John W. Crabb and Robert G.

Salomon
Miyagi, Mary E. Rayborn, Joe G. Hollyfield,
Xiaorong Gu, Susan Gillette Meer, Masaru

Age-related Macular Degeneration
Autoantibodies, Biomarkers for
Carboxyethylpyrrole Protein Adducts and
Lipids and Lipoproteins:
doi: 10.1074/jbc.M305460200 originally published online August 15, 2003
2003, 278:42027-42035. J. Biol. Chem.

10.1074/jbc.M305460200 Access the most updated version of this article at doi:

. JBC Affinity Sites Find articles, minireviews, Reflections and Classics on similar topics on the
Alerts:

When a correction for this article is posted
When this article is cited
to choose from all of JBC's e-mail alerts Click here
Supplemental material:

http://www.jbc.org/content/suppl/2003/09/10/M305460200.DC1.html

http://www.jbc.org/content/278/43/42027.full.html#ref-list-1
This article cites 29 references, 8 of which can be accessed free at

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

1
6
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m


b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

1
6
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

Carboxyethylpyrrole Protein Adducts and Autoantibodies,
Biomarkers for Age-related Macular Degeneration*
S
Received for publication, May 23, 2003, and in revised form, August 7, 2003
Published, JBC Papers in Press, August 15, 2003, DOI 10.1074/jbc.M305460200
Xiaorong Gu, Susan Gillette Meer, Masaru Miyagi, Mary E. Rayborn, Joe G. Hollyfield,
John W. Crabb**, and Robert G. Salomon
From the Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106 and Cole Eye Institute,
The Cleveland Clinic Foundation, Cleveland, Ohio 44195
Age-related macular degeneration (AMD) is a slow,
progressive disease with both genetic and environmen-
tal risk factors. Free radical-induced oxidation of doco-
sahexaenoate (DHA)-containing lipids generates -(2-
carboxyethyl)pyrrole (CEP) protein adducts that are
more abundant in ocular tissues from AMD than normal
human donors. To understand better the role of oxida-
tive damage in AMD, we have synthesized CEP-modified
proteins, produced anti-CEP antibodies, and initiated
analysis of CEP immunoreactivity and autoantibodies
in human plasma. A highly selective rabbit polyclonal
anti-CEP antibody was raised that binds CEP 1000 times
more strongly than carboxypropylpyrrole, a close struc-
tural analogue. The CEP adduct uniquely indicates ox-
idative modification fromDHA derivatives because CEP
protein modifications cannot arise from any other com-
mon polyunsaturated fatty acid. Immunocytochemistry
localized CEP to photoreceptor rod outer segments and
retinal pigment epithelium in mouse retina and demon-
strated more intense CEP immunoreactivity in photore-
ceptors from a human AMD donor compared with
healthy human retina. The mean level of anti-CEP im-
munoreactivity in AMD human plasma (n 19 donors)
was 1.5-fold higher (p 0.004) than in age-matched con-
trols (n 19 donors). Sera from AMD patients demon-
strated mean titers of anti-CEP autoantibody 2.3-fold
higher than controls (p 0.02). Of individuals (n 13)
exhibiting both antigen and autoantibody levels above
the mean for non-AMD controls, 92% had AMD. These
results suggest that together CEP immunoreactivity
and autoantibody titer may have diagnostic utility in
predicting AMD susceptibility.
Age-related macular degeneration (AMD)
1
is the most com-
mon cause of legal blindness in the elderly population in devel-
oped countries (1), with about 35% of people 75 years or older
having some degree of AMD (2). Due to the aging population,
the prevalence of AMD in the United States is predicted to
double in the next 25 years (3). Macular degeneration is char-
acterized by the breakdown of photoreceptor and retinal pig-
ment epithelial (RPE) cells in the small central portion of the
retina (2 mm in diameter) that is responsible for high acuity
