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Precise quantitative determination of human blood lipids by thin-layer and triethylaminoethylcellulose column chromatography
ANALYTICAL
BIOCHEMISTRY
Precise
Quantitative by
38, 437-445 (1970)
Determination
Thin-Layer
and Column
of Human
Blood
Lipids
Triethylaminoethylcellulose Chromatography
II. Plasma Lipids JOHN D. TURNER Department
of
Medicine,
Baylor
College
of Medicine,
Houston,
Texas
Duarte,
California
770%
AND
GEORGE Division
of Neurosciences, City
of Hope
ROUSER Medical Center,
91010
ReceivedMarch 25, 1970 Two-dimensional thin-layer chromatography (TLC) originally developed for the quantitative analysis of brain lipids (1) has been applied to plasma and erythrocytes of various mammalian species (24). In an accompanying paper (5), we describe improved resolution of human red cell lipids by the use of two-dimensional TLC alone or combined with ion-exchange cellulose column chromatography. In this report, we describe the application of these procedures with modifications necessary to obtain accurate and precise quantitative analysis of human plasma lipids. MAl’ERIALS
AND METHODS
Separation of Human Plasma and Erythrocytes Blood was obtained from healthy human subjects after an overnight fast. Disodium ethylenediaminetetrartcetate (EDTA, 1 mg/ml of whole blood) was used as anticoagulant. The blood was centrifuged at 1500g for 10 min (4’C). The plasma was removed and centrifuged again at 7700g for 10 min (4°C). The supernatant plasma was decanted and either extracted immediately or frozen and stored (-20’ or -70°C) until extracted. When erythrocytes were to be harvested for washing, an initial centrifugation at 809 for 15 min (4°C) was used, the platelet-rich plasma (PRP) transferred to another tube, and the cells and PRP centrifuged 437
438
TURNER
AND
ROUSER
again at 1500g for 10 min (4°C). The pooled plasma was then subjected to 77OOg for 10 min (4%) while the red cells were washed as described in the accompanying paper (5). Lipid, Extraction
Analytical-grade solvents were redistilled in glass and deoxygenated immediately prior to use. The antioxidant butylated hydroxytoluene (BHT) was added to solvent (0.1 mg/liter) used for extraction. For extraction of plasma lipids (2)) each milliliter of plasma was added to 8.3 ml of cold methanol in a flask (previously flushed with nitrogen) in an ice bucket containing wet ice. The mixture was stirred magnetically with a Teflon-coated stirring bar for 1 min and 16.7 ml of cold chloroform was added to give a final chloroform/methanol ratio of 2/l. Stirring was continued for 5 min under nitrogen. The insoluble residue was removed by filtration through a sintered-glass filter. All glassware and the residue were rinsed repeatedly with chloroform/methanol 2/l. The rinsings and the extracts were combined, solvents were evaporated, an aliquot for solids was weighed, and the volumes were measured accurately as described for erythrocytes (5). Removal
of Nonlipicl
Contaminants
Water-soluble nonlipid contaminants were separated from the lipids by Sephadex column chromatography (6) using the modifications described in the accompanying paper (5). Thin-Layer
Chromatography
Initially, silica gel H (Merck) with 10% (w/w) added magnesium silicate prepared as described previously (7) was used as the adsorbent for TLC. More recently, Silica Gel H with added magnesium acetate was used because improved and more reproducible resolution was obtained. The details of slurry preparation, chromatographic conditions, and solvent systems employed are described in the accompanying paper (5). Plasma total lipid extracts were chromatographed after separation of nonlipid contaminants by Sephadex column chromatography. Analysis
of Neutral
Lipids
after
TLC
Cholesterol, sterol ester, and triglycerides were separated by one-dimensional TLC using the hexane/ether solvent systems (5) ; the spots were visualized by spraying the plate with distilled water, circled, and, after the plate had dried, removed for analysis. Cholesterol and sterol ester were determined using the method of Hanel and Dam (8). Triglycerides were eluted from the adsorbent first, followed by spectrophotometric assay using a modified Carlson procedure (9).
ANALYSIS
Phosphorus
OF
HUMAN
Analysis
Determination in quadruplicate by a modification of the original accompanying paper (5).
PLASMA
439
LIPIDS
of Spots after
TLC
of phosphorus in spots was carried out procedure (1) that is described in the
Triethylaminoethylcellulose
Column
Chromatography
Standard-grade TEAE-cellulose (The Brown Co., Berlin, New Hampshire) was washed and dried, and 2.5 (i.d.) X 20 cm columns were prepared, converted to the hydroxyl form, and tested with azulene for column performance as described previously (i’,lO,ll). The bed was washed with 4 column volumes each of chloroform/methanol l/l and deoxygenated chloroform. The sample was then applied in chloroform and elution accomplished with chloroform, chloroform/methanol 9/l, and chloroform/ methanol 4/l containing aqueous ammonia and potassium acetate as described under “Results.”
