Effects of nutrients (in food) on the structure and function of the nervous system: update on dietary requirements for brain. Part 2 : macronutrients.

Among polyunsaturated omega-3 fatty acids, ALA (alpha-linolenic acid) provided the first coherent multidisciplinary experimental demonstration of the effect of diet (one of its major macronutrient) on the structure, the biochemistry, the physiology and thus the function of the brain. In fact, DHA (docosahexaenoic acid) is one for the major building structures of membrane phospholipids of brain and absolute necessary of neuronal function. It was first demonstrated that the differentiation and functioning of cultured brain cells requires not only ALA, but also the very long polyunsaturated omega-3 (DHA) and omega-6 carbon chains. Then, it was found that ALA acid deficiency alters the course of brain development, perturbs the composition of brain cell membranes, neurones, oligodendrocytes and astrocytes, as well as sub cellular particles such as myelin, nerve endings (synaptosomes) and mitochondria. These alterations induce physicochemical modifications in membranes, lead to biochemical and physiological perturbations, and results in neurosensory and behavioural upset. Consequently, the nature of polyunsaturated fatty acids (in particular omega-3, ALA and DHA) present in formula milks for infants (premature and term) conditions the visual, neurological and cerebral abilities, including intellectual. Dietary omega-3 fatty acids are involved in the prevention of some aspects of ischemic cardiovascular disease (including at the level of cerebral vascularization), and in some neuropsychiatric disorders, particularly depression, as well as in dementia, including Alzheimer's disease and vascular dementia. The implication of omega-3 fatty acids in major depression and bipolar disorder (manic-depressive illness) is under evaluation. Their dietary deficiency (and altered hepatic metabolism) can prevent the renewal of membranes and consequently accelerate cerebral ageing; nonetheless, the respective roles of the vascular component on one hand and the cerebral parenchyma itself on the other have not yet been clearly elucidated. Low fat diet may have adverse effects on mood. The nature of the amino acid composition of dietary proteins contributes to cerebral function; taking into account that tryptophan plays a special role. In fact, some indispensable amino acids present in dietary proteins participate to elaborate neurotransmitters (and neuromodulators). The regulation of glycaemia (thanks to the ingestion of food with a low glycaemic index ensuring a low insulin level) improves the quality and duration of intellectual performance, if only because at rest the brain consumes more than 50% of dietary carbohydrates, approximately 80% of which are used only for energy purpose. In infants, adults and aged, as well as in diabetes, poorer glycaemic control is associated with lower performances, for instance on tests of memory. At all ages, and more specifically in aged people, some cognitive functions appear sensitive to short term variations in glucose availability. The presence of dietary fibbers is associated with higher alertness ratings and ensures less perceived stress. Although an increasing number of genetic factors that may affect the risk of neurodegenerative disorders are being identified, number of findings show that dietary factors play major roles in determining whether the brain age successfully of experiences neurodegenerative disorders. Effects of micronutrients have been examined in the accompanying paper.

Effects of nutrients (in food) on the structure and function of the nervous system: update on dietary requirements for brain. Part 1: micronutrients.

