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Quantitative analysis of tissue neutral lipids by thin-layer chromatography
AB.4LYTICAL
RIOCHEMISTRT
Quantitative
lo,
23-31
Analysis
of
Thin-Layer ELINOR From
the Departme&
(1965)
Tissue
Neutr,al
Lipids
by
Chromatography1
LEVIN
CHARLOTTE
AND
oj Surgery, University Portland, Oregon Received
March
oj Oregon
HEAD Medical
School,
30, 1964
INTRODUCTION
A method is presented for the quantitative radioassay and concentration of monoglycerides, diglycerides, triglycerides, free fatty acids, sterols, sterol esters, and hydrocarbons using the technique of thin-layer chromatography (TLC) applied to the neutral lipid of the carrageenin granuloma. Using the method of TLC, the separation and identification of the constituent lipid classes of fats, oils, and waxes was reported by Mangold and Malins (1, 2) and the concentration of several lipid classes of feces and fecaliths was obtained by Williams et al. (3). Snyder and Stephens (4), using @-labeled tripalmitin and Cl”- and H3-labeled fatty acids, and Brown and Johnston (5)) using Cl”-labeled a-monopalmitin, palmitic acid, and tripalmitin, reported excellent recovery of the radioactivity when these commercially obtained compounds were separated as single components or as mixtures by the method of TLC. Although these authors (4, 5) state that their techniques were being used successfully by them in the separation and radioassay of tissue lipids, the details of suitable methods for the separation and radioactive assay of Gssue neutral lipids employing TLC have not been reported in the literature. The solvent system used for the TLC separations by Snyder and Stephens (4) does not separate the sterols from the diglycerides or the monoglycerides from phospholipids, while the solvent system used by Brown and Johnston (5) does not separate tripalmitin from the st,erol esters and hydrocarbons or the 1,2,-diglycerides from sterols, and the presence of a-glyceryl ethers and cardiolipin in the monoglyceride fraction was ‘This investigation was supported in part by a PHS from the Division of General Medical Sciences, Public part by a grant from the American Heart Association Research Fund. 23
research grant RG-6483: Health Service, and in and the Life Insurance
24
LEVIN
AND
HEAD
not reported. Since these investigators (4, 5) apparently used radioactive fatty acids and/or tripalmitin as tracers in their tissue studies, the separation of some of these groups, such as the sterols and diglycerides, is not as critical as it is when C14-labeled acetate is used as the tracer. In addit’ion, the concentration of the separated lipid classes by weighing or by chemical means cannot be determined when the radioactivity of the separated lipids is ascertained by scraping the silica gel into vials and using Cab-0-Si12 or a liquid scintillator (4, 5). The solvent systems used for the separation of the neutral lipids are those described by Mangold and Malins (1, 2) for the separation of complex lipid mixtures into lipid classes, but the procedure differs in that a preliminary separation of the total lipid into neutral and phospholipid was made and that certain neutral lipids, not distinctly separated by the first solvent system used, were extracted and were isolated using a second solvent system. The use of two solvent systems for the separation of the neutral lipids was found to be expedient since a single solvent system as described by other authors (l-5) does not effectively separate all of the major classes of neutral lipids as distinct entities. A preliminary separation of the total lipid into neutral and phospholipid was made because the radioassay and lipid content of these two main classes of lipids may be pertinent in some investigations and because the components of both classes may be determined without loss of one of these main fractions. EXPERIMENTAL
Source and Preparation of Lipids
After the in vitro incubation of 3.5 gm of the carrageenin granuloma minces in 20 ml of oxygen-saturated Krebs-Ringer phosphate buffer, pH 7.4, containing 1 mg of glucose per ml, 4 PC of sodium-l-C14-acetate,3 and 50 pmoles of sodium acetate, the tissue was centrifuged from the incubation medium and was homogenized in absolute alcohol in an all-glass Kontes homogenizer. A zero time control was obtained by centrifugation of a tissue preparation in the cold immediately following the addition of C14-acetate. Aliquots of the homogenates were removed for determination of dry weight and for DNA by the diphenylamine method of Dische (6). After the lipid was extracted with alcohol overnight at room temperature, twice with alcohoI:ether (3:1), and 2 Cab-0-Sil, thixotropic gel powder, La Grange, Ill. SSodium-l-C’4-acetate and C”-benzoic Nuclear Corporation, Boston, Mass.
