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Simultaneous evaluation of phospholipids, free fatty acids, free cholesterol, triglycerides and esterified cholesterol by thin layer chromatography and densitometry
271
CLINICA CHIMICA ACTA
CCA 4742
SIMULTANEOUS FREE
EVALUATION
CHOLESTEROL,
BY THIN
LI:CLhNO
LAYER
OF PHOSPHOLIPIDS,
TRIGLYCERIDES
CHROMATOGRAPHY
FREE
AND ESTERIFIED
FATTY
ACIDS,
CHOLESTEROL
AND DENSITOMETRY
GXSBARRO
lstituto “L. da Vincz”, (I
v. v. Eossombvoni 2, 50136
Flormcc
Irtaly)
3, 1971)
A method of evaluating the basic fractions of lipids in blood plasma is described. This is carried out by ozonizing a thin layer of silica gel following chromatography of the total lipid extract. The spots are detected using Schiff’s reagent and integrated with an automatic densitometer used in transmission. In addition, the normal values of lipid fractions in the plasma of subjects of both sexes, aged between 18 and 25 years, are established.
IiYTRODUCTION
The ozonization principle was applied to the support after the lipids had been separated in order to draw a parallel with ozonization after electrophoresis of the lipoproteins on cellulose acetate. In fact, if Kohn’s method’ of staining lipoproteins as modified by Berg and coworkers2~3 is used, application of the same principle to detect separated lipid fractions would appear to be justified. However, a valid objection could be made, i.e. ozonization following electrophoresis takes place in the presence of all plasma fractions, whereas ozonization of the chromatoplates is brought about in a lipid extract free from protein, glycoprotein, etc. On the other hand, the objection ceases to be valid when ozonization of lipoprotein following electrophoresis on cellulose acetate is carried out by eliminating interferences caused by non-lipid substances with Kunkel’s reagent as used by Grabner and co-workersI. In order to quantify the chromatographic spots a Joyce-Loebl Chromoscan was used without diaphanizing the chromatoplates. The reason for this is that, as will be demonstrated later, Merck’s TLC plates are suitable for direct scanning and give linear results of integration requiring no correction.
Clin. Chim.
Acta,
37
(1972)
271-27j
272
GASBARRO
REAGEKTS
AND
MATERIALS
Merck GR reagents were used throughout. Merck F-254 Thin Layer Chromatography (TLC) silica gel plastic sheets. F.I.R.M.A. S.p.A. Turbo-rooo mixer. BauschLomb 600-E spectrophotometer. JoyceeLoehl “Chromoscan” densitometer. Becker
409 Gas chromatograph.
EXPERIMENTAL
Preparation, of the lipid extract Mix 0.75 ml methanol with 1.5 ml chloroform in a centrifuge tube. Blow rapidl! and with force into the mixture 0.05 ml of plasma obtained with EDTA-h’a,. Mix well. After leaving the sealed test tubes to stand for 15 min centrifuge for 335 min at 3000 rev. /min. Transfer mixture to another test tube and add 0.25 ml H,O. Mix using TurboIOOO mixer for I min and then centrifuge for 335 min at 3000 rev./min. Eliminate water (upper phase) with a Pasteur pipette attached to a suction pump. The lower phase is evaporated with an air-blower while the test tubes are left standing on a thermostatic plate heated to 57”. The residue is collected using about 0.5 ml of chloroform
and re-evaporated
described above. The residue is finally dissolved with 3-4 drops of chloroform test tube hermetically sealed.
as
and the
Thin layer chromatogra$hy Merck TLC plastic sheets are cut into rectangles measuring 8 x IO cm. Using a chromatography glass chamber measuring appr. 6 x 12 x 15 cm,
in
which a sheet of filter paper has been placed, a pre-run is carried out with the following mixture : 17.0 ml Petroleum benzine (B.r. 60/80” C) 3.0 ml GR diethyl ether 0.1 ml Glacial GR acetic acid The support is taken out of the chamber and left to dry. The chloroform
extract
is then spread on it with a paint brush of the kind used for painting in water colours. Care should be taken to form a strip measuring 5 x IO mm. If the extract is deposited in a narrow line, the phospholipid spot will be too dense and therefore difficult to scan. At this stage, chromatography is carried out after the mixture has been changed. The run is interrupted when the front of the solvent has reached the upper edge of the chromatoplate. Ozonization and detection of Spots The chromatoplate is taken from the chamber and ozonized for at least 15 min. Flush once with 0.1% hydrochloric acid for 20~30 sec. Stain with Schiff’sreagent for at least 15 min, and then flush with 0.5 0’ ,0 nitric acid three times for IO min each time. I+& once with 0.1:/bhydrochloric acid for 20-30 sec. Dry with filter paper and air-dry thoroughly. In order to prevent oxidation that may cause the colouring of the
EVALUATIOh‘
OF LIPIDS
Ih’ PLASMA
273
Pig. I. Densitometric scanning obtained with a Chromoscan. Transmission: Wedge zoD, Cam 5-077~k, Aperture 0.5 x j mm, Gain 1 :I, Filter Lamp Q-J, Substrate : plastic sheets Silica gel Merck F/Zjl. F. phospholipids : rol; N, NEFA; T, triglycerides; CE cholesterol esters.
