
IO (1964)
188-191
4.25
0.91
jQ.70~IJ8)
(2.p8.24
traces (O-0.92)
0.14 jtr. -0.z9)
4,” (2.92-6.11)
traces (a-tr
)
I .46 (0.~~~Z.10)
1.03 (a.64-1,4oj
a.63 (0.48 -o.Bz) traces (o-ir.) I.64 (r.os-z.4ri
traces jn-tr.) 0.30 (wo-0.3:) tlzes (tr. -0.42)
0.2g (a-0.q
23,39 (zr.za-26.90)
24.m (I9.69-28.60)
4.09 (3.43-5.73)
(3-32-6494
6.01 (1.35-8.46)
tracts (ii”. -0.1
traces jtr. - 0.30)
0.28 (tr. - 3.73)
5.00
j)
traces (O-2 ,osj
traces (tr* -0.47)
p’
7.5: (b-7WPf
15.553,331
31.31 i27w%To)
32.46 13~.38-33.50)
13.35 (20.~wz5.05)
P5-95-91.3q
20.70
traces
(o--o.clS) traces +.43j
0.25 (tr. -c.61) il.31 k-54-7&) 31.98 (z&21)-41.42) 20.86 (‘5.4YW4)
traces (0 a.gq
traces (o-0.83)
traces
traces (“-0 42)
(n-0
I.64 !LOj-I.@
sg.12 (23 3 j-Zj.62)
53)
2.07 @. - 5.07)
5.94
5.15 (q-%68)
(3.82-8.00) cl.63
traces [o-a .8 I)
The highest quantities are those of olefc, palmitic, linoleic, palmitoleic, stearic and arxchidic acids; the myristic and cicosalrienoic acid content is low (about: I-Z% on the average) and the amount of the remainingacids (someof which were
absent in sctne cases)is even
less.
each d the three types cf fluidexamincd -exudates,trausndatcs and cdcma -the cases show differences as regards both the total fatty acid content and the percentage of individual acids. These differences, however, do no; depend on the site In
SHORT
190
COMMUNICATIONS
SERUM
I
EXUDATE
I
A-
l”
Fig. I. Chromatograms of a sample of edema, of transudate and of exudate. Above each chromatogram is that of the corresponding serum. In the sample of exudate and its corresponding serum, heptadecanoic acid has been added.
of origin of the fluid (pleura, peritoneum, subcutis) and have no relationship with the length of the fatty acid chain nor with their saturation degree. For the sake of we have shown in Table I the mean and &pme v&es .- hrevitv. _‘_.__,, ..- __-._ -.__.... ___ obtained
in each group of subjects
both in the blood serum and in the fluid.
In Fig. I are represented the chromatograms of a sample case from each of the three groups. The comparative examination of the blood serum and fluids shows on the whale a very similar aspect in the fatty acid composition. In individual cases, however, some Clin. Chim. Acta, IO (1964) I88--1gI
SHORT COMMUNICATIONS -L
5
4
5
6
7
191 1(
mu
E
t
Ill
ml
111
art
M
M
M
In
In
Ill
in In Jn
In
M -
In
In_
ICL
Fig. 2. Percentual values of myristic, palmitic, palmitoleic, stearic, oleic, linoleic, eicosatrienoic and arachidic acids in the 16 patients with transudates. The shaded column represents the value in serum, the blank column that in exudate.
differences in the fatty acid content and in the percentages of single fatty acids are to be found. This is seen clearly in Fig. 2, which represents graphically the percentage values of the main fatty acids in the different transudates and their corresponding sera. Institute of Clinical Medicine, University of Rome (Italy) *
C. PIETROPAOLO
L. PISANO G. CALI’
E. FIORENTINO Received March Isth, 1964 * Director: Prof. L.
CONDORELLI.
C&n. Chim.
Acta,
10 (1964)
r&-r91