The distribution of labeled palmitic acid into the diglycerides and triglycerides of rat adipose tissue

After in vitro incubation of rat epididymal fat pads with radioactive palmitic acid, the distribution of the label in the different lipid classes and in different triglycerides was determined by silica gel and silver nitrate-silica gel thin-layer chromatography (TLC). The radioactivity of the diglycerides was approximately half of the triglycerides. This ratio did not change with alteration in the time of incubation. It remained unaltered even after a subsequent 10-min incubation in a nonradioactive medium. When the fat pads were incubated, first with¹⁴C-, then with³H-labeled palmitic acid, the³H/¹⁴C ratio was slightly lower in diglycerides than in triglycerides. The fully saturated molecules contained 38% of the radioactivity of triglycerides. Addition of oleic acid or norepinephrine to the labeled palmitic acid-containing medium decreased this value. Subsequent incubation with these compounds did not alter the distribution of radioactivity.     

The incorporation of fatty acids of different chain length into liver and biliary lipids in the perfused rat liver

In an attempt to correlate the incorporation of fatty acids (FA) of different chain length into liver and biliary lipids’ isolated rat livers were perfused for 2 h with Krebs-Ringer bicarbonate containing 1% albumin and 10 μmol of [1-¹⁴C]-labeled FA: C₂’ C₈’ C₁₀’ C₁₂’ C₁₆’ and C_18∶1. One to 1.36 μmol of medium-chain fatty acids (MCFA’ C₈’ C₁₀’ and C₁₂) and 6.6 μmol of long-chain FA (LCFA) were incorporated into liver lipids’ 40% of the latter into phosphatidylcholine (PC). ¹⁴C-acetate (13 nmol) was incorporated into biliary cholesterol; ¹⁴C-MCFA contributed only 3.2–5 nmol; LCFA did not lead to newly synthesized cholesterol. Newly synthesized liver PC (2.75 to 3.25%) and newly synthesized liver cholesterol (6.5 to 10%) were secreted into bile. The specific radioactivity of biliary PC after infusion of all-saturated FA was 3.8–6.8 times higher than that of liver PC; for C_18∶1 it was only 1.7-fold. The specific radioactivity of biliary cholesterol’ as compared to liver cholesterol’ was 12 times higher for C₂ and five times higher for MCFA. This indicates that a considerable proportion of the newly synthesized lipids was secreted into bile prior to significant mixing with preexisting liver PC and cholesterol pools. liver PC contained 8% of unchanged ¹⁴C−C₁₂; while ¹⁴C−C₁₀ was not detected. Biliary PC’ in contrast’ contained 18% of unchanged ¹⁴C−C₁₂ and 3% ¹⁴C−C₁₀. These results suggest that after prolonged infusion of medium-chain triacylglycerols/longchain triacylglycerols to patients’ biliary PC may become enriched with MCFA. In addition’ the oxidation of these FA may provide C-2 units which increase cholesterol synthesis.     

The structure of rat liver triglycerides

The fatty acid compositions at the 1-, 2-, and 3-positions³ of rat liver triglycerides were determined by using pancreatic lipase and diglyceride kinase. The distribution of acids between the 1- and 3-positions is not random; rather each position has a characteristic composition. The relative abundance of species and positional isomers in the triglyceride mixture was predicted by using values from the stereospecific analysis and assuming that the composition of each position is independent of the other two. The total triglyceride was resolved into species by using TLC with silver nitrate and Silica Gel G, and the relative amounts corresponded closely with those predicted on the basis of this assumption. The major species were isolated, and the distribution of their fatty acids among the three glyceride positions was determined. From these data the relative amount of each positional isomer was calculated. The results indicate that the esterification of fatty acids at each position proceeds with a specificity that is not correlated with the composition of the other positions with the molecule. The relative abundance of the different liver triglyceride species is also found to be related in part to the composition of the 1,2-diglyceride units found in the lecithins of this tissue. Presented at the AOCS Meeting, Philadelphia, October 1966.     

