Blood and brain fatty acid contents in aged rats supplemented with n-3 long-chain polyunsaturated fatty acids

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1 Blood and brain fatty acid contents in aged rats supplemented with n-3 long-chain polyunsaturated fatty acids Anne Létondor 1,2,3, Benjamin Buaud 3, Carole Vaysse 3, Laurence Fonseca 3,Coralie Herrouin 3, Benjamin Servat 1,2,3, Sophie Layé 1,2, Véronique Pallet 1,2, Serge Alfos 1,2* 1 Univ. Bordeaux, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux, France. 2 INRA, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux, France. 3 ITERG, Institut des Corps Gras, Pessac, France. This work was supported by the Conseil Régional d Aquitaine, the Ministère de l Agriculture, de l Agroalimentaire et de la Forêt ACTIA, Association Nationale de la Recherche et de la Technologie ANRT, Société Lesieur and ONIDOL.

2 What do we know about brain and lipids? LIPIDS main components of the brain 50% of its dry weight POLYUNSATURATED FATTY ACIDS (PUFA) PUFA 35% of the total fatty acids (TFA) n-3 LC-PUFA (DHA) : critical roles in the maintenance of cognitive functions such as learning and memory during normal and pathological aging (Denis et al., 2013; Su 2010; Cunnane et al., 2009) ability of n-3 PUFA to modulate brain functions = closely dependent on their levels in brain membranes, which are related to dietary intake (Guesnet and Alessandri, 2011; Horrocks and Farooqui, 2004) the major n-3 LC-PUFA needs in the brain = covered by the circulating pool of fatty acids, which itself depends on the dietary intake (Rapoport and Igarashi, 2009; Rapoport, 2013)

3 What do we know about brain and lipids? POLYUNSATURATED FATTY ACIDS (PUFA) the abundance of n-3 LC-PUFA is brain region-specific (Xiao et al., 2005) PFC HPC DHA o preferentially enriched in the prefrontal cortex (PFC) and hippocampus (HPC) brain areas involved in memory processes o present in the striatum (STR) but at a lower proportion STR the abundance of n-3 LC-PUFA is dependent on the phospholipid classes (Dyall et al., 2007) DHA o o phosphatidylcholine (PC) = % of total PL DHA = 3-5 % of TFA phosphatidylethanolamine (PE) = % of total PEPL DHA = % of TFA PE PC

4 What do we know about brain, lipids and aging? AGING IS ASSOCIATED WITH CHANGES IN LIPID COMPOSITION OF BRAIN MEMBRANES These changes depend on the brain regions and on the phospholipid classes but the data differ from one study to another Brain regions most studies have shown lower DHA contents in o the whole brain of aged rodents (Barcelo-Coblijn et al., 2003; Labrousse et al., 2012; Arranz et al., 2013) o certain brain regions of aged rats such as the HPC (Favrelière et al., 2003), cerebral CX (Little et al., 2007) some studies have measured higher DHA content in specific areas of aged rats such as the midbrain, STR, medulla or cerebellum (Xiao et al., 2005) Phospholipid classes a decrease in the DHA contents of PE and PC in the HPC and PFC has already been observed both in aged animals and in elderly people (Dyall et al., 2007; Latour et al., 2013; Soderberg et al., 1991) A DECREASE OF THE N-3 PUFA BRAIN CONTENT = ASSOCIATED WITH MEMORY AND COGNITIVE IMPAIRMENTS IN AGED ANIMALS AND ELDERLY PEOPLE (DENIS ET AL., 2013; SU ET AL., 2010) EFFECT OF N-3 PUFA ENRICHED DIETS IN AGED RODENTS n-3 PUFA can prevent age-related changes of the brain fatty acid composition, thus helping to maintain cognitive functions during aging (Ramirez et al., 2001; Dyall et al., 2007; Little et al., 2007; Labrousse et al., 2012)

5 What is the aim of this study? From the perspective of a strategy of preventive nutrition to delay brain aging, it is crucial to well understand the evolution of the brain DHA content during aging. A number of studies have reported several correlations between the circulating fatty acid pool and the n-3 LC- PUFA brain content in adult rats (Tu et al., 2013; Stark, 2008) and humans (Kuratko and Salem, 2009) but this relationship could vary according to the brain region and the phospholipid class, and could be modified with aging identify reliable blood biomarkers of the brain DHA status taking into account age, specific brain regions and specific phospholipid classes The aim of this study was to determine the relationship between the blood fatty acid composition with the composition of several brain regions during aging and n-3 LC-PUFA supplementation in aged vs adult rats

