54 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 55268

METABOLISM OF I-PALMITOYL DIOLEIN AND 3-PALMITOYL DIOLEIN

BY ADIPOSE TISSUE*

W. R. WIiIGHT AND S. B. TOVE

Depavtments of Biochemistry and Animal Science, North Carolina State University,

Raleigh, N.C. (U.S.A.)

(Iieceived May asrd, 1966)

SUMMARY

Stereospecific metabolism of triglycerides was investigated by incubating homo- genates of adipose tissue with I-palmitoyl diolein and 3-palmitoyl diolein, each labeled with 14C at the carboxyl group of the palmitic acid. No evidence for stereospecific hydrolysis was obtained from the specific activity of the fatty acids, monoglycerides and diglycerides. However, approximately twice as much label was incorporated into the z-position of the tissue triglycerides from I-palmitoyl diolein as from 3-palmitoyl diolein, suggesting a stereospecific transfer of a fatty acid from the r-position of a triglyceride to the z-position of a triglyceride precursor.

INTRODUCTION

The observation that fatty acids are not esterified randomly among the three alcohol positions of glycerol in natural glycerides provides a continual stimulus for the investigation of specificity in lipid metabolism. The possibility of stereospecific metabolism of triglycerides is raised by the finding that r,a-diglycerides** (D-digly- cerides) are the preferred substrate in triglyceride synthesis2 and the more recent findings that the fatty acid composition of the r-position of triglycerides differs from that of the 3-position3+. Stereospecific hydrolysis of triglycerides has been investi- gated with pancreatic lipase, lipoprotein lipase and wheat germ lipase6y6. This report describes an investigation of the metabolism of enantiomeric triglycerides by mouse adipose tissue.

MATERIALS AND METHODS

Substrate synthesis The triglyceride substrates used in this study were stereospecifically labeled

3-palmitoyl diolein and I-palmitoyl diolein, each enantiomer containing 14C at the

* Published with the approval of the Director of the North Carolina Agricultural Experiment Station as Paper No. 2206 of the Journal Series. ** The numbering of the positions of glycerol is in accord with the convention of HIRSCHMAN~.

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METABOLISM OF TRIGLYCERIDE ENANTIOMERS BY ADIPOSE TISSUE 55

carboxyl carbon of palmitic acid. These substrates were synthesized from n-mannitol

and r.-mannitol respectively. o-Mannitol was obtained from commercial sources, and L-mannitol was pre-

pared by reduction of L-mannose that was synthesized by the method of SOWDEN 7

without isolation of the I-deoxy-I-nitro-L-mannitol intermediate. Reduction of the t-mannose was accomplished in the following manner. To a solution of IZO g of impure L-mannose in I 1 of 0.5% NaOH was added a solution of 32 g sodium borohydride in 500 ml of 0.5% NaOH. The solution was stirred for 3 h at room temperature and, after acidification with acetic acid, taken to dryness on a rotary evaporator. The residue was refluxed for 3 h with methanol containing 5% HCl and the NaCl filtered from the hot solution. The mannitol solution was then taken to dryness on a rotary evaporator, dissolved in water, and passed through a 4.8 x 65 cm column of Amberlite IR-112 (hydrogen form). The eluate from the column was taken to dryness, and the residue dissolved in a minimal amount of water and poured into IO vol. of abs. ethanol. The evaporation procedure was repeated until the L-mannitol precipitated when the aqueous solution was poured into the alcohol. A yield of 19.5 g of L-mannitol was obtained that melted at 189-191’, [c~]D~~+o.z (water); reported* m.p. 190-191’, [Cr]D25+o.21 (water).

The D- and L-mannitols were converted to 2,3-isopropylidene glycerol and 1,2- isopropylidene glycerol respectively by the procedure of BAER~ and the products isolated by distillation. The 2,3-isopropylidene glycerol boiled at 37O/o.3 mm, [a]~” -13.3 (ether) and the r,z-isopropylidene glycerol boiled at 43-45’/1 mm, [c~]n~~+14.2 (ether). The values reported9 for r,2-isopropylidene glycerol were b.p. 77-@“/IO mm; [a]n+13.98 (in substance).

Radioactive palmitoyl chloride was prepared by equilibrating1o [r-14C]palmitic acid with palmitoyl chloride (Fluka). The isopropylidene glycerols were esterified” with the %-labeled palmitoyl chloride and the resulting monoacyl acetals hydrolyzed to monoglycerides by the borate method of MARTIN’~. To minimize acyl migration, the monoglycerides were converted without isolation to triglycerides by treatment with oleoyl chloridell. Oleoyl chloride was prepared from oleic acid using oxalyl chloride as the chlorinating agent 13.

