Preferential labeling of phosphatidylcholine during phospholipid synthesis by bovine mammary tissue

Bovine mammary cells and tissue synthesize de novo the classes of phospholipids, found in mammary tissue and milk, from various precursor molecules. Several short term experiments were carried out in vitro, using labeled precursors, i.e., 1-¹⁴C-fatty acids; 2-¹⁴C-acetate; U-¹⁴C-glycerol; 1,2-¹⁴C-choline; 1,2-¹⁴C-ethanolamine; 2-¹⁴C-serine; and Me-¹⁴C-methionine. All the phospholipid classes were labeled. The specific activity of tissue phosphatidylcholine was consistently three to six-fold greater than that of phosphatidylethanolamine. The results indicated that stepwise methylation of phosphatidylethanolamine with labeled methyl group of methionine was occurring to a minor extent, as was a negligible amount of choline exchange. Serine was incorporated into phosphatidylserine and sphingomyelin. Significant quantities of labeled phosphatidylserine were decarboxylated to phosphatidylethanolamine. Apparently phosphatidylcholine was synthesized de novo from choline via phosphorylcholine and CDP-choline. Based on the present observations and other data, it is suggested that there may be two pools of phosphatidylcholine in lactating mammary cells.     

Sphingolipid biosynthesis by particulate fractions of normal and “Quaking” mouse brain

No differences were detected in the ability of brain particulate fractions isolated from “Quaking” and normal mice for the incorporation of:¹⁴C-L-serine into 3-keto-dihydrosphingosine; 1-¹⁴C-stearoyl acid and 1-¹⁴C-stearoyl CoA into ceramide and¹⁴C-choline from CDP¹⁴C-choline into sphingomyelin.     

Biosynthesis of phospholipids in subcellular particles from cultured cells of human tissue

A time study of the incorporation of³²P_i into the phospholipids of HeLa, KB, human heart, and liver tissue-culture cell lines has been carried out. The incorporation of³²P_i at various time-intervals into the phospholipids of nuclei, mitochondria, and microsomes of HeLa and KB cells was investigated. The labeling of the isotope into the phospholipids was divided into three groups. The first had two components: phosphatidyl inositol and polyglycerol phosphatides, which showed the greatest incorporation of the isotope as demonstrated in the specific activity values and the percentage of total radioactivity after 15 to 30 minutes of incubation. A second group was composed of the major phospholipids of all tissue-culture cell lines studied, phosphatidyl choline, and phosphatidyl ethanolamine. At first, there was a delayed labeling of these phospholipids; however, after one hour of incubation, a rapid increase was shown in the incorporation of³²P_i. A third group of lipids containing sphingomyelin and phosphatidyl serine demonstrated low specific activity values. The phospholipids of the subcellular fractions, nuclei, mitochondria, and microsomes, had a high degree of incorporation of the isotope into the individual phospholipids and probably represented an active process in the membranes of these cellular units or a renewal of the biological membrane structures. Part of a thesis submitted to the Graduate School of the University of North Dakota in partial fulfillment for the degree of Doctor of Philosophy.     

Chemical composition of subcellular particles from cultured cells of human tissue

Chemical composition of subcellular components of HeLa, KB, human heart and liver tissue-culture cell lines have been studied. The concentration of RNA, protein and phospholipid (μg/μg of DNA) of total subcellular particles was similar for all four cell lines studied. The greatest RNA concentration and lowest protein concentration is found in the microsomes as compared to the other subcellular fractions of HeLa and KB cells. The lipid P/Protein N ratio of mitochondria was greater than the other subcellular fractions from tissue-culture cell lines studied. Phosphatidyl choline and phosphatidyl ethanolamine are the major phospholipids with the former more predominant in all of the subcellular fractions of tissue-culture cells studied. Phosphatidyl inositol, phosphatidyl serine, sphingomyelin and polyglycerol phosphatide were shown to be present. Phosphatidyl choline composition (per cent of total lipid-P) is greatest in the microsomes when compared with the other subcellular fractions obtained from all of the cell lines studied except the nuclear fraction of human liver cells. Correspondingly, the mitochondrial fraction for all of the tissue culture cell lines contains the greatest composition of phosphatidyl ethanolamine except for the human liver and heart cells. The mitochondrial fraction contains the lowest amount of phosphatidyl inositol. Polyglycerol phosphatide is mainly present in the mitochondrial fraction of the tissue-culture cells. Part of a thesis submitted to the Graduate School of the University of North Dakota in partial fulfillment for the degree of Doctor of Philosophy.     

