A unique antioxidant activity of phosphatidylserine on iron-induced lipid peroxidation of phospholipid bilayers

The relationship between the antioxidant effect of acidic phospholipids, phosphatidic acid (PA), phosphatidylglycerol (PG) and phosphatidylserine (PS), on iron-induced lipid peroxidation of phospholipid bilayers and their abilities to bind iron ion was examined in egg yolk phosphatidylcholine large unilamellar vesicles (EYPC LUV). The effect of each acidic phospholipid added to the vesicles at 10 mol% was assessed by measuring phosphatidylcholine hydroperoxides (PC-OOH) and thiobarbituric acid-reactive substances. The addition of dipalmitoyl PS (DPPS) showed a significant inhibitory effect, although the other two acidic phospholipids, dipalmitoyl PA (DPPA) and dipalmitoyl PC (DPPG), did not exert the inhibition. Neither dipalmitoyl PC (DPPC) nor dipalmitoyl phophatidylethanolamine (DPPE) showed any remarkable inhibition on this system. None of the tested phospholipids affected the lipid peroxidation rate remarkably when the vesicles were exposed to a water-soluble radical generator. The iron-binding ability of each phospholipid was estimated on the basis of the amounts of iron recovered in the chloroform/methanol phase after separation of the vesicle solution to water/methanol and chloroform/methanol phases. EYPC LUV containing DPPS, DPPA, and DPPG had higher amounts of bound iron than those containing DPPC and DPPE, indicating that these three acidic phospholipids possess an iron-binding ability at a similar level. Nevertheless, only DPPS suppressed iron-dependent decomposition of PC-OOH significantly. Therefore, it is likely that these three acidic phospholipids possess a significant iron-binding ability, although this ability per se does not warrant them antioxidative activities. The ability to suppress the iron-dependent decomposition of PC-OOH may explain the unique antioxidant activity of PS.     

Rate of restoration of cardiolipin and other major phospholipids during liver regeneration in rat

The rate of restoration of liver phospholipids, especially cardiolipin, during liver regeneration after two-thirds partial hepatectomy, was studied. Preoperative content of cardiolipin was not restored during the first week of regeneration, while this was the case for the other major liver phospholipids. The observations are discussed in relation to the morphogenesis of mitochondria during liver regeneration.     

The role of lipids in heme synthesis

The effect of lipids on protoheme ferrolyase, which combines ferrous iron with protoporphyrin, was investigated. The enzyme extracted with 1% Na cholate from acetone-dried powder of chicken erythrocyte stroma showed high enzymic activity, while that extracted with 0.4 M KCl showed little activity. The former contained lipids, the main components of which were lecithin and phosphatidylethanolamine, whereas the latter contained little lipid. Crude lipids of several sources restored the enzyme activity of 0.4 M KCl extracts. The activating effects of purified lipids, especially phospholipids which showed the strongest activation, were further examined. Phospholipids were divided into three groups: the choline-containing group, lecithin and sphingomyelin, was inhibitory or slightly accelerative on heme synthesis; the acidic phospholipids, namely phosphatidylethanolamine, cardiolipin, phosphatidic acid and phosphatidylinositol, were strong activators and the intensity of activation was in the order of the acidity of the phospholipids; and the lysophospholipids, namely lysolecithin, lysophosphatidylethanolamine and sphingosylphosphorylcholine, activated the heme synthesis most effectively. In the presence of cholate, choline-containing lipids were highly effective, while acidic phospholipids were inhibitory and lysophospholipids were neutral. Palmitic acid showed slight stimulative effect. Tripalmitin was neutral or inhibitory. Anionic, cationic and neutral synthetic detergents were slightly stimulative in low concentration and inhibitory in high concentration. An activation mechanism of phospholipids was proposed in which the hydrophilic anionic part of lipid in the lipoprotein enzyme molecule attracts ferrous iron. After being stripped of solvation water, the ferrous iron is transferred to the hydrophobic part of the enzyme molecule to be inserted into porphyrin.     

