A novel assay of cellular stearoyl-CoA desaturase activity of primary rat hepatocytes by HPLC.
The stearoyl-CoA desaturase (SCD) activity is involved in regulation of metabolism, energy storage, and membrane fluidity. However, only few cellular assays have been developed. We describe a simple and robust method to quantitate SCD activity and its inhibition in primary rat hepatocytes. Hepatocytes assimilate stearic acid, with or without modification by SCD, into its lipid pool. To measure the extent of this conversion primary rat hepatocytes were cultivated 4h or overnight with [1-(14)C]18:0 and extracellular fatty acids were washed out. Total cell lipids were then hydrolyzed and extracted. Recoveries of 18:0 were secured with a modified Folch method by addition of 0.1% Triton X-114 to the samples. The extracted fatty acids were dissolved in 85% ethanol and separated by reverse phase HPLC, which took 10 min including column recovery time. [1-(14)C]18:0 and [1-(14)C]18:1(n9) were detected and quantified by on-line flow scintillation analysis. Incubation of the cells with SCD inhibitors resulted in decreased ratios of 18:1/18:0 in dose-dependent manners. The improvements enabled us to establish a novel robust assay based solely on HPLC analysis of cellular SCD activity, which was developed in 12-well format.
Su
Chao
C
Research Department, Biovitrum AB, Tomtebodavägen 23A, SE-112 76, Stockholm, Sweden. chao.su@biovitrum.com
Gullberg
Hjalmar
H
Simko
Hanna
H
Luthman
Marguerite
M
Edlund
Per-Olof
PO
Lundbäck
Thomas
T
D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005227:Fatty Acids / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D022781:Hepatocytes / Q000201:enzymology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D013229:Stearic Acids / Q000737:chemistry / Q000378:metabolism; D013230:Stearoyl-CoA Desaturase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005227:Fatty Acids; D013229:Stearic Acids; C031183:stearic acid; D013230:Stearoyl-CoA Desaturase