available acid were
from
Packard
obtained
Instrument
from New
Co.,
England
NEUTRAL
LIPID
ASSAY
BY
TLC
25
once with chloroform:methanol (2: 1) for 15-30 min periods, the extracts were pooled and taken to near dryness in a vacuum under nitrogen and the residue was extracted with petroleum ether :chloroform (6: 1). This extract was washed with water in a separatory funnel five times to remove contaminating material and was then dried with anhydrous sodium sulfate. The total lipid was fractionated into neutral and phospholipid by acetone precipitation before further separation by TLC. Aliquots of the total lipid, neutral lipid, and phospholipid were taken for determination of lipid weight and radioactivity. Chromatography The apparatus and silica gel G used for TLC was purchased from Brinkmann Instruments Co.* A slurry of silica gel G, prepared by mixing 25 gm of silica gel G and 55 ml of distilled water, was applied in a layer, 250 p thick, on 20 X 20 cm glass plates. The plates were heated at 90-95°C for 2 hr and were stored in a desiccator. Individual neutral lipids were purchased from commercial sources. Glyceryl monooleate, diglycerides, tristearin, triolein, stearic acid, oleic acid, cholesterol, cholesteryl palmitate, squalene, batyl distearin, chimyl dipalmitate, and chimyl, selachyl, and batyl alcohols were spotted on the chromatoplates as single components and mixtures. After development with hexane (b.p. 65-69°C) :diethyl ether:glacial acetic acid (9O:lO:l) (solvent system I), the positions of the separat.ed lipids were noted under ultraviolet light after spraying the chromatogram with a solution of 0.2% 2’,7’-dichlorofluorescein (DCF) in 95% ethanol. As found by Mangold and Malins (1, 2)) the cholesterol and diglycerides move together in this solvent system while the monoglycerides and phospholipids remain at the origin. It was found that hexane:diethyl ether: glacial acetic acid (30:70:1) (solvent system II) gave excellent separation of the monoglycerides, cholesterol, and diglycerides while the phospholipid remained at the origin, In solvent system II, two components were observed for the diglycerides which corresponded to 1,2- and 1,3diglycerides when compared to purchased diglycerides containing these isomers. The major neutral lipid classes found in the carrageenin granulomas were determined by comparing the components of the separated granuloma lipids to those of the single and mixed reference compounds in solvent systems I and II (see Table 1). For the quant.itative separation of the 3-, 6-, and g-day carrageenin granuloma neutral lipid fractions, the solvent volume was reduced under nitrogen so that the lipid concentration was about 25 mg/ml. The lipid 4Great
Neck,
Long
Island,
New
York.
26
LEVIN
AND
HEAD
TABLE 1 OF GR.~NuLOMA NEUTR.4L LIPID FRACTIONS THIN-LAYER CHROMATOGRAPHY~
IDENTITY
Fraction
No.*
IA (origin) 2A 2: 2B 2c 3 4 5 6 7 1:
iklajor
neutral
SEPARATED
lipid
class
BY
in fraction
Phospholipid Monoglycerides, a-glyceryl ethers Sterols Diglycerides Unknown Free fatty acids Triglycerides Sterol esters Hydrocarbons, as squalene, etc.
o The diglycerides of the a-glyceryl ethers and long-chain fatty acid aldehydes are found between the triglycerides and sterol esters in solvent system I. However, these lipids were not found in the carrageenin granulomas. b Fractions l-7 were separated using solvent system I. Fractions 1 and 2 were rechromatographed using solvent system II to give fractions lA, 2A, 2B, and 2C.
was applied to the chromatoplates in lOO-~1 aliquots using a micropipet until a total of 9 to 13 lOO-~1 aliquots were placed in discrete spots across the plate. An additional lOO-,uI aliquot was applied to one side of the plate, slightly apart from the rest, to serve as a guide in marking the plates, and the chromatoplates were developed using solvent system I. The neutral lipids were found to be fluorescent under ultraviolet light without staining, as was observed by Williams et al. (3), so that only the guide sample was stained with DCF and was used as an additional marker in locating the lipid fractions. After the plates were marked into component lipids, each lipid fraction was removed by scraping the silica gel off across the plate with a razor blade and collecting it in 15-ml centrifuge tubes. The origin and first fraction above the origin (fractions 1 and 2 in Table 1) were collected together. The lipid was extracted by stirring the silica gel three times with 10 ml of chloroform: methanol (2:l) and once with methanol, followed each time by centrifugation and decantation of the extract into tared scintillator vials. Lipid weight was determined by evaporating off the solvents and reweighing the vials to constant weight after drying in a desiccator. The scintillator solution, consist.ing of toluene containing 0.4% 2,5-diphenyloxazole (PPO) and 0.01% 1,4-bis-2- (5-Phenyloxazolyl) -Benzene (POPOP) , was added to the vials after they reached constant weight for determination of radioactivity in a Packard Tri-Carb scintillation spectrometer. The results in disintegrations per minute (dpm) were obtained by the method of internal standards using a Cl”-benzoic acid standard3 made
NEt-TRAL
LIPID
ASSAY
BY TLC
27
up in toluene. The extracts of the combined fraction (fractions 1 and 2 of Table 1) were not placed in tared vials, but were evaporated under nitrogen to a volume of 2 ml and were replat,ed by spotting 7 to 12 lOO,ul aliquots across the chromatoplate. After development in solvent system II, the lipids were marked, removed, and processed as previously described. At the same time that the lipid was applied to the chromatoplates for fract,ionation, lOO-~1 a1iquot.s of the total neutral lipid were measured directly into a t.ared vial and ont.o a silica gel plate for extraction without development. The lipid weight and radioactivity of all of the fractions were calculated as that found in the total granuloma by use of the appropriate dilution factors. Since the total neutral lipid fractions contained some phospholipid due to incomplete precipitation by the acetone method, a det.ermination of phosphorus by the King method (7) on all of the extracts showed that only the origin of the chromatoplates in each solvent system contained phosphorus and that 88% of the phosphorus was recovered from the origin after development in solvent system I (fract,ion 1 in Table 1) and that 73% of the phosphorus was recovered from the second chromatoplate (fraction 1A in Table I). A determination of free and esterified cholesterol by the method of Sperry and Webb (8) on each extract showed that only fractions 2B and 6 contained cholesterol, indicating that the separations were discrete and that the marking of the chromatograms and removal of the silica gel was accurate. RESULTS
AND DISCUSSION
For fifteen granuloma neutral lipids examined, the radioactivity and lipid weight of the total neutral lipid spotted on silica gel G (without developing) was compared to that of the total neutral lipid measured directly and was found to be 95% (range, 92-1OOoJo)for radioactivity and 104% (range, 9%111%) for lipid weight. These results show that the radioactivity and lipid can be recovered from the silica gel G. The sum of the radioact,ivity and lipid weight of the nine lipid classes isolated by TLC compared to that of the total neutral lipid measured directly was 96% (range, 83108%) for radioactivity and 106% (range, 96-118%) for lipid weight. The recovery of the lipid weight was probably high due to traces of silicic acid in the methanol extracts. Since the wet and dry tissue weight differed significantly between the granulomas of the ages studied, the radioactivity of the neutral lipid classes isolated from five 8-day carrageenin granulomas and a zero time control is shown in Table 2 as dpm/mg DNA (disintegrations per minute/mg deoxyribonucleic acid) so that the results between granulomas might later be compared. Since the radioactivity found in the zero
28
LEVIN
AND
HEAD
TABLE RADIOACTIVITY
Expt.
