Quantitation of free sphingosine in liver by high-performance liquid chromatography.
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.
Merrill
A H
AH
Department of Biochemistry, Emory University, Atlanta, Georgia 30322.
Wang
E
E
Mullins
R E
RE
Jamison
W C
WC
Nimkar
S
S
Liotta
D C
DC
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
C012491:phytosphingosine; D013110:Sphingosine; C005682:safingol