Fatty acid contaminants were removed from the triglyceride enantiomers by passage through a column of Amberlite-IRA-400 (hydroxyl form). The triglycerides were then chromatographed on florisil 14. Thin-layer chromatography on silica gel using heptane-isopropyl ether-acetic acid (6:4:0.3, v/v) as the developing solvent showed single spots corresponding to known triglycerides. Analysis for ester groups’5 and for glycerol I8 following hydrolysis gave expected values (Table I).

To demonstrate that acyl migration of the labeled palmitate to the 2-position had not occurred during synthesis, 500 mg (8.7 * 10~ counts/min) of each enantiomer

TABLE I

ESTER AND GLYCEROL ANALYSIS OF I-PALMITOYL DIOLEIN AND 3-PALMITOYL DIOLEIN

Each value is an average of duplicate determinations.

Sample

I-Palmitoyl diolein 3-Palmitoyl diolein

Amount Ester Glycerol (przoles) (#umoles/3) (fLmoles)

28.8 27.1 28.2 21.8 21.3 20.8

Biochim. Biophys. Acta, 137 (1967) 54-58

56 W. R. WRIGHT, S. B. TOVE

were subjected to pancreatic lipolysis r7y18. The resulting monoglycerides representing the fatty acid in the z-position were isolated by chromatography on florisil and found to have no appreciable radioactivity, i.e., 138 counts/min for the monoglycerides from I-palmitoyl diolein and 164 counts/min for those from 3-palmitoyl diolein.

Tissue preparation and incubatiom

Single epididymal fat pads from 5 mice were placed in 5 ml of ice cold 0.15 M KC1 and homogenized for I min in a micro-unit of a Servall Omni-mixer with 85 mg of labeled I-palmitoyl diolein. The contralateral pads from the same mice were similarly homogenized with labeled 3-palmitoyl diolein. Duplicate incubations were carried out on a rotary shaker at 40’ in 3 ml of solution containing z ml of homo- genate, IO pmoles of d-glycerophosphate, 2. ,umoles of ATP, 3 ,umoles of MgCl,, 0.1

pmole of CoA and 125 ,umoles of potassium phosphate buffer at pH 7.0. After 30 min incubation, 5 ml of 5% HCl were added.

Separation and analytical procedures

The acidified incubation mixtures were extracted with chloroform-methanol (2 : I, v/v) and the fatty acids removed from the neutral lipids by passage of the chloro- form phase through a column of alkaline silicic acidlg. The acids were then eluted from the column with a solution of 2% formic acid in etherla. The neutral lipids were separated into monoglycerides, diglycerides and triglycerides by chromatography on florisil 14.

A major portion of each triglyceride fraction was subjected to pancreatic lipo- lysis17>18. The monoglycerides were isolated from this lipolysis mixture by florisil chromatography after removal of the free fatty acids by IRA-400 resin.

For specific-activity determinations the ester groups of the glyceride fractions were measured by the hydroxamate method of SNYDER AND STEPHENS’~. Free fatty acids were measured spectrophotometrically by the procedure of DUNCOMBE 20. Radioactivity in the lipid samples was measured in a liquid scintillation spectrometer

TABLE II

RELATIVE SPECIFlC ACTIVITY OF FREE FATTY ACIDS AND GI_YCERIDES FclIJ_OT?‘ING INCUBATION OF I-PAUvlITOYL DIOLEIN AND Q-PALMITOYL DIOLEIN WITH HOMOGENATES OF ADIPOSE TISSUE

Each enantiomeric triglyceride was incubated with homogenates of epididymal fat pads as de- scribed in the text. The reaction mixture contained IO pmoles of cc-glycerophosphate, 2 pmoles of .kTP, 3 ,umoles of M&l,, o. I pmole of CoA and 125 ,umoles potassium phosphate buffer (pH 7.0) in a final volume of 3 ml. The incubation was carried out at 40” for 30 min. The values shown are __-- the means and standard deviations (z/d2/n- I) of 5 experiments. Rela.tivc specific activity is the ratio of the specific activity of an individual lipid fraction to the specific activity of the corre- sponding triglyceride fraction.