The objective of this update is to give an overview of the effects of dietary nutrients on the structure and certain functions of the brain. As any other organ, the brain is elaborated from substances present in the diet (sometimes exclusively, for vitamins, minerals, essential amino-acids and essential fatty acids, including omega- 3 polyunsaturated fatty acids). However, for long it was not fully accepted that food can have an influence on brain structure, and thus on its function, including cognitive and intellectuals. In fact, most micronutrients (vitamins and trace-elements) have been directly evaluated in the setting of cerebral functioning. For instance, to produce energy, the use of glucose by nervous tissue implies the presence of vitamin B1; this vitamin modulates cognitive performance, especially in the elderly. Vitamin B9 preserves brain during its development and memory during ageing. Vitamin B6 is likely to benefit in treating premenstrual depression. Vitamins B6 and B12, among others, are directly involved in the synthesis of some neurotransmitters. Vitamin B12 delays the onset of signs of dementia (and blood abnormalities), provided it is administered in a precise clinical timing window, before the onset of the first symptoms. Supplementation with cobalamin improves cerebral and cognitive functions in the elderly; it frequently improves the functioning of factors related to the frontal lobe, as well as the language function of those with cognitive disorders. Adolescents who have a borderline level of vitamin B12 develop signs of cognitive changes. In the brain, the nerve endings contain the highest concentrations of vitamin C in the human body (after the suprarenal glands). Vitamin D (or certain of its analogues) could be of interest in the prevention of various aspects of neurodegenerative or neuroimmune diseases. Among the various vitamin E components (tocopherols and tocotrienols), only alpha-tocopherol is actively uptaken by the brain and is directly involved in nervous membranes protection. Even vitamin K has been involved in nervous tissue biochemistry. Iron is necessary to ensure oxygenation and to produce energy in the cerebral parenchyma (via cytochrome oxidase), and for the synthesis of neurotransmitters and myelin; iron deficiency is found in children with attention-deficit/hyperactivity disorder. Iron concentrations in the umbilical artery are critical during the development of the foetus, and in relation with the IQ in the child; infantile anaemia with its associated iron deficiency is linked to perturbation of the development of cognitive functions. Iron deficiency anaemia is common, particularly in women, and is associated, for instance, with apathy, depression and rapid fatigue when exercising. Lithium importance, at least in psychiatry, is known for a long time. Magnesium plays important roles in all the major metabolisms: in oxidation-reduction and in ionic regulation, among others. Zinc participates among others in the perception of taste. An unbalanced copper metabolism homeostasis (due to dietary deficiency) could be linked to Alzheimer disease. The iodine provided by the thyroid hormone ensures the energy metabolism of the cerebral cells; the dietary reduction of iodine during pregnancy induces severe cerebral dysfunction, actually leading to cretinism. Among many mechanisms, manganese, copper, and zinc participate in enzymatic mechanisms that protect against free radicals, toxic derivatives of oxygen. More specifically, the full genetic potential of the child for physical growth ad mental development may be compromised due to deficiency (even subclinical) of micronutrients. Children and adolescents with poor nutritional status are exposed to alterations of mental and behavioural functions that can be corrected by dietary measures, but only to certain extend. Indeed, nutrient composition and meal pattern can exert either immediate or long-term effects, beneficial or adverse. Brain diseases during aging can also be due to failure for protective mechanism, due to dietary deficiencies, for instance in anti-oxidants and nutrients (trace elements, vitamins, non essential micronutrients such as polyphenols) related with protection against free radicals. Macronutrients are presented in the accompanying paper.

An important source of omega-3 fatty acids, vitamins D and E, carotenoids, iodine and selenium: a new natural multi-enriched egg.

As natural eggs can contribute significantly to overcoming dietary deficits, we have designed and studied the composition of multiple-enriched eggs (Benefic eggs) whose composition is close to the natural egg. They are obtained by feeding laying hens in the usual way, but using additional autoclaved linseed, minerals, vitamins and lutein to provide the extra components. These eggs have greater nutritional value than standard. Thus 100 g of these eggs contains 6 times more of the omega-3 fatty acid ALA (15% of the French recommended daily allowance (RDA)), 3 times more DHA (100% of RDA), 3 times more vitamin D (30% of RDA), 4 times more folic acid (70% of RDA), 6 times more vitamin E (66% of RDA), 6 times more lutein and zeaxanthine (70% of international recommendation), 2.5 times more iodine (100% RDA), and 4 times more selenium (45% RDA). As the content of omega-6 fatty acids remains unchanged, the omega-6/omega-3 ratio is lower, and thus improved. These eggs contain a little less cholesterol and, like standard eggs, are rich in vitamin B12 (160% of RDA) and vitamin A (25% of RDA), plus vitamin B2 (riboflavin), vitamin B5 (pantothenic acid) and phosphorus. Proteins quality is indeed excellent. These eggs are interesting for everybody, and particularly appropriate for older people. The nutritional value of enriched eggs (similar to the multiple-enriched eggs of this study) has been assessed in animals and in human volunteers in terms of their influence on blood lipids. They improve the blood concentration of omega-3 fatty acids, HDL cholesterol, LDL cholesterol and triglycerides.