In vitro incorporation of acetate-1-14C into the phospholipids of rabbit and human endometria

Endometria from nonpregnant and 6-day pregnant rabbits and from humans in the proliferative and secretory phases were incubated with 1-¹⁴C-acetate.¹⁴CO₂ was collected, and subsequently the amounts, specific radioactivities, and in some cases the fatty acid compositions of the isolated phospholipids were determined. Phosphatidyl choline was the phospholipid present in highest amount in endometria from both nonpregnant and pregnant rabbits, and in human endometria; this phospholipid also showed the highest degree of incorporation of¹⁴C-acetate. Pregnancy in the rabbit seemed to decrease the incorporation of¹⁴C-acetate into most of the endometrial phospholipid classes. In humans, the incorporation of acetate into phosphatidyl choline and phosphatidyl ethanolamine was lower in the secretory than the proliferative endometria. Of the fatty acids, linoleic acid in phosphatidyl choline and phosphatidyl ethanolamine of the rabbit endometria showed a significant relative increase during pregnancy and palmitoleic acid showed a decrease. This investigation was supported by a grant from the Ford Foundation.     

Phospholipids of the pulmonate land snailCepaea nemoralis (L.)

The phospholipids of the snailCepaea nemoralis, comprising the major lipid fraction (65%) in this terrestrial pulmonate, were investigated by thin-layer and column chromatography. Detailed gas chromatographic analyses of liberated fatty acid fractions and amino acid analyses of the water soluble moieties of isolated phospholipid classes were carried out. Phosphatidyl choline (47%) and phosphatidyl ethanolamine (21%) were found to be the predominant phospholipid classes, while phosphatidyl serine (8%), phosphatidyl inositol (6%), diphosphatidyl glycerol (3%), ceramide amino-ethylphosphonate (7%), lysophosphatidyl choline (1%), and phosphatidic acid (1%) were present in lesser amounts. In the phosphatidyl ethanolamine and phosphatidyl serine fractions, minor quantities of plasmalogen analogues were detected. Fatty acid profiles of the various phospholipid classes appeared to be strikingly diverse, e.g. a characteristic component, such as linoleic (18∶2ω6) acid, ranging from 3–54%. In vivo radioisotope studies using 1-¹⁴C-acetate demonstrated the high biosynthetic rate of all phospholipid classes and their respective fatty acid fractions. Results are discussed in relation to data on the phospholipids from other invertebrate species.     

Effect of low methionine,choline deficient diets upon major unsaturated phosphatidyl choline fractions of rat liver and plasma

To see how the metabolism of specific phosphatidyl choline fractions might be affected when only a limited source of methyl groups was available, rats were fed for 7 days a low methionine, cholinedeficient diet or one supplemented with either choline or methionine. Prior to killing, they were injected with¹⁴C-methyl methionine and liver and plasma phosphatidyl choline isolated and separated by argentation chromatography into 3 major unsaturated fractions. Fatty acid composition and radioactivity of the fractions were determined. Deficient rats had reduced total liver phosphatidyl choline when compared with the supplemented groups, but the proportions of 20∶4 and 22∶6 fatty acids in the total phosphatidyl choline were unchanged. Plasma phosphatidyl choline also was reduced sharply by the deficiency, as was its proportion of 20∶4 fatty acid. Specific activities of the liver 22∶6, 20∶4, and 18∶2 phosphatidyl choline fractions showed that deficient rats had less radioactivity in their 20∶4 and 18∶2 phosphatidyl choline than did the supplemented animals. Plasma phosphatidyl choline fractions presented a similar pattern. Feeding methionine or choline nearly doubled radioactive methyl group incorporation into the 20∶4 phosphatidyl choline fraction of liver and plasma, while incorporation into the 22∶6 phosphatidyl choline was reduced or unchanged. The results suggested that, in the rat, limited availability of methyl groups altered the metabolism of liver and plasma phosphatidyl choline fractions. Methionine, as a source of labile methyl groups, appears necessary for the normal synthesis of certain unsaturated phosphatidyl choline fractions (particularly 20∶4 phosphatidyl choline). Transmethylation of phosphatidyl ethanolamine molecular species to the corresponding phosphatidyl choline species may be an important reaction in normal lipid metabolism and transport. Relative affinities for incorporation of the labeled methyl groups into the phosphatidyl choline fractions of either deficient or supplemented rats were: 22∶6>20∶4>18∶2.     