The effect of elaidic acid incorporation upon the lipid composition of ehrlich ascites tumor cells and of the host’s liver

The incorporation of elaidic acid into Ehrlich ascites tumor cells (EATC) upon feeding the host an elaidic acid-rich diet has been investigated in the present study. The EATC lipids contained only one-half the concentration of elaidic acid found in the lipids of either the host livers or of livers from normal mice. On the other hand, elaidic acid incorporation into tumor cells was close to that of ascites fluid. This incorporation was mainly into phospholipids; the highest into choline phospholipids and ethanolamine phospholipids. Some changes in the EATC fatty acid composition were noted due to this incorporation. EATC phospholipids had reduced polyunsaturated fatty acids as compared with oleic acid-grown cells. The same was true with respect to ascites fluid phospholipids, but neutral lipids were not altered. Tumor development was accompanied by an increase in elaidic acid of the host’s liver. Elaidic acid incorporation into tumor cells resulted in a reduction in the amount of all major lipids in the tumor. In contrast, elaidic acid had no effect on lipid composition of livers from normal mice and-tumor bearing mice, and also had no effect upon the lipids of the ascites fluid that bathes the tumor cells. The incorporation of elaidic acid into the lipids of EATC, normal liver and host liver did not affect the relative composition of phospholipids in these tissues. The development of the tumor did result in decreases in triacylglycerols and esterified cholesterol, and increases in phospholipids and free cholesterol in the livers of host animals.     

Measurement of lipid peroxidation in vivo: A comparison of different procedures

A study was undertaken to investigate whether some of the methods commonly used to detect lipid peroxidation of cellular membranes in vivo correlate with each other. The study was performed with the livers of bromobenzene-intoxicated mice, in which lipid peroxidation develops when the depletion of glutathione (GSH) reaches a threshold value. The methods tested and compared were the following: i) measurement of the malondialdehyde (MDA) content of the liver; ii) detection of diene conjugation absorption in liver phospholipids; iii) measurement of the loss of polyunsaturated fatty acids in liver phospholipids; and iv) determination of carbonyl functions formed in acyl residues of membrane phospholipids as a result of the peroxidative breakdown of phospholipid fatty acids. Correlations among the values obtained with these methods showed high statistical significances, indicating that the procedures measure lipid peroxidation in vivo with comparable reliability. Analogously, the four methods appeared also to correlate when applied to in vitro microsomal lipid peroxidation.     

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.     

Tissue lipid responses to 3-hydroxy-3-methylglutaric acid with different dietary fats

Simultaneous administration of 3-hydroxy-3-methylglutaric acid (HMG) for 4 weeks to rats fed 20% saturated fats prevented rise of serum cholesterol, triglycerides, and phospholipids. Except phospholipids, other liver lipids were significantly decreased. HMG administration for 4 weeks along with atherogenic diet significantly decreased cholesterol and phospholipids of serum, liver, aorta, and heart. The phospholipids of epididymal fat and brain were also significantly lowered. The triglyceride levels in serum, liver, and epididymal fat were significantly decreased. The maximal hypolipidemic effect of HMG was observed in serum. U.S. Patent No. 3629449, dated December 21, 1971, on “Process of combatting hypercholesterolemia.” A part of the work submitted to Aligarh Muslim University for Ph.D. degree.     

Phospholipid composition of liver in rats fed high levels of 13-cis retinoic acid

The composition of liver phospholipids was studied in rats fed for 4 weeks diets containing 0, 100 or 300 mg 13-cis retinoic acid per kg diet. There was a significant decrease in phosphatidylcholine content, whereas the levels of phosphatidylethanolamine were slightly increased in liver phospholipids of rats fed 13-cis retinoic acid. The fatty acid composition of total phospholipids, PC, PE, and PI+PS fractions revealed a general increase in the levels of 18∶2 and 20∶3ω6, whereas the levels of 20∶4ω6 and C₂₂ fatty acids were reduced in most of the hepatic phospholipids isolated from rats fed 13-cis retinoic acid containing diets. A decrease in the double-bond index of fatty acids was also observed in phospholipids of rats fed 13-cis retinoic acid. The data suggest that high levels of 13-cis retinoic acid may possibly be influencing the activities of microsomal desaturating and chain-elongating enzymes in the liver.     

