title: Quantitation of free sphingosine in liver by high-performance liquid chromatography.
abstract: Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt.
authors.affiliation: Department of Biochemistry, Emory University, Atlanta, Georgia 30322.
authors.forename: A H
authors.initials: AH
authors.lastname: Merrill
authors.forename: E
authors.initials: E
authors.lastname: Wang
authors.forename: R E
authors.initials: RE
authors.lastname: Mullins
authors.forename: W C
authors.initials: WC
authors.lastname: Jamison
authors.forename: S
authors.initials: S
authors.lastname: Nimkar
authors.forename: D C
authors.initials: DC
authors.lastname: Liotta
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005260:Female; D008099:Liver / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D013110:Sphingosine / Q000031:analogs & derivatives / Q000032:analysis* / Q000592:standards
chemical_list: C012491:phytosphingosine; D013110:Sphingosine; C005682:safingol