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.     

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.     

Reversibility of cholinephosphotransferase in lung microsomes

The effect of cytidine 5′-monophosphate (CMP) on the incorporation of cytidine 5′-diphosphate (CDP) [methyl-¹⁴C]choline or [1-¹⁴C]dipalmitoylglycerol into phosphatidylcholine (PC) catalyzed by rabbit lung microsomal CDP choline:1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied. In the presence of 0.85 mM CMP and nonsaturating diacylglycerol concentration, the incorporation of CDP[¹⁴C]choline into PC was markedly stimulated, but the incorporation of [¹⁴C]dipalmitoylglycerol into PC was inhibited. This was due to the increase of endogenous diacylglycerol generated from microsomal PC by the cholinephosphotransferase reverse reaction. However, the newly synthesized PC was not readily hydrolyzed in the presence of CMP. The results of this study suggest that the endogenous membranous diacylglycerol is utilized more preferentially for PC synthesis than the exogenous diacylglycerol and that the newly synthesized PC could rapidly equilibrate with the endogenous membrane PC pool.     

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.     

Phosphatidylethanolamine metabolism in rats fed a low methionine,choline-deficient diet

The metabolism of phosphatidylethanolamine (PE) was studied in male rats fed a low methionine diet for 7 days with or without supplemental choline. Groups of animals were injected with 2-¹⁴C-ethanolamine and killed at intervals thereafter up to 72 hr. Liver phospholipids were isolated, and PE and phosphatidylcholine (PC) were separated by argentation chromatography into diene (18∶2), tetraene (20∶4) and hexaene (22∶6) fractions. Fatty acid composition and the distribution of radioactivity and specific activity in the total phospholipids and in the fractions were determined. Choline deficiency did not affect total liver phospholipid, but it did increase the amount of PE and decreased that of PC. The major effect of the deficiency on phospholipid fatty acids was to decrease the proportion of PE arachidonate and to increase that of docosahexaenoate. Ethanolamine incorporation into liver PE of deficient rats was only slightly less than in the controls, but loss of the radioactivity from the PE was slower. Ethanolamine radioactivity appearing in the PC of deficient rats was about half that of the controls, even though specific activities of the PE precursors were similar to the control rats. Choline deficiency increased the biological half-lives of the total PE and its fractions. Although the proportion of PE tetraenoic fraction was reduced, the total amount of this liver PE fraction in deficient rats was not affected. However the amount of hexaenoic fraction was doubled, and it accounted for most of the increased quantity of liver PE seen in deficient animals. The results suggested that in choline deficiency PE synthesis was delayed but not appreciably suppressed, and that limited availability of methionine for methylating the PE fractions in their proper proportions affected the concentrations of the PE fractions and impaired their normal conversion to PC. Presented in part at the AOCS Meeting, Houston, May 1971.     

Lipid synthesis by perfused lung

An isolated lung ventilated with pulses of negative pressure and perfused through the pulmonary vasculature was utilized for the study of 3-sn-phosphatidylcholine synthesis. The perfusion fluid consisted of a Krebs-Ringer phosphate buffer with 6% bovine serum albumin, pH 7.4, and the appropriate substrate. The simultaneous incorporation of (1-¹⁴C) palmitate and (2-³H) glycerol and the simultaneous incorporation of (CH₃-¹⁴C) choline and (CH₃-³H) methionine were examined. From these experiments it is concluded: 1) lung tissue incorporates (2-³H) glycerol into 3-sn-phosphatidylcholine to a greater extent than any other lipid examined; 2) both choline and methionine contribute to the synthesis of 3-sn-phosphatidylcholine, and 50–70% of the label in its nitrogen base is derived from choline and 30–50% from methionine; and 3) a high PO₂ appears to reduce the synthesis of 3-sn-phosphatidylcholine.     

Effect of pentadecan-2-one on lipid metabolism in HeLa cells

HeLa cells exposed to trace amounts of pentadecan-2-one showed changes in metabolism of 1-¹⁴C-palmitate. These changes consisted of an increased incorporation of radioactivity into the triglycerides and free fatty acids and a decreased¹⁴C incorporation into the ether moiety of alk-1-enyl acyl phosphoglycerides. Chemical analysis of the several lipid fractions showed a threefold increase in triglyceride content but no change in the amount of alk-1-enyl acyl or diacyl phosphoglycerides in the treated cells. Pentadecan-2-one added to the culture medium apparently gains entrance to the cell since both pentadecan-2-one and pentadecan-2-ol were detected in the ketone-treated cells and their culture medium.     