The incorporation of 2-aminoethylphosphonic acid into rat liver diacylglyceroaminoethylphosphonate

Rats intravenously administered (¹⁴C) 2-aminoethylphosphonic acid (AEP) incorporated ca. 16% of the total injected compound into liver lipids. Thin layer chromatography and selective chemical and enzymatic hydrolysis of the labeled lipids demonstrated that essentially all of the radioactivity was in one compound, diacylglycerol-AEP, the phosphonate analog of phosphatidylethanolamine. Lipids from kidneys, heart, skeletal muscle, adipose, pancreas and brain were examined and found to contain less than 2% collectively of the total injected radioactivity. The residues from the tissues contained ca. 3.2% of the total injected (¹⁴C) AEP.     

Relative incorporation of linoleic and arachidonic acid in phospholipids and triglycerides of different rat tissues

Fat-deficient rats were fed different amounts of methyl linoleate for increasing periods of time. The fatty acid composition of triglycerides and phospholipids of epididymal fat pad, epirenal fat depot, intestinal fat depot, liver, and the pool of heart, kidney, lungs and pancreas was determined. The distribution of the total amount of linoleic and arachidonic acid incorporated into phospholipids and triglycerides per rat was calculated. Phospholipids and triglycerides of depot tissues presented different fatty acid compositions. Although the phospholipids of liver and the pool of heart, kidney, lung and pancreas specifically incorporated linoleic acid at the beginning they very rapidly attained a rather steady composition, whereas triglycerides went on incorporating the acid. The amount of linoleic acid incorporated into the phospholipids of depot tissues was rather small. The triglycerides undoubtedly contributed in the highest proportion to the total pool of linoleic acid. However, the highest proportion of arachidonic acid was found in the total pool of phospholipids. The total amount of linoleic acid incorporated into the phospholipids was an approximately lineal function of the amount of phospholipids independent of period of administration and doses of methyl linoleate. Besides presenting two lineal functions of the amount of phospholipids, arachidonic acid showed a vertical increase coincident with a vertical decrease of the amount of eicosa-5,8,11-trienoic acid. At this period no change in the amount of the phospholipid was shown. This phenomenon is explaioned as a possible direct replacement of eicosatrienoic acid by arachidonic acid.     

The effect of 3-methylindole on the uptake and incorporation of14C-choline into phospholipids in lung tissue slices

3-Methylindole (3MI) is the causative agent in the development of acute bovine pulmonary edema. Microscopic studies revealed a structural disruption in the lamellar bodies of type II cells, indicating an abnormal metabolism of phospholipid in the lung of 3MI treated animals. In the present study, lung slices from 4 goats were used to investigate the changes in phosphatidylcholine metabolism induced by 3MI. Eighteen slices were cut from each healthy lung and divided into control and 3MI groups. After a 4-hr pretreatment with 3MI (.19 or .57 mM) or carrier, the level of incorporation of¹⁴C-choline into phosphatidylcholine, sphingomyelin and their water soluble intermediates was studied. The uptake of¹⁴C-choline and its incorporation into phosphatidylcholine and sphingomyelin was depressed by 3MI treatment. In the water soluble fractions, the radioactivity increased in free choline and CDP-choline while it decreased in P-choline. This suggests that choline kinase and the P-choline transferases have become relatively more rate limiting and may play a role in the depressed de novo synthesis of phosphatidylcholine induced by 3MI.     

The selective utilization of diglyceride species into maize triglycerides

The maize triglycerides were resolved into species by silver nitrate thin layer chromatography. The distribution of the fatty acids among the 1, 2 and 3 positions of each triglyceride species was determined by stereospecific analysis. From these data the relative amounts of each positional isomer were calculated. The results indicate that esterification of the fatty acids at each position proceeds with a specificity that is correlated with the composition of the other positions of the triglyceride.     

Behavior of diglycerides and conjugated fatty acid triglycerides in reverse-phase chromatography

The behavior of conjugated fatty acid triglycerides and diglycerides on reverse-phase chromatography was studied. Trieleostearin is a geometric isomer of trilinolenin. The conjugated double bond arrangement in trieleostearin enhances its hydrophobic interaction with the stationary phase and causes it to be eluted later than trilinolenin. In separation of “critical pairs” of tri- and diglycerides, diglycerides elute later than triglycerides due to the longer fatty acid constituent. Position isomers of 1,2- and 1,3-diglycerides can be separated by reverse-phase high-performance liquid chromatography.     

