OBSERVATIONS ON THE SILICIC ACID CHROMATOGRAPHY OF THE NEUTRAL LIPIDES OF RAT LIVER,

BEEF LIVER, AND YEAST*

BY EDWARD J. BARRON AND DONALD J. HANAHAN

(FTWI the Department of Biochemistry, University of Washington, Seattle, Waahington)

(Received for publication, November 11, 1957)

Within the past several years the application of silicic acid to the chromatographic fractionation of simple and complex lipides has received increased attention. In particular this adsorbent has been used in the separation of phospholipides by McKibbin (I), Lea et aE. (Z), and Hanahan et al. (3). As shown by Borgstrom (4), who used model lipide compounds, and Fillerup and Mead (5), who employed mainly the naturally occurring lipide of plasma, the various neutral lipides can also be separated on silicic acid by judicious choice of solvents. Lovern (6), working with the lipides of the haddock flesh, obtained quite sharp peaks on silicic acid chromatography but subsequently found that these components did not necessarily represent pure fractions. In a study of the lipides of human hair, Nicolaides and Foster (7) have reported a partial fractionation on silicic acid with benzenediethyl ether mixtures.

In a consideration of the various studies carried out on neutral lipide separations on silicic acid, it seemed that insufficient characterization data were available on the individual fractions thus obtained. Further- more, in view of our experience, petroleum ether as the main non-polar solvent was neither as effective nor as reproducible as desired. In a pre- liminary note from this laboratory (8), it was reported that pure n-hexane appeared suitable as the major non-polar solvent, and some of the char- acteristics of fractions obtained by silicic acid chromatography of certain neutral lipides were presented. The present communication provides further details on this study of the chemical nature of the neutral lipides from rat liver, beef liver, and bakers’ yeast (Sacchuromyces cerevisiae).

EXPERIMENTAL

Material and Methods-Mallinckrodt’s silicic acid, reagent grade, 100 mesh, suitable for chromatographic analysis, was used throughout the

* This work was supported by grants from the American Cancer Society and the National Science Foundation. A part of this study was reported at the Forty-eighth annual meeting of the American Society of Biological Chemists at Chicago, April 15-19 (1957).

493

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494 CHROMATOGRAPHY OF XEUTK4L LIPIDES

study. This adsorbent was used either as obt,ained from a freshly opened bottle or preferably was dried for 12 hours at 110” before use. A com- mercial grade of n-hexane, obtained from Phillips Petroleum Company,’ was redistilled over potassium permanganate to which a small amount of acetone had been added to dissolve t,he permanganat,e. The fraction distilling between 67.5-69.5” was collected and used in these experiments. Merck’s reagent grade anhydrous ether, t,hiophene-free benzene, and anhydrous methanol were used without further purification.

Beef liver was obtained as soon as possible after the animal was killed and the lipide extraction initiated within 1 hour. Adult rats of the Sprague-Dawley t,ype were killed, the liver was rapidly removed, and the extraction started immediately. Bakers’ yeast (8. cerevisiae) was generously provided through the courtesy of Standard Brands Incorporated. In all three instances the extraction and isolation of the lipides were per- formed as described previously (3). In brief this involved an alcohol-ether extraction at room temperature, concentration of the extract, and re- ext,raction with ether. Finally, a separation by acetone addition was made into an acetone-soluble fraction (neutral lipides) and an acetone- insoluble fract,ion (phospholipides) . In most experiments, this technique yielded a neutral lipide fraction that contained approximately 4 per cent phospholipides.

Preparation of Column-The design and operation of the columns were essentially the same as described before (3).