No.
4 7 12 13 16 Ave. Stand. dev. Zero time control
INCORPORATED
Total neutral lipid
3689 4470 4742 4938 4722 4512 489 44
INTO NEUTR.4L GRANULOMAS ON 2A
2B
MG
S
2c DG
157 197 211 210 206 196 23 2
1173 1249 1293 1352 1419 1297 94 <1
146 173 243 199 166 185 37 <1
2 LIPID A
DNA
CLASSES BASIS”
OF ~-DAY
D3P”‘mg YA Unknown
139 165 47 113 123 117 44 0
FFA
356 301 175 296 241 274 69 <1
CARRAGEENIN
5
6
7
TG
SE
HC
1065 1371 1971 1795 2129 1661 435
131 145 215 223 31 149 78 0
37 43 47 22 36 37 10 0
a Abbreviations: DPM/mg DNA = disintegrations per minute/mg deoxyribonucleic acid. MG = monoglycerides. S = sterols. DG = diglycerides. FFA = free fatty acids. TG = triglycerides. SE = sterol esters. HC = hydrocarbons.
time control is presumably due to contamination of the lipid extracts with free CY4-acetate, the absence of any appreciable radioactivity in the neutral lipid classes of the zero time control indicates that none of the radioactivity present in the neutral lipid classes of the granuloma is due to free C14-acetate. Seventy-five per cent of the radioactivity of the zero time total lipid was found in the phospholipid fraction, and 147% was found in the neutral lipid fraction. Of the 44 dpm/mg DNA found in the total neutral lipid fraction, 28 dpm was in the phospholipid contaminating the neutral lipid fraction and less than 3 dpm was found in all of the neutral lipid classes. It appears that the radioactivity of the zero time lipids is lost during the chromatographic procedures, apparently due to incomplete removal of (Y-acetate from the silica gel G with the solvents used for lipid extraction. The loss of W-acetate has also been observed in this laboratory when unwashed zero time granuloma total lipids were separated into neutral and phospholipid fractions by sicilic acid chromatography. From these fractions 92% of the lipid weight and 94% of the lipid phosphorus were recovered, with only a 2% recovery of the radioactivity. The distribution of the radioactivity and content of the neutral lipid classes of a series of 3- and g-day carrageenin granulomas as per cent of that in the total neutral lipid is given in Tables 3 and 4. Considering the biological variation in the development of granulomas between animals, variation in the results between granulomas of the same age group was expected. Although the variation is most marked in fractions containing small amounts of radioactivity and lipid weight, the results
NEUTRAL
LIPID
ASSAY
TABLE DISTRIBUTION AND
OF ~-DAY
W-ACETATE CARRAGEENIN
Expt.
AS LIPID
INTO NEUTRAL LIPID CLASSES SEPARSTEU BY THIN-LAYER
PER CENT OF TOTAL RADIOACTIVITY Noutr;l
lipid
2A
2B
2c
MG
S
DG
granulomas : 5 6 10 11 15 Ave. Stand. dev.
4.2 3.5 3.2 3.9 4.4 3.8 0.5
51.9 51.1 45.0 49.0 40.5 47.5 4.4
2.5 1.3 2.3 3.3 2.2 2.3 0.7
0.0 3.1 3.0 3.0 4.0 2.6 1.5
granulomas: 4 7 12 13 16 Ave. Stand. dev.
4.3 4.4 4.4 4.3 4.4 4.4 0.1
31.8 27.9 27.3 27.4 30.0 28.8 2.0
4.0 3.9 5.1 4.0 3.5 4.1 0.6
3.8 3.7 1.0 2.3 2.6 2.7 1.1
No.
29
TLC
3
INCORPORATED GRANULOM~S
CHROMATOGRAPHY
BY
Unknown
fraction* 4
OF
NEUTRAL
6
7
TG
SE
HC
4.0 1.4 4.0 2.2 4.5 3.2 1.3
26.0 26.9 22.7 26.1 25.2 25.4 1.6
2.1 2.0 2.2 3.1 3.8 2.6 0.8
0.4 0.6 0.7 0.4 1.3 0.7 0.4
9.6 6.7 3.7 6.0 5.1 6.2 2.2
28.9 30.7 41.0 36.3 45.1 36.4 6.8
3.5 3.2 4.5 4.5 0.7 3.3 1.6
1.0 1.0 1.0 0.5 0.8 0.9 0.2
FFA
3
5
3-day
8-day
a Abbreviations: free fatty acids.
TG
Mg = monoglycerides. S = sterols. = triglycerides. SE = sterol esters.
I)G = diglycerides. HC = hydrocarbons.