Graded dietary levels of RRR-gamma-tocopherol induce a marked increase in the concentrations of alpha- and gamma-tocopherol in nervous tissues, heart, liver and muscle of vitamin-E-deficient rats.

The effect of dietary RRR-gamma-tocopherol supplementation on serum and tissue alpha- and gamma-tocopherol concentrations was studied in vitamin-E-deficient rats fed diets containing adequate levels of RRR-alpha-tocopherol and graded levels of RRR-gamma-tocopherol over a 60 day period. Feeding rats with a RRR-alpha-tocopherol-supplemented diet induced in forebrain, sciatic endoneurium, skeletal muscle, heart and liver a marked increase in alpha-tocopherol concentration. In contrast, feeding rats with a diet containing the same level of RRR-gamma-tocopherol induced a small increase in gamma-tocopherol concentrations in brain, sciatic endoneurium, skeletal, muscle, heart and liver and a slight but significant decrease in alpha-tocopherol concentration in all tissues examined. In rats fed diets containing a constant level of RRR-alpha-tocopherol and graded levels of RRR-gamma-tocopherol, the concentrations of alpha-tocopherol in all tissues were much higher than those in rats fed a control diet containing RRR-alpha-tocopherol alone. The higher the gamma/alpha ratio, the more the alpha-tocopherol concentrations increased. Significant positive linear regressions were found between the gamma/alpha ratio and the alpha- and gamma-tocopherol concentrations in most of the tissues examined. These results indicate that when gamma-tocopherol was supplied continuously in the diet gamma-tocopherol accumulated significantly in the tissues but to a much smaller extent than when rats were fed with RRR-alpha-tocopherol. These experiments also indicate that gamma-tocopherol did not depress the serum and tissue alpha-tocopherol concentrations. On the contrary, gamma-tocopherol supplements induced a marked increase in alpha-tocopherol concentrations in the serum and tissues. These results suggest that there is a relationship between alpha- and gamma-tocopherol levels in vivo and that the biopotency of alpha-tocopherol should be reevaluated especially when high levels of gamma-tocopherol were present in the diet.

Ketone body utilization for energy production and lipid synthesis in isolated rat brain capillaries.

Isolated brain capillaries from 2-month-old rats were incubated for 2 h in the presence of [3-14C]acetoacetate, D-3-hydroxy[3-14C]butyrate, [U-14C]glucose, [1-14C]acetate or [1-14C]butyrate. Labelled CO2 was collected as an index of oxidative metabolism and incorporation of label precursors into lipids was determined. The rate of CO2 production from glucose was slightly higher than from the other substrates. Interestingly, acetoacetate was oxidized at nearly the same rate as glucose. This shows that ketone bodies could be used as a source of energy by brain capillaries. Radiolabelled substrates were also used for the synthesis of lipids, which was suppressed by the addition of albumin. The incorporation of [U-14C]glucose in total lipids was 10-times higher than that from other precursors. However, glucose labelled almost exclusively the glycerol backbone of phospholipids, especially of phosphatidylcholine. Ketone bodies as well as glucose were incorporated mainly into phospholipids, whereas acetate and butyrate were mainly incorporated into neutral lipids. The contribution to fatty acid synthesis of various substrates was in the following order: butyrate greater than or equal to acetate greater than ketone bodies greater than or equal to glucose. All precursors except glucose were used for sterol synthesis. Glucose produced almost exclusively the glycerol backbone of phospholipids.

Effect of lipid peroxidation on Na+,K(+)-ATPase, 5'-nucleotidase and CNPase in mouse brain myelin.