spots to fade, the support is sprayed in ethanol.
with a solution
550 nm (Ilford), cholcste-
I;C, free
of 20% vinyl polymers dissolved
Densitometry The thin layer is now ready to be evaluated mission with a 550 nm filter (maximum For further explanations RESULTS
AND
absorption
using the Chromoscan
in trans-
is 559 nm).
see legend to Fig. I
DISCUSSION
Five lipid fractions Phospholipids Free cholesterol
are obtained
from the deposited sample, i.e.
Free fatty acids (N.E.I’.A.) Triglycerides Cholesterol esters The separation of lipid fractions is very satisfactory
(Fig.
2).
Scanning in trafasmissiow Merck I;. 254 plastic sheets, though not transparent, are nevertheless suitable for scanning in transmission without being diaphanized. Electrophoresis of the lipoproteins on cellulose acetate was carried out using the method as described by Berg and co-workers. Percentage values were recorded with scanning in transmission following complete transparency. Scanning of the same sample was carried out inserting it between the two glass plates of the densitometer with a strip of silica gel. Results were identical in both tests.
274
GASBARRO
Beer’s law is followed up toconcentrations of lioomg o/; of any of the fivelipid groups detected with Schiff’s reagent after 0,. In order to standardize the method, chromatography of graded solutions of the same extract should be carried out. Comparison between the amounts left to migrate and scanned results was acceptable, with variations that never exceed 5O/;O (Fig. 3).
Percentage values obtained from quantification of the scanned surfaces are given below and may be considered normal for subjects whose ages varied from 18 to 25 years. 14.0.-23.00/& Phospholipids 7.~11.5~1’ Free cholesterol 0.6.--- 2.07; NEFA Triglycerides 9.5---37.50:: Cholesterol esters 35.0~60.00/ The fluctuations shown above were obtained by examining 156 subjects aged between 18 and 25 years, of whom 76 were male and 80 female. Values in the subjects were normal with regard to glucose, uric acid, electrophoresis of protein, total cholesterol, phospholipids and triglycerides. Values were obtained by traditional methods+10. At the same time, total lipids were quantified using both vanillinll and a method comprising a single acid dichromate reagent 12. Accurate anamnesis enabled the children of subjects with arteriosclerosis, from the group examined.
Clin. Chive. Acta, 37 (1972) 271-z7.j
diabetes
and h~peruricaem~a
to be excluded
EVALUATION
OF LIPIDS
27.5
IN PLASMA
CONCLUSION
When values obtained using densitometry are compared with those obtained with GLC, the resultant difference never exceeds 3%. The simplicity of the method permits mass screening. The lipid fractions are determined rapidly and with precision. The 3 traditional methods used so far can therefore simple method as, for example, is electrophoresis.
be transformed
into one
REFERENCES I
J. KOHP;, Natwe, 189 (1961) 312. P.KUDOLPH,ME~. Welt, 17(1966) ‘93. G. BERG ASD G. H. WILLITHAL, lilin. Wochschr., 43 (7965) IIo9. W.GRABSER, W.GRONAUER,G.BERG AND D.BERGR'ER,C~~~. Chim.Acta,z8(Ig7o) D. WATSON, Clin. Chim. Acta, 5 (1960) 637. D. B. ZILVERSMIT AND A. K. DAVIS, J. Lab. Clin. Med., 3.5 (1950) 155. G. ROGATZKI, Arch. Eisenhiittenwesen. 12 (1938) 195. M. EGGSTEIN, lilzn.Wochschr., 44 (1966) 267. 111.ECGSTEIN AND F. H. KREUTZ, Klin. Wochschv., 44 (1966) 262. F. H. SCHMIDT AND K. VOX DAHL, Z. h'lin.Chem., 6 (1968) 156. N. ZOELLZIER AND K. KIRSCH, Z.Ges. I%%'$.l%fed.,135 (1962) j4j. J. S. AMEXTA, Cl&i. Chew., 16 (1970) 3x9.
2 G. BERCASD 3 4
5 6
7 8 g IO II 12
Clin. Chim.
