Phospholipid hydroperoxide glutathione peroxidase activity and vitamin E level in the liver microsomal membrane: effects of age and dietary alpha-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.

Dietary polyunsaturated fatty acids and aging modulate glutathione-related antioxidants in rat liver.

The effects of dietary polyunsaturated fatty acids (PUFA) on the age-dependent changes in liver glutathione antioxidant system were investigated in male Wistar rats fed diets supplying either balanced amounts of linoleic acid and alpha-linolenic acids (control) or deficient in alpha-linolenic acid [n-3) deficient]. The animals were studied at the age of 6 or 24 mo. Glutathione antioxidative metabolism was markedly affected by aging. Cytosolic concentration of reduced glutathione (GSH) was lower (P < 0.01), whereas that of oxidized glutathione (GSSG) as well as the GSSG:GSH ratio were greater (P<0.001) in the 24-mo-old as compared with the 6-mo-old rats, regardless of the diet. Glutathione peroxidase activities were lower (P < 0.001) in 24-mo-old rats, though more markedly in those fed the control diet. The lipid composition of rat liver microsomal membranes was strongly modified by both diet and aging. The age-related changes mainly involved the polyunsaturated fatty acids. These results suggest that the nature of dietary PUFA and not only their degree of unsaturation affects the cellular glutathione-dependent antioxidant system and thus may modify the age-related changes in metabolic reactions.

Essential fatty acid deficiency in the pig: effects on eicosanoid basal levels and in vitro synthesis by the small intestine.

The influence of nutritional essential fatty acid (EFA) deficiency on arachidonate metabolism by porcine small intestine has been studied. Great care was exercised in the manipulation of the jejunal wall to avoid artefactual metabolism of arachidonate. Thus, jejunal wall was frozen in liquid nitrogen after organ removal and washing, and subsequently lyophilized. This lyophilized tissue was used as starting material for all experiments, including organic solvent extractions (for basal level determinations) and reconstitution in aqueous buffer (for neosynthesis experiments). Feeding pigs with a low linoleate diet for 12 weeks resulted in a 36% diminution in the % of arachidonate in jejunal phospholipids. Basal levels of 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha), thromboxane B2 (TXB2), PGF2 alpha, PGE2, PGD2 and leukotriene B4 (LTB4) were not altered in the EFA-deficient state. However, we observed a significant lowering of the synthesis of each of these eicosanoids (except LTB4) by the EFA-deficient jejunum during brief (15s) in vitro neosynthesis experiments. The origin of arachidonate as a substrate of PG endoperoxide synthase, also named PGH synthase or cyclooxygenase (Cox) in these neosynthesis experiments is probably a non-esterified fatty acid pool since, (1) neosynthesis was not inhibited by the phospholipase A2 (PLA2) inhibitor parabromophenacylbromide, and (2) substantial amounts of arachidonic acid were found in the jejunum, frozen or lyophilized. Cox activity of the lyophilized jejunum and Cox content of liver and intestine microsomes were not modified in the EFA-deficient state.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of an n-3 fatty acid-deficient diet on the adenosine-dependent melatonin release in cultured rat pineal.

We studied the effect of a diet deficient in n-3 fatty acids on the adenosine-dependent melatonin release from cultured rat pineal gland after stimulation by 5'-N-ethylcarboxamidoadenosine (NECA), an A2 adenosine agonist. Experiments were conducted with 2-month-old rats raised on semipurified diets containing either peanut oil (n-3 deficients) or peanut plus rapeseed oil (controls). The proportion of docosahexaenoic acid (22:6 n-3) in the pineal total lipid fraction and in phosphatidylcholine and phosphatidylethanolamine was significantly decreased in n-3-deficient rats. This was compensated for partially by an increase in 22:4 n-6 and 22:5 n-6 levels. The activity of the cultured rat pineal, in terms of cyclic AMP content and N-acetylserotonin and melatonin release in the medium, was lower after stimulation by 10(-5) mol/L NECA in the group fed peanut oil than in the group fed peanut plus rapeseed oil. The increased ratio of n-6/n-3 fatty acids in pineal total lipids and the major glycerophospholipids (phosphatidylcholine and phosphatidylethanolamine) may have an important influence on the rat pineal responses. The results are discussed in the context of changes in membrane-bound proteins, including enzymes and/or receptors involved in the rat pineal gland function.

Lipid intake during pregnancy in developing countries: possible effect of essential fatty acid deficiency on fetal growth.

There is a strong epidemiological association between poor nutritional status and low-birthweight (LBW) newborns. According to the World Health Organization (WHO), 95% of total LBW in the world are born in developing countries. Nevertheless, the nutritional etiology of intra uterine growth retardation (IUGR) is still controversial and not yet established largely because of contradictory nutritional studies in pregnancy. In 1981 Menon et al described an animal model of IUGR due to mild deficiency in essential fatty acids (EFA, linoleic and alpha linolenic acids), with a strong correlation between EFA intake during pregnancy and fetal growth. According to the WHO reports in the last decade, there was a dramatic deficiency of lipid intakes (less than 10% of total caloric amounts) in the majority of developing countries while the EFA requirements alone of normal nourished women are evaluated at 6% of total caloric amounts during pregnancy. A mild deficiency in dietary EFA may be a limiting factor in fetal growth processes in humans as it has been shown in animals. Such a mechanism could be easily verified. Research proposals are made in an attempt to test this hypothesis in developing countries with possible applications in further nutritional interventions in pregnancy.