In the presence of H2O2, solutions of Fe2+ were applied to brain homogenate and isolated myelin from adult SWV control mice and the shiverer dysmyelinating mutant mouse as a source of a reactive oxygen species (Fenton reaction). Under these conditions, lipid peroxidation was initiated and measured as thiobarbituric acid-reactive oxidation products (TBAR). This was accompanied by 85% inhibition of myelin-associated Na+,K(+)-ATPase and 25% inhibition of 5'-nucleotidase. In contrast, CNPase activity was not altered. Studies on the shiverer mutant brain revealed that in spite of hypomyelination and prevalence of premature, myelin-like membranes in the homogenate, the myelin-related enzymes reacted as normal enzymes to peroxidation. Differences in the resistance of Na+,K(+)-ATPase to peroxidation in the brain homogenate and myelin suggest that the myelin enzyme is extremely sensitive to reactive oxygen toxicity.

Condensation activity for polyunsaturated fatty acids with malonyl-CoA in rat brain microsomes. Characteristics and developmental change.

Condensation activities for gamma-linolenic acid (18:3(n-6)), octadecatetraenoic acid (18:4(n-3)) and eicosapentaenoic acid (20:5(n-3)) with malonyl-CoA were measured and compared with the condensation activities for 16:0-CoA, 18:1-CoA, 18:2(n-6)-CoA and 18:3(n-3)-CoA in rat brain microsomes of various ages. The age-dependence of condensation activities for 18:3(n-6), 18:4(n-3) and 20:5(n-3) showed a maximum at 1- to 2-month-old and were still higher at 3-month-old 2- to 3-fold than the activities in microsomes of pups. Conversely, the age-dependence of condensation activity for 16:0-CoA showed a peak around 1 month-old, but decreased at 3-month-old to the level of the activities in pups. The condensation activity for 20:5(n-3) was inhibited by 18:3(n-6) or 18:4(n-3) and the inhibition was not competitive. The condensation of 18:3(n-6) was also inhibited by 18:4(n-3) in the same manner. A physiological implication of the inhibition system at the substrate level was discussed.

Uptake of dietary RRR-alpha- and RRR-gamma-tocopherol by nervous tissues, liver and muscle in vitamin-E-deficient rats.

The time course of RRR-alpha-tocopherol and RRR-gamma-tocopherol uptake by liver, muscle and selected nervous tissues was studied in vitamin-E-deficient rats fed diets containing either RRR-alpha-tocopherol or RRR-gamma-tocopherol over a 60 day period. Feeding rats with a RRR-alpha-tocopherol-supplemented diet induced in brain, cerebellum, sciatic endoneurium and muscle a marked and regular increase in alpha-tocopherol concentration. In addition, the tocopherol concentration in liver reached a plateau very rapidly. In contrast, feeding rats with a diet containing the same level of RRR-gamma-tocopherol induced a very small increase in gamma-tocopherol concentration in brain, cerebellum, sciatic endoneurium and muscle, no change in alpha-tocopherol concentration of brain and muscle and a slight but significant decrease in alpha-tocopherol concentration in sciatic endoneurium and cerebellum. These results indicate that when gamma-tocopherol was supplied continuously in the diet gamma-tocopherol accumulated significantly in the tissues but to a much lesser extent than when rats were fed with RRR-alpha-tocopherol. These results also show that in the tocopherol-deficient rat, gamma-tocopherol does not significantly affect the residual alpha-tocopherol concentrations in brain or cerebellum, except poorly in sciatic endoneurium.

A spin-label study of sciatic nerves from quaking, jimpy and trembler mice.

The spin labels, 5-nitroxide stearic acid and 16-nitroxide stearic acid were incorporated into whole sciatic nerves dissected from normal, quaking, jimpy and trembler mice. With 5-nitroxide stearic acid, we have studied the thermal variation of the maximal apparent coupling constant (T) between 0 degrees C and 50 degrees C. Within this range of temperatures, we obtained identical values of 2 T for nerves from normal and jimpy mice, whereas 2 T was smaller for nerves from quaking and trembler mice. With 16-nitroxide stearic acid, composite spectra were recorded, particularly in the high-field range. A line characteristic of myelin was clearly observed in the spectra of nerves from normal and jimpy mice; its intensity was somewhat less in nerves from quaking mice and much less in spectra from trembler mice. A shoulder in the principal highfield line of the spectrum is modified only with nerves from jimpy mice. The results agree well with those obtained by electron microscopy, which reveal normal myelination in nerves from jimpy mice, a slight modification of the myelin from those of quaking mice and a practically complete demyelination in peripheral nerves from trembler mice. However, the structure of the nerves of jimpy mice also seems to be modified at an, as yet, undetermined level.