FFA
=
between granulomas of the same age group are fairly consistent. When the results are compared between age groups, some of the lipid classes show definite differences in radioactivity and content at the two time periods and the values of one age group do not overlap with the values of the other group even though there was considerable variation within the group. Determination of the lipid content by weighing is tedious and time consuming and traces of silicic acid in the methanol extracts and the small lipid content of some of the fractions tend to introduce errors in the gravimetric determination of weight, Calorimetric methods are now being utilized to determine the lipid content of those neutral lipids found to be of importance in our invest,igations on connective tissue growth. Some of the lipid classes isolated by TLC may contain lipid of another class which is found in trace amounts in tissue; for example, the o(glyceryl ethers are found with the monoglycerides. If a class is suspected
30
LEVIN
AND
HEAD
TABLE DISTRIBUTION CARRAGEENIN
4
OF LIPID WEIGHT OF NEUTRAL LIPID CLASSES OF 3- AND 8- DAY GRANULOMAS SEPARATED BY THIN-LAYER CHROMATOGRAPHY AS PER CENT OF TOTAL NEUTRAL LIPID WEIGHT
Neutral lipid fractiona
Expt. No.
2A
2B
MG
s
lipid 2c DG
Neupl Unknown
fytiona
5
6
7
FFA
TG
SE
HC
43.3 56.0 46.5 52.2 37.4 47.1 7.3
11.7 6.5 8.6 7.1
3.3 2.7 2.0 2.1 3.3 2.7 0.6
35.7 33.1 46.0 40.3 41.6 39.3 5.1
2.7 5.7 3.3 5.1 3.3 4.0 1.3
3&y granulomas: 5 6
3.1
10 11
1.8 2.3 2.6 2.3 0.5
19.7
2.5
3.2
4
2.5
7
2.9
12 13 16
2.5 1.8 2.2 2.4 0.4
23.1 32.3 28.5
4.0 6.3 3.2 4.1 4.3 4.4 1.2
15
Ave. Stand. dev.
1.9
23.4 18.2
4.8 3.3
0.0 0.8
3.1 1.3
11.0
1.1
7.9
19.9
3.8 3.8
19.6
5.3
0.6 1.2 0.7 0.5
4.0 8.1 4.8 3.0
0.8
12.7
16.7
9.0 8.5 2.0
S-day granulomas :
Ave. Stand. dev.
29.5 31.3
28.9 3.4
1.1
9.7
0.4 0.4 0.8 0.7 0.3
6.5
8.1 5.8 8.6 2.8
a Abbreviations: Mg = monoglycerides. S = sterols. DG = diglycerides. free fatty acids. TG = triglycerides. SE = sterol esters. HC = hydrocarbons.
1.3 2.7 1.2 1.3 1.1 1.5
0.7
FFA =
of containing trace lipids or is of special interest, such a fraction can be investigated further for component lipids by TLC using other solvent systems and/or adsorbents or by chemical means. Although the phospholipid contaminant of the total neutral lipid was easily separated from the individual neutral lipids by the method described, silicic acid chromatography is probably preferable to acetone precipitation since a neutral lipid fraction practically free of phospholipid would be obtained. Borgstrom (9) reported that the neutral lipid found in the phospholipid fraction is about the same for both methods. The neutral lipids separated by silicic acid chromatography, unlike those separated by acetone precipitation, do not fluoresce under ultraviolet light, so that location of the separated lipids on the chromatoplate is difficult unless sprayed lightly with 0.02% DCF or it may be possible to locate the lipids by viewing the chromatoplate with back lighting and using a stained guide for reference.
NEUTRAL
LIPID
ASSAY
BY
TLC
31
The results obtained on the radioactive assay and content of the neukal lipid classes of the carrageenin granuloma demonstrate the feasibility of using TLC for the quantitative separation and recovery of radioactivity and content of the neutral lipid classes from biological materials. SUMMARY
,4 method is described for the quantitative separation of the neutral lipid fraction of carrageenin granulomas int.0 the major neutral lipid classes, i.e., monoglycerides, diglycerides, triglycerides, free fatty acids, sterols, sterol esters, and hydrocarbons, using the technique of thinlayer chromatography. The radioactivity and lipid content found in the total neutral lipid fraction were quantitatively recovered in the separated neutral lipid classes. Although the method described was applied to the separation of the neutral lipid from carrageenin granulomas for studies of lipid changes during connective tissue growth, the results demonstrate that thin-layer chromatography can be applied successfully in the quantitative separation and recovery of the major neutral lipid classes of tissue particularly when a complete survey of the lipid content and incorporation of radioactive tracers into the neutral lipid classes of t,issue is desired. REFERENCES 1. MANGOLD, H. Ii., AND MALINS, D. C., J. Am. Oil Chemists’ Sot. 37, 383 (1960). 2. MALINS, D. C., AND MANGOLD, H. K., J. Am. Oil Chemists’ Sot. 37, 576 (1960). 3. WILLIAMS, J. A., SHARMA, A., MORRIS, L. J., AND HOLMAN, R. T., Proc. Sot. Exptl. Biol. Med. 105, 192 (1960). 4. SNYDER, F., AND STEPHENS, N., Anal. Biochem. 4, 128 (1962). 5. BROWN, F. L., AND JOHNSTON, J. M., J. Lipid Res. 3, 480 (1962). 6. DISCHE, Z., in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.),
Vol. 1, p. 287. Academic Press, New York, 1955. E. J., Biochem. J. 26, 292 (1932). 8. SPERRY, W. M., AND WEBB, M., J. Biol. Chem. 187, 97 (1950). 9. BORGSTFGM, B., Acta Physiol. Scnnd. 25, 101 (1952).