6 Fatty acids g/100 diet LA 1.1 Material and methods ALA 0.1 EPA - DHA - n-6/n w.o. n=10 13 m.o. n=10 / gp fat-free diet + lipids (5% by w.) peanut/rapeseed/sunflower oils (60/25/15 by vol.) Control (CTL) 5 months Control (CTL) EPA/DHA fat-free diet + lipids (5% by w.) fish/rapeseed/sunflower oils (50/20/30 by vol.) 6 m.o. Adult CTL 18 m.o. Aged CTL Aged EPA/DHA plasma RBC* HPC STR PFC total fatty acid composition Lepage & Roy, GC PE and PC fatty acid composition Peuchant and TLC, GC PE and PC fatty acid composition Folch and TLC, GC Fatty acids g/100 diet LA 1.2 ALA 0.1 EPA 0.7 DHA 0.7 n-6/n *RBC = red blood cells; PI = phosphatidylinositol; PS = phosphatidylserine

7 Results Fatty acid composition of the plasma total lipids * % OF TOTAL FATTY ACIDS ** ## ** ** ## ## The values are the means ± SEM. A student s t-test was performed after Bonferroni correction for pairwise comparisons (α = 0.025): Aged CTL vs Adult CTL,and Aged EPA/DHA vs Aged CTL. ## P<0.01 and P<0.001 vs Adult CTL; * P<0.025, ** P<0.01 and P<0.001 vs Aged CTL. EFFECT OF AGE (Aged CTL vs Adult CTL) MUFA -21% (16:1n-7 18:1n-9 18:1n-7) n-6 PUFA +28% LA +18% ARA +41% n-3 PUFA +26% EPA +26% n-3 DPA +74% DHA +23% EFFECT OF DIET (Aged EPA/DHA vs Aged CTL) SFA +8% (16:0) MUFA -38% (18:1n-9 18:1n-7) n-6 PUFA -19% LA +14% ARA -44% n-6 DPA -47% n-3 PUFA x6 ALA +35% EPA x36 n-3 DPA x5 DHA x4

8 Results Fatty acid composition of the RBC PE % OF TOTAL FATTY ACIDS * ## The values are the means ± SEM. A student s t-test was performed after Bonferroni correction for pairwise comparisons (α = 0.025): Aged CTL vs Adult CTL,and Aged EPA/DHA vs Aged CTL. ## P<0.01 and P<0.001 vs Adult CTL; * P<0.025 and P<0.001 vs Aged CTL. EFFECT OF AGE (Aged CTL vs Adult CTL) n-6 DPA -24% n-3 DPA +43% RBC PC same changes described PE > PC EFFECT OF DIET (Aged EPA/DHA vs Aged CTL) MUFA -29% (18:1n-9) n-6 PUFA -29% LA +29% ARA -25% n-6 DPA -81% n-3 PUFA x3.5 EPA x38 n-3 DPA x2.5 DHA x2.5

9 Results Fatty acid composition of the HPC PE % OF TOTAL FATTY ACIDS The values are the means ± SEM. A student s t-test was performed after Bonferroni correction for pairwise comparisons (α = 0.025): Aged CTL vs Adult CTL,and Aged EPA/DHA vs Aged CTL. # P<0.025 and P<0.001 vs Adult CTL; * P<0.025 and P<0.001 vs Aged CTL. EFFECT OF AGE (Aged CTL vs Adult CTL) MUFA +14% (16:1n-7) EFFECT OF DIET (Aged EPA/DHA vs Aged CTL) n-6 PUFA -23% LA +59% ARA -16% n-6 DPA -74% n-3 PUFA +27% EPA x8 n-3 DPA x5 DHA +22% HPC PC same changes described PE > PC

10 Results Fatty acid composition of the STR PE ** # % OF TOTAL FATTY ACIDS # ** The values are the means ± SEM. A student s t-test was performed after Bonferroni correction for pairwise comparisons (α = 0.025): Aged CTL vs Adult CTL,and Aged EPA/DHA vs Aged CTL. # P<0.025, ## P<0.01 and P<0.001 vs Adult CTL; ** P<0.01 and P<0.001 vs Aged CTL. EFFECT OF AGE (Aged CTL vs Adult CTL) SFA -9% MUFA +15% (18:1n-9 18:1n-7) n-3 PUFA -7% n-3 DPA +38% DHA -13% STR PC same changes described PE > PC EFFECT OF DIET (Aged EPA/DHA vs Aged CTL) n-6 PUFA -26% LA +30% ARA -23% n-6 DPA -67% n-3 PUFA +25% EPA n-3 DPA x4 DHA +19%

