A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e , V o l . 3 , N o . 2 C o p y r ig h t © 1 9 7 3 , I n s t i t u t e f o r C l in ic a l S c ie n c e

Separation o f Plasm a Lipids by Thin-Layer Chrom atography

MARGE BREWSTER, Ph.D. AND HOWARD QUITTNER, M.D.

University of Arkansas Medical Center, Little Rock, AR 72201

ABSTRACT

A rapid comprehensive thin-layer chromatographic separation of the major plasma lipid classes on commercial silicic acid glass fiber paper is described. For screening purposes, oxidized fractions are rapidly visualized for qualitative interpretation. Standard colorimetric procedures for each lipid class are per­formed upon the migration areas cut from the strips. This provides quantitative measurements from only 0.03 ml of plasma. The separation procedure is easily accomplished in the routine laboratory and quantitation is practical in batch analyses.

In trod u ction

Lipid assays performed directly on plasma or serum have been limited to total cholesterol1’17 and to free cholesterol after precipitation as the digitonide.16 Phospho­lipids have been determined as inorganic phosphate present in a Folch extract7 of plasma following strong acid hydrolysis.8 Triglycerides have been assayed as esters14 or as free glycerol after saponification.6 Be­cause of phospholipid interference in both types of triglyceride assays, procedures have included an absorbent treatment to remove phospholipids15 or have employed selective extraction of triglycerides into non-polar solvents such as heptane, nonane or isopropanol, which do not extract phos­pholipids.11

The work-time and sample volumes re­quired for a complete set of assays usually limits the analysis of plasma lipids to only one or two types of determinations.

Thin-layer chromatographic separation of serum lipid components has also been uti­lized in research.10’13 Since these techniques have entailed thin-layer plate preparation and lengthy elutions before quantitation, adoption as routine clinical laboratory pro­cedures has not occurred.

To measure the major plasma lipids rapidly and comprehensively, a thin-layer separation on commercial silicic acid glass- fiber sheets and quantitation of these sepa­rated classes in situ by colorimetric tech­niques has been developed.

Principle

A Folch extract of serum is separated into its neutral lipid components by means of a silicic acid impregnated glass-fiber medium in an ascending chromatographic system. The components are visualized by iodine vapors, marked, and cut out with scissors. Free and esterified cholesterol and phos-

7 9

8 0 B R E W S T E R AND Q U IT T N E R

EsterifiedCholesterol

Triglyceride

Diglyceride

Free Fatty Acid_

F i g u r e 1. Thin-layer separation of plasma lipid extracts on ITLC-SA as described in text. Patients show (left to right): ele­vated cholesterol esters, triglycerides and free cho­lesterol; normal; elevated cholesterol esters and free cholesterol, slightly ele­vated triglyceride.

Free Cholesterol

Monoglyceride

Phospholipid —

pholipids are determined directly from the fiber spots. Triglycerides are eluted and then assayed colorimetrically.

R e a g e n ts

Chromatography solvent (hexane: di­ethyl ether: glacial acetic acid, 100 :8 :2 , v /v /v ) diethyl ether* is purchased in small cans. The solvent is prepared just before use and discarded after one hour.

Standard solutions are dissolved in chlo­roform at a concentration of 10 mg per ml.f Standards are stored tightly capped at 4°C but are brought to room temperature before measuring.

Ammonium sulfate solution, 10 g per dl.

* Baker.f Cholesterol oleate, cholesterol, tristearin, lec­

ithin, and oleic acid may be obtained from Applied Science Labs, State College, PA.

S p e c ia l A p p a r a tu sThe following are needed: (1 ) chroma­

tography chamber,® (2 ) silicic acid— glass fiber sheets 5 X 20 cm and 20 X 20 cm,f(3 ) developing chamber with cover for 20 X 20 cm plates,! (4 ) disposable 5 jul pipettes,§ (5 ) drying oven to operate at 80 to 100°C and 180°C, (6 ) heating blocks 60°C and 180 to 200°C,|| (7 ) centrifuge tubes, conical screw cap, graduatedll and screw cap with teflon liner** and (8 ) liquid scintillation vials with foil-lined screw caps.if

* Gelman #51325 , Gelman Instrument Co., Ann Arbor, MI.

f Gelman ITLC-SA , # 5 1 4 3 3 and #51432 .| Scientific Products #C 5310-30.§ DisPo capillary pipets, Scientific Products

#P4518-5.¡1 Lab-Line Instruments, Inc., Melrose Park, IL .If Kimble #45166.00 Kimble #45066-C .i f Kimble #74500 .