Enzymatic enrichment of conjugated linoleic acid isomers and incorporation into triglycerides

A method was developed for the enrichment of either the cis9,trans11 or the trans10,cis12 isomer of conjugated linoleic acid (CLA) from a synthetic CLA mixture consisting predominantly of these isomers in equal amounts. Lipases were screened for their ability to selectively esterify one isomer at a significantly greater rate than the other isomer. An immobilized lipase from Rhizomucor miehei was nonselective, but a lipase from Geotrichum candidum esterified the cis9,trans11 isomer more rapidly than the trans10,cis12 isomer. This selectivity was exploited at the kilogram scale to prepare an ester fraction with a content of 91% cis9,trans11 CLA and an unreacted free fatty acid fraction consisting of 82% trans10,cis12 CLA, based on total CLA content. The components of the reaction mixture were separated by molecular distillation. Each enriched fraction was then incorporated into palm oil triglycerides by interesterification with the non-selective lipase from R. miehei. Two triglyceride fats resulted, which were enriched in either cis9,trans11 CLA (26.5% cis9,trans11 and 1.7% trans10,cis12) or trans10,cis12 CLA (3.5% cis9,trans11 and 22.9% trans10,cis12).     

The effect of bleomycin on the uptake and incorporation of [14C] choline into phospholipids in hamster lung tissue slices

Intratracheal administration of the anticancer drug bleomycin to hamsters produced an increase in the uptake and incorporation of [¹⁴C] choline into phospholipids of lung slicesin vitro. The stimulatory effect is opposite to the results obtained previously using [¹⁴C] acetate and would appear to occur distal to cytidine diphosphocholine. Although alternate explanations are possible, the results are consistent with morphological evidence, published by others, indicating an increase in lung phospholipid following bleomycin treatment, and illustrate the significance of precursor selection when evaluating the effects of xenobiotics on phospholipid synthesis.     

Separation of monoglycerides,diglycerides, and triglycerides by liquid-liquid extraction

Summary Ternary-equilibrium data were obtained at 60°C. for mono- and dilaurin, and di- and trilaurin with aqueous alcohols. Distribution between phases and solvent selectivity are shown graphically. Variation of solvent polarity by water addition caused a shift in the phase boundary curve so that either Type 1 or Type 2 diagrams could be obtained. Pure mono-, di-, and trilaurin mixtures, and commercial monoglycerides were studied as quaternary systems for monoglyceride distribution with 50 wt.-% aqueous ethanol. Pure and commercial systems showed similar distribution tendencies even with differences in molecular weight and degree of unsaturation. Solvent selectivity data (β) for quaternary systems were predicted from ternary systems studied with a common solvent. Aqueous ethanol will separate mixtures of mono-, di-, and triglycerides by selective liquid-liquid extraction; however the literature favors more complex systems based on two immiscible solvents. Continuous countercurrent extraction of commercial monoglycerides by 40% aqueous ethanol solution in a four-stage agitated column gave a 15% over-all stage efficiency. Cooling the extract to 0°C. precipitated 96.6% of the extract solids. Flow diagrams were prepared for batch and continuous extraction processes. The findings reported in this paper were included in a thesis submitted by J. A. Monick to the Graduate Division of the College of Engineering of New York University in partial fulfillment of the requirements for the degree of Doctor of Engineering Science. Presented at the fall meeting, American Oil Chemists’ Society, in Philadelphia. Pa., October 10–12, 1955.     

Incorporation ofcis-octadecenoic acids into the rat liver mitochondrial membrane phospholipids and adipose tissue triglycerides

The incorporation of the dietarycis 18∶1 (n−12) andcis 18∶1 (n−10) into liver mitochondrial membrane phospholipids and adipose tissue trigly cerides was studied in 4 groups of rats fed diets containing 10 weight percent (wt%) of fat with the following contents of octadecenoic acids: 50%cis 18∶1(n−12) +9%cis 18∶1 (n−9); 25%cis 18∶1 (n−12)+32%cis 18∶1 (n−9); 50%cis 18∶1 (n−10)+10%cis 18∶1 (n−9); or 54%cis 18∶1 (n−9). Dietary linoleic acid was 3 wt% in all 4 groups. In the mitochondrial membranes, the isomeric octadecenoic acids were primarily incorporated into the 1-position of phosphatidylcholines and phosphatidylethanolamines at the expense of saturated fatty acids. The maximal incorporations observed in the 1-position of phosphatidylethanolamines were 4.8% 18∶1 (n−12) and 8.9% 18∶1 (n−10). No effects on the contents of polyunsaturated fatty acids in the phospholipids were seen. In the adipose tissue, the isomeric octadecenoic acids were incorporated at a level of 13%cis 18∶1 (n−12) or 23%cis 18∶1 (n−10), paralleled by a reduction in the content of oleic acid. Presented in part at the 9th Scandinavian Symposium on Lipids, Visby, Sweden, June 1977.     