vision. Specific gene mutations cause early onset, juvenile
forms of macular degeneration, and although genetic factors
are also associated with late onset macular degeneration, the
identity of AMD susceptibility genes remains unclear (3). AMD
is a slow, progressive disease with environmental risk factors
that have been associated with oxidative damage (4), for exam-
ple, cigarette smoking and perhaps diet and lifetime light ex-
posure (1). Presently no cure exists for AMD; however, the
progression of the disease can be slowed with antioxidant vi-
tamins and zinc for select individuals (5). Recently, we pro-
vided direct evidence that oxidative protein modifications gen-
erated from docosahexaenoate (DHA)-containing lipids,
namely carboxyethyl pyrrole (CEP) adducts, are more abun-
dant in AMD than in normal Bruchs membrane/RPE/choroid
tissues (6). CEP adducts were also found in extracellular de-
posits (i.e. drusen) behind the retina considered to be signifi-
cant risk factors for the development of AMD (6).
CEP protein adducts belong to a family of 2-(-carboxyalkyl)
pyrrole adducts (Fig. 1) generated from the oxidation of poly-
unsaturated fatty acids (PUFA) (7). Thus, oxidative fragmen-
tation of linoleic or arachidonic acid produces 9-hydroxy-12-
oxododec-10-enoic acid (HODA) or 5-hydroxy-8-oxooct-6-enoic
acid (HOOA), respectively, which react with protein to generate
2-(-carboxyheptyl)pyrrole (CHP) or 2-(-carboxypropyl)pyr-
role (CPP) adducts, respectively. The CHP and CPP adducts
have been detected in oxidized low density lipoprotein and
human plasma (7). Furthermore, the esters of HODA and
HOOA with phosphatidylcholine (HODA-PC and HOOA-PC)
are biologically active products of low density lipoprotein oxi-
* This work was supported by National Institutes of the Health
Research Grants GM21249, HL53315 (to R. G. S.), EY2362, EY14240
(to J. G. H.), EY6603, and EY14239 (to J. W. C.). The costs of publica-
tion of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
S The on-line version of this article (available at http://www.jbc.org)
contains additional Experimental Procedures for the preparation of all
new compounds and
1
H NMR spectra of new compounds and Figs. S19.
Submitted this work in partial fulfillment of the requirements for a
doctoral degree in chemistry from Case Western Reserve University.
Present address: Dept. of Biochemistry and Molecular Biology, Uni-
versity of North Dakota, Grand Forks, ND 58202-9037.
** To whom correspondence may be addressed: Cole Eye Institute
(i31), Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland OH
44195. Tel.: 216-445-0425; Fax: 216-445-3670; E-mail: crabbj@ccf.org.
To whom correspondence may be addressed: Dept. of Chemistry,
Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH
44106-7078. Tel.: 216-368-2592; Fax: 216-368-3006; E-mail: rgs@po.
cwru.edu.
1
The abbreviations used are: AMD, age-related macular degenera-
tion; BHT, butylated hydroxytoluene; BSA, bovine serum albumin;
CEP, 2-(-carboxyethyl)pyrrole; CHP, 2-(-carboxyheptyl)pyrrole;
CPP, 2-(-carboxypropyl)pyrrole; DHA, docosahexaenoic acid; DOHA,
4,7-dioxoheptanoic acid; HODA, 9-hydroxy-12-oxodec-10-enoic acid;
HOHA, (E)-4-hydroxy-7-oxohept-5-enoic acid; HOOA, 5-hydroxy-8-
oxooct-6-enoic acid; HSA, human serum albumin; KLH, keyhole limpet
hemocyanin; MALDI-TOF MS, matrix-assisted laser desorption ioniza-
tion-time-of-flight mass spectrometry; PBS, phosphate-buffered saline;
PC, phosphatidylcholine; PP-ACA, 2-pentylpyrrolated 6-aminocaproic
acid; PUFA, polyunsaturated fatty acid; RPE, retinal pigment epithe-
lium; TBS, Tris-buffered saline; ELISA, enzyme-linked immunosorbent
assay; GPDH, glyceraldehyde-3-phosphate dehydrogenase; MS/MS,
tandem mass spectrometry.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 43, Issue of October 24, pp. 4202742035, 2003
2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 42027