Studies on the role of phospholipids in the triglyceride cycle: II. Turnover of plasma phospholipids in ethionine-treated dogs

The disappearance of native labeled plasma phospholipids (nPLP³²) was abruptly halted in the plasma of normal mongrel dogs shortly after givingdl-ethionine by intraperitoneal injection. In other dogs, chronic feeding of ethionine over a period of three days prior to these measurements prolonged the turnover time of plasma phospholipids. This impairment in the turnover of plasma PL brought about by ethionine administration occurs regardless of sex differences in lipid metabolism or other physiological differences relating to the nutritional status of the animal. The amount of nPLP³² found in the liver at the end of the turnover experiments was also measured. These data suggest that phospholipid transport from plasma to liver is not impaired by ethionine. The possible significance of an ethionine-induced choline deficiency which impairs transport of triglycerides and phospholipids from the liver to plasma is considered in regard to the role of phospholipids in the triglyceride cycle. Presented in part at the 58th Annual AOCS Meeting, New Orleans 1967.     

Coordinate packaging of newly synthesized phosphatidylcholine and phosphatidylglycerol in lamellar bodies in alveolar type II cells

Methylamine, a weak base, inhibits packaging of newly synthesized phosphatidylcholine (PC) in lamellar bodies in 20–22 h cultured alveolar type II cells, suggesting a role for acidic pH of lamellar bodies. In this study, we tested if (i) the packaging of PC is similarly regulated in freshly isolated type II cells and (ii) methylamine also inhibits the packaging of other surfactant phospholipids, particularly, phosphatidylglycerol (PG). The latter would suggest coordinated packaging so as to maintain the phospholipid composition of lung surfactant. During the short-term metabolic labeling experiments in freshly isolated type II cells, methylamine treatment decreased the incorporation of radioactive precursors into PC, disaturated PC (DSPC), and PG of lamellar bodies but not of the microsomes, when compared with controls. The calculated packaging (the percentage of microsomal lipid packaged in lamellar bodies) of each phospholipid was similarly decreased (∼50%) in methylamine-treated cells, suggesting coordinated packaging of surfactant phospholipids in lamellar bodies. Equilibrium-labeling studies with freshly isolated type II cells (as is routinely done for studies on surfactant secretion) ± methylamine showed that in methylamine-treated cells, the secretion of PC and PG was decreased (possibly due to decreased packaging), but the phospholipid composition of released surfactant (measured by radioactivity distribution) was unchanged; and the PC content (measured by mass or radioactivity) of lamellar bodies was lower, but the PC composition (as percentage of total phospholipids) was unchanged when compared with control cells. We speculate that the newly synthesized surfactant phospholipids, PC, DSPC, and PG, are coordinately transported into lamellar bodies by a mechanism requiring the acidic pH, presumably, of lamellar bodies.     

Interferon α/β induces changes in the metabolism of polyenoic phospholipids and diacylglycerols in the livers of suckling mice