PI ;3 11 12 -
“;.“ Or
(2)C/A/M/HAc/H20 3/4/l
/ l/O.5
FIG. 1. Two-dimensional TLC of a plasma total lipid extract (600 pg applied) after separation of nonlipid contaminants from other lipids by Sephadex column chromatography. See text for details. &characterized lipid classes are designated by numbers. Spots difficult to reproduce photographically are circled. Abbreviations for solvents : C = chloroform; M = methanol ; A = acetone; HAc = glacial acetic acid. Percentage of total lipid phosphorus for each lipid class -C standard deviation of four determinations: PE = phosphatidyl ethanolamine, 1.9 C 0.1; PC = phosphatidyl choline, 68.9 t 0.04; Sph = sphingomyelin, 16.6 * 02; LPC = lysophosphatidyl choline, 7.8 + 0.2; PI = phosphatidyl inositol, 1.7 t 0.1; LPE = lysophosphatidyl ethanolamine, 0.9 + 0.05 ; LPL = less polar lipid, 0.51 + 0.01; (1) 139 -I 0.02; (2) 0.16 + 0.01; (3) 0.00; (4) 0.00; (5) 0.00; (6) 0.00; (7) 0.06 + 0.05; (8) 0.00; (9) 0.00; (10) 0.00; (11) 0.13 -+ 0.01; (12) 0.24 f 0.01; (13) 0.04 -c 0.01; Or = origin, 0.19 + 0.01. Recovery of lipid phosphorus was 100.5%.
440
TURNER
AND
ROUSEX
RESULTS
Lipid Components Visualized by Direct TLC of Lipid Extracts
One-dimensional TLC of plasma total lipid extracts after separation of nonlipid contaminants by Sephadex column chromatography demonstrated free and esterified cholesterol and triglycerides to be the major less polar lipids in all plasma extracts. (BHT migrated just below sterol ester when using the hexane/diethyl ether solvent systems.) Two-dimensional TLC using alkaline magnesium silicate adsorbent gave excellent resolution of most of the polar lipids of plasma. Optimum resolution was achieved by using Silica Gel H (20 gm) spread as a slurry in water (60-65 ml) containing 1.5 gm magnesium acetate with chloroform/methanol/28% aqueous ammonia 65J25/5 in the first dimension and chloroform/acetone/methanol/acetic acid/water 3/4/1/l/0.5 in the second dimension (Fig. 1). In addition to the readily identifiable major lipid classes, a number of uncharacterized components were also noted TABLE 1 Major Lipid Components of Plasma from Healthy Human Subjects Fasting plasma” Lipid
Replicate A Phospholipid
Phosphatidyl ethanolamine Phosphatidyl choline Sphingomyelin Lysophosphatidyl choline Phosphatidyl inositol Lysophosphatidyl ethanolamine Minor component& Recovery of phosphorus (%) Total lipid P (mg/lOO ml)
1.21 72.33 14.02 7.59 2.04 0.26
f f f * * +
2.55 100.43 7.10 f 0.04 Neutral
Free cholesterol (mg/lOO ml) Ester cholesterol (mg/lOO ml) Triglycerides (mg/lOO ml)
0.03 0.67 0.31 0.16 0.05 0.02
41 103 52
Replicate B
Fasting plasma* mean values
classc
1.24 72.11 14.04 7.60 2.03 0.27
rf: 0.05 _+ 1.09 + 0.25 i 0.13 + 0.06 f 0.03
2.71 99.48 7.08 zk 0.03
1.55 68.49 16.58 7.53 1.62 1.05
* It f f f f
0.47 2.19 1.96 1.15 0.33 0.61
3.18 f 0.47 99.54 7.07 f 0.84
lipids
41 101 49
41 f 4.5 109 * 10.1 40 f 14.0
a H.F., 39 year old healthy male; standard deviation of 4 determinations. * Mean values for 9 healthy subjects (seven subjects aged 23-24 years, one 11 year old female, and one 39 year old marathon runner); standard deviation of 9 subjects. 0 Expressed as per cent total lipid phosphorus f standard deviations; includes diacyl, alkyl, and alkenyl forms of the phospholipid class. d See Table 2 for values for individual minor components.
N.P. N.P. N.D. 0.12 f 0.16 0.14 + 0.18 0.20 I!Z 0.11 3.23
0.04 + 0.02 0.47 f 0.38 N.P. 0.09 f 0.05 2.19
11 12 13 Origin Total minor components
0.07 0.15 0.05 0.16 2.30
N.P. N.P. * zk + f
0.08 0.02 0.06 0.11
0.56 + 0.29 1.28 f 0.11 N.D. 0.03 f 0.03 N.D. N.D. N.P. N.D. N.D.
0.10 0.17 0.03 0.13
f 0.06 + 0.03
zk 0.07
+ f + k
0.13 f 0.10 0.23 + 0.11 N.P. 0.18 f 0.10 3.19
N.P. N.D.
0.51 1.83 0.02 0.16 N.D. 0.04 N.P. 0.08 0.01
Date sample drawn 3/6/68 4/17/68 0.05 0.09 0.09 0.11
z!z 0.11
f f rt f
0.02 f 0.02 0.03 f 0.04 N.P. 0.13 rt 0.06 2.68
N.P. N.P.
0.64 1.49 0.06 0.11 N.D. 0.20 N.P. N.D. N.D.