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

1
6
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

dation and are present in atherosclerotic plaques (811). The
detection of CPP or CHP does not uniquely indicate the oxida-
tive damage of linoleic or arachidonic acid or their esters,
because these protein adducts can also arise from other com-
mon PUFAs. In contrast, only DHA gives rise to 2-(-carboxy-
ethyl)pyrrole (CEP) adducts, by oxidative cleavage to 4-hy-
droxy-7-oxohept-5-enoic acid (HOHA) and reaction of the latter
with protein (Fig. 1). Indeed, we have shown recently (12) that
HOHA-PC is a minor oxidative product from DHA-PC and that
it can generate peptide-bound CEP adducts. Although rare in
most human tissues, DHA is present in 80 mol % of the
polyunsaturated lipids in photoreceptor outer segments (13).
The abundance of DHA in photoreceptors, the high photooxi-
dative stress in retina, and the fact that DHA is the most
oxidizable fatty acid in humans (13) suggests that DHA oxida-
tion products may have possible utility as biomarkers for AMD
susceptibility.
Here we explore further the role of oxidative fragmentation
of DHA in AMD. We report methods for preparing CEP-modi-
fied proteins and characterization of a rabbit polyclonal anti-
CEP antibody. In addition, we present evidence that CEP im-
munoreactivity and autoantibody titers are elevated in human
plasma from AMD donors relative to age-matched normal
controls.
EXPERIMENTAL PROCEDURES
MaterialsChicken egg ovalbumin (grade V), bovine serum albumin
(BSA, fraction V), human serum albumin (HSA, fraction V), and arachi-
donic acid were purchased from Sigma; the dipeptide Ac-Gly-Lys-OMe
was purchased from Bachem, Torrance, CA; and keyhole limpet hemo-
cyanin (KLH) was purchased from Calbiochem. All organic chemicals
were analytical grade and purchased from Aldrich. Pentylpyrrole ami-
nocaproic acid-modified albumin (PP-ACA-BSA), 2-(-carboxypropyl)
pyrrole-modified albumin (CPP-HSA), a 2-(-carboxyheptyl)pyrrole-
modified albumin (CHP-HSA), and rabbit polyclonal anti-CPP and anti-
CHP antibodies were prepared as described previously (7).
Human Plasma PreparationBlood was collected into 7-ml vacu-
tubes containing EDTA (10.5 mg), from AMD and normal healthy
donors at the Cole Eye Institute, Cleveland Clinic Foundation. Cells
were removed by centrifugation at 2500 rpm (1300 g) for 10 min, and
the plasma was transferred to plastic vials containing butylated hy-
droxytoluene (BHT; 1 mg/ml), leupeptin (35 M), pepstatin (5 M), and
aprotinin (0.1 trypsin inhibitor unit/ml). Plasma was then quench-
frozen in liquid nitrogen and stored at 80 C until used. Usually, BHT
and protease inhibitors were added to the plasma samples 35 h after
blood was drawn.
Preparation of -Carboxyethylpyrrole-modified Peptide and Pro-