Suckling mice were injected daily from birth for 10 days with potent preparations of mouse interferon α/β. Interferon treatment resulted in a markedly lower concentration of polyunsaturated fatty acids (20∶4ω6 and 22∶6ω3) in the two principal liver phospholipids, phosphatidylcholine and phosphatidylethanolamine, than in livers of control-treated mice. This effect appeared to correlate with a low level of synthesis of polyunsaturated phospholipids in the livers of interferon-treated mice. Thus, in control mice, synthesis of species of polyunsaturated phospholipids increased markedly in the first 10 days of life, whereas in 10-day-old interferontreated mice, the level of synthesis of species of polyunsaturated phospholipids was comparable to that in newborn mice. In parallel, a marked increase in the diacylglycerol content without change of its renewal was observed in the livers of interferon-treated mice. We suggest that interferon treatment results in an inhibition of one of the processes that leads to activation of the enzymatic systems responsible for the synthesis of species of polyunsaturated phosphatidylcholine and phosphatidylethanolamine in the liver of suckling mice. It seems likely that these results are related to the inhibition of liver cell maturation and the marked cell necrosis that are observed in interferon-treated suckling mice.     

Effects of acute administration of chlorinated water on liver lipids

An acute administration of chlorinated water to rats caused “fatty liver” and indicated a more than 2-fold increase in liver triacylglycerols at 2 days after administration. The acyl group composition of triacylglycerols and phospholipids in both liver mitochondria and liver whole homogenate were also altered by the chlorine treatment. Among the phospholipid acyl groups, there was an increase in the proportion of 20∶4 but a decrease in most other polyunsaturated acyl groups. The acyl group changes were more obvious with phosphatidylcholines than with phosphatidylethanolamines. Other phospholipids, including cardiolipin in the mitochondrial membranes, were not greatly altered. Both morphological and biochemical changes were maximum at 2 days after the treatment and were fully recovered after 10 days. The disturbance of a number of enzymatic processes in the liver membranes may account for a large part of the changes observed.     

Postnatal changes in the phospholipid composition of livers from young lambs

The total lipids were extracted from the livers of newborn lambs, from the livers of lambs during the first week after birth and from the livers of adult sheep. After separation from the nonphospholipids on columns of silicic acid the phospholipids were analyzed by thin layer chromatography and quantitative gas liquid chromatography. In all samples phosphatidyl choline and phosphatidyl ethanolamine together accounted for about 80% of the total liver phospholipids. The phosphatidyl choline-phosphatidyl ethanolamine ratio in the livers of the newborn lambs was markedly less than the ratio in the livers of the adult sheep. Moreover there was a pronounced increase in the phosphatidyl cholinephosphatidyl ethanolamine ratio in the livers of the lambs during the first week after birth. In the liver phospholipids of the lambs the concentration of phosphatidyl inositol was lower and the concentrations of phosphatidyl serine and sphingomyelin were greater than the corresponding concentrations in the liver phospholipids of the adult sheep. It is proposed that the change in the phosphatidyl choline-phosphatidyl ethanolamine ratio in the livers of the lambs during the first week after birth is due, at least in part, to the marked change that occurs in the linoleic acid-arachidonic acid ratio in the tissues of the lamb during this period.     

Phospholipid requirement of alkylglycerol monooxygenase from rat liver microsomes

The alkylglycerol monooxygenase catalyzing the cleavage of the ether bond in alkylglycerol resides in rat liver microsomes. The enzyme preparation was freed of phospholipids by sodium deoxycholate treatment followed by gel filtration in the presence of deoxycholate. The removal of phospholipids markedly decreased the alkylglycerol monooxygenase activity. The activity of the delipidated enzyme, however, could be completely restored by the addition of phospholipid vesicles without detergent. When individual phospholipids were added, anionic phospholipids such as phosphatidylglycerol and diphosphatidyl-glycerol were the most effective. These findings, along with our previous observation of a similar effect of liposomes on the purified enzyme, indicate that the amphipathic nature of the protein is responsible for the lipid dependence of enzymatic activity.     