Z/11/69
0.05 0.20 0.04 0.15 2.71
N.P. 0.00
0.53 1.48 0.07 0.07 0.02 0.06 0.01 0.03 0.004
0.10 0.27 0.00 0.23 3.18
0.00 0.00
0.79 1.33 0.05 0.19 0.03 0.08 0.01 0.07 0.03
f 0.14 f 0.47
+ 0.09 f 0.18
k! 5
+ 0.07 rt 0.08
E
i!
E E
8
% 5 2 u2 8 x
0.29 0.33 0.05 0.06 0.04 0.08
f + f f + k
Fasting plasma”
N.D. spot present but
rt: 0.05 & 0.17 5 0.06 + 0.04 + 0.49
ir: 0.08 _+ 0.41 4 0.06 _+ 0.06 + 0.03 + 0.08 + 0.01 f 0.04 zk 0.01
Mean + S.D.
Human Subjects
0 Expressed as per cent total lipid phosphorus f standard deviations. b G.S., 23 year old healthy female. c Mean values for 9 healthy subjects; standard deviations of values for 9 subjects. d Includes diacyl, alkyl, and alkenyl forms of phospholipid class; see Fig. 1 for location of spots. Abbreviations: did not contain detectable quantities of phosphorus; N-P., spot not present.
* 0.13 + 0.02
+ 0.13
f 0.17 + 0.36 + 0.21
N.P. N.P.
0.47 1.91 0.24 N.P. 0.07 N.D. N.P. 0.07 0.01
3/5/68
Fasting plasmab
Componentsa of Plasma from Healthy
9 10
* 0.79 f 0.17 f 0.01 f 0.04 2~ 0.03 rt 0.11 + 0.03
S/24/67
Phospholipid
0.49 0.90 0.02 0.05 0.04 0.06 0.03 N.D. N.P.
classd
2. Minor
Solvent front (less polar lipid) 1 2 3 4 5 6 7 8
Phospholipid
TABLE
442
TURNER
in total lipid extracts of plasma seen to be complex mixtures. Phospholipid
and Neutral
AND
ROUSER
(Fig. 1). Thus, plasma lipid extracts are Lipid
after TLC
Determination
Replicate and average values for the major phospholipid lipid phosphorus, cholesterol, sterol ester, and triglyceride in plasma are shown in Table 1 and minor phospholipid Table 2; values for an individual sample are given in Figure 1. Precautions necessary to obtain reproducibility determinations are described in the accompanying paper TEAE-Cellulose
Column
classes, total concentrations components in the legend for of phosphorus (5).
Chromatography
The most satisfactory elution sequence for plasma lipids (Table 3) was found to be chloroform, chloroform/methanol 9/l, and chloroform/ methanol 4/l containing salt and ammonia to give fractions composed of neutral lipid, choline lipid, and other phospholipids (Fig. 2). Only phosphatidyl ethanolamine, lysophosphatidyl ethanolamine, and phosphatidyl inositol could be identified with certainty in the final fraction. Many minor phospholipid components are seen in the last fraction. Some but not all of the uncharacterized components are visualized by direct TLC of total lipid extracts (Fig. 1). The major water-soluble phosphate esters (glycerylphosphorylglycerol, glycerylphosphorylglycerylphosElution
TABLE 3 of Plasma Lipids from TEAEXellulose
Fraction No.
(Hydroxyl
Eluting vdumesC
Solvent?
1
Chloroform
8
2
Chloroform/methanol
3d
0.1 M potassium acetate in chloroform/methanol 4/l containing 20 ml 2870 aq. ammonia/liler
9/l
8 8
Form) Columnsa
Lipid classes eluted (less polar) lipids (cholesterol, sterol esters, triglycerides, and BHT) Choline lipids (phosphatidyl choline, lysophosphatidyl choline, sphingomy elk) Other phospholipids (phosphatidyl ethanolamine, lysophosphatidyl ethanolamine, phosphatidyl inositol, uncharacterized components) Neutral
a See text for details. b BHT (0.1 mg/liter) added to all solvents; all solvents deoxygenated immediately prior to use. c In column volumes; 1 column volume about 75 ml for a 2.5 (i.d.) X 20 cm column. d Salt removed from fraction by Sephadex column chromatography.
ANALYSIS
OF
HUMAN
YLASMA
LIPIDS
TEAE COLUMN FRACTIONS
FIG. 2. Two-dimensional tions from human plasma for Fig. 1 for abbreviations. contain phosphorus.
