The Incorporation of '4C-Glycerol into Different Species of Diglycerides and Triglycerides in Rat Liver Slices EDWARD E. HILL, WILLIAM E. M. LANDS and SISTER P. M. SLAKEY1, Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48104 ABSTRACT ing the specificity for certain diglycerides in the The relative rates of de novo synthesis enzymic acylation of diglycerides to produce of species of diglycerides and triglycerides triglycerides have been reported (5, 6); how- from 14C-glycerol were examined in rat ever, the significance of these studies Was ob- Tiver slices. Diglycerides containing one scured by differences in solubility of different or two double bonds per molecule and diglycerides. Likewise, no appreciable specifi- triglycerides containing four or more city has been observed in the formation of double bonds per molecule represented phosphatidic acid by acylation of glycerol-3- 70% and 60% respectively of the newly phosphate (7) or in the hydrolysis of phos- synthesized diglycerides and triglycerides. phate from phosphatidic acid (8). The newly synthesized triglycerides were In the present study the de novo synthesis more unsaturated than the endogenous of diglycerides and triglycerides from a4C- triglycerides. Our results suggest that a glycerol was examined to see if all types of nonrandom synthesis of species of diglyc- triglyceride were formed at similar rates. Our erides occurred followed by an almost results indicate that a degree of selectivity random utilization of the various diglyc- occurred in the synthesis of triglycerides by eride species for the biosynthesis of tri- rat liver, and that the de novo synthesis of glycerides. triglyceride did not lead to a duplication of the pre-existing distribution of molecular species INTRODUCTION of triglycerides. N LIVER 1,2-DtGLYCERIDES can be considered EXPERIMENTAL I to serve as a common precursor for the bio- synthesis of glyceryl esters triglycerides, leci- Incorporation of 1'C-Glycerol into Liver Lipids thin and cephalin. Since the formation of all Male Sprague-Dawley rats (175-240 g) were three types of lipid requires reaction at only sacrificed by decapitation. The body cavity was the 3 position, the ordered structure of the di- immediately opened, and the liver was per- glycerides would be expected to remain as a fused with cold Locke's solution (9) devoid common feature of the product glyceryl esters. of calcium containing 0.027 M trisodium citrate Saturated and unsaturated fatty acids are pre- until the liver attained a creamy color. Liver dominantly present at the 1 and 2 positions slices were prepared with a Briggs-Stadie respectively of triglycerides, lecithin and cepha- microtome. lin (1-3), but differences in fatty acid com- Liver slices (2 to 3) were incubated in Krebs- position of these types of lipids have been Ringer bicarbonate buffer (9) containing one- observed. For example, stearic and arachidonic half the normal amount of calcium with 100 acids, which are major components of rat liver ~moles of ATP, 0.2 mg of reduced coenzyme lecithin and cephalin, are minor components A and 10/,c of 1-14C-glycerol (10 #c//~m). The in rat liver triglycerides (1, 4). Such struc- final volume of the incubation mixture was 7.5 tural studies indicate that the distribution of ml. Incubations were performed at 37C with fatty acids in glyceryl esters is not a random constant shaking in an atmosphere of 5% process, but rather that mechanisms exist in carbon dioxide and 95% oxygen. Liver slices the cell which control both the positional oc- were removed at indicated time intervals, currence and the extent of occurrence of a rinsed, and immediately frozen between 2 given fatty acid in different glyceryl esters. blocks of dry ice. Attempts to demonstrate the reactions which Extraction of Lipids control the location of a given fatty acid have not been overly successful. Experiments test- After the weight (200-400 rag) of the frozen tissue had been recorded, the sample was homogenized in 5 mi of chloroform-meth- 1Present address: Saint Dominic College, Saint Charles, anol (1:1, v/v) containing 100/d of a solution Illinois 6017~. of 1% 2,6-di(t-butyl) 4-methylphenol in chlo- 411 412 EDWARD E. HILL, WILLIAM E. M. LANDS AND SISTER P. M. SLAKEY reform with a Virtis microhomogenizer for 1 rechromatographed on thin layer plates as de- rain. An additional 2.5 ml of chloroform was scribed above; to give a radiochemical purity added, and the homogenization was continued of greater than 95%. for approximately 30 seconds. Additional Similarly, the isolated diglycerides were chloroform-methanol (2:1) was added to give found to be contaminated with small amounts a final volume of 20 ml, and the samples were of cholesterol and an unidentified impurity. allowed