PIP: This literature review presents the hypothesis that low birthweights (LBW) common in developing countries may be related to a low intake of essential fatty acids (EFA). There are 19 million babies with LBW born yearly in developing countries, most categorized as intrauterine growth retardation (IUGR). Smoking and pregnancy-induced hypertension are the only known risk factors for IUGR. EFAs are fatty acids that are like vitamins, because humans do not have enzymes to make them and require them in the diet. Mild EFA deficiency caused 25-30% LBW in rats. EFAs are needed for formation of all cell membranes and, particularly, for nervous system development in the last trimester of pregnancy and early postnatal growth. Excess saturated fats, high ambient temperatures, and high carbohydrate diets, all prevalent in many tropical developing countries, compete for EFAs in metabolism. Many women in developing countries subsist on diets with 15% or fewer calories from lipids, far lower than the 30% recommended by WHO, or the calculated 14 grams/day of EFA in a 2000 calorie diet recommended in pregnancy. Of about 30 studies reported on attempts to improve birth weights, 4 studies achieved weight gains by feeding pregnant women nutritional supplements containing EFAs. 2 types of research studies were suggested: determining red cell membrane lipid content in LBW infants and their mothers in developing countries and a controlled trial of nutritional supplements with protein, carbohydrate, and EFAs.

Simultaneous changes in lipid composition, fluidity and enzyme activity in piglet intestinal brush border membrane as affected by dietary polyunsaturated fatty acid deficiency.

The effects of dietary polyunsaturated fatty acid (PUFA) deficiency on intestinal brush border membrane (BBM) fluidity, lipid composition and 5'-nucleotidase activity were examined in piglets. Cholesterol/phospholipid and sphingomyelin (SM)/phosphatidylcholine (PC) ratios were unaffected. However, fluidity was decreased in the external regions and also tended to decrease in the core of the PUFA-deficient pig membrane lipid bilayer. Therefore, the change in the membrane physical properties seemed to be due to the large diet-induced alteration in the phospholipid (PL) fatty acid composition and to the concomitant decrease in PC and increase in phosphatidylserine levels. In the membrane total PL, the arachidonic acid level was slightly lowered, while linoleic, eicosapentaenoic and docosahexaenoic acid levels markedly decreased. PC was mainly concerned by the altered distribution of unsaturated fatty acids, but not SM. However, a significant decrease in (n-6)/(n-3) ratio occurred in the latter. These structural changes were associated with a higher 5'-nucleotidase activity in the intestinal BBM of PUFA-deficient as compared to control piglets.

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.

Effects of a low dietary linoleic acid level on intestinal morphology and enterocyte brush border membrane lipid composition.

The influence of low dietary linoleic acid level (an essential fatty acid deficiency) on the intestine mucosal morphology and the purified brush border membrane (BBM) lipid composition was investigated in the rat. Electron micrographs and morphometric measurements showed that villi and crypt sizes as well as the ultrastructure of epithelial cells were altered. Cholesterol (CHOL) and phospholipid (PL) levels, CHOL/PL ratio and PL class distribution were not changed by the low linoleate diet. However, the fatty acid composition of phospholipids was markedly modified in the enterocyte BBM, showing elevated amounts of palmitoleic (16:1n-7), oleic (18:1n-9) and 5,8,11-eicosatrienoic (20:3n-9) acids and, by contrast, depressed linoleic (18:2n-6) and arachidonic (20:4n-6) acid levels. Although the underlying mechanisms remain unknown the results obtained suggest that essential fatty acids (EFA) could be directly involved in the trigger action of the observed alterations, as regards both their dynamic (metabolic) and structural roles.

Evidence for a structurally specific role of essential polyunsaturated fatty acids depending on their peculiar double-bond distribution in biomembranes.

ESR spectrometry with 5-, 7-, 10-, and 12-doxylstearate probes and a combined index considering separately the double-bond numbers of essential and nonessential fatty acids were used to investigate the structural role of the double bonds of polyunsaturated fatty esters in membrane phosphoglycerides. Purified brush border membrane vesicles were prepared from the jejunum of piglets receiving either high (HLA) or low (LLA) dietary levels of linoleic acid (18:2 n-6). In the LLA as compared to the HLA group, there were no significant modifications of (a) the relative contents of cholesterol, phospholipid, and protein and of (b) the phosphoglyceride class distribution, contrasting with very large changes in the fatty acid compositions of each phosphoglyceride. These changes were characterized by an increase in nonessential monoene and triene (18:1 n-9 and 20:3 n-9) and a decrease in essential diene (18:2 n-6) in LLA- as compared to HLA-fed piglets. The essential tetraene 20:4 n-6 remained rather constant despite an overall nonsignificant increase in the LLA group. The total double-bond number (TDBn) was not significantly affected, contrasting with the variations in the double-bond numbers of essential and nonessential fatty acids (DBn(EFA) and DBn(nonEFA), respectively). The combined DBn(EFA)/DBn(nonEFA) index was 1.7-3.3 times lower in LLA than in HLA membrane phospholipids. It was concluded that the diet was able to affect the double-bond distribution in the upper and inner half-parts of the membrane leaflet without changing the total number of double bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

The effect of tropical ambient temperature on growth and metabolism in pigs.