T H IN -L A Y E R SEP A R A TIO N O F PL A SM A L IP ID S 8 1

ProcedureSe p a r a t io n

Plasma or serum is extracted as per Bragdon3 and the extract is stored at 4°C, tightly-capped until used. By this proce­dure, the lipids of 0.5 ml plasma are ob­tained in 9 ml chloroform.

One hundred to 500 ¡¿g lipid (usually 0.5 to 1 ml extract) is dried in a glass centri­fuge tube at 60°C under N2. The dried lipid is dissolved in 5 to 10 ¡A chloroform. This volume is applied as a 1 cm streak to an ITLC-SA sheet, 3 cm from the lower edge, using a 5 /xl disposable pipette. More chloroform is added to the tube, and this aliquot is added to the one already spotted. This procedure is repeated until approx­imately 25 fj, 1 are applied. In adjacent 1 cm streaks, standards are applied. On a 20 X 20 cm sheet, it is advisable to leave 1 to 1.5 cm free at each lateral edge and to allow 1 cm between samples, allowing 9 samples to be run simultaneously.

While the applied samples are being air- dried, the chromatography solvent is pre­pared ( enough to bring the solvent level to a depth of 2 cm in the chamber). The sheet is placed in the chamber and removed after exactly 20 minutes of migration. It is imme­diately dried in an 80 to 100° C oven to prevent diffusion of the spots.

D e t e r m in a t io n

The sheet is placed in a covered chroma­tography chamber containing several iodine crystals. Within 5 minutes, each lipid frac­tion can be seen (figure 1). The strip is removed from the chamber, and the spots are quickly circled with a pencil. The iodine is allowed to volatilize at room tem­perature for 1 hour or in an oven at 80 to 100°C for 15 minutes. Blank areas are also circled and carried through each assay. The circled areas are cut with scissors into strips approximately 4 x 6 mm. Esterified choles­terol, free cholesterol and phospholipid strips are placed in screw-cap test tubes

to be used for each assay. The spots are handled with forceps to avoid contamina­tion with skin lipids.

Triglyceride strips are placed in counting vials containing 3 ml chloroform, capped, mixed, and allowed to stand at room tem­perature 30 to 60 minutes. Vials containing strips of a blank area and 150 or 300 /xg triolein standards in 3 ml chloroform are similarly prepared. Two ml of each chloro­form eluate are transferred to a screw-cap test tube and dried under N2 at 60° C. Tri­glycerides are determined by the ferric hy- droxamate colorimetric procedure.14

Free and esterified cholesterol strips of unknowns, blanks and 50 and 100 ¡>,g cho­lesterol standards are assayed directly for cholesterol by the Parekh-Jung procedure.9 After color development, the tubes are cen­trifuged at 5000 RPM for 10 minutes, and the supernatant fluid is poured into cuvets. Phospholipid strips of unknowns and blank, along with 2 and 5 fig inorganic phosphate standards, are hydrolyzed in a heating block (180 to 200°C) with perchloric acid. Phosphate is then determined as molybde­num blue by the Fiske-SubbaRow tech­nique.8 After color development, the tubes are centrifuged at 5000 RPM for 10 min­utes, and the supernatant fluid is poured into cuvets. Assayed phosphate is converted to phospholipid by multiplying X 25.*

R e c o v e r y

If it is desired, the accuracy of these determinations can be checked by assaying an 0.5 ml aliquot of liquid extract for total lipid by the dichromate method.4

Recovery (R ) is calculated as:

FC + EC X 1.87t + TG + PLR X 100Total Lipid FC = free cholesterol, EC = esterified cholesterol, TG = triglyceride and PL = phospholipid.* Molecular weight ratio of lecithin: phospho­

rous.f Molecular weight ratio of cholesterol oleate:

cholesterol.