The adsorption columns measured 35 mm. in diameter and 400 mm. in length, in which 60 gm. of silicic acid, when fully packed down, occupied a length of approximately 155 mm. Before actual preparation of t.he column, it is advisable to wash the silicic acid in succession with the fol- lowing solvents: diethyl ether, 15 per cent benzene in hexane, and finally with n-hexane. It is convenient to carry out the washing (with a t,otal of 150 ml. of each of the solvents) and filtration in a sintered glass Btichner funnel. After the final washing, a slurry of t,he adsorbent in hexane was poured into the column, which was fitted at the bottom with a tightly packed glass wool plug or a perforated, fritted gIass disk plus a t,hin layer of glass wool, and the acid was packed under nitrogen pressure t,o a constant volume. The flow rat,e maintained in t,he elution operat,ion was 2 to 2s ml. per minute, and for a 60 gm. column t,he volume of the eluate collected, by fraction collector, was 10 ml. When total weight was desired, a total of 50 ml. of the eluates was obtained by pooling of fract,ions. The loading factor for t,hese runs was a maximum of 18 to 20 mg. of total solids per gm. of silicic acid. In general, any overloading was indicated by failure to separate the sterol and diglyceride.

1 Bartlesville, Oklahoma.

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E. J. BARRON AND D. J. HAXAHAN 495

Total weights on aliquots were obtained by a vacuum drying at 100” for 4 hours. n’eutral equivalents were determined by titration of the sample in ethanol with methanolic 0.01 N NaOH (COz-free). Glycerol was assayed by a periodate oxidation procedure 79), cholesterol essentially by the Sperry-Schoenheimer method (lo), and phosphorus by King’s technique (11). Unsaturation was assayed by a micro-Wijs adsorption procedure. In characterization of the glycerides, the fractions were hydrolyzed under nitrogen for 4 hours in 0.5 N ethanolic KOH, the ethanol removed under nitrogen, and the hydrolysate acidified with 6 N HCl. The free fatty acids were extracted with diethyl ether, washed well with water, and assayed for total weight, neutral equivalent, and unsaturation. The aqueous fraction from this extraction was used for the assay of glycerol. The beef liver sterol esters were hydrolyzed under nitrogen for 6 hours in 0.5 N ethanolic KOH, while the rat liver sterols required a longer reflux of 24 hours in 1.0 s ethanolic KOH for complete hydrolysis. The hydroly- sate was diluted with water to a 40 per cent ethanol concentration and extracted four times with fresh charges of diethyl ether. The ether frac- tion was washed well with water and assayed for total cholesterol. The water-soluble fraction was acidified, and the fatty acids were extracted and treated .as above.

Sugar was detected qualitatively by the Molisch test, acetals were de- tected by a modified Feulgen reaction (12), and amino acids were detected by the ninhydrin reagent. The Liebermann-Burchard qualitative tests for sterol were performed by dissolving the sample in 2 ml. of CHC& and adding 10 drops of acetic anhydride plus 2 drops of concentrated HzS04. Any test that registered negative in 2 minutes was rechecked after an additional 10 minute period in the dark. It was helpful to use the Lieber- mann-Burchard reaction as a check on the elution pattern of cholesterol esters and cholesterol.

RESULTS AND DISCUSSION

Fractionation Data-The nature of the fractionation pattern of the neutral lipides of rat liver, beef liver, and yeast is graphically depicted in Figs. 1, 2, and 3, respectively. The data on the composition of the major fractions are recorded in Tables I, II, and III. When the solvents indi- cated in Tables I, II, and III were used, 95 to 100 per cent of the lipide applied to a column could be recovered. The most reproducible combina- tion of solvents for fractionation of the neutral lipides from these sources and the typical fractions were hexane, hydrocarbons; 15 per cent benzene in hexane, sterol esters; 5 per cent diethyl ether in hexane, triglycerides plus any free fatty acids; 15 or 20 per cent diethyl ether in hexane, free sterol; 30 per cent diethyl ether in hexane, diglycerides; 50 per cent diethyl

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496 CHROMATOGRqPHY OF NEUTRAL LIPIDES

ether in hexane, unidentified component; and 90 to 100 per cent diethyl ether in hexane, monoglycerides. If it is suspected that diglyceride may be

5 15 25 35 45 55 65 75 t FRACTIONS (50 ml.)

5

Fro. 1. Chromatography of beef liver neutral lipides on silicic acid. Solvents were hexane (H), benzene (B), diethyl ether (E), and’methanol (M). The compo- sition of the various fractions iz recorded in Table I.

I

160-

140-

10 20 -30 40 50 60 70 80 90 0 FRACTIONS (50 ml.)