Zinc deficiency-induced changes in the composition of microsomal membranes and in the enzymatic regulation of glycerolipid synthesis

The effects of zinc deficiency and/or castration on the lipid composition of microsomal membranes of liver, small intestine and testes were studied in rats. The results showed that feeding a zinc-deficient diet to castrated rats decreased phospholipid content and consequently increased the cholesterol-to-phospholipid ratio in liver microsomes. An increase in cholesterol-to-phospholipid ratio occurred also in small intestine and testes microsomes from rats fed the zinc-deficient diet. It is postulated, therefore, that zinc deficiency alters the lipid composition and fluidity of microsomal membranes. Zinc deficiency also affected the activities of the enzymes involved in the formation of triglycerides and phospholipids. There was a large increase in total and specific activity of phosphatidate phosphatase and the changes in the total activity of choline phosphotransferase correlated well with the changes observed in serum or liver triglycerides and phospholipids. Stearoyl CoA desaturase, which is a control enzyme for hepatic lipogenesis, was also increased by more than 200% in zinc-deficient states, as was the diglyceride content of hepatic microsomes. These results indicate that the increased synthesis of triglycerides and phospholipids in zinc deficiency may be due to the increased availability of substrates as well as to increased activities of the enzymes involved in these processes.     

Quantitation of phosphatidyl N-methyl- and N,N-Dimethylaminoethanol in rat liver

The contents of phosphatidyl N-methyl-and N,N-dimethylaminoethanol were determined in the liver of rats injected with (Me-C¹⁴) methionine. Total phospholipids were extracted from aliquots of the liver and fractionated by two-dimensional thin layer chromatography after addition of carrier phosphatidyl-N-methyl-and N,N-dimethylaminoethanol. The radioactivity present in the two phosphatide spots was determined and used to calculated total disintegrations per min/100 g body wt. The remainder of the livers was pooled, and total phospholipids were isolated and subjected to acid hydrolysis. N-methyl- and N,N-dimethylaminoethanol were purified by thin layer chromatography, and their specific activity was determined after quantitation by gas liquid chromatography and radioactivity measurement. The liver contents of phosphatidyl N-methyl- and N,N-dimethylaminoethanol were determined by dividing disintegrations per min/100 g body wt by the specific activity of N-methyl- or N,N-dimethylaminoethanol.     

Effects of dietary triglyceride on the properties and lipid composition of plasma lipoproteins: Acute experiments in rats fed safflower oil

Male rats were administered 1.5 ml safflower oil by gastric intubation 0, 4, and 8 hr after a 16 hr fast. Plasma, liver, and adipose tissue were collected 16 hr after the last fatty meal. Rats fasted for 16 hr served as controls. Following fat feeding, the fatty acid composition of the very low density lipoprotein, triglyceride, and hepatic triglyceride were similar, as were the percentages of 18:2 in the very low density lipoprotein and hepatic cholesteryl esters. The phospholipids of liver and plasma lipoproteins were similar in the control groups, except that more 16:0 was present in the plasma lipoproteins. After fat feeding, the plasma lipoproein phospholipids were enriched with 18:2 more than were the hepatic phospholipids. Furthermore, the percentage of 18:2 in phospholipid was much less than in triglyceride or cholesteryl esters. Clearly, esterified lipids of liver and plasma lipoproteins (very low density lipoprotein, low density lipoprotein, and high density lipoprotein), and to a lesser extent, adipose tissue, were enriched with 18:2 derived from dietary triglyceride fatty acid even 16 hr after the terminal meal. A major proportion of the very low density lipoprotein isolated by ultracentrifugation in zonal rotors from plasma of fat fed animals had a faster rate-zonal mobility than did the very low density lipoprotein isolated from plasma of control animals. The very low density lipoprotein isolated from plasma of fat fed rats contained fewer moles of phospholipids, cholesterol, and cholesteryl esters, relative to triglyceride than did the very low density lipoprotein from plasma of animals not receiving safflower oil. The molar ratio triglyceride:phospholipid:cholesterol:cholesterol esters in the very low denity lipoprotein was 100:42.0:22.1:44.5 in the control group and 100:35.4:17.8:19.5 in the fat fed animals. It is postulated that an important biochemical mechanism by which dietary triglyceride fatty acids consumed by the animal over a long period of time alter plasma concentrations of triglyceride, phospholipids, and cholesterol esters is the directive influence of plasma free fatty acid, derived from dietary triglyceride, on the secretion of very low density lipoprotein lipids by the liver.     

