Document Detail


Determination of lipid hydroperoxides in native low-density lipoprotein by a chemiluminescent flow-injection assay.
MedLine Citation:
PMID:  8418885     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Lipid hydroperoxides have been implicated in the pathogenesis of atherosclerosis. This work was therefore set up to obtain a fast and specific chemiluminescent assay for measuring hydroperoxides in native low-density lipoprotein (LDL). The apparatus was a complete HPLC system including two pumps, an autosampler, a computer and a chemiluminescent detector with a T-mixing coil in the place of the column. Samples were injected from the autosampler and mixed with luminescent reagent (3 microM luminol and 1 microM microperoxidase in 0.1 M carbonate buffer (pH 10)) in the T-piece. To generate a calibration curve, linoleic acid hydroperoxide was obtained by incubating soybean lipoxygenase with linoleic acid. The calculated conjugated diene concentration was in good agreement with the nominal linoleic acid hydroperoxide concentration. The chemiluminescence was linear with the amount of linoleic acid hydroperoxide injected and the detection limit was about 3 pmol linoleic acid hydroperoxide. The chemiluminescence induced by copper-oxidized LDL was linear with concentration; the detection limit, when compared with linoleic acid hydroperoxide, was similar. The reproducibility of the linoleic acid hydroperoxide and of oxidized LDL hydroperoxide was examined in single pools. The coefficient of variation on the triplicates of each pool was about 3%. The titre of the linoleic acid hydroperoxide and oxidized LDL peroxides was quite stable for at least 10 days when stored under argon at 4 degrees C in the presence of EDTA. The mean value of the LDL hydroperoxides in 16 control subjects was 145.20 +/- 98.81 pmol/mg LDL protein. In conclusion, the microperoxidase-luminol-dependent chemiluminescence flow-injection assay is a rapid, sensitive and selective method for measuring lipid hydroperoxides in native LDL.
Authors:
L Cominacini; A M Pastorino; A McCarthy; M Campagnola; U Garbin; A Davoli; A De Santis; V Lo Cascio
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Biochimica et biophysica acta     Volume:  1165     ISSN:  0006-3002     ISO Abbreviation:  Biochim. Biophys. Acta     Publication Date:  1993 Jan 
Date Detail:
Created Date:  1993-02-10     Completed Date:  1993-02-10     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0217513     Medline TA:  Biochim Biophys Acta     Country:  NETHERLANDS    
Other Details:
Languages:  eng     Pagination:  279-87     Citation Subset:  IM    
Affiliation:
Istituto di Semeiotica e Nefrologia Medica, Università di Verona, Italy.
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MeSH Terms
Descriptor/Qualifier:
Chemiluminescent Measurements*
Chromatography, High Pressure Liquid / methods
Humans
Kinetics
Linoleic Acid
Linoleic Acids / chemistry
Lipid Peroxidation
Lipid Peroxides / analysis*
Lipoproteins, LDL / blood*,  chemistry
Lipoxygenase / chemistry
Luminol
Peroxidases
Chemical
Reg. No./Substance:
0/Linoleic Acids; 0/Lipid Peroxides; 0/Lipoproteins, LDL; 2197-37-7/Linoleic Acid; 521-31-3/Luminol; EC 1.11.1.-/Peroxidases; EC 1.11.1.-/microperoxidase; EC 1.13.11.12/Lipoxygenase

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