teinsPaal-Knorr reactions of -dicarbonyl compounds with primary
amines provided an efficient route to 2-(-carboxyalkyl)pyrroles (7).
Syntheses of -ketoaldehydes, 4,7-dioxoheptanoic acid (DOHA), and its
phosphatidylcholine ester (DOHA-PC) were developed as described in
the Supplemental Material to allow unambiguous production of 2-(-
carboxyethyl)pyrrole (CEP) and the corresponding PC ester. First, a
Grignard reagent was produced from 2-(2-bromoethyl)-1,3-dioxolane,
followed by selective acylation of this organomagnesium derivative with
3-carbomethoxypropionyl chloride. Saponification of the resulting eth-
ylene ketal keto ester delivered a carboxylic acid that, when coupled
with 2-lysophosphatidylcholine in the presence of dicyclohexylcarbodi-
imide and N,N-dimethyl-aminopyridine, gave a stable precursor that
yielded DOHA-PC upon hydrolysis. Transketalization of the ethylene
ketal with acetone provided a mixture containing DOHA, presumably
in equilibrium with the corresponding hemiacylal based on the
1
H NMR
spectrum that exhibits two aldehydic hydrogen singlets (Supplemental
Fig. S5). Paal-Knorr condensation of DOHA or DOHA-PC with the
dipeptide acetyl-Gly-Lys-O-methyl ester generated the CEP-dipeptides,
DOHA-dipeptide, or CEP-PC-dipeptide that were fully characterized by
1
H NMR (12).
1
H NMR and
13
C NMR spectra were recorded at 300 and
75 MHz, respectively, and solvents and chromatography conditions for
purification of synthetic products were as described elsewhere (12). All
reactions in an inert atmosphere were under argon unless otherwise
specified. As described in detail in the Supplemental Material, Paal-
Knorr condensation with DOHA was used to prepare CEP-modified
keyhole limpet hemocyanin (CEP-KLH), bovine serum albumin (CEP-
BSA), human serum albumin (CEP-HSA), and glyceraldehyde-3-phos-
phate dehydrogenase (CEP-GPDH). Protein concentrations were deter-
mined using the Pierce bicinchoninic acid protein assay (14), and
pyrrole concentrations were determined using the Ehrlich reagent,
4-(dimethylamino)benzaldehyde (15), and the above CEP-dipeptide as a
reference standard.
Identification of CEP-modified Residues in HSACEP-HSA was
reduced in 400 mM ammonium bicarbonate containing 8 M urea and 10
mM dithiothreitol for 30 min at room temperature under argon and then
thiol groups were alkylated by the addition of iodoacetamide (to 50 mM).
Following alkylation the preparation was dialyzed overnight against 10
FIG. 1. Generation of 2-(-carboxyalkyl)pyrrole epitopes. Oxidative fragmentation of polyunsaturated fatty acids generates a host of
oxidation products, including the hydroxy--oxoalkenoic acids HODA, HOOA, and HOHA, which give rise to a host of protein modifications,
including the family of carboxyalkylpyrrole protein adducts CHP, CPP, and CEP.
CEP Biomarkers for AMD 42028

