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Quantitative determination of serum triglycerides by glass-fiber paper chromatography
.?.NALYTICAL
BIOCHEMIS’IXY
Quantitative
8,
158-163
Determination Glass-Fiber
KAROLY From
the
(1964)
of
Paper
Serum
Triglycerides
by
Chromatography1
G. PINTER, JAMES G. HAMILTON, AND 0. NEAL MILLER
Nutrition and Metabolkm Laboratory of the Department and the Department of Biochemistry, Tulane University, of Medicine, New Orleans, Louisiana Received
September
of Medicine School
9, 1963
The quantitative determination of serum triglycerides in recent years has been accomplished by several direct methods. A frequently used method is silicic acid column chromatography to separate triglycerides from cholesterol esters and phospholipids (l-3) and the chemical determination of ester groups in the triglyceride-containing fraction (4). Another commonly used method is the chemical determination of glycerol of lipid extracts after removal of phospholipids with silicic acid or zeolite (5). Indirect methods, such as estimation of total ester groups and total fatty acids, are often used because of the simplicity of these determinations. In these instances triglycerides are determined as the difference between total fatty acids and fatty acids of cholesterol est.ers plus phospholipids (6). The separation of triglycerides from a mixture of lipids has been accomplished by thin-layer chromatography; recently a procedure which makes possible the quantitative estimation of esters by this method was developed (7). Although a method of separating triglycerides by glassfiber paper has been reported (S), this method provided no means of quantitative estimation. The method described below serves as a rapid and reproducible tool for the quantitative determination of serum triglycerides and can be conveniently used for a large number of samples run at one t,ime. METHODS
(a) Separation
AND
MATERIALS
and Determination
of ,Triglycerides
Blood samples are collected in nonheparinized trifuged, and serum separated. To Teflon-capped ‘Supported try
by the
Grants
of USPHS,
HE
Board. 158
04150
and
tubes, immediately cenlo-ml culture tubes are by
the Special
Dairy
Indus-
CHROMATOGRAPHY
OF
SERUM
TRIGLYCERIDES
159
added 0.5 ml of serum and 4.5 ml of ethanol-isopropyl ether solution (2: 1). This mixture is vigorously shaken in a Burrel Wrist-Action shaker for 30 min. The tubes are then centrifuged at 1500 rpm for 5 min. Twomilliliter aliquots of extracts are dried under nitrogen stream and the Iipids redissolved in 0.2 ml of hexane. Then 0.1 ml of lipid extract is applied on the glass-fiber chromatography paper on a horizontal line 4 cm long. The paper is allowed to air dry for 10 min. Known quantities of triglycerides (corn oil or trilinolein) are simultaneously chromatographed and used to construct a standard curve. The lipids are separated by ascending chromatography using a mixture of isooctane, 100 ml, and isopropyl acetate, 1 to 3 ml, as the developing solvent (9). The separation is accomplished within 15 min and the glass-fiber paper is dried again, then exposed to iodine vapors for 5-10 set in a tank containing iodine crystals in order to visualize the various separated lipids. The lipid materials appear as light yellow bands against the white background of the paper. The triglyceride fractions of the unknown, the standards, and a blank from the same sheet of paper are then cut out and placed into the reaction tubes. The calorimetric determination is done by the modified hydroxylaminolysis method (10). To each reaction tube is added 2 ml of ethanolic hydroxylamine. The tubes are then placed in a water bath at 65°C for a 20-min period. The tubes are then cooled for 5 min at room temperature and l-ml aliquots are transferred to 12 X 100 mm colorimeter tubes. To each of these tubes, 2.5 ml of ferric perchlorate solution is added, mixed thoroughly, and allowed to stand for 30 min at room temperature. The optical density of each sample is measured in a Bausch