Analyse structurale et métabolique du bis (monoacylglycerol) phosphate dans les cellules utérines stromales de rate

= Structural and metabolic analysis of bis(monoacylglycerol)phopshate in rate uterine stromale cells

• Auteur / Autrice : Céline Luquain
• Direction : Christian Laugier
• Type : Thèse de doctorat
• Discipline(s) : Biochimie
• Date : Soutenance en 2000
• Etablissement(s) : Lyon, INSA
• Ecole(s) doctorale(s) : École doctorale Interdisciplinaire Sciences-Santé (Villeurbanne ; 1995-....)
• Partenaire(s) de recherche : Laboratoire : LBP - Laboratoire de Biochimie et Pharmacologie (Lyon, Rhône1996-2003)

Mots clés

Mots clés contrôlés

Acides gras

Endocytose

Biochimie

Résumé

L'objectif de ce travail a été de caractériser un phospholipide (PL) inconnu ne représentant que 0,7% des PL cellulaires, mais contenant plus de 10% de l'acide docosahexaenoique (DHA) cellulaire et incorporant sélectivement du DHA mais pas d'acides gras polyinsaturés (AGPI) de la série n-6. La spéctrométrie de masse par bombardement d'atomes rapides (FAB-MS) nous a permis de préciser sa structure. Il s'agit d'un bis (monoacylglycérol) phosphate (ou BMP). Le BMP purifié par chromatographie sur couche mince est résolu en trois pics distincts par HPLC. L'analyse par spéctrométrie de masse en ionisation electrospray du BMP isolé des cellules contrôles, montre que les deux premiers pics de BMP contiennent les mêmes espèces moléculaires (principalement 18:1 (n-9)/22:6(n-3)) alors que le troisième pic contient principalement des espèces moléculaires en 18:1(n-9)/18:1(n-9).

The goal of this work was to characterize an unknown phospholipid (PL) representing only 0. 7 % of cellular PL but acylated by more than 10 % of the cellular docosahexaenoic acid (DHA) and in comparating selectively DHA but not polyunsaturated fatty acids (PUFA) from the n-6 series. Fast Atom Bombardment Mass spectrometry (FAB-MS), allowed us to precise it structure. It was a bis(monoacylglycerol)phosphate (or BMP). BMP purified by thin layer chromatography was resolved into 3 peaks by HPLC. Mass spectrometry analysis by electrospray of BMP from control cell showed that the first two peaks contain the same molecular species (mainly 18:1 (n-9)/ 22:6 (n-3)) whereas the third peak contains mainly 18:1 (n-9)/18: 1 (n-9) molecular species. Stereo-configurational analysis of these three compounds showed an sn-l:sn-1’ configuration for compound 2 and 3, what was unique for a PL, whereas s the first compound was a mixture or n- l :sn-1' and sn-3: n-1 ' BMP. RMN analysis of the three peaks indicated that the two FA was esterified on the carbon wearing the primary alcohol of each glycerol, whereas the two FA of the compound 1 were esterified, one on the carbon wearing the primary alcohol and the other on the carbon wearing the secondary alcohol. We developed an original method for the quantification of BMP by HPLC, using the derivatization of free hydroxyls by a f1uorescent probe. This method allowed measuring the level of this PL in our cellular model (530 ± 40 ng/ 106 cells). This level was dramatically increasing when cells are cultivated in minima conditions which induced autophagy.