Differential trypsin effect upon (1-14C) acetate incorporation into choline and ethanolamine glycerophosphatides of rat brain and liver

Preincubation of rat brain and liver slices in a medium (5% glucose, 5% fructose, 1% albumin, 1% trypsin, 10 mM phosphate buffer pH 6.0) used to pretreat brain tissue for the separation of cell types was found to uncouple the incorporation of (1-¹⁴C) acetate into ethanolamine phosphoglycerides from that of the choline phosphoglycerides. Incorporation into ethanolamine phosphoglycerides was stimulated in both brain (330%) and liver (780%) slices, while the incorporation of (1-¹⁴C) acetate into choline phosphoglycerides was reduced for both brain (71%) and liver (63%) slices, compared to control values from nonpreincubated material. With (1-¹⁴C) linolenic acid as a precursor, no significant differences were found in incorporation into ethanolamine phosphoglycerides and choline phosphoglycerides.     

The incorporation of ethanolamine into ether-containing lipids in rat brain

After intracerebral injection of C¹⁴-ethanolamine into rats, the ethanolamine phosphoglycerides were isolated and hydrolyzed with mild alkali and acid. The specific radioactivity of the diacyl, acyl alkenyl, and acyl alkyl glyceryl-3-phosphorylethanolamine, the diacyl and acyl alkenyl glyceryl-3-phosphorylcholine, and sphingomyelin was determined at 0.5, 2, 24, and 48 hours. The specific radioactivity-time relationships show that the ethanolamine plasmalogen is not a precursor for the glyceryl ether form but suggest that acyl alkyl glyceryl-3-phosphorylethanolamine is desaturated to form some of the acyl alkenyl glyceryl-3-phosphorylethanolamine. The radioactivity in the choline portion of the choline phospholipids was very low. Presented at the AOCS Meeting, Cincinnati, October 1965.     

The in vivo incorporation of labeled linoleic acid, α-linolenic acid and arachidonic acid into rat liver lipids

The incorporation of 1-¹⁴C-linoleic acid, 1-¹⁴C-α-linolenic acid and 1-¹⁴C-arachidonic acid into rat liver lipids was measured and the per cent distribution of radioactivity into the different lipid fractions determined. Normal rats were injected into the portal vein with the labeled solutions during a one minute period. Livers were quickly frozen, pulverized, and the lipids extracted and fractioned by thin layer chromatography. No significant differences were observed in the amounts of labeled fatty acids incorporated per gram of rat liver. While 1-¹⁴C-linoleic acid and 1-¹⁴C-α-linolenic acid were found in appreciable amounts in the 1,2 diacylglycerol fraction, about one fifth as much 1-¹⁴C-arachidonic acid was esterified in this fraction. 1-¹⁴C-arachidonic acid was the leading acid esterified in the phospholipid fractions.     

Absorption of Di- and triglycerides by intestinal slices in vitro

The uptake by hamster intestinal rings of labeled 1,3-diolein and triolein in bile salt emulsions was studied. About 6% of triolein was taken up from emulsions containing glycerides and fatty acid in 6 mM sodium taurodeoxycholate. Lesser uptake was noted when triolein was emulsified with lecithin, cholesterol and bile salt; lowest uptake (3%) was observed from triolein-lecithincholesterol emulsions prepared without bile salt. Absorption of 1,3-diolein from bile salt emulsion was greater and acylation to triglyceride was observed. Diglycerides and triglycerides in small quantity may be absorbed intact from a micellar phase.     