Distribution of radioactivity in myelin lipids following subcutaneous injection of [14C]stearate.

Blood fatty acids are an important parameter for the synthesis of brain myelin as exogenous stearic acid is needed: after subcutaneous injection to 18-day-old mice this labelled stearic acid is transported into brain myelin and incorporated into its lipids. However the acid is partly metabolized in the brain by elongation (thus providing very long chain fatty acids, mainly lignoceric acid) or by degradation to acetate units (utilized for synthesis of medium chain fatty acids as palmitic acid, and cholesterol). These metabolites are further incorporated into myelin lipids. The myelin lipid radioactivity increases up to 3 days; most of the activity is found in phospholipids; their fatty acids are labelled in saturated as well as in polyunsaturated homologues but sphingolipids, especially cerebrosides, contain also large amounts of radioactivity (which is mainly found in very long chain fatty acids, almost all in lignoceric acid). The occurrence of unesterified fatty acids must be pointed out, these molecules unlike other lipids, are found in constant amount (expressed in radioactivity per mg myelin lipid).

Nervonic acid biosynthesis by erucyl-CoA elongation in normal and quaking mouse brain microsomes. Elongation of other unsaturated fatty acyl-CoAs (mono and poly-unsaturated).

Biosynthesis of nervonic acid by enzymatic elongation of erucyl-CoA has been studied in mouse brain microsomes. The substrate and cofactor requirements have been measured. Malonyl-CoA and reduced nicotine-adenine-dinucleotide phosphate are required, but not FMN, FAD or NADH. The effect of protein concentration, incubation time, ATP and CoA has been determined; the reaction products were checked by gas-liquid chromatography with automatic counting of the eluate. Very little activity was found in hydroxylated fatty acids. In the presence of phosphotransacetylase (which impedes the de novo microsomal system), the main reaction product was nervonic acid. It is concluded that nervonic acid is biosynthesised by elongation using a two-carbon unit from malonyl-CoA. The same enzyme biosynthesises saturated and mono-unsaturated very long chain fatty acids. The elongation capacity of "quaking" microsomes is reduced to 30% of the normal value with both erucyl-CoA and behenyl-CoA. Elongation of trans isomer (brassidyl-CoA) and poly-unsaturated homologue (clupanodonyl-CoA) was compared to elongation of erucyl-CoA in both normal and mutant mice. Both unsaturated acyl-CoAs are elongated under the same conditions as erucyl-CoA in brain: the poly-unsaturated acyl-CoA is elongated more actively than the mono-unsaturated acyl-CoA in the mutant.

Interactive effects of dietary (n-3) polyunsaturated fatty acids and chronic ethanol intoxication on synaptic membrane lipid composition and fluidity in rats.

The influence of dietary polyunsaturated fatty acids on fatty acid composition, cholesterol and phospholipid content as well as 'fluidity' (assessed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) probes) of brain synaptic plasma membranes (SPM) and their interactions with chronic ethanol effects were studied in rats fed for two generations with diets either devoid of (n-3) fatty acids (sunflower oil diet), rich in alpha-linolenic acid (soya oil diet) or in long chain (n-3) fatty acids (sunflower + cod liver oil diet). Results were compared with rats fed standard lab chow. Sunflower oil led to an increase in the (n-6)/(n-3) ratio in the membranes with an increase of the 'fluidity' at membrane apolar level; sunflower + cod liver oil decreased the (n-6)/(n-3) ratio without affecting membrane 'fluidity' while no difference was seen between the SPM of rats fed soya oil and standard diet. After 3 weeks alcohol intoxication in rat fed the standard diet: oleic alpha-linoleic acids and cholesterol levels were increased, arachidonic acid and the double bond index/saturated fatty acids were decreased and there was a decrease of 'fluidity' in the lipid core of the SPM. Soya oil almost totally abolished these usually observed changes in the SPM fatty acids composition but increased oleic acid and cholesterol without any change in fluidity. Sunflower oil led to the same general alterations of fatty acid as seen with standard diet but to a greater extent, with decrease of the 'fluidity" at the apolar level and in the region probed by TMA-DPH. When sunflower oil was supplemented with cod liver oil, oleic and alpha-linoleic acids were increased while the 'fluidity' of the apolar core of SPM was decreased. So, the small changes in fatty acid pattern seem able to modulate neural properties i.e. the responses to a neurotoxic like ethanol. A structurally specific role of PUFA is demonstrated by the pernicious effects of the alpha-linolenic acid deficient diet which are not totally prevented by the supply of long chain (n-3) PUFA.