2. C/A/M/HAc/H20
3141 I / I/ 0.5
TLC of TEAE-cellulose (hydroxyl form) column fraclipid extract. See text for details and discussion and legend The uncharacterized circled spots in fraction 2 did not
phorylglycerol) obtained by alkaline deacylation indicate the uncharacterized acidic lipids to be related to bisphosphatidic acid and diphosphatidyl glycerol. Spot 1 (Fig. 1) has the correct migration for semilysobisphosphatidic acid. Plasma lipid extracts gave different results when the TEAE elution sequence described for erythrocytes (5) was used. With plasma lipids, acidic phospholipids were found in the chloroform/methanol 2/l and all subsequent eluates. This failure of separation is similar to that observed for acidic lipid mixtures which have been converted entirely to the magnesium salts. DISCUSSION
Better resolution of polar lipids is obtained by two-dimensional TLC than by the commonly used one-dimensional procedure. Not only is it possible to separate all the major phospholipid classes present in plasma, but also a number of previously unrecognized minor components are visualized. A further increase in resolution and the detection and determination of more minor components is achieved by TEAE-cellulose (hydroxyl form) column chromatography followed by two-dimensional TLC. With the modified procedure for determination of phosphorus in spots after TLC (5)) improvement of sensitivity makes possible the routine and accurate analysis of even minor components. Plasma phospholipid values obtained by one-dimensiona TLC (12-15) are in relatively good agreement with our data for phosphatidyl ethanolamine, phosphatidyl choline, lysophosphatidyl choline, and sphingomyelin.
444
TURNER
AND
ROUSER
Most investigators have not reported values for the other lipid classes we have measured routinely with the high-resolution two-dimensional TLC system. Kunz and Kosin (15)) using a one-dimensional system, detected minor plasma phospholipid components the identifications of which do not agree with ours by two-dimensional TLC and column chromatography. We have not observed phosphatidic acid, diphosphatidyl glycerol (cardiolipin), or phosphatidyl serine in normal human plasma, whereas we regularly observe lysophosphatidyl ethanolamine. TLC adsorbent containing calcium sulfate was not found to give the compact spot size and resolution of silica gel mixed with magnesium silicate. Adsorbent containing magnesium silicate spread with water was introduced in 1964 (16) and improvement of resolution with some solvents was noted later (7) when the adsorbent was spread in a potassium hydroxide solution to introduce potassium silicate. We have obtained about the same resolution of plasma polar lipids with adsorbent containing either alkaline magnesium silicate or magnesium acetate, although complete separation of lysophosphatidyl ethanolamine is achieved more regularly with the latter. Minor phospholipid components are detected with both. In contrast, Broekhuyse (17) using alkaline magnesium silicate but different solvent mixtures, failed to obtain the same high resolution and detected only a few minor components. Careful matching of adsorbent, activity (water content), and solvent proportions is thus stressed as essential. SUMMARY
Two-dimensional thin-layer chromatography (TLC) alone or following ion-exchange (TEAE) cellulose column chromatography is shown to provide improved separation of human plasma polar lipids. With both procedures, a number of uncharacterized phospholipids occurring in small amounts are detectable in human plasma. Determination in quadruplicate of phosphorus in spots after two-dimensional TLC is shown to be a rapid, reproducible procedure for accurate analysis of plasma phospholipid composition, standard deviations for major components being in the l-2% range. ACKNOWLEDGMENTS This work was supported in part by U. S. Public FR-65425, and NS-66237, a grant from the Houston from Mr. Ben May of Mobile, Alabama. The authors gratefully acknowledge the technical Ince, Jauhree Sparks, and Richard Baldwin.
Health Heart
Service Grants HE-05435, Association, and support
assistance
of Pamela
REFERENCES 1. ROUSER, 2: NELSON,
G.,
SIAKOTOS,
G. J., Lipids
A. N., AND FLEISCHER, 2, 323 (1967).
S., Lipids
1, 85 (1966).
King,
Ann
ANALYSIS
OF
HUMAN
PLASMA
445
LIPIDS
3. NELSON,
G. J., Lipids 2, 64 (1967). 4. NELSON, G. J., Biochim. Biophys. Actu 144, 221 (1967). 5. TURNER, J. D., AND ROUSER, G., Anal. Biochem. 38, 423 (1970). 6. SIAKOTOS, A. N., AND ~IJSER, G., J. Am. Oil Chemists’ Sot. 42, 913 (1965). 7. ROUSER, G., ~ITCHEVSKY, G., AND YAMAMOTO, A., in ‘Lipid Chromatographic Analysis”
(G. V. Marinetti,
ed.), Vol. I, p. 99. Dekker, New York,
8. HANEL, H. K., AND DAM, H., Acta Chem. Stand. 9, 677 (1955). 9. CARLSON, L. A., J. Atheroscler. Res. 3, 334 (1963). 10. ROUSER, G., KRITCHEVSKY, G., HELLER, D., AND LIEBER, E., J. Am. Oil
1967.
Chemists’
Sot. 40, 425 (1963). 11. SIMON,
G., AND R,QUSER, G., Lipids 4, 607 (1969). 12. VIKROT, O., Acta Med. Sand. 175, 442 (1964). 13. H~~GDAHL, A., AND VIKROT, O., Acta Med. Scund. 178, 637 (1965). 14. GJONE, R., AND ORNING, 0. M., &and. J. Clin. Lab. Invest. 18, 209 (1966). 15. KUNZ, F., AND KOSIN, D., Wiener Klin. Wochschr. 41, 764 (1968). 16. ROUSER, G., GALLI, C., LIEBER, E., BLANK, M. L., AND PRIVETT, 0. S., J. Am. Chemists’ Sot. 41, 836 (1964). 17. BROEKHUYSE, R. M., Clin. Chim. Actu 23, 457 (1969).