Polyenoic acid metabolism in cultured human skin fibroblasts

The incorporation of [1-¹⁴C]linoleic acid, and [1-¹⁴C]linoleic acid into cellular lipids of cultured human skin fibroblasts was studied. Cultured cells took up both labeled fatty acids at nearly the same rate and incorporated them into a variety of lipid classes. At the end of 1 hr incubation with [1-¹⁴C]linoleic acid, radioactivity was found in the triacylglycerol (TG) and choline phosphoglyceride (CPG) pools preferentially. Incorporation into the TG fraction decreased rapidly, while the uptake into CPG, serine phosphoglyceride (SPG), and ethanolamine phosphoglyceride (EPG) fractions increased progressively with longer incubation times. Similar results were obtained with [1-¹⁴C]linoleic acid as precursor. At the end of 24 hr, desaturation and chain elongation of 18∶3 n−3 was more extensive than conversion of 18∶2 n−6 to higher polyenoic acids. During pulse-chase experiments with either fatty acid precursor, the incorporated radioactivity was progressively lost from cellular lipids, particularly from the TG and CPG fractions, but continued to increase in the SPG and EPG pools. The similar labeling pattern of cellular phospholipids with linoleic or linolenic acids, and data from pulse-chase studies suggest that a direct transfer of fatty acids from CPG to EPG is a likely pathway in fibroblast cultures. Incorporation into the EPG pool during the pulse-chase experiments paralleled extensive desaturation and elongation of linoleic acid into 20∶4 n−6, and 22∶4 n−6; and of linolenic acid into 22∶5 n−3 and 22∶6 n−3.     

Fatty acid biosynthesis and incorporation into lipid classes in seeds and seed tissues of flax

Fatty acid metabolism in developing flaxseeds was studied by incubating whole seeds or isolated seed tissues in buffered solutions of 1-¹⁴C-acetate, 2-¹⁴C-malonate and¹⁴CO₂. Lipid classes were separated by thin layer chromatography, and fatty acid labeling in phospholipids, diglycerides and triglycerides was determined by combined thin layer and gas liquid chromatographic techniques. Incorporation of¹⁴C from acetate into embryo lipids was very rapid with phospholipids and 1,2-diglycerides becoming highly labeled in treatment times as short as 5 min. Triglycerides were labeled more slowly. Phospholipid radioactivity was largely associated with the phosphatidyl choline fraction. Oleic acid had the highest specific activity of all major fatty acids in short treatment periods. This was followed in decreasing order of activity by palmitic, linoleic, stearic and linolenic acids. As the treatment period was lengthened to 90 min or longer, linoleic and linolenic activities were markedly increased. Use of malonate or CO₂ rather than acetate as the substrate increased the labeling of the saturated acids. Incorporation of¹⁴C from acetate into lipids of endosperm tissues and whole flax seeds was slower than incorporation into embryo lipids. Stearate had the highest specific activity of the fatty acids in endosperm and whole seeds. Presented in part at the AOCS Meeting in New York, October 1968.     

Lipid labeling with32P-orthophosphate and14C-acetate in marine copepods

Freshly collectedCalanus pacificus were maintained in sea water containing 25 μCi/ml [³²P]orthophosphate or 1 μCi/ml [¹⁴C]acetate at 10 C for 24 hr. The animals took up label from the environment and incorporated it into various lipid fractions. After incubation with [¹⁴C]acetate the order of specific activity of the different lipid classes was: phospholipids > free fatty acids > wax esters > triglycerides. Argentation thin layer chromatography of the fatty acid methyl esters showed that ca. 50% of the activity was in saturated fatty acids and 34% in polyunsaturated acids. When the animals were exposed to [³²P]orthophosphate, lysophosphatidyl choline became most heavily labeled, followed by lysophosphatidyl ethanolamine, sphingomyelin, phosphatidyl ethanolamine, and phosphatidyl choline. Comparison of the data obtained with those available for decapods and mammals revealed striking similarities between these phylogenetically distant groups. It is believed that labeling the lipids of marine and freshwater planktonic crustaceans in this way will provide much information about the metabolism of lipids in these organisms.     

