Mechanisms responsible for increasing immunoreactivity of apolipoprotein A-I with storage: the role of oxidation.
Atherogenesis has been linked to low concentrations of high-density lipoprotein and its principal carrier protein, apolipoprotein (apo) A-I, in serum. We measured apo A-I by enzyme-linked immunosorbent assay, using polyclonal antiserum raised against purified apo A-I and conjugated to peroxidase. During storage at 4 degrees C and above, the immunoreactivity of apo A-I increased so that measured values in stored serum were difficult to interpret. The immunoreactivity of serum apo A-I also increased substantially (up to 50-fold) during exposure to NaIO4. This increase was apo A-I concentration dependent, was linearly dependent on the concentration of NaIO4 up to 200 mmol/L at pH 7.4 and 25 degrees C, and was not ascribable to nonspecific binding of antibody-peroxidase conjugate to the reaction wells. Treatment with NaIO4 caused apo A-I immunoreactivity to increase to a peak or plateau, then gradually decline. In 100 mmol/L NaIO4 (pH 7.4, 25 degrees C), this plateau occurred 30-60 min after oxidation commenced. When frozen serum and stored nonfrozen serum were both oxidized with NaIO4 under the same conditions, the apo A-I from the frozen serum had a significantly (P = 0.0004) greater increase in immunoreactivity. Sera from different patients with different apo A-I concentrations did not have proportionately increased immunoreactivity after treatment with NaIO4. Our findings suggest that the increase in apo A-I immunoreactivity on storage at 4 degrees C or higher involves atmospheric oxidation, and they raise the possibility of in vivo oxidized and nonoxidized forms of apo A-I.
Wang
X L
XL
Department of Cardiovascular Medicine, Prince Henry Hospital, University of New South Wales, Sydney, Australia.
Dudman
N P
NP
Wang
J
J
Wilcken
D E
DE
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000097:blood* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005189:False Positive Reactions; D005615:Freezing; D006801:Humans; D007452:Iodates; D008075:Lipoproteins, HDL / Q000097:blood*; D010084:Oxidation-Reduction; D013048:Specimen Handling
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D007452:Iodates; D008075:Lipoproteins, HDL; C032285:sodium iodate