Supplementary Material For s1

Supplementary material for

Efficient docosahexaenoic acid uptake by the brain from a structured phospholipid Mayssa Hachem1, Alain Géloën1, Amanda Lo Van1, Baptiste Foumaux1, Laurence Fenart2, Fabien Gosselet2, Pedro Da Silva3, Gildas Breton4, Michel Lagarde1, Madeleine Picq1, and Nathalie Bernoud-Hubac1*

1 Université de Lyon, INSERM UMR 1060, CarMeN laboratory, IMBL/INSA-Lyon, F- 69621, Villeurbanne, France.

2 Université Lille Nord de France, Lille, U. Artois, LBHE, EA 2465, Lens, IMPRT-IFR114, Lille, France.

3 Université de Lyon, INRA UMR 203, BF2I laboratory, INSA-Lyon, F-69621 Villeurbanne, France.

4 Polaris, 29170 Pleuven, France.

*e-mail: [email protected] Fig. S1

In vitro model of the blood-brain barrier (BBB). The in vitro BBB model consists in a co- culture of brain capillary endothelial cells (BCECs) and glial cells. Differentiated BCECs display most of the characteristics observed in vivo. These cells indeed form complex tight junctions; they also express a high electrical resistance, a low permeability for hydrophilic molecules (such as sucrose or inulin), specific transporters for molecules (such as amino acids and glucose), specific enzymatic activities (ɤ-glutamyl transpeptidase, monoamine oxidase) and P-glycoprotein, specific receptors for low density lipoproteins, transferrin.

The luminal (1.5 ml) and abluminal (2.5 ml) culture media used for the co-culture is

DMEM supplemented with 10 % (v/v) calf serum (CS) and 10 % (v/v) horse serum (HS), 2 mM glutamine, 50 μg.ml-1 gentamycin, and 1 ng.ml-1 of basic fibroblast growth factor is then added every other day. Under these conditions, endothelial cells form a confluent monolayer in 12 days Endothelial cells

Fig. S2

Radioactivity of brain endothelial cells (BCECs) lipids. 4 hours after incubation with

[14C]AceDoPC (filled dark bars), [14C]PC-DHA (filled grey bars), [14C]DHA (open bars),

BCECs were rinsed with phosphate buffer PBS and were recovered by scraping. The radioactivity was quantified using liquid scintillation counting. Results are expressed as percentage of 14C initial radioactivity and presented as means ± SEM of four values. Groups were compared by analysis of variance (ANOVA) followed by Bonferroni test. Data with different superscript letters are significantly different at P < 0.05 Abluminal medium – labeled AceDoPC

Abluminal medium – labeled DHA

Fig. S3

Distribution of radioactivity among the highest labeled lipid classes from the abluminal medium 4 hours after incubation with A [14C]AceDoPC or B [14C]DHA. Lipid classes were separated by preparative thin-layer chromatography (silica gel G 0.75 mm) by an elution system: chloroform / methanol / aqueous methylamine solution (40%) (60:20:5 vol/vol/vol). The radioactivity of lipid classes was determined by liquid scintillation.

Results are expressed as percentage of 14C total radioactivity, presented as means ± SEM of four values. Light hatched bars: lysoPC, filled dark bars: AceDoPC, open bars: free fatty acids (FFA) Brain

Fig. S4

Distribution of radioactivity within lipid classes extracted from organs as a function of time after intravenous injection of [14C]DHA: 1 h (open bars), 24 hours (filled grey bars), 48 hours (filled dark bars) in A brain and B heart. PC, PE, NL+FFA were identified. Lipid classes were separated by preparative thin-layer chromatography (silica gel G 0.75 mm) by an elution system: chloroform / methanol / aqueous methylamine solution (14%) (60:20:5 vol/vol/vol). The radioactivity of lipid classes was determined by liquid scintillation.

Results represent means +/- SD of three values Table S1

Endothelial permeability coefficient values obtained for LY with or without human plasma,

DHA, PC-DHA and AceDoPC. The labeled DHA, PC-DHA and AceDoPC solutions at 5

μM were prepared in DMEM containing 1% (v/v) human plasma. Lucifer Yellow (LY) is used as a paracellular marker allowing the evaluation of the compound effect on the integrity of the BBB. This small hydrophilic molecule presents a low cerebral penetration and its low endothelial permeability coefficient reveals the endothelial cell monolayer integrity. Transport study with LY was performed on triplicate filters only coated with collagen (insert Transwell PC 0.4 μm pore size). The monolayer integrity was checked before experimentation with a control experiment using LY (Pe = 0.79 x 10-3 cm.min-1). A control experiment with DMEM containing 1% (v/v) human plasma was performed with a

LY Pe value of Pe = 0.62 x 10-3 cm.min-1 (insert Transwell PC 0.4 μm)