Incorporation of [ 3H]Valproic Acid into Lipids in GT1–7 Neurons

Abstract

Valproic acid (2-propylpentanoic acid, valproate, VPA), an 8-carbon, branched chain fatty acid, is effectively used in the treatment of mania and epilepsy. The biochemical mechanisms by which this drug has its therapeutic effects are not yet established. The purpose of this study was to partially characterize the incorporation of [ 3H]VPA into phospholipids of GT1–7 neurons, an immortalized hypothalamic cell line. GT1–7 neurons were grown to confluence in culture dishes, and then were incubated with various concentrations of [ 3H]VPA between 10 and 400 μg/mL for various times up to 20 hr. Total lipids were extracted and phospholipids were separated from neutral lipids using TLC. Our results indicate that [ 3H]VPA (10 μg/mL) was incorporated into phospholipids of GT1–7 neurons in a time-dependent and saturable manner over 300 min. Subsequent separation of the lipid fraction by TLC indicated that 44.4% of the radioactivity taken up by the cells was incorporated into phospholipids and neutral lipids. One of the phospholipids migrated with a slightly lower R f value than authentic phosphatidylcholine. Our results show that the incorporation of VPA into phospholipids and glycerides was linear with VPA concentrations from 10 to 400 μg/mL. Finally, we synthesized 1-acyl-2-valproyl- sn-glycero-3-phosphocholine and validated its structure with nuclear magnetic resonance and electrospray mass spectrometry to verify the structure of this compound, confirming that this compound is structurally possible. We conclude that VPA is incorporated into lipids in GT1–7 neurons and discuss the possible effects of valproyl phospholipids on neuronal functional properties.