Raymond Christon

Modification of fluidity and lipid-protein relationships in pig intestinal brush-border membrane by dietary essential fatty acid deficiency.

The effect of dietary essential fatty acid (EFA) deficiency on the dynamic molecular organization of pig intestinal brush-border membrane (BBM) was studied using purified BBM vesicles. A 6 week dietary treatment of weaning piglets induced a typical EFA-deficient pattern in the lipid composition of both plasma and epithelial membranes. In pigs fed on the EFA-deficient diet, the plasma 20:3(n - 9)/20:4(n - 6) ratio progressively increased and reached a stable value after 3 weeks of experiment, whereas it remained low (less than 0.2) in controls. In the intestinal BBM, the cholesterol/protein, phospholipid/protein and consequently the cholesterol/phospholipid ratios, as well as the phospholipid class distribution, were unchanged. In particular, the sphingomyelin/phosphatidylcholine (SM/PC) molar ratio was not affected. However, the fatty acid composition of phospholipid main classes was markedly modified, leading to decreased lipid fluidity and to a large change in membrane protein behaviour with EFA deficiency. These findings could be interpreted in terms of reduced lipid-protein interactions. Moreover, the increasing gradient of fluidity which took place within the lipidic matrix from its surface was modified by the dietary treatment, as fluidity was lowered by EFA deficiency at different depths of the layer.

Diet-induced structural and functional modifications in the pig liver endoplasmic reticulum membrane: effect of polyunsaturated fatty acid deficiency

Abstract Weaned 39-day-old female piglets were fed diets containing either corn-soybean oil (control) or hydrogenated coconut oil (essential fatty acid (EFA) deficient) for 12 weeks. EFA deficiency produced increased cholesterol (Chol) and decreased phospholipid (PL) contents relative to protein, and thereafter an enhanced Chol/PL molar ratio in the liver endoplasmic reticulum (E.R.) membrane. The primary changes included modified PL distribution, with significant decrease in phosphatidylcholine (PC) and increase in phosphatidylinositol (PI) and phosphatidylserine (PS) levels. Furthermore, fatty acid profiles of the four main PL classes were altered by EFA deficiency. In particular, linoleic, arachidonic, and docosapentaenoic acid levels were largely reduced, whereas palmitoleic and oleic acid levels were increased. The biosynthesis of 5, 8, 11-eicosatrienoic acid (from oleic acid) was strongly stimulated in EFA-deficient pig liver E.R. membranes. The lower polyunsaturated fatty acid level of these latter induced a decreased peroxidability as measured by thiobarbituric assay. Moreover, the modified lipid composition due to EFA deficiency was followed by a decrease in membrane fluidity and an alteration in the activity of several membrane proteins. Liver E.R. membrane from EFA-deficient piglets exhibited a decreased Ca −+ uptake, although passive Ca ++ efflux was unaffected. NADH-Cyt.b 5 and NADH-Cyt.b 5 reductase activity were enhanced, whereas NADPH-Cyt. P450 electron transferring system and some of the liver detoxifying enzyme activities were significantly depressed.

Nutrition and biomembranes: additional information concerning the incidence of dietary polyunsaturated fatty acids on membrane organization and biological activity

One of the important questions in biomembranes now is: Do the essential fatty acids (polyunsaturated fatty acids of the n-6 and n-3 series) play an original structural role in the arrangement of the lipid matrix capable, in particular, of triggering modifications of intrinsic protein activities? Preliminary results from our laboratories are presented in rat and piglet fed standard or essential fatty acid-deficient diets. The relative amounts of 18:2 (n-6) and 20:4 (n-6) in total fatty acids of hepatic microsome or enterocyte brush border membrane phospholipids are closely dependent on the type of diet (a globally decreasing effect with deficiency), whereas no differences were observed with relative amounts of cholesterol, phospholipids, and proteins. This effect of deficiency on membrane fatty acids has to be compared to the decreasing specific activities of microsome NADPH-cytochrome c reductase or aniline hydroxylase (studied in rat), to the increasing order of the structure of both membrane microsome and brush border lipid matrix (studied in both rat and piglet), and to the increasing mobility (or accessibility) of the membrane-protein surface-bonded spin-label (studied in the piglet brush border membrane), suggesting a probably defective protein-lipid fit in the case of deficiency. These results could favor conformational change in the whole membrane structure (i.e. proteins and lipids). The specificity of these effects remains to be assessed.