FIG. 2. Chromatography of rat liver neutral lipides on silicic acid. Solvents were hexane (II), benzene (B), diethyl ether (E), and methanol (M). The composition of the various fractions is recorded in Table II.

present in the lipide mixture, as indicated by either fatty acid or glycerol test on free sterol fraction, it was found most advantageous to use 15 per cent diethyl ether in hexane for optimal removal of the sterol, with 30 per cent diethyl ether employed for removal of diglyceride. Otherwise 20 per cent diethyl ether in hexane was the solvent of choke, as it allowed a

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E. J. BARBON AND D. J. HANAHAN 497

faster removal of the free sterol fraction. In Figs. 1, 2, and 3, the 3 per cent methanol in diethyl ether removed bile acid-like compounds, while 8 per cent methanol in diethyl ether removed any phosphorus-containing lipides. The recoveries quoted in Tables I, II, and III are based on the total amount of lipide obtained through the 90 to 100 per cent ether elution.

Beef Liver and Rat L&P--AS illustrated by the data in Tables I and II, the major components of beef and rat liver neutral lipides, respectively, were cholesterol esters, triglycerides, and cholesterol. Of considerable interest, a significant amount of diglyceride was found in rat liver only, and a smaller but definitive amount of monoglyceride was also detected in both sources. Mead and Fillerup (13) have reported the occurrence and

FRACTION (50 ml.)

FIG. 3. Chromatography of yeast neutral lipides on ailicic acid. Solvents were hexane (H), benzene (B), diethyl ether (E), and methanol @A). The composition of the various fractions is recorded in Table III.

also the separation of di- and monoglycerides in plasma by silicic chroma- tography. Through the use of 10 per cent ether in petroleum ether, 25 per cent ether in petroleum ether, and 100 per cent ether, they were able to obtain free sterol, diglyceride, and monoglyceride, respectively. In our experiments it was found that 15 per cent ether in hexane separated the sterol, 30 per cent ether in hexane removed the diglyceride, and 90 to 100 per cent ether in hexane would remove monoglyceride (some monoglyceride will be removed at a slow rate in 70 per cent ether). Although in the evidence presented here the sterol esters and the hydrocarbons were not as well separated as desired, the collection of smaller volume eluates would improve this fractionation. Also in order to obtain separation of the hydrocarbons, it was necessary to submit the hexane-benzene mixture to a preliminary drying with dried silicic acid.

Another aspect of the results was the finding that the triglyceride frac-

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498 CHROMATOGRAPHY OF NEUTRAL LIPIDES

tion contained a significant amount of free fatty acids (unesterified acids). Although Mead and Filler-up (14), in a study of the plasma lipides, have

TABLE I Composition of Fractions Obtained from Chromatography of

Beef Liver Neutral Lipides on S&Xc Acid

1.18 gm. of lipide were applied to 60 gm. of silicic acid. The total recovery was 1.20 gm. (101 per cent).

Solvent

Hexane

15% ben- zene in hexane

Pigment, hydro- carbons

Sterol esterst Unsaturation,

rnM GlyceroI, Y0 Fatty acid, y0

total esters Neutral equiv-

alent Unsaturation,

ClEYesterol, 70 total ester

Fatty acid/- cholesterol, molar ratio

Unaccounted material, y0 total est,er

-

3

-

Trace

11.2 2.4

O.OW 36.0

;oa

1.2

54.7

0.96

9.3

T

S c ,olvent tiethy ther ir 3exane

-

L ;

cr Len

5

20

30

50 loo

-

54 ;p a Wl.

3cM

oa

6cM

6cM 6cM -

_-

1

)

1

1 1 -

Components

Triglyceridest Fatty acid, y0 Neutral equiv-

alent Unsaturation,

mai Glycerol, y. Fatty acid/glyc

erol, molar ratio

Free fatty acids? Unaccounted

weight, y0 t,o- tal fraction

Free sterolt Cholesterol; y0 Unsaturation,

ME “C. No detectable

material Unidentified Monoglyceride

-r 2

1

1

41.9 88.0 96

0.84

9.4 2.91

6.8 6.3

34.8 cm

1.03

46-148

3.2 3.1

* All fractions gave negat.ive reactions for phosphorus, ninhydrin-reactive ma- terial, and carbohydrates. At no time was any sterol detected in the triglyceride fraction.

