Abstract
Incubation of human serum or high density lipoprotein (HDL) at 37 degrees C in the presence of Fe2+, Fe2+/Fe3+, or Mn2+ results in the increased immunoreactivity (up to 12-, 40-, and 80-fold, respectively) of specific apoA-I epitopes identified as 3D4 and 6B8, while Mg2+, Ca2+, or Cu2+ have minimal or nonsignificant effects. The effect of Mn2+ on the 3D4 epitope requires a specific association with lipids since it can be observed with HDL but not with apoHDL, even in the presence of other lipoproteins. The increase in immunoreactivity noted with Fe2+/Fe3+ or Mn2+ can be blocked with either EDTA or antioxidants (GSH and ascorbic acid), suggesting that it takes place during a peroxidative reaction of the lipids. The peroxidation of lipids which accompanies the increase in immunoreactivity does cross-link apoA-I both with itself and with apoA-II but does not cleave the molecule. The apoA-I-containing lipoproteins which float between 1.18 and 1.22 g/ml and have a pre B-electrophoretic migration are characterized by a very low immunoreactivity with monoclonal antibody 3D4 but are 10-fold or more responsive to Mn2+ treatment than other lipoprotein subfractions, thus demonstrating heterogeneity under oxidative conditions. Proteoliposomes containing apoA-I, cholesterol, and dilinoleyl-lecithin are sensitive to Mn2+ treatment, but not those made with dioleyl- or dimyristoyl-lecithins. However, the increase in 3D4 immunoreactivity is weak and transient and is followed by the disappearance of the epitope caused by cross-linking. We conclude that lipid peroxidation can specifically cross-link apoA-I and change its conformation and antigenicity.
Highlights
Incubation of human serum or high density lipopro- is either purified Apolipoprotein A-I (apoA-I) [1, 2] or fresh high density lipoprotein (HDL) [3]
We demonstrate that this process takes place under clonal antibody 3D4 bauret 10-foldor more responsive the conditions where the peroxidation of polyunsaturated to Mn2+treatment than other lipoprotein subfractions,lipids occurs and that rietquires a specificassociation between demonstrating heterogeneity under oxidactiovne- apoA-I and the lipids
We initially observed that one series of mAbs obtained by immunization with purified apoA-I [1]did react with epitopes which increased in immunoreactivity with storage of serum a t 4 "C [2]
Summary
MDA was prepared from 1,1,3,3-tetramethoxyropanbey acid hydrolysis according to the method of Kwon and Watts [20]. Fromthe blank gradient,1-mlfractions containing 1mM EDTA and 0.02% NaN3at 4 "C overnight to remove were removed and the density and position of each determined to any unreacted MDA. In order to assess the occurrence of lipid peroxidationduring incubation of plasma and HDL fractions at +4 "C and 37 "C in the presence or absence of 1mM MnClz for 48 h, the thiobarbituric acid assay was performed as described by Buege and Aust [22].The TBAR products were measured in 2 0 0 4 aliquots of plasma and 1800-111 aliquots of HDL fractions These samples were made up to 2.0ml and contained 0.01 M sodium phosphate, pH 7, and the following antioxidants: 0.5 mg/ml glutathione, 1 mM ascorbic acid, 1 mM from the top of each plasma gradient and corresponding fractions EDTA, and 0.1 mg/ml, butylated hydroxytoluene.
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