82 B R E W S T E R AND Q U IT T N E R

F

■mg M

B

'IP

•c

f

■m

B

o f l B

*

A A *

i I I I I I

F i g u r e 2 . E ffect of ether on lipid separation. H exane:ace­tic acid = 100 ml: 2 ml in all three solvents. Ether content = 11 ml ( I ) , 8 ml ( I I ) , 5 ml ( I I I ). A = phospholipids, B = free cholesterol, C — free fatty acids, D = triglycerides, E = esterified cholesterol and F = solvent front.

Recovery should be between 90 and 100 percent if the assays are correct.

S c r e e n i n g P r o c e d u r e

If quantitative values are not required ( i.e., if it is only important to know whether or not triglycerides are normal or elevated), the thin-layer sheet is migrated and dried as described in the separation section. It is then dipped in 10 percent ammonium sul­fate for five minutes, drained and imme­diately suspended in an oven at 180°C for 15 minutes. This procedure chars all of the lipid fractions present2 and relative amounts of each lipid fraction can be estimated.

S o u rc e s o f E r ro rThe only significant sources of error arise

from inattention to meticulous techniques in the preparation of the lipid extracts ( glassware preparation, specimen handling,

storage, etc.). Storage of lipid extracts for more than two weeks leads to a significant increase in diglycerides with an equivalent decrease in triglycerides. This hydrolysis is accelerated in specimens with marked ele­vations of triglyceride.

D isc u ss io nIn figure 1 is shown the separation ob­

tained from plasma extracts of three pa­tients. As much as two mg of lipid can be separated, although 100 to 500 /xg is rec­ommended.

The silicic acid sheets change with age, and the amount of ether in the chromatog­raphy solvent can be varied to achieve the best separation. In figure 2 is illustrated the effect of varying ether proportions with fresh sheets. As can be seen in solvent I, too much ether causes cholesterol esters to run into the solvent front with the hydro-

T H IN -L A Y E R SEP A R A TIO N O F PL A SM A LI1MUS 8 3

100

50

F i g u r e 3. Concentration- vs-density for various lip-

were spotted on a sheet of ITLC-SA, ammonium sul­fate dipped and charred as described in the text.

10

vs-density tor various lip- ids. Pure lipid standards 25

snntfpirl nn a slippf nf

• m •

«D O

Lecithin Tristearin Cholesterol Cholesterol CholesterolPalmiate Oleate

carbons and very apolar plate contami­nants. Too little ether, as seen with solvent III, allows minimal separation of triglyc­eride and free cholesterol. Extremely ele­vated triglycerides would merge these two bands. Solvent II is the one chosen for optimum separation.

The ferric hydroxamate procedure was selected for triglyceride assay because it yields nominal blank values with ITLC-SA sheets. Although the assay utilizing saponi­fication, periodate oxidation, and Hantzch condensation12 is more sensitive, high blanks render it unsatisfactory.

Efforts to quantitate lipid fractions by densitometry of ammonium sulfate charred strips have so far been unsuccessful. In fig­ure 3 is emphasized the variability in den- sity-vs-concentration relationships for each lipid fraction. Using a double-beam scan­ning densitometer* in either the reflectance or transmittance mode, non-linear curves are obtained for all fractions except free

* Schoeffel #SD-3000.

cholesterol. The procedure can be used quantitatively only if numerous standards are included in each run and a manual graph constructed for each lipid fraction. For rapid screening, the charring method works quite well. It is especially useful for fluid assays, such as pleural fluid, when type rather than amount of lipid is re­quired.

By the screening technique, as little as five /¿g of each lipid can be seen ( figure 3 ). The quantitative colorimetric techniques require that a minimum of 0.5 ml lipid extract be applied to the thin-layer. This represents the extract of approximately 0.03 ml of normal plasma. ( One ml plasma yields 18 ml lipid extract.)