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

1
6
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

mM ammonium bicarbonate, and about 260 pmol (17 g) was digested
with 0.2 g of trypsin (Promega) (16). CEP-modified tryptic peptides
were sought using peptide mass mapping methods (17, 18) and a
Voyager DE Pro MALDI-TOF mass spectrometer (PE Biosystems,
Framingham, MA) based on a mass addition of 122.037 to the monoiso-
topic residue weight of lysine. Putative CEP-modified peptides detected
by MALDI-TOF MS were sequenced by tandem mass spectrometry
using a PE Sciex API 3000 triple quadrupole electrospray instrument
fitted with a Protana nanospray interface (17).
Preparation of Anti-CEP AntibodiesA Pasteurella-free, New Zeal-
and White rabbit was inoculated subcutaneously along the back and
rear legs with about 340 g (7.1 nmol) of CEP-KLH (1 mol of
pyrrole/mol of KLH, 2.7 mg/ml KLH in PBS). Initial injections were
administered with Freunds complete adjuvant, and booster injections
were given every 21 days with incomplete Freunds adjuvant. Antibody
titers were monitored 10 days after each inoculation by ELISA. The
rabbit was exsanguinated after 92 days and a total of 5 inoculations.
The IgG fraction of anti-CEP antibodies was purified using immobilized
protein G (19).
ELISAThree ELISA procedures were employed and are termed
here as Methods AC (7, 19). Method A was used to determine the titer
of anti-CEP antisera. Method B was used to characterize cross-reactiv-
ities and to quantify carboxyalkyl pyrrole immunoreactivities. Method
C was used to titer anti-CEP autoantibodies in human plasma.
ELISA Method AAnti-CEP antibody titer in rabbit serum was
measured as for other anti-carboxyalkyl pyrrole antibodies (7) with the
following modifications. Baxter 96-well ELISA plates were coated with
CEP-BSA containing 1.5 mol of pyrrole per mol of protein by incubating
a 4.3 g/ml protein solution in PBS (100 l/well) at 37 C for 1 h in a
moist chamber. The coating solution was washed away with PBS (3
300 l), and the wells were incubated with blocking solution (1.0%
chicken egg ovalbumin in PBS, 300 l) at 37 C for 1 h. The blocking
solution was washed away with 0.1% ovalbumin in PBS (300 l), and
then various dilutions of rabbit serum were added (100 l) in PBS
containing 0.2% ovalbumin. Ovalbumin (0.2%) in PBS without serum
and nonimmune rabbit serum, diluted as above, were employed as
controls. Following incubation at 37 C for 1 h, the wells were washed 3
times with PBS containing 0.1% ovalbumin and then incubated for 1 h
at 37 C with 100 l of alkaline phosphatase-conjugated goat anti-
rabbit IgG (1:1000 dilution). The wells were again washed 3 times with
PBS containing 0.1% ovalbumin, and substrate p-nitrophenyl phos-
phate (Bio-Rad) (0.9 mg/ml, pH 9.6) was added (100 l/well) and incu-
bated 30 min at room temperature. The absorbance at 405 nm was
determined relative to 650 nm with a Bio-Rad 450 automated Micro-
plate reader.
ELISA Method BFor competitive binding studies to measure cross-
reactivities between anti-CEP and anti-CPP antibodies, CEP-BSA,
CEP-GDPH, and CPP-BSA were used as coating agents, CEP-HSA and
CPP-HSA were used as reference standards, and ELISAs were per-
formed as described for studies with the anti-CPP antibodies (7). To
quantify CEP, CPP, and CHP immunoreactivity, samples of DHA,
arachidonic acid, linoleic acid, or DHA-PC that had been oxidized in the
presence of HSA were analyzed by ELISAs using anti-CEP, anti-CPP,
and anti-CHP antibodies as described previously (7, 19). To quantify
CEP immunoreactivity in human plasma, ELISA of plasma from AMD
patients and healthy volunteers was performed as in the inhibition
assays using a plasma dilution factor of 0.2. To compare intra- and
inter-assay variability, triplicate assays of plasma from a single AMD
patient were run on a single plate, and this experiment was repeated on
three different days.
ELISA Method CTo determine anti-CEP autoantibody titer in hu-
man plasma, a 96-well plate was coated with CEP-BSA (100 l). As a
blank, BSA (2%, 100 l) was used. The plate was incubated for 1 h at
37 C, washed with PBS (10 mM, 300 l) 3 times, and blocked with
ovalbumin (1%, 300 l) for 1 h at 37 C. Then the plate was washed once