7. KING,
RIOCHEMISTRT
Quantitative
lo,
23-31
Analysis
of
Thin-Layer ELINOR From
the Departme&
(1965)
Tissue
Neutr,al
Lipids
by
Chromatography1
LEVIN
CHARLOTTE
AND
oj Surgery, University Portland, Oregon Received
March
oj Oregon
HEAD Medical
School,
30, 1964
INTRODUCTION
A method is presented for the quantitative radioassay and concentration of monoglycerides, diglycerides, triglycerides, free fatty acids, sterols, sterol esters, and hydrocarbons using the technique of thin-layer chromatography (TLC) applied to the neutral lipid of the carrageenin granuloma. Using the method of TLC, the separation and identification of the constituent lipid classes of fats, oils, and waxes was reported by Mangold and Malins (1, 2) and the concentration of several lipid classes of feces and fecaliths was obtained by Williams et al. (3). Snyder and Stephens (4), using @-labeled tripalmitin and Cl”- and H3-labeled fatty acids, and Brown and Johnston (5)) using Cl”-labeled a-monopalmitin, palmitic acid, and tripalmitin, reported excellent recovery of the radioactivity when these commercially obtained compounds were separated as single components or as mixtures by the method of TLC. Although these authors (4, 5) state that their techniques were being used successfully by them in the separation and radioassay of tissue lipids, the details of suitable methods for the separation and radioactive assay of Gssue neutral lipids employing TLC have not been reported in the literature. The solvent system used for the TLC separations by Snyder and Stephens (4) does not separate the sterols from the diglycerides or the monoglycerides from phospholipids, while the solvent system used by Brown and Johnston (5) does not separate tripalmitin from the st,erol esters and hydrocarbons or the 1,2,-diglycerides from sterols, and the presence of a-glyceryl ethers and cardiolipin in the monoglyceride fraction was ‘This investigation was supported in part by a PHS from the Division of General Medical Sciences, Public part by a grant from the American Heart Association Research Fund. 23
research grant RG-6483: Health Service, and in and the Life Insurance
24
LEVIN
AND
HEAD
not reported. Since these investigators (4, 5) apparently used radioactive fatty acids and/or tripalmitin as tracers in their tissue studies, the separation of some of these groups, such as the sterols and diglycerides, is not as critical as it is when C14-labeled acetate is used as the tracer. In addit’ion, the concentration of the separated lipid classes by weighing or by chemical means cannot be determined when the radioactivity of the separated lipids is ascertained by scraping the silica gel into vials and using Cab-0-Si12 or a liquid scintillator (4, 5). The solvent systems used for the separation of the neutral lipids are those described by Mangold and Malins (1, 2) for the separation of complex lipid mixtures into lipid classes, but the procedure differs in that a preliminary separation of the total lipid into neutral and phospholipid was made and that certain neutral lipids, not distinctly separated by the first solvent system used, were extracted and were isolated using a second solvent system. The use of two solvent systems for the separation of the neutral lipids was found to be expedient since a single solvent system as described by other authors (l-5) does not effectively separate all of the major classes of neutral lipids as distinct entities. A preliminary separation of the total lipid into neutral and phospholipid was made because the radioassay and lipid content of these two main classes of lipids may be pertinent in some investigations and because the components of both classes may be determined without loss of one of these main fractions. EXPERIMENTAL
Source and Preparation of Lipids
After the in vitro incubation of 3.5 gm of the carrageenin granuloma minces in 20 ml of oxygen-saturated Krebs-Ringer phosphate buffer, pH 7.4, containing 1 mg of glucose per ml, 4 PC of sodium-l-C14-acetate,3 and 50 pmoles of sodium acetate, the tissue was centrifuged from the incubation medium and was homogenized in absolute alcohol in an all-glass Kontes homogenizer. A zero time control was obtained by centrifugation of a tissue preparation in the cold immediately following the addition of C14-acetate. Aliquots of the homogenates were removed for determination of dry weight and for DNA by the diphenylamine method of Dische (6). After the lipid was extracted with alcohol overnight at room temperature, twice with alcohoI:ether (3:1), and 2 Cab-0-Sil, thixotropic gel powder, La Grange, Ill. SSodium-l-C’4-acetate and C”-benzoic Nuclear Corporation, Boston, Mass.