t Per cent of total lipide applied to column; material in methanol-ether (M-E) fractions not included (see Fig. 1).

indicated a separation of free fatty acids from triglycerides by use of 3 per cent ether in petroleum ether, it was not possible under the conditions of our experiments to obt,ain comparable fractionation. However, this may be attributed to differences in the source of lipides and the solvents employed for chromatography. At present it is not known whet,her these acids arise from the degradation of neutral or phospholipides during the

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E. J. BARROS AKD D. J. HAXAHAS 499

TABLE II Composition of Fractions Obtained from Chromatography of Rat

Liver Neutral Lipides on Silicic Acid

0.827 gm. of lipides applied to 60 gm. of silicic acid. Total recovery was 0.812 gm. (98 per cent).

Solvent

Hexane

15yo hen eene in hexanc

Components*

Pigments, hydro- carbonst

Sterol esters* Unsaturation,

rnM Glycerol, y. Fatty acid, y.

total ester Neutral equiv-

alent Unsaturation,

ClELterol, y. total ester

Fatty acid/- cholesterol, molar ratio

Unaccounted material, y. ester fraction

3.0

6.8 Jot rur

0.0047 40.0

02

1.06

55.3

0.93

4.7

olvent liethy! ther ir h?xam

er ceai

5

15

30

50 100

I Components

Triglycerideat Fatty acid, y. Neutral equiv-

alent Unsaturation,

rnM Glycerol, y. Fatty acid/glyc

erol, molar ratio

Free fatty acidst Unaccounted ma-

terial, y. total fraction

Free sterolt Cholesterol, To

Diglyceridet Fatty acid, y. Neutral equiv-

alent Unsaturation,

maf Glycerol, y. Fatty acid/glyc

erol, molar ratio

Unaccounted weight, y.

Unknownt Monoglyceridest

0)

66.0 83.2

270

0.83

9.6 2.93

1.5 3.2

16.3 98.6

5.8 82

300

1.2

11.8 2.07

. 6.2

0.8 0.9

* All fractions gave negative reactions for phosphorus, ninhydrin-reactive ma- terial, and carbohydrates. At no time was any sterol detected in the triglyceride fraction.

t Per cent of total lipide applied to column; material in methanol-ether @M-E) fractions not included (see Fig. 2).

isolation procedure or are present as normal components. From the ob- servations of Pairbairn (15), it would appear highly unlikely that unesteri-

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500 CHROMATOGRAPHY OF SEUTRAL LIPIDES

fied acids would be present to any significant extent in the original tissue. As recommended by Borgstriim (4), these acids may be removed from the

TABLE III Composition of Fractions Obtained from Chromatography of Yeast

Neutral Lipides on Silicic Acid

1.12 gm. of lipides applied to 60 gm. of silicic acid. Total recovery was 1.17 gm. (104 per cent). - -

$4 ,lvent

Solvent iethyl ;g components* .her in components i exam2

$1 3 - -

$1. rr cm1 ml. Hexane 150 Pigments, hydro- 2.0 20 ool Free sterolt + 3.4

carbonst Diglyceridet 10.3 15yo ben- 3x Sterol esterst 1.8 Ergosterol, y. 25

zene in fraction hexane Fatty acids, y. 51.4

5% di- 3Oc Triglycerideat 12.1 fraction ethyl Fatty acid/glyc- 2.97 Neutral equiv- 253 ether in erol, molar alent hexane ratio Unsaturation, 0.48

Free fatty acidst 58.9 mhc Neutral equiv- 64 Glycerol, y. 8.9

alent Fatty acid/glyc 2.09 Unsaturation, 0.6 erol, molar

mdl ratio Unaccounted 2.1

weight, Y. 50 60( Unknownt 4.0 90 6ol Monoglyceridet 7.0

Fatty acids, y. 75.5 Neutral equiv- 240

lent Unsaturation, 0.85

Glzrol, % 30.0 Fatty acid/glyc- 0.99

erol, molar ratio

- * All fractions gave negative reactions for phosphorus, ninhydrin-reactive mate-

rial, and carbohydrates. At no time was any sterol detected in the triglyceride fraction.

t Per cent of total lipide applied to column; material in methanol-ether (M-E) fractions not included (see Fig. 3).

triglycerides by washing with ethanolic 50 per cent alkali. However, in the latter case diglycerides and especially monoglycerides should be absent inasmuch as they may be removed along with the free acids.

