Claude Leger

Essential fatty acids in trout serum lipoproteins, vitellogenin and egg lipids

This paper describes evidence of (n−3) and particularly of 22∶6 (n−3) fatty acid enrichment in trout lipoproteins as well as in vitellogenin, egg lipovitellin and oil globule. Among the lipoproteins, HDL and LDL were the main forms of blood lipid transport, whereas phospholipids and cholesteryl esters are the preferential chemical carriers for (n−3) fatty acid transport. However, cholesteryl esters were less important as esterified fatty acid carriers than in man. Taken together with the data obtained in mammals, our results suggest that there may be a relationship between EFA activity and the distribution of the EFA among the lipoprotein lipid fractions in vertebrates, irrespective of the EFA series. Administration of an (n−3) fatty acid deficient diet for three months prior to trout spawning produced a significant increase in egg lipid content, primarily as a result of the increase of the oil globule composed almost exclusively of triacylglycerols. This diet decreased the 22∶6 (n−3), as well as the (n−3) fatty acid contents of lipoproteins, lipovitellin, vitellogenin and the oil globule. In contrast, the (n−3) fatty acid level was always higher in lipoproteins and lipovitellin than in the vitellogenin and the oil globule. Moreover, the relative levels of 22∶6 (n−3) and total (n−3) fatty acids were quite similar in lipoproteins and lipovitellin on the one hand, and in vitellogenin and the oil globule on the other. These findings suggest a direct relationship between the two forms of plasma lipid transport and the two egg compartments. During ovogenesis, dietary lipids seemed to be diverted from the adipose tissue and essentially deposited in the egg.

Effect of dietary fatty acids differing by chain lengths and ω series on the growth and lipid composition of turbot Scophthalmus maximus L.

1. A previous paper (Gatesoupe et al., 1977) showed that turbot had a specific requirement for omega 3HPUFA since equivalent dietary amounts of 18:3 omega 3 or omega 3HPUFA (0.55% of the diet) did not lead to the same growth performances. 2. In the present paper, we demonstrated that fish given a high level of dietary 18:3 omega 3 (3.7% of the diet), without omega 3HPUFA, presented better growth than those offered a lower level of 18:3 omega 3, and almost the same performances as fish receiving 0.57% omega 3HPUFA. 3. This suggested that turbot, like trout, might be able to use the 18:3 omega 3 as a precursor of the omega 3 series. Furthermore, according to the present relatively short-term experiment, elongation-desaturation reactions of the omega 3FA did not appear to be reduced with low dietary omega 3FA levels. 4. On the other hand, these types of reactions seemed to be totally missing with the 18:2 omega 6. Thus, it may be assumed that there was no direct relationship between growth and omega 3 elongating-desaturating activities, and that omega 3 lowering fish body content was not the cause, or at least not the only cause, of poor growth in long-term experiments.

Effects of a (n−3) polyunsaturated fatty acid-deficient diet on profiles of serum vitellogenin and lipoprotein in vitellogenic trout (Salmo gairdneri)

During the 6 months of vitellogenesis, 3-year-old female trout (Salmo gairdneri) were fed either an enriched (E) or an (n−3) polyunsaturated fatty acid (PUFA)-deficient (D) diet; serum vitellogenin (VG) and lipoproteins (d<1.21 g/ml) were analyzed at the third month of vitellogenesis (September) and at ovulation (December). The serum content of high density lipoproteins (HDL), the major protein class, maintained a mean value of 1500 mg/dl at both stages and with both diets. On the contrary, very low density lipoproteins (VLDL) were 90% higher during vitellogenesis than at spawning time, whereas excess vitellogenin circulated at this period (6580 mg/dl serum with diet E). The diet deficient in (n−3) lowered serum vitellogenin content by 16% in September and by 26% in December. The degree of (n−3) PUFA incorporation moderately decreased in low density lipoproteins (LDL) and in HDL with the (n−3)-deficient diet. The effect was more pronounced for 20∶5. On the other hand, essential 22∶6 was incorporated into vitellogenin at the same rate in September as in December with diet E (23% and 25%, respectively), whereas after a 3-month deficiency, the percentage fell to 12%; this percentage rose again to 19% at spawning time. These findings show that, although stored (n−3) PUFA were not exhausted after a 6-month dietary deficiency, the incorporation of essential fatty acids (EFA) into vitellogenin during the early stages of oogenesis was low, suggesting changes in egg composition that may influence hatching.

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.

Fatty acid composition of lipids in the trout—I. Influence of dietary fatty acids on the triglyceride fatty acid desaturation in serum, adipose tissue, liver, white and red muscle

Abstract 1. 1. The present study examined the influence of 6 and 10% lipid-diets on the fatty acid composition of triglyceride body compartments and the influence of two diets differing by their n-6 and n- 3 HPUFA content on the bioconversion of 18:2 n- 6 and 18:3 n- 3 . 2. 2. A 6% fat diet caused a high level of 18:1 n- 9 as previously reported in fish and mammals for low fat diets. 3. 3. The lipid content of trout serum was very high (about 2000 mg/100 ml) but the apolar (triglycerides + cholesteryl esters) and more polar (phospholipids + free cholesterol) components in trout serum were balanced as in man. 4. 4. A useful index [P + Σ(n−x)]/P , where P is the precursor of the n − x series, allows us to determine the relative importance of the Δ9, or Δ6, desaturation. 5. 5. The n-3 HPUFA seem to present a feedback effect on the n-x and n-6 FA bioconversions as reported by some authors whereas the Δ6 desaturation of 18:1 n-9 was blocked. 6. 6. The general decreasing importance of the Δ6-desaturation in the compartments is as followed: liver, serum, intestine and muscle, adipose tissue.