and Lomb Spectronic 20 at 530 rnp. Reagents: Stock Ferric Perchlorate-ferric perchlorate (5 gm) is dissolved in 10 ml of 70% HClO, and 10 ml of water, then diluted to 100 ml with 98% ethanol and stored in refrigerator. Reagent Ferric Perchlorate -4 ml of stock ferric perchlorate and 3 ml of 70% HClO, are diluted to 100 ml with 98% ethanol. Alkaline Hydroxylamine-equal volumes of a 4% ethanolic hydroxylamine solution (2.0 gm dissolved in 2.5 ml of water diluted to 50 ml with 98% ethanol) and of an 8% ethanolic NaOH (4 gm dissolved in 2.5 ml of water diluted to 50 ml with 98% ethanol) are mixed in a stoppered cylinder. NaCl was separated by centrifugation and the supernatant was decanted for use. (b) Proof
of Separation
of Triglycerides
As proof of the identification of the triglyceride fraction both radioactive synthetic lipids were employed. Carboxyl-labeled (Ftripalmitin, trilinolein, 4XFcholestero1, 4-CY-cholesteryl palmitate, 4-(IF-ChoIesteryl linolenate, 1-C14-fatty acids, carboxyl-labeled CF-phosphatidyl-
160
PINTER,
HAMILTON,
AND
MILLER
choline, and ethanolamine were added to individual lipid mixtures (serum extracts). All of these samples were chromatographed and counted in the scintillation counter (11). The per cent recoveries of radioactivity at the appropriate Rf values are given in Fig. 1. Solvent:
TG
Isooctane Isopropyl
FA
IOOml acetate
C
I ml
CL
PC
1. Recovery of added radioactivity from lipid mixtures (serum extracts). The following @‘-containing materials were used: TG (tripalmitin and triolein), FA (palmitic and linoleic acid), C (cholesterol), CL (cholesteryl linoleate) and PC (phosphatidyl choline). FIG.
The recovery of known amounts of nonradioactive triglyceride which were added to serum samples was investigated and the per cent recovery consistently ranged between 93.&106.0% with an average recovery of 97%. To demonstrate that monoglycerides have a different Rf value with the solvent used than the triglycerides, known amounts of triglyceride were added to a diglyceride and to a monoglyceride solution and chromatographed. The recovery of triglyceride from these mixtures was consistently 98 -c- 30/o, showing that the triglyceride fraction was not contaminated with monoglycerides or diglycerides and only the ester bonds of triglycerides were involved in hydroxylaminolysis. Standard curves were constructed using increasing concentrations of standard pure trilinolein or corn oil lipids both before and after chromatography. These curves demonstrated a linear relationship between triglycerides and optical density (Fig. 2). The reproducibility of the method was determined by repeated analysis (ten times) of two serum samples, one with a high and one with a
CHROMATOGRAPHT
OF
SERUM
It51
TRIGLYCERIDES
low triglyceride content. The values for the two sera were shown to be 112.8 +- 3.2 mg/lOO ml and 600.8 -t 26.8 mg/lOO ml, thus indicating that the method is reproducible within ~3.5%.
Trrltndem
Slandwd
Chromotogrophed - - - Not chnxwtoprophed
--.-.-.-
loo
200
300 pg
FIG.
trations
2. Linearity employed.
of standards
Chromatographed Not chromotopraphed
400
503
Triglyceride
demonstrates
recovery of triglycerides
at concen-
The accuracy of the method was tested finally by comparing the analysis of ten human sera analyzed in duplicates, with both silicic acid column (1) chromatography and glass-fiber paper chromatography. These data, shown in Table 1, demonstrate good agreement between the two methods. TABLE COMPARISON
OF TRIGLYCERIDE
1
CONCENTRATION
OF TEN
Silicic acid column and glass-fiber paper chromatography cate. Serum Column
32 43 53 59 68
Glass-fiber
triglycerides,
paper
38 50 51 67 75
(c) Application
mg/lOO
SERUM
LIPID
EXTRACTS
was done on each in dupli-
ml COlUlllll
Glass-fiber
100
110
148 200 280 366
150 181 262 382
~rqer
of the Method
Three volunteers who had fasted overnight were fed a 500-cal emulsified diet containing 25 gm of gIycery1 monolinoleate (or 25 gm of glyceryl monostearate) and 55 gm of dry skim milk solids in 600 ml of water.