Acta,
zgg.
37 (1972) 271-275
CLINICA CHIMICA ACTA
CCA 4742
SIMULTANEOUS FREE
EVALUATION
CHOLESTEROL,
BY THIN
LI:CLhNO
LAYER
OF PHOSPHOLIPIDS,
TRIGLYCERIDES
CHROMATOGRAPHY
FREE
AND ESTERIFIED
FATTY
ACIDS,
CHOLESTEROL
AND DENSITOMETRY
GXSBARRO
lstituto “L. da Vincz”, (I
v. v. Eossombvoni 2, 50136
Flormcc
Irtaly)
3, 1971)
A method of evaluating the basic fractions of lipids in blood plasma is described. This is carried out by ozonizing a thin layer of silica gel following chromatography of the total lipid extract. The spots are detected using Schiff’s reagent and integrated with an automatic densitometer used in transmission. In addition, the normal values of lipid fractions in the plasma of subjects of both sexes, aged between 18 and 25 years, are established.
IiYTRODUCTION
The ozonization principle was applied to the support after the lipids had been separated in order to draw a parallel with ozonization after electrophoresis of the lipoproteins on cellulose acetate. In fact, if Kohn’s method’ of staining lipoproteins as modified by Berg and coworkers2~3 is used, application of the same principle to detect separated lipid fractions would appear to be justified. However, a valid objection could be made, i.e. ozonization following electrophoresis takes place in the presence of all plasma fractions, whereas ozonization of the chromatoplates is brought about in a lipid extract free from protein, glycoprotein, etc. On the other hand, the objection ceases to be valid when ozonization of lipoprotein following electrophoresis on cellulose acetate is carried out by eliminating interferences caused by non-lipid substances with Kunkel’s reagent as used by Grabner and co-workersI. In order to quantify the chromatographic spots a Joyce-Loebl Chromoscan was used without diaphanizing the chromatoplates. The reason for this is that, as will be demonstrated later, Merck’s TLC plates are suitable for direct scanning and give linear results of integration requiring no correction.
Clin. Chim.
Acta,
37
(1972)
271-27j
272
GASBARRO
REAGEKTS
AND
MATERIALS
Merck GR reagents were used throughout. Merck F-254 Thin Layer Chromatography (TLC) silica gel plastic sheets. F.I.R.M.A. S.p.A. Turbo-rooo mixer. BauschLomb 600-E spectrophotometer. JoyceeLoehl “Chromoscan” densitometer. Becker
409 Gas chromatograph.
EXPERIMENTAL
Preparation, of the lipid extract Mix 0.75 ml methanol with 1.5 ml chloroform in a centrifuge tube. Blow rapidl! and with force into the mixture 0.05 ml of plasma obtained with EDTA-h’a,. Mix well. After leaving the sealed test tubes to stand for 15 min centrifuge for 335 min at 3000 rev. /min. Transfer mixture to another test tube and add 0.25 ml H,O. Mix using TurboIOOO mixer for I min and then centrifuge for 335 min at 3000 rev./min. Eliminate water (upper phase) with a Pasteur pipette attached to a suction pump. The lower phase is evaporated with an air-blower while the test tubes are left standing on a thermostatic plate heated to 57”. The residue is collected using about 0.5 ml of chloroform
and re-evaporated
described above. The residue is finally dissolved with 3-4 drops of chloroform test tube hermetically sealed.
as
and the
Thin layer chromatogra$hy Merck TLC plastic sheets are cut into rectangles measuring 8 x IO cm. Using a chromatography glass chamber measuring appr. 6 x 12 x 15 cm,
in
which a sheet of filter paper has been placed, a pre-run is carried out with the following mixture : 17.0 ml Petroleum benzine (B.r. 60/80” C) 3.0 ml GR diethyl ether 0.1 ml Glacial GR acetic acid The support is taken out of the chamber and left to dry. The chloroform
extract
is then spread on it with a paint brush of the kind used for painting in water colours. Care should be taken to form a strip measuring 5 x IO mm. If the extract is deposited in a narrow line, the phospholipid spot will be too dense and therefore difficult to scan. At this stage, chromatography is carried out after the mixture has been changed. The run is interrupted when the front of the solvent has reached the upper edge of the chromatoplate. Ozonization and detection of Spots The chromatoplate is taken from the chamber and ozonized for at least 15 min. Flush once with 0.1% hydrochloric acid for 20~30 sec. Stain with Schiff’sreagent for at least 15 min, and then flush with 0.5 0’ ,0 nitric acid three times for IO min each time. I+& once with 0.1:/bhydrochloric acid for 20-30 sec. Dry with filter paper and air-dry thoroughly. In order to prevent oxidation that may cause the colouring of the
EVALUATIOh‘
OF LIPIDS
Ih’ PLASMA
273
Pig. I. Densitometric scanning obtained with a Chromoscan. Transmission: Wedge zoD, Cam 5-077~k, Aperture 0.5 x j mm, Gain 1 :I, Filter Lamp Q-J, Substrate : plastic sheets Silica gel Merck F/Zjl. F. phospholipids : rol; N, NEFA; T, triglycerides; CE cholesterol esters.