Lipid fraction I-Palmitoyl diolein 3-Palmitoyl diolein (countslmin per ,umole) (ceuntslmin per pmole)

Free fatty acids 49.2 i 5.6 47.0 + 7.’ Monoglycerides 24.5 + 5.2 30.5 f 3.2 Diglycerides 44.6 zt 5.0 37.6 ;L 5.’ 2-Monoglycerides 23.8 & 3.0* II.4 : 3.0* z-Monoglycerides (zero time) 3.0** 1.5**

* Significantly different (P < 0.01) ; all other fractions not significantly different (P < 0.05). ** Average of 2 experiments.

Biochim. Biophys. Acta, 137 (1967) 54-58

METABOLISM OF TRIGLYCERIDE ENANTIOMERS BY ADIPOSE TISSUE 57

using a solution of 0.5% diphenyl oxazole and 0.03%, r,+bis-2(5-phenyl-oxazolyl)-

benzene in toluene as the scintillation medium.

RESULTS

The results of five incubations of the stereospecifically labeled triglyceride enantiomers with homogenates of adipose tissue are summarized in Table II. In order to minimize the variation resulting from endogenous triglycerides, the values are expressed as relative specific activity, i.e., specific activity of each fraction divided

by the specific activity of the corresponding triglyceride fraction. Comparison of the relative specific-activity values of free fatty acids, mono-

glycerides and diglycerides revealed no statistically significant differences (P < 0.05) between the triglyceride enantiomers. Although these data provide no evidence for major stereospecific hydrolysis of triglycerides by adipose tissue, the experimental variation was large andmay have masked stereospecific hydrolysis of alowermagnitude.

In contrast, evidence for stereospecific transfer of palmitate from the primary hydroxyls to the z-position of glycerol is apparent from the relative specific activity of the 2-monoglycerides. About twice as much palmitic acid was transferred from I-palmitoyl diolein to the 2-position of the triglycerides as was transferred from 3- palmitoyl-diolein (Table II).

To reconfirm the initial absence of radioactivity in the z-position of the labeled triglycerides, samples were taken from two of the incubation mixtures immediately before incubation. Specific activity of the triglycerides of these samples and of their a-monoglycerides was determined. These data indicate that the amount of 1°C in the z-position of the enantiomers was negligible (Table II).

DISCUSSION

The stereospecific activity of enzymes has been demonstrated so frequently that it is now an expected occurrence. It is noteworthy, therefore, that stereospecific hydrolysis of triglycerides has yet to be demonstrated despite the fact that a D-diglyc- eride is a precursor of triglyceride synthesis a. TATTRIE et aL6 showed that pancreatic lipase hydrolyzed fatty acids esterified at the r-position and 3-position of triglycerides at equal rates. KARNOVSKY AND WOLFER obtained the same results with pancreatic lipase, lipoprotein lipase and wheat germ lipase. Similarly, the results of the studies reported herein fail to demonstrate stereospecific hydrolysis of triglycerides by homo- genates of adipose tissue.

On the other hand, we have obtained evidence for the stereospecific exchange of a fatty acid from the r-position of a triglyceride to the z-position of an unidentified triglyceride precursor. Although the mechanism of the stereospecific transfer is un- known, it can be accounted for by either of two systems. First, a stereospecific hydrolysis of the fatty acid at the r-position of the triglyceride followed by its re- esterification with an endogenous triglyceride precursor. The nature of the reesterifi- cation process would be unimportant as long as the liberated palmitic acid could esterify the s-position of the triglyceride precursor. It is clear, however, from the data of Table II that were such a system to occur, it must take place in a small metabolic compartment in which the products of the stereospecific hydrolysis are not in rapid

Biochim. Biophys. Acta, 137 (1997) 54-58

58 W. R. WRIGHT, S. B. TOVE

equilibrium with the majority of the free fatty acids and partial glycerides of the homogenate.

The second system would involve a direct stereospecific transfer of a fatty acid from the r-position of the triglyceride to the z-position of a triglyceride precursor without the release of the fatty acid. Such a reaction would not necessitate activation of a free fatty acid and would not require ATP. Direct transesterification from one hydroxyl group to another has been reported. GLOMSET~~ found that a fatty acid occupying the z-position of lecithin could be directly transferred to the hydroxyl group of cholesterol and ERBLAND AND MARINETTI~~ reported a system in which 2

moles of lysolecithin yielded lecithin and glycerophosphorylcholine without the requi- rement of ATP.

ACKNOWLEDGEMENT

This work was supported in part by research grant AM 2483 from the National Institutes of Health, Department of Health, Education and Welfare.

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