Sodium-dependent high-affinity uptake of taurine by isolated rat brain capillaries.

Transport of taurine has been demonstrated in capillary preparations from adult rat brains using [3H]taurine. Taurine transport is mediated by a saturable high-affinity system which is entirely dependent on sodium ions. The apparent maximal influx (Vmax) and half-saturation concentration (Km) corresponded to 1.06.10(-4) mumol/min per mg protein and 27.5 microM, respectively. Competition experiments in the presence of sodium ion showed that [3H]taurine uptake was strongly inhibited by 0.1 mM unlabeled structural analogues of taurine such as beta-alanine and hypotaurine as well as unlabeled taurine. gamma-Aminobutyric acid (GABA) (0.1 mM) inhibited the uptake of labeled taurine by 30%, whereas isethionic acid, L-methionine, L-2,4-diaminobutyric acid, glycine, L-cysteinesulfonic acid and cystamine did not exhibit any inhibitory effect. The results suggest that the Na+ gradient is the principal source of energy for taurine transport into isolated brain capillaries. This transport system may play an active role in the regulation of taurine concentration in the brain extracellular space.

Alterations of cholesterol synthesis precursors (7-dehydrocholesterol, 7-dehydrodesmosterol, desmosterol) in dysmyelinating neurological mutant mouse (quaking, shiverer and trembler) in the PNS and the CNS.

In brain, levels of cholesterol, desmosterol and 7-dehydrodesmosterol are reduced in shiverer and quaking, but not in trembler 60-day-old dysmyelinating mutant mice. Very interestingly, 7-dehydrocholesterol is not altered in any mutant. The amount of cholesterol is similar in the different normal control mouse strains and in rat. In contrast, levels of precursors are not the same. In sciatic nerve, cholesterol is slightly reduced in shiverer, reduced 2-fold in quaking, and dramatically reduced in trembler (10-fold). 7-Dehydrocholesterol is affected in all mutants.

Effect of dietary alpha-linolenic acid on functional characteristic of Na+/K(+)-ATPase isoenzymes in whole brain membranes of weaned rats.

The influence of dietary fatty acids on Na+ sensitivity and ouabain affinity of Na+/K(+)-ATPase isoenzymes of whole brain membranes were studied in weaned rats fed for two generations with diets either devoid of alpha-linolenic acid (sunflower oil diet) or rich in alpha-linolenic acid (soya oil diet). The (n--3) deficiency induced by the sunflower oil diet led to an increase in the (n--6)/(n--3) molar ratio in whole brain membranes. Na+/K(+)-ATPase isoenzymes were discriminated on the basis of their differential affinities for ouabain. In rats fed sunflower oil diet, the ouabain titration displayed three inhibitory processes with markedly different affinities: low affinity (alpha 1); high affinity (alpha 2); and very high affinity (alpha 3). Membranes of rats fed soya oil diet exhibited only two inhibitory processes, i.e., low affinity (likely alpha 1+ alpha 2) and high affinity (likely alpha 2+ alpha 3) with the low affinity form intermediate between the sunflower alpha 1 and alpha 2 forms, and the high affinity form intermediate between the sunflower alpha 2 and alpha 3 forms. In fact, the Na+ response shows that the three isoenzymes have different Na+ sensitivities. Regardless of the diet, alpha 1 has a similar Na+ sensitivity (less than 1 mM), whilst alpha 2 and alpha 3 are more sensitive in soya oil membranes compared to sunflower oil membranes (5.1 vs. 7.2 mM and about 11 vs. 22.5 mM, respectively). Thus, sodium appears to be a better criterion of heterogeneity than ouabain.