spots to fade, the support is sprayed in ethanol.
with a solution
550 nm (Ilford), cholcste-
I;C, free
of 20% vinyl polymers dissolved
Densitometry The thin layer is now ready to be evaluated mission with a 550 nm filter (maximum For further explanations RESULTS
AND
absorption
using the Chromoscan
in trans-
is 559 nm).
see legend to Fig. I
DISCUSSION
Five lipid fractions Phospholipids Free cholesterol
are obtained
from the deposited sample, i.e.
Free fatty acids (N.E.I’.A.) Triglycerides Cholesterol esters The separation of lipid fractions is very satisfactory
(Fig.
2).
Scanning in trafasmissiow Merck I;. 254 plastic sheets, though not transparent, are nevertheless suitable for scanning in transmission without being diaphanized. Electrophoresis of the lipoproteins on cellulose acetate was carried out using the method as described by Berg and co-workers. Percentage values were recorded with scanning in transmission following complete transparency. Scanning of the same sample was carried out inserting it between the two glass plates of the densitometer with a strip of silica gel. Results were identical in both tests.
274
GASBARRO
Beer’s law is followed up toconcentrations of lioomg o/; of any of the fivelipid groups detected with Schiff’s reagent after 0,. In order to standardize the method, chromatography of graded solutions of the same extract should be carried out. Comparison between the amounts left to migrate and scanned results was acceptable, with variations that never exceed 5O/;O (Fig. 3).
Percentage values obtained from quantification of the scanned surfaces are given below and may be considered normal for subjects whose ages varied from 18 to 25 years. 14.0.-23.00/& Phospholipids 7.~11.5~1’ Free cholesterol 0.6.--- 2.07; NEFA Triglycerides 9.5---37.50:: Cholesterol esters 35.0~60.00/ The fluctuations shown above were obtained by examining 156 subjects aged between 18 and 25 years, of whom 76 were male and 80 female. Values in the subjects were normal with regard to glucose, uric acid, electrophoresis of protein, total cholesterol, phospholipids and triglycerides. Values were obtained by traditional methods+10. At the same time, total lipids were quantified using both vanillinll and a method comprising a single acid dichromate reagent 12. Accurate anamnesis enabled the children of subjects with arteriosclerosis, from the group examined.
Clin. Chive. Acta, 37 (1972) 271-z7.j
diabetes
and h~peruricaem~a
to be excluded
EVALUATION
OF LIPIDS
27.5
IN PLASMA
CONCLUSION
When values obtained using densitometry are compared with those obtained with GLC, the resultant difference never exceeds 3%. The simplicity of the method permits mass screening. The lipid fractions are determined rapidly and with precision. The 3 traditional methods used so far can therefore simple method as, for example, is electrophoresis.
be transformed
into one
REFERENCES I
J. KOHP;, Natwe, 189 (1961) 312. P.KUDOLPH,ME~. Welt, 17(1966) ‘93. G. BERG ASD G. H. WILLITHAL, lilin. Wochschr., 43 (7965) IIo9. W.GRABSER, W.GRONAUER,G.BERG AND D.BERGR'ER,C~~~. Chim.Acta,z8(Ig7o) D. WATSON, Clin. Chim. Acta, 5 (1960) 637. D. B. ZILVERSMIT AND A. K. DAVIS, J. Lab. Clin. Med., 3.5 (1950) 155. G. ROGATZKI, Arch. Eisenhiittenwesen. 12 (1938) 195. M. EGGSTEIN, lilzn.Wochschr., 44 (1966) 267. 111.ECGSTEIN AND F. H. KREUTZ, Klin. Wochschv., 44 (1966) 262. F. H. SCHMIDT AND K. VOX DAHL, Z. h'lin.Chem., 6 (1968) 156. N. ZOELLZIER AND K. KIRSCH, Z.Ges. I%%'$.l%fed.,135 (1962) j4j. J. S. AMEXTA, Cl&i. Chew., 16 (1970) 3x9.
2 G. BERCASD 3 4
5 6
7 8 g IO II 12
Clin. Chim.
Acta,
zgg.
37 (1972) 271-275