Incorporation of 1,2-14C-ethanolamine into subfractions of rat liver phosphatidylethanolamines and phosphatidylcholines

The incorporation of 1,2-¹⁴C-ethanolamine into the liver phosphatidylethanolamines (PE) and phosphatidylcholines (PC) in female rats was studied. These phosphatides were fractionated according to their degrees of unsaturation and the specific activities of monoenoic, dienoic, tetraenoic and hexaenoic fractions were measured at intervals of 1, 20, 60 and 300 min after injection of the labeled precursor. Hexaenoic and dienoic PE incorporated and lost the label rapidly. Although the labeled precursor was incorporated into tetraene PE at a similar rate, this fraction attained a relatively low specific activity that remained essentially constant between 10 and 300 min after injection of the label. Hexaenoic PC had the highest specific activity among the PC fractions at all time periods. Estimations of the rate of loss of radioactivity in the hexaenoic PE fraction and its appearance in hexaenoic PC indicate that the docosahexaenoic acid is conserved, possibly by being reincorporated into PE after becoming a part of the hexaenoic PC species. The high rate of turnover of the hexaenoic PE also suggests that this fraction might have some special role in endogenous choline synthesis.     

Uptake of [14C] choline and incorporation into lung phospholipid by the isolated perfused rat lung

We have used the isolated perfused lung (IPL) preparation from the rat to determine whether uptake of choline from the vascular compartment could limit the rate of synthesis of phosphatidylcholine (PC). The uptake of choline was rapid and did not saturate at a concentration of 10 mM. The rate of incorporation of choline into phospholipid was saturated above 0.1 mM choline. Whereas, uptake and incorporation were depressed at 4 C, uptake was neither dependent on the extracellular sodium concentration nor inhibited by equimolar concentrations of hemicholinium-3 (HC-3). We could find no evidence that uptake might limit synthesis of lung lecithin and conclude that uptake is either by free diffusion, or by a carrier-mediated process with a very high Km.     

Dietary fat effect on incorporation and release of lipids and cholesterol by rat intestinal slices

The effect of saturated and unsaturated fats on in vitro formation and release of lipids and cholesterol from¹⁴C acetate by rat intestinal tissue was investigated. The rats were fed a basal diet enriched with either 25% corn oil or lard and then sacrificed after a 10- or 25-day feeding period. It was observed that a similar¹⁴C lipid content but a greater¹⁴C cholesterol content was found in the intestinal tissue of rats fed corn oil than in rats fed lard for 10 days. After a longer period of feeding of 25 days, the intestinal tissue¹⁴C cholesterol level was decreased in the corn oil fed rats without any significant effect on other lipids. These data suggest that corn oil in some way influences cholesterol biosynthesis depending upon its degree of unsaturation and the period of time for which it is fed. The decrease at the later time might involve some mechanism which aids in getting rid of accumulated tissue cholesterol. Less¹⁴C lipid and¹⁴C cholesterol were released by the intestinal tissue of rats fed the unsaturated fat as compared with those fed the saturated fat, suggesting a possible role in vivo in reducing blood lipids and blood cholesterol levels. Robert A. Welch Foundation.     

Ratio of in vivo incorporation of3H arachidonic acid and14C linoleic acid into liver lipids from normal and diabetic rats

Normal and streptozotocin diabetic rats were injected via the portal vein with a labeled solution containing³H arachidonic acid and¹⁴C linoleic acid (³H/¹⁴C ratio, 0.5) during a 1 min period. Livers were quickly frozen, pulverized, and the lipids extracted and fractioned by thin layer chromatography. The incorporation of³H and¹⁴C into liver lipids was measured and the percentage distribution of radioactivity into the different lipid fractions was determined. The incorporation of¹⁴C linoleic acid and³H arachidonic acid into liver lipids is apparently reduced in rats with severe diabetes. The higher³H/^14C ratio found in the 1,2 diglycerides from diabetic rats may be explained by the apparently smaller incorporation of¹⁴C linoleic acid or by an isotopic dilution attributable to the great availability of this acid in diabetic rats. On the other hand, the higher³H/¹⁴C ratio observed in triglycerides and phospholipids from diabetic rats, due to a relatively large incorporation of³H arachidonic acid into this fraction, may be explained by the affinity of the enzymes involved in their synthesis for some 1,2-diglyceride units. Insulin was unable to correct the changes observed in the diabetic rats.