Dietary alpha-linolenic acid deficiency in adult rats for 7 months does not alter brain docosahexaenoic acid content, in contrast to liver, heart and testes.

In adult rats, 22:6(n - 3) dietary deficiency does not affect brain membranes, but has a significant effect on some other visceral organs. 60-day-old male rats fed a diet containing sufficient amounts of both linoleic and alpha-linolenic acid were divided into three groups. One group continued the same diet; the second was fed a diet containing 2% sunflower oil, the third was fed 10% sunflower oil (sunflower oil contains linoleic acid, but trace amount of alpha-linolenic acid). Animals were killed different times after receiving the new diets (1 to 31 weeks). For animals fed the diets containing only sunflower oil, deficiency in cervonic acid content (DHA, docosahexaenoic acid, 22:6(n - 3)) was not detected in whole brain, myelin or nerve endings within 31 weeks. In contrast, this acid progressively declined in liver, heart and testes up to 3 weeks and remained nearly stable thereafter. In parallel to the reduction of cervonic acid content, 22:5(n - 6) content increased in liver and heart, but not in testes. It also increased in brain, nerve endings and myelin from week 3, 6 and, 9 respectively. These results suggest that brain cervonic acid is highly preserved or is maintained at the expense of other organs.

Structure determination of the polymorphism of acylgalactosylceramide in rat brain by gas chromatography/mass spectrometry and proton magnetic resonance.

This paper describes the structure of acylcerebrosides isolated from rat brains. Three fractions (acylglycosylceramides I, II, III) were resolved according to their decreasing RF values on TLC. GLC analysis of acylglycosylceramides II and III indicates that their ester-linked fatty acids are short and rather unsaturated, while amide-linked fatty acids are longer and hydroxylated. Sugar GLC analysis indicates that acylglycosylceramides II and III contain only galactose. To determine the substitution position of the acyl group on the galactose moiety, the free hydroxyl groups of acylglycosylceramide were protected with dihydropyran, deacylated and subjected to permethylation. The methylated galactoside acetates obtained after hydrolysis and reduction were then analyzed by gas chromatography/mass spectrometry. Acylglycosylceramides II and III turned out to be complex mixtures of 2-O-acyl-, 3-O-acyl-, 4-O-acyl- and 6-O-acylgalactosylceramides. Moreover, the abundance of alpha-methylgalactoside reveals the existence of unsubstituted galactose, suggesting that some ester-linked fatty acids could be esterified to the hydroxyl group of hydroxy fatty acids linked to sphingosine. NMR spectrometry was used to confirm this ester linkage. The key spectral feature of the fatty acid-galactose linkage (4.45 ppm) did move to 4.15 ppm after saponification of acylglycosylceramide II; on the other hand, acylglycosylceramide III contained only the spectral feature 4.15 ppm, corresponding to a high percentage of unsubstituted galactose and consistent with the presence in the molecule of a fatty acid esterified by the omega-OH group of the hydroxy fatty acid (3.95 ppm).

Effect of increasing amounts of dietary fish oil on brain and liver fatty composition.

Increasing dietary fish oil in rat had the following effect on brain lipids: Arachidonic acid regularly decreased; eicosapentanenoic acid, normally nearly undetectable, was present; 22:5(n - 3), dramatically increased but remained below 1% of total fatty acids; cervonic acid was increased by 30% at high fish oil concentration. Saturated and monounsaturated fatty acids were not affected regardless of chain-length. In contrast, in the liver, nearly all fatty acids (saturated, monounsaturated and polyunsaturated) were affected by high dietary content of fish oil, but liver function was normal: serum vitamin A and E, glutathione peroxidase, alkaline phosphatase, transaminases were not affected. Serum total cholesterol, unesterified cholesterol and phosphatidylcholine were slightly affected. In contrast, triacylglycerols were dramatically reduced in proportion to the fish oil content of the diet.