Oil
BIOCHEMISTRY
Precise
Quantitative by
38, 437-445 (1970)
Determination
Thin-Layer
and Column
of Human
Blood
Lipids
Triethylaminoethylcellulose Chromatography
II. Plasma Lipids JOHN D. TURNER Department
of
Medicine,
Baylor
College
of Medicine,
Houston,
Texas
Duarte,
California
770%
AND
GEORGE Division
of Neurosciences, City
of Hope
ROUSER Medical Center,
91010
ReceivedMarch 25, 1970 Two-dimensional thin-layer chromatography (TLC) originally developed for the quantitative analysis of brain lipids (1) has been applied to plasma and erythrocytes of various mammalian species (24). In an accompanying paper (5), we describe improved resolution of human red cell lipids by the use of two-dimensional TLC alone or combined with ion-exchange cellulose column chromatography. In this report, we describe the application of these procedures with modifications necessary to obtain accurate and precise quantitative analysis of human plasma lipids. MAl’ERIALS
AND METHODS
Separation of Human Plasma and Erythrocytes Blood was obtained from healthy human subjects after an overnight fast. Disodium ethylenediaminetetrartcetate (EDTA, 1 mg/ml of whole blood) was used as anticoagulant. The blood was centrifuged at 1500g for 10 min (4’C). The plasma was removed and centrifuged again at 7700g for 10 min (4°C). The supernatant plasma was decanted and either extracted immediately or frozen and stored (-20’ or -70°C) until extracted. When erythrocytes were to be harvested for washing, an initial centrifugation at 809 for 15 min (4°C) was used, the platelet-rich plasma (PRP) transferred to another tube, and the cells and PRP centrifuged 437
438
TURNER
AND
ROUSER
again at 1500g for 10 min (4°C). The pooled plasma was then subjected to 77OOg for 10 min (4%) while the red cells were washed as described in the accompanying paper (5). Lipid, Extraction
Analytical-grade solvents were redistilled in glass and deoxygenated immediately prior to use. The antioxidant butylated hydroxytoluene (BHT) was added to solvent (0.1 mg/liter) used for extraction. For extraction of plasma lipids (2)) each milliliter of plasma was added to 8.3 ml of cold methanol in a flask (previously flushed with nitrogen) in an ice bucket containing wet ice. The mixture was stirred magnetically with a Teflon-coated stirring bar for 1 min and 16.7 ml of cold chloroform was added to give a final chloroform/methanol ratio of 2/l. Stirring was continued for 5 min under nitrogen. The insoluble residue was removed by filtration through a sintered-glass filter. All glassware and the residue were rinsed repeatedly with chloroform/methanol 2/l. The rinsings and the extracts were combined, solvents were evaporated, an aliquot for solids was weighed, and the volumes were measured accurately as described for erythrocytes (5). Removal
of Nonlipicl
Contaminants
Water-soluble nonlipid contaminants were separated from the lipids by Sephadex column chromatography (6) using the modifications described in the accompanying paper (5). Thin-Layer
Chromatography
Initially, silica gel H (Merck) with 10% (w/w) added magnesium silicate prepared as described previously (7) was used as the adsorbent for TLC. More recently, Silica Gel H with added magnesium acetate was used because improved and more reproducible resolution was obtained. The details of slurry preparation, chromatographic conditions, and solvent systems employed are described in the accompanying paper (5). Plasma total lipid extracts were chromatographed after separation of nonlipid contaminants by Sephadex column chromatography. Analysis
of Neutral
Lipids
after
TLC
Cholesterol, sterol ester, and triglycerides were separated by one-dimensional TLC using the hexane/ether solvent systems (5) ; the spots were visualized by spraying the plate with distilled water, circled, and, after the plate had dried, removed for analysis. Cholesterol and sterol ester were determined using the method of Hanel and Dam (8). Triglycerides were eluted from the adsorbent first, followed by spectrophotometric assay using a modified Carlson procedure (9).
ANALYSIS
Phosphorus
OF
HUMAN
Analysis
Determination in quadruplicate by a modification of the original accompanying paper (5).
PLASMA
439
LIPIDS
of Spots after
TLC
of phosphorus in spots was carried out procedure (1) that is described in the
Triethylaminoethylcellulose
Column
Chromatography
Standard-grade TEAE-cellulose (The Brown Co., Berlin, New Hampshire) was washed and dried, and 2.5 (i.d.) X 20 cm columns were prepared, converted to the hydroxyl form, and tested with azulene for column performance as described previously (i’,lO,ll). The bed was washed with 4 column volumes each of chloroform/methanol l/l and deoxygenated chloroform. The sample was then applied in chloroform and elution accomplished with chloroform, chloroform/methanol 9/l, and chloroform/ methanol 4/l containing aqueous ammonia and potassium acetate as described under “Results.”
