Abstract
Atherosclerosis manifests a state of increased oxidative stress characterized by comparable
lipid and protein oxidation in the affected arterial wall. While oxidative modification of low
density lipoprotein (LDL) has been extensively studied, increasing attention has been focused
recently on oxidation of high-density lipoproteins (HDL) and its functional consequences in
relation to atherosclerosis. Oxidative modification is thought to generate “dysfunctional”
HDL that has lost anti-atherosclerotic activities, including the ability to remove cholesterol
from lipid-laden cells. Therefore, there has been much interest in the detection of oxidized
HDL. Unfortunately, available methods to detect oxidized HDL are limited at present, in part
because oxidative modification of HDL is a complex process and ‘oxidized HDL’ is not a
chemically defined entity. What is known however is that conversion of methionine (Met)
residues of apolipoprotein (apo) A-I to methionine sulfoxide (MetO) is a process that occurs
commonly as HDL undergoes oxidative modification. For example, human apoA-I+16
(containing MetO86 or MetO112) and apoA-I+32 (MetO86 plus MetO112) are generated when
apoA-I reacts with lipid hydroperoxides formed as a consequence of the lipoprotein being
exposed to 1e−oxidants. The formation of MetO in apoA−I induced by 2e−oxidants (i.e.,
hydrogen peroxide, hypochlorous acid or myeloperoxidase/hydrogen peroxide/chloride
system) is associated with an impaired ability of the apolipoprotein to facilitate reactions
relevant to reverse cholesterol transport. In addition, a previous study has suggested the
plasma content of apoA-I+32 to be increased in certain subjects that have an increased risk to
develop cardiovascular disease (CVD). Moreover, the MetO content in circulating,
HDL−associated apoA−I is elevated in type 1 diabetes, a disorder commonly associated with increased oxidative stress and a risk factor for atherosclerosis. Therefore, in the present study, an existing HPLC method was applied to HDL samples from
the Fletcher−Challenge study, a nested case control study, to test the potential usefulness of
MetO-containing apoA-I as a marker of oxidative stress and/or CVD in a general population.
Plasma samples whose HDL contained detectable apoA-I+16 and/or apoA-I+32 had
significantly elevated levels of F2-isoprostanes, a marker of in vivo lipid oxidation, consistent
with MetO-containing apoA-I being a useful marker of in vivo protein oxidation. Despite this
however, there was no significant difference between controls and cases in their
concentrations of HDL apoA-I+16 and apoA-I+32 or F2-isoprostanes, suggesting that markers
of protein and lipid oxidation are not associated with the risk of coronary heart disease
(CHD) in this general population.
A limitation of the Fletcher−Challenge study was that only 22% of the 534 HDL samples
analyzed contained apoA-I+16 and/or apoA-I+32. In addition, the HPLC−based method used is
expensive and time−consuming and may lack the sensitivity needed for apolipoproteins to
clinical studies. Thus, a mouse monoclonal anti-human apoA-I+32 antibody (MOA−1) was
raised using HPLC−purified apoA-I+32 as immunogen. A sensitive ELISA was then
developed using a commercial anti-human apoA-I monoclonal antibody as capture and
biotinylated MOA−1 as detection antibody, respectively. The assay detected lipid−free
HPLC−purified human apoA-I+32 in a concentration-dependent manner and with a
significantly lower limit of detection (i.e., 3 ng/mL) than the HPLC method (1 μg/mL). The
ELISA also detected lipid-free apoA-I modified by 2e-oxidants (hydrogen peroxide,
hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in
buffer or human plasma. Moreover, the extent of recognition of MetO by MOA−1 increased
with increasing numbers of MetO in apoA−I, as assessed by the experiments with
H2O2−oxidized forms of apoA−I mutants, in which one, two or three Met residues were replaced with Leu. Their detection was concentration-dependent, reproducible, and exhibited
a linear response over a physiologically plausible range of concentrations of oxidized HDL.
In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by
hydroxyl radicals or metal ions, or LDL modified by 2e-oxidants. Furthermore, MOA−1 did
not detect other Met−containing proteins oxidized by either hypochlorous acid or hydrogen
peroxide. Taken together, the results showed that recognition of oxidized proteins by
MOA−1 is limited to MetO contained in apoA−I.
Finally, in a pilot study, plasma samples obtained from subjects with coronary artery disease
(CAD) proven by angiography, and samples from CAD patients undergoing percutaneous
coronary intervention (PCI) were analyzed by the ELISA. The preliminary data obtained
showed elevated levels of MetO-containing apoA-I in plasma samples of CAD patients
compared to those of corresponding control subjects. Unexpectedly, levels of MetOcontaining
apoA-I decreased PCI compared to before PCI. A possible explanation for these
results is that HDL−associated apoA−I become displaced by acute phase proteins, such as serum amyloid A, in response to PCI.
In summary, the ELISA developed here specifically detects apoA-I containing MetO in HDL
and human plasma. As such it may provide a useful tool for investigating the relationship
between oxidized HDL and CAD.
