Hydrolysis of very-low-density lipoproteins labeled with a fluorescent triacylglycerol: 1,3-dioleoyl-2-(4-pyrenylbutanoyl)glycerol.
The fluorescent triacylglycerol (DPBG) 1,3-dioleoyl-2-(4-pyrenylbutanoyl)glycerol was incorporated into plasma very-low-density lipoproteins (VLDL) to form DPBG-VLDL. In the presence of albumin, the addition of milk lipoprotein lipase to DPBG-VLDL hydrolyses DPBG together with the VLDL triacylglycerol and pyrenyl fatty acids are transferred to albumin. As a consequence the monomer fluorescence increases while that of the excimer decreases [Mantulin, W. W., Massey, J. B., Gotto, A. M., Jr & Pownall, H. J. (1981) J. Biol. Chem. 256, 10815-10819]. The relationship of the intensity of the excimer at 475 nm to that of the monomer at 396 nm was measured before and after lipolysis of VLDL by milk lipoprotein lipase. These fluorescent changes parallel the release of free fatty acids from VLDL and their uptake by albumin. The rate of increase of monomer to excimer fluorescence was dependent upon the enzyme, substrate and albumin concentration. The lipolysis reaction, as monitored by fluorescence changes, followed Michaelis-Menten kinetics with a Km of 1.7 M for milk lipoprotein lipase. The use of the fluorescent triacylglycerol probe increases the sensitivity of the technique by a factor 50-80 compared to a technique previously reported using a fluorescent phospholipid. The present method is applicable to 2-10 micrograms triacylglycerol corresponding to about 50-100 microliters of newborn plasma or 30-50 microliters normal adult plasma. The use of an Airfuge ultracentrifuge for VLDL isolation, in conjunction with that of DPBG as a fluorescent probe enables a rapid study of VLDL lipolysis on minimal sample amounts. It can therefore be easily applied to normal and dyslipoproteinemic samples and to the newborns.
D000418:Albumins / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008053:Lipid Mobilization; D008071:Lipoprotein Lipase / Q000378:metabolism; D008079:Lipoproteins, VLDL / Q000378:metabolism*; D013379:Substrate Specificity; D014280:Triglycerides
D000418:Albumins; D005227:Fatty Acids; D005456:Fluorescent Dyes; D008079:Lipoproteins, VLDL; D014280:Triglycerides; C046906:1,3-dioleoyl-2-(4-pyrenylbutanoyl)glycerol; D008071:Lipoprotein Lipase