Fatty acid composition of lipids in the trout—II. Fractionation and analysis of plasma lipoproteins

Abstract 1. 1. Blood level of lipoproteins was studied in adult male trout. Blood samples were collected in April, i.e. 3–4 months after the spawning period. 2. 2. Samples from 15 fish were pooled for analysis. The main classes of lipoproteins: chylomicrons, VLDL, LDL, HDL were present, but their electrophoretic mobility was different from that of human lipoproteins. 3. 3. The proportions of VLDL, LDL, HDL were respectively 26, 51 and 23% of total lipoproteins. The eventual correlation of the predominance of LDL and the annual reproductive cycle has not been elucidated yet. 4. 4. The major lipids in the different classes of lipoproteins were TG in VLDL and PL in LDL and HDL. The level of total cholesterol was 235 mg/100 ml plasma; about half of it was unesterified in all classes of lipoproteins. The NEFA contents of the plasma fractions d 1.21 g/ml were negligible as compared to those of the esterified forms of fatty acids. 5. 5. The level of essential fatty acid 22:6 n- 3 in all classes of lipoproteins was much higher than in the dietary lipids. It is suggested that a part of the dietary 18:3 n- 3 was elongated and desaturated in the intestinal epithelium and incorporated into chylomicrons and VLDL.

Specific distribution of fatty acids in the triglycerides of rainbow trout adipose tissue. Influence of temperature

In the trout, the unsaturated fatty acids are preferentially located in the β-position and the saturated fatty acids in the α-position of triglycerides. This fatty acid distribution is retained even with diets containing lard. The fish are, therefore, able to modify completely the fatty acid distribution of dietary triglycerides. There is no retention of the β-monoglyceride structure during the biosynthetic processes. However, the modification of the dietary fatty acid distribution by the trout seems to be more difficult at 18 C than at 10C.

Some properties of pancreatic lipase in Salmo gairdnerii rich.: Km, effects of bile salts and Ca2+, gel filtrations

1. The Michaelis constant (Km) of the trout pancreatic lipase is 1.3 x 10(-6) M tributyrin, calculated by the interface concentration of the emulsion. This value is lower than that of porcine pancreatic lipase. 2. The lipase hydrolyses tributyrin in a Ca2+ free medium. Conversely, Ca2+ is essential for the lipolysis of triolein. The cation might be an effector of the reaction, but it seems to remove the inhibition of the enzyme by its product. 3. The curves of the lipase activity according to bile salt concentration seem to suggest the existence of a colipase, that we have not evidenced yet by direct procedures. 4. The apparent molecular weight of the lipase seems to be lower in the trout than in the species studied so far.

Immobilized colipase affinities for lipases B, A, C and their terminal peptide (336―449): the lipase recognition site lysine residues are located in the C-terminal region

Zonal high-performance affinity chromatography has been used in order to study the interactions between pig isolipases A, B and C and the terminal peptide chain fragment 336-449 of the pig lipase on the one hand, and the homolog colipase bound to the inert LiChrosorb diol support on the other. A mathematical treatment led the to assessment of the dissociation constant of the lipase-colipase complex using isolipases or the terminal peptide as eluted acceptors and colipase as silica-bound ligand (Mahé, N., Léger, C.L., Linard, A. and Alessandri, J.-M. (1987) J. Chromatogr. 395, 511-521). A higher affinity of isolipase B as compared to isolipases A and C towards colipase was observed (KD, respectively, of 0.68, 11 and 12 microM) at pH 6.5. Under the same chromatographic conditions, the terminal peptide chain interacted with the bound colipase (KD 0.70 microM, close to that of isolipase B). The chromatographic behaviors of both native and chemically modified lipase and terminal peptide were very similar. In particular, guanidination of lysine residues of both peptide and isolipase B led to the loss of interactions with colipase. The same result was observed with the peptide preincubated in the presence of increasing amounts of free colipase. Accordingly, it is suggested that, firstly, a preferential association of isolipase B to colipase could take place and, secondly, the colipase recognition site of lipase could be located in the C-terminal region, the conformational structure of the terminal peptide not being affected by the enzymic cleavage and, therefore, being largely independent of the rest of the polypeptide molecule. On the other hand, a lower colipase affinity for isolipases A or C than for isolipase B or the C-terminal peptide could tentatively be attributed to a non-local (distant) disturbing effect of the negatively charged glycan chain, as sialic acid is present in both isoforms A and C. Finally, the present paper confirms and extends earlier studies on lipase-colipase interactions.

Effect of essential fatty acid deficiency on lipid composition of basolateral plasma membrane of pig intestinal mucosal cells

The effect of essential fatty acid (EFA) deficiency on the lipid composition of basolateral plasma membranes (BPM) from intestinal mucosal cells was investigated in weaning pigs fed control or EFA-deficient diets for 12 weeks. The phospholipid and cholesterol contents relative to protein were similar in both groups, showing a cholesterol/phospholipid molar ratio of 0.6. The distribution of phospholipid classes was also unaffected by the diet. In contrast, fatty acid profiles of the two phospholipid main classes, phosphatidylcholine and phosphatidylethanolamine were altered by EFA deficiency. Linoleic acid (18∶2n−6) was largely reduced, whereas arachidonic acid (20∶4n−6) only slightly decreased in EFA-deficient pigs. The unsaturation index was essentially maintained by high levels of oleic acid (18∶1n−9) and by conversion of oleic acid to 5,8,11-eicosatrienoic acid (20∶3n−9). Finally, during the period of EFA deficiency, the lipid composition of BPM of the intestinal mucosal cells was little affected, suggesting a preferential uptake of 20∶4n−6 and (or) precursor mobilized from other tissues. However, an effect of dietary treatment on the function of membrane-associated proteins cannot be ruled out.