available acid were
from
Packard
obtained
Instrument
from New
Co.,
England
NEUTRAL
LIPID
ASSAY
BY
TLC
25
once with chloroform:methanol (2: 1) for 15-30 min periods, the extracts were pooled and taken to near dryness in a vacuum under nitrogen and the residue was extracted with petroleum ether :chloroform (6: 1). This extract was washed with water in a separatory funnel five times to remove contaminating material and was then dried with anhydrous sodium sulfate. The total lipid was fractionated into neutral and phospholipid by acetone precipitation before further separation by TLC. Aliquots of the total lipid, neutral lipid, and phospholipid were taken for determination of lipid weight and radioactivity. Chromatography The apparatus and silica gel G used for TLC was purchased from Brinkmann Instruments Co.* A slurry of silica gel G, prepared by mixing 25 gm of silica gel G and 55 ml of distilled water, was applied in a layer, 250 p thick, on 20 X 20 cm glass plates. The plates were heated at 90-95°C for 2 hr and were stored in a desiccator. Individual neutral lipids were purchased from commercial sources. Glyceryl monooleate, diglycerides, tristearin, triolein, stearic acid, oleic acid, cholesterol, cholesteryl palmitate, squalene, batyl distearin, chimyl dipalmitate, and chimyl, selachyl, and batyl alcohols were spotted on the chromatoplates as single components and mixtures. After development with hexane (b.p. 65-69°C) :diethyl ether:glacial acetic acid (9O:lO:l) (solvent system I), the positions of the separat.ed lipids were noted under ultraviolet light after spraying the chromatogram with a solution of 0.2% 2’,7’-dichlorofluorescein (DCF) in 95% ethanol. As found by Mangold and Malins (1, 2)) the cholesterol and diglycerides move together in this solvent system while the monoglycerides and phospholipids remain at the origin. It was found that hexane:diethyl ether: glacial acetic acid (30:70:1) (solvent system II) gave excellent separation of the monoglycerides, cholesterol, and diglycerides while the phospholipid remained at the origin, In solvent system II, two components were observed for the diglycerides which corresponded to 1,2- and 1,3diglycerides when compared to purchased diglycerides containing these isomers. The major neutral lipid classes found in the carrageenin granulomas were determined by comparing the components of the separated granuloma lipids to those of the single and mixed reference compounds in solvent systems I and II (see Table 1). For the quant.itative separation of the 3-, 6-, and g-day carrageenin granuloma neutral lipid fractions, the solvent volume was reduced under nitrogen so that the lipid concentration was about 25 mg/ml. The lipid 4Great
Neck,
Long
Island,
New
York.
26
LEVIN
AND
HEAD
TABLE 1 OF GR.~NuLOMA NEUTR.4L LIPID FRACTIONS THIN-LAYER CHROMATOGRAPHY~
IDENTITY
Fraction
No.*
IA (origin) 2A 2: 2B 2c 3 4 5 6 7 1:
iklajor
neutral
SEPARATED
lipid
class
BY
in fraction
Phospholipid Monoglycerides, a-glyceryl ethers Sterols Diglycerides Unknown Free fatty acids Triglycerides Sterol esters Hydrocarbons, as squalene, etc.
o The diglycerides of the a-glyceryl ethers and long-chain fatty acid aldehydes are found between the triglycerides and sterol esters in solvent system I. However, these lipids were not found in the carrageenin granulomas. b Fractions l-7 were separated using solvent system I. Fractions 1 and 2 were rechromatographed using solvent system II to give fractions lA, 2A, 2B, and 2C.
was applied to the chromatoplates in lOO-~1 aliquots using a micropipet until a total of 9 to 13 lOO-~1 aliquots were placed in discrete spots across the plate. An additional lOO-,uI aliquot was applied to one side of the plate, slightly apart from the rest, to serve as a guide in marking the plates, and the chromatoplates were developed using solvent system I. The neutral lipids were found to be fluorescent under ultraviolet light without staining, as was observed by Williams et al. (3), so that only the guide sample was stained with DCF and was used as an additional marker in locating the lipid fractions. After the plates were marked into component lipids, each lipid fraction was removed by scraping the silica gel off across the plate with a razor blade and collecting it in 15-ml centrifuge tubes. The origin and first fraction above the origin (fractions 1 and 2 in Table 1) were collected together. The lipid was extracted by stirring the silica gel three times with 10 ml of chloroform: methanol (2:l) and once with methanol, followed each time by centrifugation and decantation of the extract into tared scintillator vials. Lipid weight was determined by evaporating off the solvents and reweighing the vials to constant weight after drying in a desiccator. The scintillator solution, consist.ing of toluene containing 0.4% 2,5-diphenyloxazole (PPO) and 0.01% 1,4-bis-2- (5-Phenyloxazolyl) -Benzene (POPOP) , was added to the vials after they reached constant weight for determination of radioactivity in a Packard Tri-Carb scintillation spectrometer. The results in disintegrations per minute (dpm) were obtained by the method of internal standards using a Cl”-benzoic acid standard3 made
NEt-TRAL
LIPID
ASSAY
BY TLC
27
up in toluene. The extracts of the combined fraction (fractions 1 and 2 of Table 1) were not placed in tared vials, but were evaporated under nitrogen to a volume of 2 ml and were replat,ed by spotting 7 to 12 lOO,ul aliquots across the chromatoplate. After development in solvent system II, the lipids were marked, removed, and processed as previously described. At the same time that the lipid was applied to the chromatoplates for fract,ionation, lOO-~1 a1iquot.s of the total neutral lipid were measured directly into a t.ared vial and ont.o a silica gel plate for extraction without development. The lipid weight and radioactivity of all of the fractions were calculated as that found in the total granuloma by use of the appropriate dilution factors. Since the total neutral lipid fractions contained some phospholipid due to incomplete precipitation by the acetone method, a det.ermination of phosphorus by the King method (7) on all of the extracts showed that only the origin of the chromatoplates in each solvent system contained phosphorus and that 88% of the phosphorus was recovered from the origin after development in solvent system I (fract,ion 1 in Table 1) and that 73% of the phosphorus was recovered from the second chromatoplate (fraction 1A in Table I). A determination of free and esterified cholesterol by the method of Sperry and Webb (8) on each extract showed that only fractions 2B and 6 contained cholesterol, indicating that the separations were discrete and that the marking of the chromatograms and removal of the silica gel was accurate. RESULTS
AND DISCUSSION
For fifteen granuloma neutral lipids examined, the radioactivity and lipid weight of the total neutral lipid spotted on silica gel G (without developing) was compared to that of the total neutral lipid measured directly and was found to be 95% (range, 92-1OOoJo)for radioactivity and 104% (range, 9%111%) for lipid weight. These results show that the radioactivity and lipid can be recovered from the silica gel G. The sum of the radioact,ivity and lipid weight of the nine lipid classes isolated by TLC compared to that of the total neutral lipid measured directly was 96% (range, 83108%) for radioactivity and 106% (range, 96-118%) for lipid weight. The recovery of the lipid weight was probably high due to traces of silicic acid in the methanol extracts. Since the wet and dry tissue weight differed significantly between the granulomas of the ages studied, the radioactivity of the neutral lipid classes isolated from five 8-day carrageenin granulomas and a zero time control is shown in Table 2 as dpm/mg DNA (disintegrations per minute/mg deoxyribonucleic acid) so that the results between granulomas might later be compared. Since the radioactivity found in the zero
28
LEVIN
AND
HEAD
TABLE RADIOACTIVITY
Expt.