PI ;3 11 12 -
“;.“ Or
(2)C/A/M/HAc/H20 3/4/l
/ l/O.5
FIG. 1. Two-dimensional TLC of a plasma total lipid extract (600 pg applied) after separation of nonlipid contaminants from other lipids by Sephadex column chromatography. See text for details. &characterized lipid classes are designated by numbers. Spots difficult to reproduce photographically are circled. Abbreviations for solvents : C = chloroform; M = methanol ; A = acetone; HAc = glacial acetic acid. Percentage of total lipid phosphorus for each lipid class -C standard deviation of four determinations: PE = phosphatidyl ethanolamine, 1.9 C 0.1; PC = phosphatidyl choline, 68.9 t 0.04; Sph = sphingomyelin, 16.6 * 02; LPC = lysophosphatidyl choline, 7.8 + 0.2; PI = phosphatidyl inositol, 1.7 t 0.1; LPE = lysophosphatidyl ethanolamine, 0.9 + 0.05 ; LPL = less polar lipid, 0.51 + 0.01; (1) 139 -I 0.02; (2) 0.16 + 0.01; (3) 0.00; (4) 0.00; (5) 0.00; (6) 0.00; (7) 0.06 + 0.05; (8) 0.00; (9) 0.00; (10) 0.00; (11) 0.13 -+ 0.01; (12) 0.24 f 0.01; (13) 0.04 -c 0.01; Or = origin, 0.19 + 0.01. Recovery of lipid phosphorus was 100.5%.
440
TURNER
AND
ROUSEX
RESULTS
Lipid Components Visualized by Direct TLC of Lipid Extracts
One-dimensional TLC of plasma total lipid extracts after separation of nonlipid contaminants by Sephadex column chromatography demonstrated free and esterified cholesterol and triglycerides to be the major less polar lipids in all plasma extracts. (BHT migrated just below sterol ester when using the hexane/diethyl ether solvent systems.) Two-dimensional TLC using alkaline magnesium silicate adsorbent gave excellent resolution of most of the polar lipids of plasma. Optimum resolution was achieved by using Silica Gel H (20 gm) spread as a slurry in water (60-65 ml) containing 1.5 gm magnesium acetate with chloroform/methanol/28% aqueous ammonia 65J25/5 in the first dimension and chloroform/acetone/methanol/acetic acid/water 3/4/1/l/0.5 in the second dimension (Fig. 1). In addition to the readily identifiable major lipid classes, a number of uncharacterized components were also noted TABLE 1 Major Lipid Components of Plasma from Healthy Human Subjects Fasting plasma” Lipid
Replicate A Phospholipid
Phosphatidyl ethanolamine Phosphatidyl choline Sphingomyelin Lysophosphatidyl choline Phosphatidyl inositol Lysophosphatidyl ethanolamine Minor component& Recovery of phosphorus (%) Total lipid P (mg/lOO ml)
1.21 72.33 14.02 7.59 2.04 0.26
f f f * * +
2.55 100.43 7.10 f 0.04 Neutral
Free cholesterol (mg/lOO ml) Ester cholesterol (mg/lOO ml) Triglycerides (mg/lOO ml)
0.03 0.67 0.31 0.16 0.05 0.02
41 103 52
Replicate B
Fasting plasma* mean values
classc
1.24 72.11 14.04 7.60 2.03 0.27
rf: 0.05 _+ 1.09 + 0.25 i 0.13 + 0.06 f 0.03
2.71 99.48 7.08 zk 0.03
1.55 68.49 16.58 7.53 1.62 1.05
* It f f f f
0.47 2.19 1.96 1.15 0.33 0.61
3.18 f 0.47 99.54 7.07 f 0.84
lipids
41 101 49
41 f 4.5 109 * 10.1 40 f 14.0
a H.F., 39 year old healthy male; standard deviation of 4 determinations. * Mean values for 9 healthy subjects (seven subjects aged 23-24 years, one 11 year old female, and one 39 year old marathon runner); standard deviation of 9 subjects. 0 Expressed as per cent total lipid phosphorus f standard deviations; includes diacyl, alkyl, and alkenyl forms of the phospholipid class. d See Table 2 for values for individual minor components.
N.P. N.P. N.D. 0.12 f 0.16 0.14 + 0.18 0.20 I!Z 0.11 3.23
0.04 + 0.02 0.47 f 0.38 N.P. 0.09 f 0.05 2.19
11 12 13 Origin Total minor components
0.07 0.15 0.05 0.16 2.30
N.P. N.P. * zk + f
0.08 0.02 0.06 0.11
0.56 + 0.29 1.28 f 0.11 N.D. 0.03 f 0.03 N.D. N.D. N.P. N.D. N.D.
0.10 0.17 0.03 0.13
f 0.06 + 0.03
zk 0.07
+ f + k
0.13 f 0.10 0.23 + 0.11 N.P. 0.18 f 0.10 3.19
N.P. N.D.
0.51 1.83 0.02 0.16 N.D. 0.04 N.P. 0.08 0.01
Date sample drawn 3/6/68 4/17/68 0.05 0.09 0.09 0.11
z!z 0.11
f f rt f
0.02 f 0.02 0.03 f 0.04 N.P. 0.13 rt 0.06 2.68
N.P. N.P.
0.64 1.49 0.06 0.11 N.D. 0.20 N.P. N.D. N.D.