Apparent relative retention of the phosphatidylethanolamine molecular species 18:0–20:5(n−3), 16:0–22:6(n−3) and the sum 16:0–20:4(n−6) plus 16:0–20:3(n−9) in the liver microsomes of pig on an essential fatty acid deficient diet

Attempts at a better understanding of the cell membrane organization and functioning need to assess the physical properties which partly depend (i) on the positional distribution of the fatty acids in the membrane phospholipids (PLs) and (ii) on the way by which the PL molecular species are affected by exogenous fatty acids. To do that, the effects of essential (polyunsaturated) fatty acid (EFA) deficiency and enrichment were studied in the liver microsomes of piglets feeding on either an EFA-deficient diet or an EFA-enriched diet containing hydrogenated coconut oil or a mixture of soya + corn oils, respectively. After derivatization, the diacylated forms of choline and ethanolamine PLs were analyzed using a combination of chromatographic techniques and fast-atom bombardment-mass spectrometry. The dinitrobenzoyl-diacylglycerol derivatives corresponding to the molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were identified. It appears that three factors brought about a marked apparent relative retention: the nature of (i) the base of the polar head, (ii) fatty acids at the sn-1 position and (iii) fatty acids at the sn-2 position. The highest apparent relative retentions were displayed by the 18:0-20:5(n-3)-PE and 16:0-22:6(n-3)-PE. It is noteworthy that the behavior of 20:3 n-9--which is synthesized during the EFA-deficient diet by the same bioconversion system as 20:4 n-6--was very similar to that of 20:4 n-6 during the formation of PC and PE molecular species and that the molecular species of PE containing 20:4(n-6) and 20:3(n-9), gathered together as metabolical homologues, were also apparently retained, particularly in association with 16:0. Present observations are consistent with some others showing retention or preferential distribution of EFA in PE and suggest that specific acyltransferase(s), ethanolamine phosphotransferase and methyltransferase would be mainly involved for PE and PC formation in liver endoplasmic reticulum. Fast-atom bombardment-mass spectrometry of intact phospholipids enables us to show that there is no very long chain dipolyunsaturated phospholipid in liver endoplasmic reticulum.

Phospholipid hydroperoxide glutathione peroxidase activity and vitamin E level in the liver microsomal membrane: effects of age and dietary α-linolenic acid deficiency

Age and diet-induced variations of phospholipid hydroperoxide glutathione peroxidase (PHGPx) activity and alpha-tocopherol concentration in the liver microsomal membrane were studied in male Wistar rats fed a semipurified diet either balanced in n-6 and n-3 polyunsaturated fatty acids (PUFA) (Control) or deprived of alpha-linolenic acid, i.e. n-3 PUFA (Deficient) over two generations. The animals were studied at the age of 6 months (adult) or 24 months (old). Both PHGPx activity and vitamin E level were significantly higher in 24-month old rats as compared to 6-month old rats. By contrast, the thiobarbituric acid reactive substances (TBARS) following stimulated in vitro peroxidation of membrane lipids were markedly lower (P < 0.01) with aging. The fatty acid composition of microsomal membrane phospholipids (PL) was also considerably modified by age. In particular, the levels of arachidonic acid and total n-6 PUFA were lower (P < 0.001) whereas n-3 PUFA levels were higher (P < 0.001) in most PL main classes. The alpha-linolenic acid deficiency markedly influenced these age-related changes. The higher PHGPx activity in the old rats as compared to the adult rats was only significant in those fed the control diet. In the 6-month old rats (but not in the 24-month old rats), the deficient diet led to a higher membrane vitamin E level and to lower TBARS production than the control diet. The results suggest that the nature of dietary PUFA may influence the age-related variations in this pair of membrane antioxidants and also in the fatty acid composition of microsomes.

Comparative chromatographic study of modifications of brush-border membrane vesicles induced by an essential fatty acid-deficient diet

The surface properties of small intestine brush-border membranes (BBMs) were examined by frontal affinity chromatography using three types of unsolubilized ligands: phlorizin polymer, immobilized lectins and linolenic acid bound to agarose gel. BBM vesicles were purified from piglets fed a corn oil diet (control diet) or a hydrogenated coconut oil diet. The second diet was representative of a deficient supply of essential polyunsaturated fatty acid (EPUFA). It induced a marked decrease in 18:2n-6 content in membrane choline phosphoglycerides and ethanolamine phosphoglycerides, whereas 20:3n-9 appeared in each class of phospholipids. Control and EPUFA-deprived BBM vesicles bound to phlorizin polymer, linolenic acid-agarose and wheat germ agglutinin (WGA) gel. In contrast, concanavalin A gel and Lens culinaris A gel exhibited a low binding capacity towards the two types of vesicles. EPUFA deficiency induced a slight decrease in binding on phlorizin polymer and a marked increase in binding on WGA gel, whereas the two types of vesicles similarly bound to linolenic acid-agarose. Desorption of phlorizin polymer-bound membranes was performed using several detergents with special regard to sodium deoxycholate (NaDOC) micelles. Sucrase activity recovery showed that the efficiency of NaDOC desorption was diminished in the case of EPUFA-deprived vesicles. EPUFA-deprived membrane domains involved in the binding would be less sensitive to the detergent attack. This assumption agrees with the putative decrease in membrane fluidity induced by the deficient diet. The possibility that fatty acid compositional changes induced by dietary lipids are extensive enough to alter some chromatographic properties of BBM vesicles is discussed.