162
PINTER,
HAMILTON,
AND
MILLER
Each individual was given both emulsions with a l-week interval between the experiments. For 8 hr following administration of the diet, the subjects were allowed to consume only coffee or tea. In another experiment two subjects received an emulsion consisting either of 50 gm of glyceryl monolinoleate or of 50 gm of glyceryl monostearate in 250 ml of skim milk. The triglyceride contents of the sera are given in Table 2. TABLE COMPARISON
2
OF SERUM TRIGLYCERIDE CONCENTRATION DURINQ POSTABSORPTIVE PERIOD IN FIVE VOLUNTEERS
EIGHT
HOUR
Each individual received both the glyceryl monolinoleate (L) and glyceryl monostearate (S) supplemented emulsion in l-week interval. Volunteers 1, 2, and 3 consumed 25 gm of the monoglycerides; volunteers 4 and 5 consumed 50 gm of the monoglycerides. Serum
Time, hr
0 2 4 6 8
mg/lOO
ml
Sub%t No.
2
1
triglycerides,
L
S
I,
S
I.
80 215 370 248 110
115 114 110 108 116
100 J18 72 68 65
62 65 68 79 52
55 76 60 44 36
S
42 40 30 36 40
4
5
L
S
L
102 142 170 240 298
100 109 105 160 167
88 104 117 105 97
S
91 98 99 96 97
DISCUSSION
The chromatographic method presented here for the separation and quantitative determination of serum triglycerides has proved itself to be both reproducible and time saving. The chromatography takes about 20 min and there is no need to elute the t’riglycerides from the glass-fiber paper. Both the thin-layer and glass-fiber paper chromatographic methods have an inherent advantage over the column chromatographic methods because contaminations of triglycerides by cholesterol esters and diand mono-glycerides easily are avoided by the temporary visualization of all these lipids when the sheets are exposed to iodine vapor. This method was employed in a preliminary investigation of the possible effect of various dietary fats on the serum triglyceride concentration. In the comparison between glyceryl monolinoleate and glyceryl monostearate consistent differences in serum triglyceride concentration were observed in the period of alimentary hyperlipemia. The unsaturated glyceryl monolinoleate caused a marked increase in concentration of serum triglycerides 2-4 hr after the administration of 25 gm of thr monoglyceride. This effect was given more pronounced when the 50 gm of
CHROMATOGRAPHY
OY
SERUAI
163
TRIGLYCERIDES
glyceryl monolinoleate was administered. Glyceryl monostearate when administered at either the 25 gm or 50 gm level had little or no effect on serum triglyceride concentration. Since these studies were conducted on nonhospitalized volunteers no attempt was made to estimate the fecal fat excretion in these experiments. Therefore no explanation is given for any mechanism involved in this observation. SUMMARY
A rapid and reproducible procedure for the quantitative determination of serum triglycerides based on their isolation by glass-fiber paper chromatography and the calorimetric estimation of the triglycerides by hydroxylaminolysis is presented and has been utilized for a series of biological experiments to illustrate the usefulness of this technique. There was a marked increase in serum triglyceride concentration after the consumption of an emulsion containing glyceryl monolinoleate. This was not observed when glyceryl monostearate was the source of fat in the emulsion. ACKNOWLEDGMENT The technical
assistance of Mr. Robert
Osburn is gratefully
acknowledged.
REFERENCES AND HORNING, E. C., J. Lipicl Res. 1, 482 (1960). BARRON, E., AND HANAHAN, D., J. Biol. Chem. 231, 493 (1958). HIRSCH, J., AND AHRENS, E., J. Sol. Chem. 233, 311 (1958). ANTONIS, ARNOLD, J. Lipid Res. 1, 485 (1960). CARLSON, L. A., AND WADSTROM, L. B., Clin. Chim. Acta 4, 197 (1959). ALBRINK, M. J., J. Lipid Res. 1, 53 (1959). VKQUE, E., AND HOLMAN, R. T., J. Am. Oil Chemists’ Sot. 39, 63 (1962). HAMILTON, J. G., AND MULDREY, J. E., J. Am. Oil Chemksts’ Sot. 38, 582 (1961). HAMILTON, J. G., SWARIWOUT, J. R., MILLER, 0. N., AND MULDREY, J. E., Biochem. Biophys. Res. Commun. 5, 226 (1961). SNYDER, F., AND STEPHENS, N., Biochim. Biophys. Acta 34, 244 (1959). PINTER, K. G., HAMILTON, J. G., AND MILLER, 0. N., Anal. Biochem. 4, 458
1. HORNING, 2. 3. 4. 5. 6.
7. 8. 9.
10. 11.
(1963).