Z/11/69
0.05 0.20 0.04 0.15 2.71
N.P. 0.00
0.53 1.48 0.07 0.07 0.02 0.06 0.01 0.03 0.004
0.10 0.27 0.00 0.23 3.18
0.00 0.00
0.79 1.33 0.05 0.19 0.03 0.08 0.01 0.07 0.03
f 0.14 f 0.47
+ 0.09 f 0.18
k! 5
+ 0.07 rt 0.08
E
i!
E E
8
% 5 2 u2 8 x
0.29 0.33 0.05 0.06 0.04 0.08
f + f f + k
Fasting plasma”
N.D. spot present but
rt: 0.05 & 0.17 5 0.06 + 0.04 + 0.49
ir: 0.08 _+ 0.41 4 0.06 _+ 0.06 + 0.03 + 0.08 + 0.01 f 0.04 zk 0.01
Mean + S.D.
Human Subjects
0 Expressed as per cent total lipid phosphorus f standard deviations. b G.S., 23 year old healthy female. c Mean values for 9 healthy subjects; standard deviations of values for 9 subjects. d Includes diacyl, alkyl, and alkenyl forms of phospholipid class; see Fig. 1 for location of spots. Abbreviations: did not contain detectable quantities of phosphorus; N-P., spot not present.
* 0.13 + 0.02
+ 0.13
f 0.17 + 0.36 + 0.21
N.P. N.P.
0.47 1.91 0.24 N.P. 0.07 N.D. N.P. 0.07 0.01
3/5/68
Fasting plasmab
Componentsa of Plasma from Healthy
9 10
* 0.79 f 0.17 f 0.01 f 0.04 2~ 0.03 rt 0.11 + 0.03
S/24/67
Phospholipid
0.49 0.90 0.02 0.05 0.04 0.06 0.03 N.D. N.P.
classd
2. Minor
Solvent front (less polar lipid) 1 2 3 4 5 6 7 8
Phospholipid
TABLE
442
TURNER
in total lipid extracts of plasma seen to be complex mixtures. Phospholipid
and Neutral
AND
ROUSER
(Fig. 1). Thus, plasma lipid extracts are Lipid
after TLC
Determination
Replicate and average values for the major phospholipid lipid phosphorus, cholesterol, sterol ester, and triglyceride in plasma are shown in Table 1 and minor phospholipid Table 2; values for an individual sample are given in Figure 1. Precautions necessary to obtain reproducibility determinations are described in the accompanying paper TEAE-Cellulose
Column
classes, total concentrations components in the legend for of phosphorus (5).
Chromatography
The most satisfactory elution sequence for plasma lipids (Table 3) was found to be chloroform, chloroform/methanol 9/l, and chloroform/ methanol 4/l containing salt and ammonia to give fractions composed of neutral lipid, choline lipid, and other phospholipids (Fig. 2). Only phosphatidyl ethanolamine, lysophosphatidyl ethanolamine, and phosphatidyl inositol could be identified with certainty in the final fraction. Many minor phospholipid components are seen in the last fraction. Some but not all of the uncharacterized components are visualized by direct TLC of total lipid extracts (Fig. 1). The major water-soluble phosphate esters (glycerylphosphorylglycerol, glycerylphosphorylglycerylphosElution
TABLE 3 of Plasma Lipids from TEAEXellulose
Fraction No.
(Hydroxyl
Eluting vdumesC
Solvent?
1
Chloroform
8
2
Chloroform/methanol
3d
0.1 M potassium acetate in chloroform/methanol 4/l containing 20 ml 2870 aq. ammonia/liler
9/l
8 8
Form) Columnsa
Lipid classes eluted (less polar) lipids (cholesterol, sterol esters, triglycerides, and BHT) Choline lipids (phosphatidyl choline, lysophosphatidyl choline, sphingomy elk) Other phospholipids (phosphatidyl ethanolamine, lysophosphatidyl ethanolamine, phosphatidyl inositol, uncharacterized components) Neutral
a See text for details. b BHT (0.1 mg/liter) added to all solvents; all solvents deoxygenated immediately prior to use. c In column volumes; 1 column volume about 75 ml for a 2.5 (i.d.) X 20 cm column. d Salt removed from fraction by Sephadex column chromatography.
ANALYSIS
OF
HUMAN
YLASMA
LIPIDS
TEAE COLUMN FRACTIONS
FIG. 2. Two-dimensional tions from human plasma for Fig. 1 for abbreviations. contain phosphorus.
