A simple test for predisposition to LDL oxidation based on the fluorescence development during copper-catalyzed oxidative modification.
When human low density lipoprotein (LDL) obtained from 10 volunteers was incubated in air at 37 degrees C in the presence of various concentrations of copper, an increase in fluorescence was observed with emission maximum at 430 nm when excitation was performed at 360 nm. The fluorescence increase was inhibited by ethylenediamine tetraacetic acid and by 4-methyl-2,6-di-tert-butylphenol. The fluorescence was found to be tightly bound to the protein moiety. Furthermore, Cu2+ modification of LDL was associated with a decrease in the reactive amino groups of apolipoprotein B and in the uptake of the lipoprotein by rabbit fibroblasts. Under our conditions, the fluorescence increase showed two consecutive periods; an inhibition period during which the fluorescence increased only weakly, and a propagation period with a rapid increase in fluorescence that was linear for at least 5 h. Both periods were influenced by copper concentration. The study also shows that the extent of fluorescence generated upon LDL oxidation varied greatly in the volunteers. Thus, while the results demonstrate that the fluorescence increase may likely monitor the extent of the apoB derivatization, the calculation of the fluorescence development rate of the propagation period together with the duration of the inhibition period may constitute a quantitative measurement of the susceptibility of apoB to be derivatized.
Cominacini
L
L
Department of Internal Medicine, University of Verona, Italy.
Garbin
U
U
Davoli
A
A
Micciolo
R
R
Bosello
O
O
Gaviraghi
G
G
Scuro
L A
LA
Pastorino
A M
AM
D000704:Analysis of Variance; D001055:Apolipoproteins B / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000378:metabolism*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
D001055:Apolipoproteins B; D008077:Lipoproteins, LDL; D003300:Copper