Three experiments involving 34 individually fed pigs were conducted in Guadeloupe (16 degrees Lat. N., 61 degrees Long. W.) to determine the effects of environmental temperature (tropical, 22 to 32 degrees C, vs thermoneutral, 17 to 21 degrees C) and feeding method (restricted vs ad libitum) on performance, carcass characteristics and physiological and metabolic responses of pigs at three weight ranges (8 to 25, 29 to 50 and 54 to 79 kg live weight). Compared with the control environment, the tropical climate increased rectal temperature and respiratory rate but depressed growth rate and efficiency of feed utilization. In addition, in the heaviest weight group, feed intake was reduced and body fat increased. Changes in metabolic status, such as increased concentrations of plasma free fatty acids, triglycerides, cholesterol and adipose tissue lipoprotein lipase activity were observed in pigs housed in the tropical environment. Moreover, in these pigs, there was a decreased plasma concentration of thyroid hormones (triiodothyronine and thyroxine). These results indicate that tropical ambient temperature markedly affects the metabolism of pigs and, therefore, probably influences their nutritional requirements.

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.

The effect of dietary essential fatty acid deficiency on the composition and properties of the liver microsomal membrane of rats.

The effect of dietary essential fatty acid (EFA) deficiency on lipid composition, fluidity and important enzyme and transport activities of liver microsomal membrane was studied in weanling rats. After 133 d of EFA deficiency, no difference was noticed in membrane phospholipid, cholesterol and protein levels, but a significant change occurred in the fatty acid composition of bilayer phospholipids. In EFA-deficient rats, linoleic (18:2(n-6] and arachidonic (20:4(n-6] acids were both severely lower while oleic (18:1(n-9], palmitoleic (16:1(n-7] and particularly 5,8,11-eicosatrienoic (20:3(n-9] acids were significantly higher than in controls. The higher level of the latter tended to compensate for the lower level of 20:4(n-6). Membrane fluidity, as estimated by the reciprocal of the order parameter S, was lower in the deficient rats than in the controls, and all the measured microsomal enzyme activities were markedly affected. NADH-Cyt b5 electron transferring system, coupled with the fatty acid desaturation system, was higher than in controls. In contrast, the cytochrome P450 complex activity was lower and some of the important liver detoxifying enzyme activities were lower due to physical-chemical changes in the microsomal membrane. Calcium uptake and Ca2+-ATPase activity were also significantly lower in EFA-deficient rats than in controls. It was concluded that fatty acid composition may be the major factor contributing to membrane fluidity and function and that EFA might play a role in regulating the intrinsic membrane protein activities.

[Influence of the environmental temperature on the metabolic use of dietary energy and nitrogen in the growing rat]. / Influence de la température ambiante sur l'utilisation métabolique de l'énergie et de l'azote alimentaires chez le rat en croissance.

Sixty-seven male rats of the Wistar CF strain were used in two trials to study the effect of ambient temperature on nitrogen and energy balances (experiment 1) and on the variations of certain biochemical parameters of metabolism (experiment 2). In both cases, the rats, housed individually in metal cages and fed ad libitum, were kept under three different temperatures : 21 degrees C (control), 3 degrees C (cold), 31 degrees C (hot). Compared to the controls, the rats at 3 degrees C showed a significant increase (P less than 0.01) in food intake and a significant decrease (P less than 0.01) in daily growth rate and feed efficiency ratio; body fat content was unchanged. Compared to the controls, the rats at 31 degrees C showed a decrease (P less than 0.01) in food intake and growth rate, a better feed efficiency ratio (P less than 0.01) and a significant increase in carcass lipid content. When compared to rats at 21 degrees C, those at 3 degrees C had a much lower nitrogen retention; at 31 degrees C this retention was only slightly lower. The urinary nitrogen excretion, increased by the cold temperature, reflected higher nitrogen catabolism, while its increase at a high temperature was probably due to a decrease in protein synthesis, and a subsequent degradation of excess ingested nitrogen. Energy retention increased (P less than 0.01) as the ambient temperature increased from 3 to 31 degrees C, and wide variations in the amounts of fixed energy in the form of proteins or lipids were observed. The balance results obtained have been discussed in relation to the variations of some biochemical parameters of metabolism such as free fatty acids, free glycerol, lipoprotein-lipase activity and in vitro utilization of labelled glucose.