Fatty acid synthesis in rat testes injected intratesticularly or incubated with 1-14C acetate

Fatty acid synthesis was studied in testes of young and adult rats either injected intratesticularly or incubated with 1-¹⁴C acetate. The pattern of¹⁴C incorporation into lipids and individual fatty acids in the two age groups was similar but results obtained with intratesticular injection differed considerably from those obtained in the in vitro studies. In the former more than 70% of the¹⁴C incorporated in total lipids was in phosphatides, with about 15% in triglycerides and only minor amounts in cholesteryl esters and free fatty acids. Most of the¹⁴C incorporated into total fatty acids was in saturated acids (predominantly 16∶0). A small amount of¹⁴C was in the higher polyenes and there was a progressive increase with time after acetate injection in the¹⁴C content of 22∶5 W6. In testes incubated with 1-¹⁴C acetate, the phosphatide, triglyceride, and free fatty acid fractions had similar amounts of¹⁴C. In the total fatty acid fraction about 40% of the incorporated¹⁴C was in saturated acids (predominantly 14∶0 and 16∶0) and about 50% in the higher polyenes. Twenty carbon polyenes and 22∶5 W6 had significant¹⁴C incorporation, but most of the¹⁴C was in 22∶4 W6. About 80% of the¹⁴C in the latter compound was in the carboxyl carbon, indicating its origin from endogenous 20∶4 W6 elongated by the added 1-¹⁴C acetate used as substrate. The¹⁴C 22∶4 was present predominantly in the triglyceride and phosphatide fractions with minor amounts in other lipids.¹⁴C-labeled compounds of retention time greater than 22∶5 were also present in all lipid fractions. Presented at the ISF-AOCS World Congress Chicago, September 1970.     

The synthesis in vivo of choline and ethanolamine phosphoglycerides in different brain areas during aging

The biosynthesis of choline and ethanolamine phosphoglycerides was tested in vivo in different brain areas of the rat during aging. Mixtures of [2−³H] glycerol and [Me-¹⁴C] choline or [2-³H] glycerol and [2-¹⁴H] ethanolamine were injected into lateral ventricle of the brain as lipid precursors and their incorporation into corresponding phospholipid was examined. A significant decrease of synthesis of both phosphoglycerides takes place in cerebral cortex and in the striatum, and is already apparent at 9 months of age with no further decrease or change therafter. No significant change takes place in the cerebellum. The unchanged absorption of injected water-soluble precursors, together with the lack of any significant change of phospholipid/protein ratio in all examined brain areas, suggests that the incorporation of both glycerol and nitrogen bases are affected by aging.     

Intravenously injected [1-14C]arachidonic acid targets phospholipids, and [1-14C]palmitic acid targets neutral lipids in hearts of awake rats

The differential uptake and targeting of intravenously infused [1-¹⁴C]palmitic ([1-¹⁴C] 16∶0) and [1-¹⁴C]arachidonic ([1-¹⁴C]20∶4n−6) acids into heart lipid pools were determined in awake adult male rats. The fatty acid tracers were infused (170 μCi/kg) through the femoral vein at a constant rate of 0.4 mL/min over 5 min. At 10 min postinfusion, the rats were killed using pentobarbital. The hearts were rapidly removed, washed free of exogenous blood, and frozen in dry ice. Arterial blood was withdrawn over the course of the experiment to determine plasma radiotracer levels. Lipids were extracted from heart tissue using a two-phase system, and total radioactivity was measured in the nonvolatile aqueous and organic fractions. Both fatty acid tracers had similar plasma curves, but were differentially distributed into heart lipid compartments. The extent of [1-¹⁴C]20∶4n−6 esterification into heart phospholipids, primarily choline glycerophospholipids, was elevated 3.5-fold compared to [1-¹⁴C]16∶0. The unilateral incorporation coefficient, k ^*, which represents tissue radioactivity divided by the integrated plasma radioactivity for heart phospholipid, was sevenfold greater for [1-¹⁴C]20∶4n−6 than for [1-¹⁴C]16∶0. In contrast, [1-¹⁴C]16∶0 was esterified mainly into heart neutral lipids, primarily triacylglycerols (TG), and was also found in the nonvolatile aqueous compartment. Thus, in rat heart, [1-¹⁴C]20∶4n−6 was primarily targeted for esterification into phospholipids, while [1-¹⁴C]16∶0 was targeted for esterification into TG or metabolized into nonvolatile aqueous components.