Determination of lipoprotein lipase activity in post heparin plasma of streptozotocin-induced diabetic rats by high-performance liquid chromatography with fluorescence detection.
The activity of lipoprotein lipase (LPL), an enzyme responsible for lipoprotein metabolism, would vary in diseases and metabolic disorders. For determination of LPL activity, a highly sensitive high performance liquid chromatography (HPLC) method using a fluorescent reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was applied to determinate the oleic acid (OA) generated from triolein by LPL activity without multiple solvents extraction step. We studied the optimal conditions of the reaction including the effect of emulsifiers, deproteinizing solvents, and the concentration of bovine serum albumin (BSA). Ten millimolar concentrations of triolein, 5% of BSA, 1% of Gum arabic (GA), and acetonitrile showed the optimum conditions for measuring the LPL activity. The accuracy values for the determination of LPL activity in 10 microL of rat post heparin plasma were 108.73 approximately 114.36%, and the intra- and inter-day precision values were within 1.28% and 2.91%, respectively. The limit of detection was about 4.53 nM (signal-to-noise ratio 3). The proposed method was applied to determination of LPL activity in post heparin plasma of normal and streptozotocininduced diabetic rats associated with 52.3% reduction. The established assay system could be used for determining LPL activity in different physiological and pathological conditions to clarify the relationship between LPL activity and diabetes mellitus.
Chou
Yu-Ching
YC
Department of Pharmaceutical Analysis, School of Pharmacy, Taipei Medical University, No. 250, Wu-Hsing St, Taipei 110, Taiwan.
Tsai
Yih-Chiao
YC
Chen
Chien-Ming
CM
Chen
Shih-Ming
SM
Lee
Jen-Ai
JA
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003921:Diabetes Mellitus, Experimental / Q000139:chemically induced / Q000201:enzymology*; D008071:Lipoprotein Lipase / Q000097:blood* / Q000378:metabolism; D008297:Male; D015394:Molecular Structure; D019301:Oleic Acid / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013311:Streptozocin
D019301:Oleic Acid; D013311:Streptozocin; D008071:Lipoprotein Lipase