M. G., WILLIAMS,
E. A.,
BIOCHEMIS’IXY
Quantitative
8,
158-163
Determination Glass-Fiber
KAROLY From
the
(1964)
of
Paper
Serum
Triglycerides
by
Chromatography1
G. PINTER, JAMES G. HAMILTON, AND 0. NEAL MILLER
Nutrition and Metabolkm Laboratory of the Department and the Department of Biochemistry, Tulane University, of Medicine, New Orleans, Louisiana Received
September
of Medicine School
9, 1963
The quantitative determination of serum triglycerides in recent years has been accomplished by several direct methods. A frequently used method is silicic acid column chromatography to separate triglycerides from cholesterol esters and phospholipids (l-3) and the chemical determination of ester groups in the triglyceride-containing fraction (4). Another commonly used method is the chemical determination of glycerol of lipid extracts after removal of phospholipids with silicic acid or zeolite (5). Indirect methods, such as estimation of total ester groups and total fatty acids, are often used because of the simplicity of these determinations. In these instances triglycerides are determined as the difference between total fatty acids and fatty acids of cholesterol est.ers plus phospholipids (6). The separation of triglycerides from a mixture of lipids has been accomplished by thin-layer chromatography; recently a procedure which makes possible the quantitative estimation of esters by this method was developed (7). Although a method of separating triglycerides by glassfiber paper has been reported (S), this method provided no means of quantitative estimation. The method described below serves as a rapid and reproducible tool for the quantitative determination of serum triglycerides and can be conveniently used for a large number of samples run at one t,ime. METHODS
(a) Separation
AND
MATERIALS
and Determination
of ,Triglycerides
Blood samples are collected in nonheparinized trifuged, and serum separated. To Teflon-capped ‘Supported try
by the
Grants
of USPHS,
HE
Board. 158
04150
and
tubes, immediately cenlo-ml culture tubes are by
the Special
Dairy
Indus-
CHROMATOGRAPHY
OF
SERUM
TRIGLYCERIDES
159
added 0.5 ml of serum and 4.5 ml of ethanol-isopropyl ether solution (2: 1). This mixture is vigorously shaken in a Burrel Wrist-Action shaker for 30 min. The tubes are then centrifuged at 1500 rpm for 5 min. Twomilliliter aliquots of extracts are dried under nitrogen stream and the Iipids redissolved in 0.2 ml of hexane. Then 0.1 ml of lipid extract is applied on the glass-fiber chromatography paper on a horizontal line 4 cm long. The paper is allowed to air dry for 10 min. Known quantities of triglycerides (corn oil or trilinolein) are simultaneously chromatographed and used to construct a standard curve. The lipids are separated by ascending chromatography using a mixture of isooctane, 100 ml, and isopropyl acetate, 1 to 3 ml, as the developing solvent (9). The separation is accomplished within 15 min and the glass-fiber paper is dried again, then exposed to iodine vapors for 5-10 set in a tank containing iodine crystals in order to visualize the various separated lipids. The lipid materials appear as light yellow bands against the white background of the paper. The triglyceride fractions of the unknown, the standards, and a blank from the same sheet of paper are then cut out and placed into the reaction tubes. The calorimetric determination is done by the modified hydroxylaminolysis method (10). To each reaction tube is added 2 ml of ethanolic hydroxylamine. The tubes are then placed in a water bath at 65°C for a 20-min period. The tubes are then cooled for 5 min at room temperature and l-ml aliquots are transferred to 12 X 100 mm colorimeter tubes. To each of these tubes, 2.5 ml of ferric perchlorate solution is added, mixed thoroughly, and allowed to stand for 30 min at room temperature. The optical density of each sample is measured in a Bausch and Lomb Spectronic 20 at 530 rnp. Reagents: Stock Ferric