Protein-Phospholipid Interactions in Nonclassical Protein Secretion: Problem and Methods of Study

Extracellular proteins devoid of signal peptides use nonclassical secretion mechanisms for their export. These mechanisms are independent of the endoplasmic reticulum and Golgi. Some nonclassically released proteins, particularly fibroblast growth factors (FGF) 1 and 2, are exported as a result of their direct translocation through the cell membrane. This process requires specific interactions of released proteins with membrane phospholipids. In this review written by a cell biologist, a structural biologist and two membrane engineers, we discuss the following subjects: (i) Phenomenon of nonclassical protein release and its biological significance; (ii) Composition of the FGF1 multiprotein release complex (MRC); (iii) The relationship between FGF1 export and acidic phospholipid externalization; (iv) Interactions of FGF1 MRC components with acidic phospholipids; (v) Methods to study the transmembrane translocation of proteins; (vi) Membrane models to study nonclassical protein release.     

Stimulation of a neutral triacylglycerol hydrolase from rat heart by phosphatidylethanolamine and lysophosphatidylethanolamine

Triacylglycerol hydrolase activity measured at pH 7.5 in a pH 5.2 precipitate fraction from rat heart was increased two-to three-fold by the presence of phosphatidylethanolamine (PE) or lysophosphatidylethanolamine (LPE). This stimulatory effect also could be obtained in assays with particulate and soluble subcellular fractions and was observed with two different methods of preparing triolein substrate emulsions. Ethanolamine and glycerophosphorylethanolamine had no effect on hydrolase activity, whereas phosphatidylcholine (PC) and acidic phospholipids such as cardiolipin were inhibitory. Palmitic acid, palmityl CoA and palmityl carnitine inhibited PE-stimulated hydrolase activity, but ethyl esters of palmitate had no effect. The preparation of acetone-ether powders resulted in a marked reduction of triacylglycerol hydrolase activity, but PE and LPE now stimulated hydrolase activity by ten-fold or greater, suggesting that these phospholipids may have an obligatory role in modulating triacylglycerol hydrolase activity. Triton X-100 also stimulated hydrolase activity in acetone-ether powders.     

Linoleic acid-induced fatty acid changes in platelet and aorta of the rat: Effect of age and cholesterol

The influence of age and cholesterol on polyunsaturated fatty acids (PUFa) levels was studied in young and old male Sprague-Dawley rats. Animals were fed a fat-free diet supplemented with 10% (by wt) safflower oil with or without 1% cholesterol for 8 wk. As a result of cholesterol feeding, proportions of linoleic acid (18∶2n−6) and dihomo-γ-linolenic acid (30∶3n−6) were increased and and that of arachidonic acid (20∶4n−6) was decreased in the liver and platelet phospholipids in 64-wk-old rats, suggesting inhibitory effects of cholesterol on 20∶4n−6 synthesis from 18∶2n−6. The prominent age-dependent effect on the levels of PUFA was a retention of C−22 n−3 PUFA, accompanied by decreased C−22 n−6 PUFA and increased 20∶3n−6 in the liver and platelet phospholipids. Ratio of 20∶3n−6/20∶4n−6 increased in 64-wk-old rats regardless of dietary cholesterol, suggesting depressed Δ5-desaturase with age. In aorta phospholipids, 20∶3n−6 content and 20∶3n−6/20∶4n−6 ratio increased with cholesterol supplementation, but not with age. These results suggest that changes of PUFA composition of platelet phospholipids with age are closely linked with changes in liver phospholipids. The 20∶4n−6 content in both platelet and aorta phospholipids is kept constant, despite other n−6 and n−3 PUFA being affected by age.