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E. J. BARRON ASD D. J. HANAHAN 501

Yeast-A most interesting pattern developed in the fractionation of the lipides of yeast. As shown in Table III, the triglycerides were composed mainly of free fatty acids. From the results with the neutral lipides of rat liver and beef liver, it is reasonable to assume that these did not arise by decomposition of the lipide in the chromatographic procedure. As pointed out by Kleinzeller (16), there appear to be some clear indications that a considerable amount of free fatty acids may be present in micro- organisms. Although the procedures for the isolation of the lipides from this source are gentle, it does not rule out entirely degradation in the extraction process. This is based on the probability that powerful lipo- lytic enzymes exist in the cells and may be solvent-activated during the isolation procedure as are the lecithinases (17), thus causing extensive degradation of the lipides.

The remainder of the constituents of the yeast neutral lipides were sterol esters, sterol (ergosterol) , diglycerides, monoglycerides, and traces of other components too small to be adequately identified. In actual practice it was found impossible to separate the sterol and diglyceride fractions, even when the loading or the solvent system was altered. This may be due to the increased polarity of ergosterol over cholesterol, which then makes the difference between these compounds (on a chromatographic basis) much less.

General CommenLs-In view of the results discussed above, it would appear that hexane is a more suitable “non-polar” solvent than petroleum ether for silicic acid chromatography of neutral lipides. Hexane is readily available in a state of high purity and with a definitive narrow boiling point range, whereas petroleum ether (b.p. 30-66”) lacks these characteris- tics. Thus the above combination of solvents (“Fractionation data”) accomplished the most reproducible and effective separation of the neutral lipides from rat liver, beef liver, and yeast. Recently, Mead2 has recom- mended the use of pentane as a substitute for petroleum ether, and this has been applied by Mukherjee et al. (18). However, because of its low boiling point (36.2”), it may pose problems similar to those encountered with petroleum ether (i.e. bubbling; drying of the column near the base).

As would be expected, the nature of the solvents and the pretreatment of the column were of considerable importance. The influence of benzene on the adsorption characteristics of the silicic acid was particularly definitive. In columns in which benzene was not included in the initial solvent system, the triglycerides were rapidly eluted with 1 per cent diethyl ether in hexane and were in general contaminated with cholesterol esters. However, in those columns wherein 15 per cent benzene in hexane was employed for removal of the sterol esters, the triglycerides were removed only with

e Personal communicstion.

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502 CHROMATOGRAPHY OF NEUTRAL LIPIDES

higher concentrations of diethyl ether in hexane and with no st.erol con- tamination. While it was observed that 3 per cent ether in hexane would slowly elute the triglycerides, the use of 5 per cent ether in hexane allowed a more rapid and reproducible elution.

There appeared to be little influence of temperature (25-28”) on the chromatographic separation; however, depending on particular laboratory conditions, it is advantageous to have water-jacketed columns for tempera- ture control. In the case of the yeast lipides, undue exposure of the column to light can cause alterations in the sterols. Although there may be a slight amount of finely divided silicic acid filtering through into the eluates, the solubility of silicic acid is very low in the solvents used. Xever- theless, silicic acid is detectable in the eluates, and in view of evidence presented by Holt and Yates (19)) it can be attributed to the dissolving effect of hydroxy-containing compounds, i.e. monoglycerides.

At this point, it is well to emphasize that lipide samples as fresh as possible should be used, and in general it has been noted that, the frac- tionated samples are much more stable than the original crude fraction. Furthermore, as has been indicated previously (3), chromatographic separation of lipide samples is by no means a panacea. However, it can be a very useful technique when used with the proper precautions and knowledge that there are inherent errors in such procedures. In addition, the use of as many criteria as possible to establish the identity of a fraction is most desirable, and use of a particular solvent, system or procedure may require some alteration, depending upon the source of material and the general composition of the samples. Thus, these procedures can serve as an initial stage for parGal or complete fractionation of certain com- ponents, an aid in st.udy of the mechanism of lipolytic degradations, or in application to certain metabolic problems.