to remain at room temperature for Since these contaminants yielded anomalous approximately 30 min. The extracts were bands in the subsequent separation of the di- washed once with 0.2 volumes of 0.58% so- glyceride fraction into species, the diglycerides dium chloride and once with "pure upper were further purified by TLC in a solvent of phase" as described by Folch et al. (10). The chloroform-acetone (96:4) with a layer of samples were evaporated to dryness using a boric acid-Adsorbosil-1 (12). Approximately rotary evaporator which was fushed with 90% of it was located in the 1,2-diglyceride nitrogen, dissolved in chloroform and stored fraction and more than 95% of the isotope was at -15C. recovered. The remainder of the radioactivity Isolation of Neutral Lipids was found in nearly equal amounts in mono- glyceride, 1,3-diglyceride and triglyceride frac- Neutral lipids were separated from polar tions. lipids using DEAE-cellulose columns as de- scribed by Rouser et el. (11 ). Diglycerides Separation of Di- and Triglycerides and triglycerides were isolated by thin-layer into Molecular Species chromatography (TLC) using Mallinckrodt The separation of diglycerides into individual silicic acid of less than 200 mesh. The plates species was achieved using thin layer plates were developed 4 cm in diethyl ether-petroleum containing 5% (w/w) silver nitrate Adsorbosil- ether (4:1). After drying for 15 min at room 1. Thin layers (0.3 mm) were activated at temperature, they were developed in diethyl 125C for 75 rnin and stored over a saturated ether-petroleum ether (12:88) to a distance solution of calcium chloride in a metal cabinet. of 18-20 cm. The lipids were visualized under Diglycerides prepared from pig liver lecithin ultraviolet light after spraying the plate with a by the action of phospholipase C (13) were solution of 0.2% 2',7'-dichlorofluorescein in added as a carrier to all diglyceride samples to methanol. The bands of triglycerides (R~ = facilitate location of the samples in subsequent 0.70) and diglycerides (R r = 0.30) were steps. The plates were developed in a solvent scraped from the plate and the lipids were system composed of dietbyl ether-petroleum eluted with methanol-ether (I :9). ether-benzene-methanol (45:35:30:2). After An aliquot of the recovered triglyceride spraying the plates lightly with a solution of fraction revealed a radiochemical purity of less 0.1% of 2',7'-dichlorofluorescein in methanol than 90% when chromatogrammed with car- five discrete bands were visible under ultra- rier diglycerides and triglycerides. Accordingly, violet light. These bands were found to con- the whole triglyceride samples were routinely tain one to five or more double bonds per TABLE I Fatty Acid Composition of the Carrier Diglyceride Resolved by Silver Nitrate-TLC Fatty acid 16:0 18:0 18:1 18:2 18:3 20:3 20:4 22:4 22:6 Band Band a Mole % designation 4 20 29 45 5 2 .... b 19 30 44 6 2 .... Monoene 5 13 35 4 49 ..... b 14 33 7 46 ..... Diene 7 10 44 4 -- 18 25 -- -- -- b 10 42 3 10 17 28 -- -- -- Triene 8 12 43 4 -- I 1 35 5 -- b 13 48 4 -- -- -- 36 -- -- Tetraene 9 17 41 3 -- 1 I 1 -- 28 b 17 41 3 -- I 4 -- -- 34 Polyunsaturated Total 16 35 20 13 3 4 4 1 4 aThe TLC-plate was divided into ten bands. Less than 3% of the total fatty acids was located in bands other than those indicated in this table. bThe second row represents a separate experiment. LIPIDS, VOL. 3, No. 5 '*C-GLYCEROL IN D~- AND TRIGLYCERIDES 413 molecule by means of GLC analysis of the methyl esters derived from the diglycerides. 50 The fatty acid composition of these bands is indicated in Table I. Less than 3% of the total diglyceride sample applied to the TLC plate was recovered in areas other than the five bands described in Table I. The method used to separate the triglyceride gg fraction of rat liver into its component species has been described in a previous communica- ~176 tion from this laboratory (14). Elution of Di- and Triglycerides from ~10 Silver Nitrate-Silicic Acid / The bands of silicic acid that contained the separated glycerides were scraped from the 1 | II thin layer plates and transferred into small so rio ,h test tubes. A solution of 1% sodium chloride Time, min in 90% methanol was added in portions (ap- proximately 0.5 ml) with vigorous mixing until Fro. l. The incorporation of "C-glycerol into the characteristic red color of the silver-di- diglycerides and triglycerides in rat liver slices. chlorofluorescein complex was destroyed. The Q) Q) Diglycerides, /k-/k Triglycerides. glycerides were extracted with 5 ml of ether- methanol (9:1). After centrifugation, the neutral lipids. The total counts present in the supernatant solution was transferred to a scin- diglyceride and triglyceride fractions were tillation vial.