2. C/A/M/HAc/H20
3141 I / I/ 0.5
TLC of TEAE-cellulose (hydroxyl form) column fraclipid extract. See text for details and discussion and legend The uncharacterized circled spots in fraction 2 did not
phorylglycerol) obtained by alkaline deacylation indicate the uncharacterized acidic lipids to be related to bisphosphatidic acid and diphosphatidyl glycerol. Spot 1 (Fig. 1) has the correct migration for semilysobisphosphatidic acid. Plasma lipid extracts gave different results when the TEAE elution sequence described for erythrocytes (5) was used. With plasma lipids, acidic phospholipids were found in the chloroform/methanol 2/l and all subsequent eluates. This failure of separation is similar to that observed for acidic lipid mixtures which have been converted entirely to the magnesium salts. DISCUSSION
Better resolution of polar lipids is obtained by two-dimensional TLC than by the commonly used one-dimensional procedure. Not only is it possible to separate all the major phospholipid classes present in plasma, but also a number of previously unrecognized minor components are visualized. A further increase in resolution and the detection and determination of more minor components is achieved by TEAE-cellulose (hydroxyl form) column chromatography followed by two-dimensional TLC. With the modified procedure for determination of phosphorus in spots after TLC (5)) improvement of sensitivity makes possible the routine and accurate analysis of even minor components. Plasma phospholipid values obtained by one-dimensiona TLC (12-15) are in relatively good agreement with our data for phosphatidyl ethanolamine, phosphatidyl choline, lysophosphatidyl choline, and sphingomyelin.
444
TURNER
AND
ROUSER
Most investigators have not reported values for the other lipid classes we have measured routinely with the high-resolution two-dimensional TLC system. Kunz and Kosin (15)) using a one-dimensional system, detected minor plasma phospholipid components the identifications of which do not agree with ours by two-dimensional TLC and column chromatography. We have not observed phosphatidic acid, diphosphatidyl glycerol (cardiolipin), or phosphatidyl serine in normal human plasma, whereas we regularly observe lysophosphatidyl ethanolamine. TLC adsorbent containing calcium sulfate was not found to give the compact spot size and resolution of silica gel mixed with magnesium silicate. Adsorbent containing magnesium silicate spread with water was introduced in 1964 (16) and improvement of resolution with some solvents was noted later (7) when the adsorbent was spread in a potassium hydroxide solution to introduce potassium silicate. We have obtained about the same resolution of plasma polar lipids with adsorbent containing either alkaline magnesium silicate or magnesium acetate, although complete separation of lysophosphatidyl ethanolamine is achieved more regularly with the latter. Minor phospholipid components are detected with both. In contrast, Broekhuyse (17) using alkaline magnesium silicate but different solvent mixtures, failed to obtain the same high resolution and detected only a few minor components. Careful matching of adsorbent, activity (water content), and solvent proportions is thus stressed as essential. SUMMARY
Two-dimensional thin-layer chromatography (TLC) alone or following ion-exchange (TEAE) cellulose column chromatography is shown to provide improved separation of human plasma polar lipids. With both procedures, a number of uncharacterized phospholipids occurring in small amounts are detectable in human plasma. Determination in quadruplicate of phosphorus in spots after two-dimensional TLC is shown to be a rapid, reproducible procedure for accurate analysis of plasma phospholipid composition, standard deviations for major components being in the l-2% range. ACKNOWLEDGMENTS This work was supported in part by U. S. Public FR-65425, and NS-66237, a grant from the Houston from Mr. Ben May of Mobile, Alabama. The authors gratefully acknowledge the technical Ince, Jauhree Sparks, and Richard Baldwin.
Health Heart
Service Grants HE-05435, Association, and support
assistance
of Pamela
REFERENCES 1. ROUSER, 2: NELSON,
G.,
SIAKOTOS,
G. J., Lipids
A. N., AND FLEISCHER, 2, 323 (1967).
S., Lipids
1, 85 (1966).
King,
Ann
ANALYSIS
OF
HUMAN
PLASMA
445
LIPIDS
3. NELSON,
G. J., Lipids 2, 64 (1967). 4. NELSON, G. J., Biochim. Biophys. Actu 144, 221 (1967). 5. TURNER, J. D., AND ROUSER, G., Anal. Biochem. 38, 423 (1970). 6. SIAKOTOS, A. N., AND ~IJSER, G., J. Am. Oil Chemists’ Sot. 42, 913 (1965). 7. ROUSER, G., ~ITCHEVSKY, G., AND YAMAMOTO, A., in ‘Lipid Chromatographic Analysis”
(G. V. Marinetti,
ed.), Vol. I, p. 99. Dekker, New York,
8. HANEL, H. K., AND DAM, H., Acta Chem. Stand. 9, 677 (1955). 9. CARLSON, L. A., J. Atheroscler. Res. 3, 334 (1963). 10. ROUSER, G., KRITCHEVSKY, G., HELLER, D., AND LIEBER, E., J. Am. Oil
1967.
Chemists’
Sot. 40, 425 (1963). 11. SIMON,
G., AND R,QUSER, G., Lipids 4, 607 (1969). 12. VIKROT, O., Acta Med. Sand. 175, 442 (1964). 13. H~~GDAHL, A., AND VIKROT, O., Acta Med. Scund. 178, 637 (1965). 14. GJONE, R., AND ORNING, 0. M., &and. J. Clin. Lab. Invest. 18, 209 (1966). 15. KUNZ, F., AND KOSIN, D., Wiener Klin. Wochschr. 41, 764 (1968). 16. ROUSER, G., GALLI, C., LIEBER, E., BLANK, M. L., AND PRIVETT, 0. S., J. Am. Chemists’ Sot. 41, 836 (1964). 17. BROEKHUYSE, R. M., Clin. Chim. Actu 23, 457 (1969).
Oil