In a recent study, LaRoche (20) has observed that an entire lipide sample containing all the neutral lipides as well as the phospholipides of salmon tissues can be separated on silicic acid by use of the solvent sys- tems described here for neutral lipides, followed by use of chloroform methanol mixtures for the phospholipides (3). In addition, LaRoche (20) observed t.hat a general separation of the neutral lipides from the phospho- lipides could be accomplished by passage of the entire lipide sample through silicic acid first in 100 per cent ether, wherein the neutral lipides are not adsorbed, with subsequent elution of the phospholipides with methanol. As has been reported by Borgstrdm (4) and confirmed here, chloroform can replace diet,hyl ether in the fractionation.

SUMMARY

The chromatographic separation of the neutral lipides of rat liver, beef liver, and yeast on silicic acid has been investigated. Hexane has

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E. J. BARRON AND D. J. HAXAHAN 503

been employed as the major non-polar solvent, and in combination with t.he following solvents a fractionation of the indicated components could be achieved: hexane, pigments, hydrocarbons; hexane-15 per cent benzene, sterol est,ers; 5 per cent ether in hexane, t.riglycerides plus free fatty acids; 15 or 20 per cent ether in hexane, free sterols; 30 per cent ether in hexane, diglycerides; and 90 to 100 per cent ether in hexane, monoglycerides.

Significant amounts of diglycerides, and to lesser extent monoglycerides, were found in rat liver but not in beef liver lipides. Monoglycerides were found in both rat and beef liver. The “triglyceride” fraction of yeast was composed mainly of free fatty acids.

BIBLIOGRAPHY

1. McKibbin, J. M., J. Biol. Chem., 220, 537 (1956). 2. Lea, C. H., Rhodes, D. N., and Stall, R. D., Biocha. J., 60, 353 (1955). 3. Hanahan, D. J., Dittmer, J. C., and Warashina, E., J. Biol. Chem., 228,685 (1957). 4. Borgstrom, B., Acta physiol. &and., 25, 101, 111 (1952). 5. Fillerup, D., and Mead, J., PTOC. Sot. Exp. Biol. and Med., 83, 574 (1953). 6. Lovern, J. A., Biochem. J., 63,373 (1956). 7. Nicolaides, N., and Foster, R. C., Jr., J. Am. Oil Chem. Sot., Xl, 404 (1956). 8. Hanahan, D. J., and Barron, E. J., Federation Proc. 18, 191 (1957). 9. Hanahan, D. J., and Olley, J., J. BioE. Chem., in press.

10. Schoenheimer, R., and Sperry, W. M., in Hawk, P. B., Oser, B. L., and Summerson, W. H., Practical physiological chemistry, Garden City, 13th edition, 589 (1954).

11. King, E. J., Biochem. J., 26,292 (1932). 12. Feulgen, R., and Grunberg, H., 2. Physiol. Chem., 257,161 (1938). 13. Mead, J. F., and Fillerup, D., J. BioZ. Chem., 227, 1669 (1957). 14. Mead, J. F., and Fillerup, D., Proc. Sot. Exp. BioZ. and Med., 86,449 (1954). 15. Fairbairn, D., J. Biol. Chem., 167, 645 (1945). 16. Kleinseller, A., Advances in Enzymol., 8, 317 (1948). 17. Hanahan, D. J., Progress in the chemistry of fats and other lipids, 4, 141 (1957). 18. Mukherjee, S., Achaya, K. T., Deuel, H. J., Jr., and Alfin-Slater, R. B., J. BioZ.

Chem., 226, 845 (1957). 19. Holt, P. F., and Yates, D. M., Biochem. J., 54, 300 (1953). 20. LaRoche, G., Thesis, University of Washington (1957).

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Edward J. Barron and Donald J. HanahanBEEF LIVER, AND YEAST

NEUTRAL LIPIDES OF RAT LIVER,CHROMATOGRAPHY OF THE

OBSERVATIONS ON THE SILICIC ACID

1958, 231:493-503.J. Biol. Chem. 

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