No.
4 7 12 13 16 Ave. Stand. dev. Zero time control
INCORPORATED
Total neutral lipid
3689 4470 4742 4938 4722 4512 489 44
INTO NEUTR.4L GRANULOMAS ON 2A
2B
MG
S
2c DG
157 197 211 210 206 196 23 2
1173 1249 1293 1352 1419 1297 94 <1
146 173 243 199 166 185 37 <1
2 LIPID A
DNA
CLASSES BASIS”
OF ~-DAY
D3P”‘mg YA Unknown
139 165 47 113 123 117 44 0
FFA
356 301 175 296 241 274 69 <1
CARRAGEENIN
5
6
7
TG
SE
HC
1065 1371 1971 1795 2129 1661 435
131 145 215 223 31 149 78 0
37 43 47 22 36 37 10 0
a Abbreviations: DPM/mg DNA = disintegrations per minute/mg deoxyribonucleic acid. MG = monoglycerides. S = sterols. DG = diglycerides. FFA = free fatty acids. TG = triglycerides. SE = sterol esters. HC = hydrocarbons.
time control is presumably due to contamination of the lipid extracts with free CY4-acetate, the absence of any appreciable radioactivity in the neutral lipid classes of the zero time control indicates that none of the radioactivity present in the neutral lipid classes of the granuloma is due to free C14-acetate. Seventy-five per cent of the radioactivity of the zero time total lipid was found in the phospholipid fraction, and 147% was found in the neutral lipid fraction. Of the 44 dpm/mg DNA found in the total neutral lipid fraction, 28 dpm was in the phospholipid contaminating the neutral lipid fraction and less than 3 dpm was found in all of the neutral lipid classes. It appears that the radioactivity of the zero time lipids is lost during the chromatographic procedures, apparently due to incomplete removal of (Y-acetate from the silica gel G with the solvents used for lipid extraction. The loss of W-acetate has also been observed in this laboratory when unwashed zero time granuloma total lipids were separated into neutral and phospholipid fractions by sicilic acid chromatography. From these fractions 92% of the lipid weight and 94% of the lipid phosphorus were recovered, with only a 2% recovery of the radioactivity. The distribution of the radioactivity and content of the neutral lipid classes of a series of 3- and g-day carrageenin granulomas as per cent of that in the total neutral lipid is given in Tables 3 and 4. Considering the biological variation in the development of granulomas between animals, variation in the results between granulomas of the same age group was expected. Although the variation is most marked in fractions containing small amounts of radioactivity and lipid weight, the results
NEUTRAL
LIPID
ASSAY
TABLE DISTRIBUTION AND
OF ~-DAY
W-ACETATE CARRAGEENIN
Expt.
AS LIPID
INTO NEUTRAL LIPID CLASSES SEPARSTEU BY THIN-LAYER
PER CENT OF TOTAL RADIOACTIVITY Noutr;l
lipid
2A
2B
2c
MG
S
DG
granulomas : 5 6 10 11 15 Ave. Stand. dev.
4.2 3.5 3.2 3.9 4.4 3.8 0.5
51.9 51.1 45.0 49.0 40.5 47.5 4.4
2.5 1.3 2.3 3.3 2.2 2.3 0.7
0.0 3.1 3.0 3.0 4.0 2.6 1.5
granulomas: 4 7 12 13 16 Ave. Stand. dev.
4.3 4.4 4.4 4.3 4.4 4.4 0.1
31.8 27.9 27.3 27.4 30.0 28.8 2.0
4.0 3.9 5.1 4.0 3.5 4.1 0.6
3.8 3.7 1.0 2.3 2.6 2.7 1.1
No.
29
TLC
3
INCORPORATED GRANULOM~S
CHROMATOGRAPHY
BY
Unknown
fraction* 4
OF
NEUTRAL
6
7
TG
SE
HC
4.0 1.4 4.0 2.2 4.5 3.2 1.3
26.0 26.9 22.7 26.1 25.2 25.4 1.6
2.1 2.0 2.2 3.1 3.8 2.6 0.8
0.4 0.6 0.7 0.4 1.3 0.7 0.4
9.6 6.7 3.7 6.0 5.1 6.2 2.2
28.9 30.7 41.0 36.3 45.1 36.4 6.8
3.5 3.2 4.5 4.5 0.7 3.3 1.6
1.0 1.0 1.0 0.5 0.8 0.9 0.2
FFA
3
5
3-day
8-day
a Abbreviations: free fatty acids.
TG
Mg = monoglycerides. S = sterols. = triglycerides. SE = sterol esters.
I)G = diglycerides. HC = hydrocarbons.