High dietary fish oil alters the brain polyunsaturated fatty acid composition.

Feeding adult rats a 17% corn-oil diet for 8 weeks did not change brain polyunsaturated fatty acids (PUFA) compared to rats fed 2.2% corn oil (with 2.2% lard added). When the corn-oil diet was supplemented with 14.5% cod liver oil or 12.5% salmon oil, the fatty acid composition of brain PUFA was significantly altered, even if alpha-tocopherol was added to the salmon-oil diet. Comparing salmon-oil- and cod-liver-oil-fed animals with corn-oil-fed animals, arachidonic acid 22:4(n-6) and 22:5(n-6) were reduced, and 20:5(n-3), 22:5(n-3) and 22:6(n-3) were increased. Liver fatty acids were also significantly altered. Thus, the brain is not protected against a large excess of very-long-chain n-3 PUFA, which increase n-3/n-6 ratio and could lead to abnormal function, and which might be difficult to reverse.

Where to find omega-3 fatty acids and how feeding animals with diet enriched in omega-3 fatty acids to increase nutritional value of derived products for human: what is actually useful ?

Omega-3 polyunsaturated fatty acids have two major field of interest. The first lies in their quantitative abundance and their role in the development and maintenance of the brain. The second is their role in the prevention of different pathologies, mainly the cardiovascular diseases, and more lately some psychiatric disorders, from stress to depression and dementia. Thus, dietary omega-3 fatty acids are very important to ensure brain structure and function, more specifically during development and aging. However, concerning essential alpha-linolenic acid (ALA), most occidental diets contain about 50 % of the recommended dietary allowances. The problem is to know which foods are naturally rich in this fatty acid, and to determine the true impact of the formulations (enriched in omega-3 fatty acids, either ALA or EPA and DHA) in chows used on farms and breeding centres on the nutritional value of the products (meat, butter, milk and dairy products, cheese, and eggs, etc), and thus their effect on the health of consumers, especially to ensure adequate quantities in the diet of the aging people. The consequences (qualitative and quantitative) of modifications in the composition of animal foods on the value of derived products consumed by humans are more marked when single-stomach animals are concerned than multi-stomach animals. Because, for example, hydrogenating intestinal bacteria of the latter group transform a large proportion of polyunsaturated fatty acids in their food into saturated fatty acids, among others, thus depriving them of any biological interest. Under the best conditions, by feeding animals with extracts of linseed and rapeseed grains for example, the level of ALA acid is increased approximately two-fold in beef and six-fold in pork, ten-fold in chicken, and forty-fold in eggs. By feeding animals with fish extracts or algae (oils) the level of DHA is increased about 2-fold in beef, 7-fold in chicken, 6-fold in eggs, and 20-fold in fish (salmon). To obtain such results, it is sufficient to respect only the physiological needs of the animal, which was generally the case with traditional methods. It is important to stress the role of fish, whose nutritional value for humans in terms of lipids (determined by omega-3 fatty acid levels) can vary considerably according to the type of fats the animals have been fed. The aim of preventing some aspects of cardiovascular disease (and other pathologies) can be achieved, or on the contrary frustrated, depending on the nature of fatty acids present in fish flesh, the direct consequence of the nature of fats with which they have been fed. It is the same for eggs, "omega- 3 eggs" being in fact similar to natural eggs, were used in the formulation of certain formula milks for infants, whose composition was closest to that of breast milk. In fact, the additional cost on the price paid by the consumer is modest compared to the considerable gain in nutritional value in terms of omega-3 fatty acids content. Interestingly, in aged people, ALA recommendations in France are increased (0.8% daily energy intake in adult, 0.9 % in aged) and DHA is multiplied by 2 (0.05 % daily energy intake in adult, 0.1 % in aged; as well as in pregnant and lactating women).