11 Results Fatty acid composition of the PFC PE % OF TOTAL FATTY ACIDS The values are the means ± SEM. A student s t-test was performed after Bonferroni correction for pairwise comparisons (α = 0.025): Aged CTL vs Adult CTL,and Aged EPA/DHA vs Aged CTL. P<0.001 vs Aged CTL. EFFECT OF AGE (Aged CTL vs Adult CTL) no statistically significant effect EFFECT OF DIET (Aged EPA/DHA vs Aged CTL) n-6 PUFA -27% ARA -21% n-6 DPA -75% n-3 PUFA +32% EPA x8 n-3 DPA x4.5 DHA +28% PFC PC same changes described PE > PC

12 Results Relationship between RBC and HPC, STR, PFC fatty acid compositions INVESTIGATION OF THE PUTATIVE RELATIONSHIPS BETWEEN THE CIRCULATING FATTY ACID POOL AND THE BRAIN FATTY ACID COMPOSITION Significant positive associations between the DHA content in the RBC PE and in the HPC and PFC PE No significant positive associations between the DHA content in the RBC PE and in the STR PE Any significant relationship between the DHA level in the RBC PC and in the 3 brain regions PC PE PL of interest as a biomarker to investigate the FA composition changes in blood and brain Correlations of DHA levels in the PE fraction between RBC and (A) HPC, (B) STR and (C) PFC, in CTL and EPA/DHA Aged rats. r: Pearson s correlation coefficient; NS: Not significant. DHA level in hippocampal PE (% of total fatty acids) DHA level in striatal PE (% of total fatty acids) DHA level in cortical PE (% of total fatty acids) A DHA level in PE of RBC membranes (% of total fatty acids) B NS DHA level in PE of RBC membranes (% of total fatty acids) C DHA level in PE of RBC membranes (% of total fatty acids) p < 0.01 r = r² = p < r = r² = 0.445

13 Results What do they mean? EFFECT OF AGE FA profile in the blood o higher total PUFA level in the plasma of Aged CTL compared to Adult CTL increase in the n-6 and n-3 PUFA levels, more particularly in ARA EPA n-3 DPA DHA since the plasma FA pool is a major source of PUFA for the brain, such an alteration in the plasma FA composition may have consequences for the bioavailability of n-3 PUFA to the brain o higher n-3 DPA (PE and PC) and lower n-6 DPA (PE) in the RBC of Aged CTL compared to Adult CTL no changes ot the total n-6 and n-3 PUFA contents FA profile in the brain o the total n-6 PUFA content remains stable during aging in both PE and PC in the 3 brain regions of Aged CTL vs Adult CTL o the total n-3 PUFA content (mainly DHA) decreases in the STR PC and PE, and in the HPC PC of Aged CTL vs Adult CTL on the basis of the plasma FA profile, the n-3 PUFA bioavailability to the brain is reduced during aging : decrease in the transport of n-3 PUFA through the BBB? o no age-related modifications of the PFC FA profile of Aged CTL vs Adult CTL PFC may be less sensitive to the effect of aging than the HPC and the STR?

14 Results What do they mean? EFFECT OF EPA/DHA SUPPLEMENTATION FA profile in the blood o accumulation of n-3 PUFA in the plasma and the RBC of Aged EPA/DHA vs Aged CTL large increase in EPA n-3 DPA DHA and reduction in ARA n-6 DPA since the plasma FA pool is a major source of PUFA for the brain, such an alteration in the plasma FA composition may have consequences for the bioavailability of n-3 PUFA to the brain FA profile in the brain o Aged EPA/DHA exhibited a n-3 PUFA content in the brain (EPA n-3 DPA DHA) close to or even higher than that measured in Adult CTL, except in the STR and PFC PC (increases not statistically significant) o the increase in n-3 PUFA content occurs mainly in HPC PE and PC of Aged EPA/DHA impact on the memory processes (HPC : highly involved in spatial memory / maintenance of spatial working memory altered by aging) o the total n-6 PUFA content is lower in Aged EPA/DHA compared to Aged CTL

15 Take home message THIS STUDY SUGGESTS Aging is associated with altered FA bioavailability leading to an increase in the blood n-3 LC-PUFA content but not indicative of the decrease in the n-3 LC-PUFA content observed in the brain The DHA content in the RBC PE was indicative of the DHA levels in the HPC and the PFC PE in EPA/DHA supplemented aged rats suggesting the possibility of using RBC PE DHA content as a reliable biomarker of the DHA status in specific brain regions involved in memory processes, within which age-related impairments are described in the framework of dietary preventive n-3 PUFA supplementation in elderly people, a current line of research to slowdown age-related memory impairment

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