FFA
=
between granulomas of the same age group are fairly consistent. When the results are compared between age groups, some of the lipid classes show definite differences in radioactivity and content at the two time periods and the values of one age group do not overlap with the values of the other group even though there was considerable variation within the group. Determination of the lipid content by weighing is tedious and time consuming and traces of silicic acid in the methanol extracts and the small lipid content of some of the fractions tend to introduce errors in the gravimetric determination of weight, Calorimetric methods are now being utilized to determine the lipid content of those neutral lipids found to be of importance in our invest,igations on connective tissue growth. Some of the lipid classes isolated by TLC may contain lipid of another class which is found in trace amounts in tissue; for example, the o(glyceryl ethers are found with the monoglycerides. If a class is suspected
30
LEVIN
AND
HEAD
TABLE DISTRIBUTION CARRAGEENIN
4
OF LIPID WEIGHT OF NEUTRAL LIPID CLASSES OF 3- AND 8- DAY GRANULOMAS SEPARATED BY THIN-LAYER CHROMATOGRAPHY AS PER CENT OF TOTAL NEUTRAL LIPID WEIGHT
Neutral lipid fractiona
Expt. No.
2A
2B
MG
s
lipid 2c DG
Neupl Unknown
fytiona
5
6
7
FFA
TG
SE
HC
43.3 56.0 46.5 52.2 37.4 47.1 7.3
11.7 6.5 8.6 7.1
3.3 2.7 2.0 2.1 3.3 2.7 0.6
35.7 33.1 46.0 40.3 41.6 39.3 5.1
2.7 5.7 3.3 5.1 3.3 4.0 1.3
3&y granulomas: 5 6
3.1
10 11
1.8 2.3 2.6 2.3 0.5
19.7
2.5
3.2
4
2.5
7
2.9
12 13 16
2.5 1.8 2.2 2.4 0.4
23.1 32.3 28.5
4.0 6.3 3.2 4.1 4.3 4.4 1.2
15
Ave. Stand. dev.
1.9
23.4 18.2
4.8 3.3
0.0 0.8
3.1 1.3
11.0
1.1
7.9
19.9
3.8 3.8
19.6
5.3
0.6 1.2 0.7 0.5
4.0 8.1 4.8 3.0
0.8
12.7
16.7
9.0 8.5 2.0
S-day granulomas :
Ave. Stand. dev.
29.5 31.3
28.9 3.4
1.1
9.7
0.4 0.4 0.8 0.7 0.3
6.5
8.1 5.8 8.6 2.8
a Abbreviations: Mg = monoglycerides. S = sterols. DG = diglycerides. free fatty acids. TG = triglycerides. SE = sterol esters. HC = hydrocarbons.
1.3 2.7 1.2 1.3 1.1 1.5
0.7
FFA =
of containing trace lipids or is of special interest, such a fraction can be investigated further for component lipids by TLC using other solvent systems and/or adsorbents or by chemical means. Although the phospholipid contaminant of the total neutral lipid was easily separated from the individual neutral lipids by the method described, silicic acid chromatography is probably preferable to acetone precipitation since a neutral lipid fraction practically free of phospholipid would be obtained. Borgstrom (9) reported that the neutral lipid found in the phospholipid fraction is about the same for both methods. The neutral lipids separated by silicic acid chromatography, unlike those separated by acetone precipitation, do not fluoresce under ultraviolet light, so that location of the separated lipids on the chromatoplate is difficult unless sprayed lightly with 0.02% DCF or it may be possible to locate the lipids by viewing the chromatoplate with back lighting and using a stained guide for reference.
NEUTRAL
LIPID
ASSAY
BY
TLC
31
The results obtained on the radioactive assay and content of the neukal lipid classes of the carrageenin granuloma demonstrate the feasibility of using TLC for the quantitative separation and recovery of radioactivity and content of the neutral lipid classes from biological materials. SUMMARY
,4 method is described for the quantitative separation of the neutral lipid fraction of carrageenin granulomas int.0 the major neutral lipid classes, i.e., monoglycerides, diglycerides, triglycerides, free fatty acids, sterols, sterol esters, and hydrocarbons, using the technique of thinlayer chromatography. The radioactivity and lipid content found in the total neutral lipid fraction were quantitatively recovered in the separated neutral lipid classes. Although the method described was applied to the separation of the neutral lipid from carrageenin granulomas for studies of lipid changes during connective tissue growth, the results demonstrate that thin-layer chromatography can be applied successfully in the quantitative separation and recovery of the major neutral lipid classes of tissue particularly when a complete survey of the lipid content and incorporation of radioactive tracers into the neutral lipid classes of t,issue is desired. REFERENCES 1. MANGOLD, H. Ii., AND MALINS, D. C., J. Am. Oil Chemists’ Sot. 37, 383 (1960). 2. MALINS, D. C., AND MANGOLD, H. K., J. Am. Oil Chemists’ Sot. 37, 576 (1960). 3. WILLIAMS, J. A., SHARMA, A., MORRIS, L. J., AND HOLMAN, R. T., Proc. Sot. Exptl. Biol. Med. 105, 192 (1960). 4. SNYDER, F., AND STEPHENS, N., Anal. Biochem. 4, 128 (1962). 5. BROWN, F. L., AND JOHNSTON, J. M., J. Lipid Res. 3, 480 (1962). 6. DISCHE, Z., in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.),
Vol. 1, p. 287. Academic Press, New York, 1955. E. J., Biochem. J. 26, 292 (1932). 8. SPERRY, W. M., AND WEBB, M., J. Biol. Chem. 187, 97 (1950). 9. BORGSTFGM, B., Acta Physiol. Scnnd. 25, 101 (1952).
7. KING,