The technique is sensitive, only one ex­traction is required and the ITLC-SA sheets are easily cut for assays. It is routinely used in our laboratory for the determination of individual lipoprotein lipids5 and for in- depth studies of disease processes where all the lipids are to be measured.

8 4 B R E W S T E R AND Q U IT T N E R

R esu m eThis method is offered as a prospective

approach to a rapid, sensitive, comprehen­sive analysis of the major lipid components of serum. The technique is applicable to other samples such as amniotic fluid, pleu­ral fluid, leukocytes, tissue culture cells or isolated lipoproteins.

AcknowledgmentAppreciation is expressed to Mrs. Marion Mori-

arty, MT(ASCP) and Mrs. Ann Kane, MT(ASCP) for their technical assistance.

R eferences1 . A b e l l , L . L . , L e v y , B . B . , B r o d i e , B . B . , a n d

K e n d a l l , F . E.: A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J. Biol. Chem. 195:357-366, 1952.

2. B o r o w s k i , E. a n d Z i m in s k i , T.: A new spray reagent replacing sulfuric acid in thin-layer chromatography. J. Chromatog. 23:480-482,1966.

3. B r a c d o n , J. H.: Extraction of lipids from serum. Lipids and the Steroid Hormones in Clinical Medicine, Sunderman, F. W. and Sunderman, F. W., Jr., eds. Lippincott, Phila­delphia, pp. 6-8, 1960.

4. B r a g d o n , J. H.: Colorimetric determination of blood lipids. J. Biol. Chem. 190:513-517,1951.

5. B r e w s t e r , M.: Lipoprotein fractionation by column chromatography. Ann. Clin. Lab. Sci. 2:301-306, 1972.

6. C a r l s o n , L. A. a n d W a d s t r o m , L. E.: Deter­mination of glycerides in blood serum. Clin. Chim. Acta 4:197-205, 1959.

7. F o l c h , J . , L e e s , M., a n d S l o a n e - S t a n l e y , G. H . : A simple method for the isolation and purification of total lipids from animal tissues. J . Biol. Chem. 226:497-509, 1957.

8. K i n g , E. J.: The colorimetric determination of phosphorous. Biochem. J. 26:292-297, 1932.

9. P a r e k h , A. C. a n d J u n g , D . H . : Cholesterol determination with ferric acetate-uranium ace­tate and sulfuric acid-ferrous sulfate reagents. Anal. Chem. 42:1423-1427, 1970.

10. P o s t m a , T. a n d S t r o e s , J . A. P . : Lipid screen­ing by thin-layer chromatography. Z . Klin. Chem. 8:520-524, 1970.

11. R o y e r , M. B. a n d K o , H . : A simplified semi­automated assay for plasma triglycerides. Anal. Biochem. 29:405-416, 1969.

12. S a r d e s a i , V. M . a n d M a n n i n g , J . A.: The determination of triglycerides in plasma and tissues. Clin. Chem. 14:156-161, 1968.

13. S k ip s k i , V. P., G o o d , J. J., B a r c l a y , M., a n d R e g g i o , R . B.: Quantitative analysis of simple lipid classes by thin-layer chromatography. Biochem. Biophys. Acta 152:10-19, 1968.

14. S n y d e r , F . a n d S t e p h e n s , N.: A simplified spectrophotometric determination of ester groups in lipids. Biochem. Biophys. Acta 34: 244-245, 1959.

15. V a n H a n d e l , E. a n d Z i l v e r s m i t , D . B.: Mi­cromethod for the direct determination of serum triglycerides. J. Lab. Clin. Med. 50:152— 157, 1957.

16. Z a k , B . , D i c k e n m a n , R. C., W h i t e , E. G., B u r n e t t , H . , a n d C h e r n e y , P. J.: Rapid esti­mation of free and total cholesterol. Amer. J. Clin. Path. 24:1307-1315, 1954.

17. Z l a t k i s , A., Z a k , B . , a n d B o y l e , A. U.: A new method for the direct determination of serum cholesterol. J . Lab. Clin. Med. 41:486— 492, 1953.

“Leadership is never found among men seeking comfort; but rather among those seeking answers.”

Anonymous