Perchlorate-ferric perchlorate (5 gm) is dissolved in 10 ml of 70% HClO, and 10 ml of water, then diluted to 100 ml with 98% ethanol and stored in refrigerator. Reagent Ferric Perchlorate -4 ml of stock ferric perchlorate and 3 ml of 70% HClO, are diluted to 100 ml with 98% ethanol. Alkaline Hydroxylamine-equal volumes of a 4% ethanolic hydroxylamine solution (2.0 gm dissolved in 2.5 ml of water diluted to 50 ml with 98% ethanol) and of an 8% ethanolic NaOH (4 gm dissolved in 2.5 ml of water diluted to 50 ml with 98% ethanol) are mixed in a stoppered cylinder. NaCl was separated by centrifugation and the supernatant was decanted for use. (b) Proof
of Separation
of Triglycerides
As proof of the identification of the triglyceride fraction both radioactive synthetic lipids were employed. Carboxyl-labeled (Ftripalmitin, trilinolein, 4XFcholestero1, 4-CY-cholesteryl palmitate, 4-(IF-ChoIesteryl linolenate, 1-C14-fatty acids, carboxyl-labeled CF-phosphatidyl-
160
PINTER,
HAMILTON,
AND
MILLER
choline, and ethanolamine were added to individual lipid mixtures (serum extracts). All of these samples were chromatographed and counted in the scintillation counter (11). The per cent recoveries of radioactivity at the appropriate Rf values are given in Fig. 1. Solvent:
TG
Isooctane Isopropyl
FA
IOOml acetate
C
I ml
CL
PC
1. Recovery of added radioactivity from lipid mixtures (serum extracts). The following @‘-containing materials were used: TG (tripalmitin and triolein), FA (palmitic and linoleic acid), C (cholesterol), CL (cholesteryl linoleate) and PC (phosphatidyl choline). FIG.
The recovery of known amounts of nonradioactive triglyceride which were added to serum samples was investigated and the per cent recovery consistently ranged between 93.&106.0% with an average recovery of 97%. To demonstrate that monoglycerides have a different Rf value with the solvent used than the triglycerides, known amounts of triglyceride were added to a diglyceride and to a monoglyceride solution and chromatographed. The recovery of triglyceride from these mixtures was consistently 98 -c- 30/o, showing that the triglyceride fraction was not contaminated with monoglycerides or diglycerides and only the ester bonds of triglycerides were involved in hydroxylaminolysis. Standard curves were constructed using increasing concentrations of standard pure trilinolein or corn oil lipids both before and after chromatography. These curves demonstrated a linear relationship between triglycerides and optical density (Fig. 2). The reproducibility of the method was determined by repeated analysis (ten times) of two serum samples, one with a high and one with a
CHROMATOGRAPHT
OF
SERUM
It51
TRIGLYCERIDES
low triglyceride content. The values for the two sera were shown to be 112.8 +- 3.2 mg/lOO ml and 600.8 -t 26.8 mg/lOO ml, thus indicating that the method is reproducible within ~3.5%.
Trrltndem
Slandwd
Chromotogrophed - - - Not chnxwtoprophed
--.-.-.-
loo
200
300 pg
FIG.
trations
2. Linearity employed.
of standards
Chromatographed Not chromotopraphed
400
503
Triglyceride
demonstrates
recovery of triglycerides
at concen-
The accuracy of the method was tested finally by comparing the analysis of ten human sera analyzed in duplicates, with both silicic acid column (1) chromatography and glass-fiber paper chromatography. These data, shown in Table 1, demonstrate good agreement between the two methods. TABLE COMPARISON
OF TRIGLYCERIDE
1
CONCENTRATION
OF TEN
Silicic acid column and glass-fiber paper chromatography cate. Serum Column
32 43 53 59 68
Glass-fiber
triglycerides,
paper
38 50 51 67 75
(c) Application
mg/lOO
SERUM
LIPID
EXTRACTS
was done on each in dupli-
ml COlUlllll
Glass-fiber
100
110
148 200 280 366
150 181 262 382
~rqer
of the Method
Three volunteers who had fasted overnight were fed a 500-cal emulsified diet containing 25 gm of gIycery1 monolinoleate (or 25 gm of glyceryl monostearate) and 55 gm of dry skim milk solids in 600 ml of water.
162
PINTER,
HAMILTON,
AND
MILLER
Each individual was given both emulsions with a l-week interval between the experiments. For 8 hr following administration of the diet, the subjects were allowed to consume only coffee or tea. In another experiment two subjects received an emulsion consisting either of 50 gm of glyceryl monolinoleate or of 50 gm of glyceryl monostearate in 250 ml of skim milk. The triglyceride contents of the sera are given in Table 2. TABLE COMPARISON
2
OF SERUM TRIGLYCERIDE CONCENTRATION DURINQ POSTABSORPTIVE PERIOD IN FIVE VOLUNTEERS
EIGHT
HOUR
Each individual received both the glyceryl monolinoleate (L) and glyceryl monostearate (S) supplemented emulsion in l-week interval. Volunteers 1, 2, and 3 consumed 25 gm of the monoglycerides; volunteers 4 and 5 consumed 50 gm of the monoglycerides. Serum
Time, hr
0 2 4 6 8
mg/lOO
ml
Sub%t No.
2
1
triglycerides,
L
S
I,
S
I.
80 215 370 248 110
115 114 110 108 116
100 J18 72 68 65
62 65 68 79 52
55 76 60 44 36
S
42 40 30 36 40
4
5
L
S
L
102 142 170 240 298
100 109 105 160 167
88 104 117 105 97
S
91 98 99 96 97
DISCUSSION
The chromatographic method presented here for the separation and quantitative determination of serum triglycerides has proved itself to be both reproducible and time saving. The chromatography takes about 20 min and there is no need to elute the t’riglycerides from the glass-fiber paper. Both the thin-layer and glass-fiber paper chromatographic methods have an inherent advantage over the column chromatographic methods because contaminations of triglycerides by cholesterol esters and diand mono-glycerides easily are avoided by the temporary visualization of all these lipids when the sheets are exposed to iodine vapor. This method was employed in a preliminary investigation of the possible effect of various dietary fats on the serum triglyceride concentration. In the comparison between glyceryl monolinoleate and glyceryl monostearate consistent differences in serum triglyceride concentration were observed in the period of alimentary hyperlipemia. The unsaturated glyceryl monolinoleate caused a marked increase in concentration of serum triglycerides 2-4 hr after the administration of 25 gm of thr monoglyceride. This effect was given more pronounced when the 50 gm of
CHROMATOGRAPHY
OY
SERUAI
163
TRIGLYCERIDES
glyceryl monolinoleate was administered. Glyceryl monostearate when administered at either the 25 gm or 50 gm level had little or no effect on serum triglyceride concentration. Since these studies were conducted on nonhospitalized volunteers no attempt was made to estimate the fecal fat excretion in these experiments. Therefore no explanation is given for any mechanism involved in this observation. SUMMARY
A rapid and reproducible procedure for the quantitative determination of serum triglycerides based on their isolation by glass-fiber paper chromatography and the calorimetric estimation of the triglycerides by hydroxylaminolysis is presented and has been utilized for a series of biological experiments to illustrate the usefulness of this technique. There was a marked increase in serum triglyceride concentration after the consumption of an emulsion containing glyceryl monolinoleate. This was not observed when glyceryl monostearate was the source of fat in the emulsion. ACKNOWLEDGMENT The technical
assistance of Mr. Robert
Osburn is gratefully
acknowledged.
REFERENCES AND HORNING, E. C., J. Lipicl Res. 1, 482 (1960). BARRON, E., AND HANAHAN, D., J. Biol. Chem. 231, 493 (1958). HIRSCH, J., AND AHRENS, E., J. Sol. Chem. 233, 311 (1958). ANTONIS, ARNOLD, J. Lipid Res. 1, 485 (1960). CARLSON, L. A., AND WADSTROM, L. B., Clin. Chim. Acta 4, 197 (1959). ALBRINK, M. J., J. Lipid Res. 1, 53 (1959). VKQUE, E., AND HOLMAN, R. T., J. Am. Oil Chemists’ Sot. 39, 63 (1962). HAMILTON, J. G., AND MULDREY, J. E., J. Am. Oil Chemksts’ Sot. 38, 582 (1961). HAMILTON, J. G., SWARIWOUT, J. R., MILLER, 0. N., AND MULDREY, J. E., Biochem. Biophys. Res. Commun. 5, 226 (1961). SNYDER, F., AND STEPHENS, N., Biochim. Biophys. Acta 34, 244 (1959). PINTER, K. G., HAMILTON, J. G., AND MILLER, 0. N., Anal. Biochem. 4, 458
1. HORNING, 2. 3. 4. 5. 6.
7. 8. 9.
10. 11.
(1963).
M. G., WILLIAMS,
E. A.,