with 0.1% ovalbumin plus 0.05% Tween 20 (300 l). The plate was
loaded with plasma, diluted 20 times with 0.2% ovalbumin plus 0.05%
Tween 20, and incubated for 1 h at room temperature. The plate was
washed 3 times with 0.1% ovalbumin plus 0.05% Tween 20 (300 l).
Secondary antibodies were added (alkaline phosphatase-conjugated
goat anti-human IgG or alkaline phosphatase-conjugated goat anti-
human IgM, diluted 1:4000 with 1% ovalbumin plus 0.05% Tween 20
(100 l)), and after 30 min the plate was washed with 0.05% Tween 20
containing 0.1% ovalbumin (3 times, 300 l), and then substrate was
added (p-nitrophenyl phosphate, 100 l/well, 0.9 mg/ml, pH 9.6, Bio-
Rad). After 60 min, the absorbance was read at 405 nm with reference
at 650 nm. The titer was defined as the ratio of plasma binding to
antigen (A) versus binding to BSA (A
o
).
CEP Immunoreactivity from the Reaction of HOHA-PC with HSA
HSA (16.5 mg, 0.25 mol) and 4-hydroxy-7-oxohept-5-enoic acid ester of
2-lysophosphatidylcholine (HOHA-PC, 5 mg, 7.9 mol) (12) were dis-
solved in PBS (5 ml), yielding a final concentration of 1.6 mM HOHA-PC
and 0.05 mM HSA. The preparation was incubated at 37 C under
argon, and aliquots were withdrawn with time. A portion of each aliquot
was hydrolyzed by 0.2 N KOH at 37 C for 30 min, neutralized with HCl,
and then dialyzed against PBS (2 1 liter) at room temperature for
24 h. Levels of CEP immunoreactivity were determined by ELISA
Method B using anti-CEP antibodies.
In Vitro Oxidation of PUFAs or DHA-PC with HSADocosahexae-
noic acid (DHA, 6.1 mol), arachidonic acid (6.6 mol), or linoleic acid
(7.1 mol) and HSA (0.5 mol) were dissolved in PBS (10 ml), and
oxidation was initiated by the addition of 20 mM ascorbate (510 l) and
0.8 mM FeSO
4
(510 l). The preparations were incubated up to 72 h at
37 C under air and aliquots were removed periodically; afterward the
oxidation reactions were quenched by the addition of EDTA (to 1 mg/ml)
and BHT (to 40 mM) followed by overnight dialysis at room temperature
against PBS (2 1 liter) containing EDTA (1 mg/ml). Prior to oxidation
of DHA-PC, DHA-PC liposomes were prepared (20). Briefly, a solution
of DHA-PC (6.3 mol) in chloroform was evaporated in vacuo, and the
resulting lipid film was hydrated in PBS (2 ml) at 37 C and the
suspension extruded through a polycarbonate membrane (100-nm pore
size, Nuclepore Co., Pleasanton, CA) to produce a clear solution of
unilamellar liposomes. The resulting DHA-PC liposomes were com-
bined with HSA in PBS (5 ml), yielding concentrations of about 0.6 mM
DHA-PC and 0.05 mM HAS, and oxidation was initiated by addition of
20 mM ascorbate (215 l) and 0.8 mM FeSO
4
(215 l). The preparation
was incubated at 37 C under air up to 24 h; aliquots were removed
periodically, and the oxidation reaction was quenched as described
above. A portion of the preparation was hydrolyzed with 0.2 N KOH at
37 C for 30 min, then neutralized with HCl, and finally dialyzed
against PBS (2 1 liter) containing EDTA (1 mg/ml) for 24 h at room
temperature.
ImmunocytochemistryImmunocytochemistry was performed to lo-
calize CEP adducts in mouse and human retina using methods de-
scribed recently (6). Mouse eyes were enucleated from BALB/c mice
immediately after sacrifice by CO
2
asphyxiation and cervical disloca-
tion. Normal and AMD human donor eye tissues were obtained through
the Eye Donor Program of the Foundation Fighting Blindness (Owings
Mills, MD). Human eyes were enucleated 1.57 h after death and frozen
in liquid nitrogen. After separating the anterior segment, posterior eye
cups were fixed overnight in freshly prepared 4% paraformaldehyde in
PBS. Retinal tissues sections (3 m thick) were incubated for 16 h at
4 C with anti-CEP antibody (diluted 1:100 in PBS containing 6% BSA).
Control mouse retinal sections were preincubated overnight with the
CEP-HSA prior to incubation with anti-CEP. Preimmune rabbit sera
served as the immunocytochemical control for analysis of human tis-
sues. After washing extensively with PBS, sections were treated with
goat anti-rabbit IgG conjugated to peroxidase ABC (Vector Laborato-
ries, Burlingame, CA, 1:200 dilution) for 1 h at room temperature.
Sections were washed with PBS and incubated in 0.05% 3,3-diamino-
benzidine (Sigma) and 0.03% hydrogen peroxide in PBS at room tem-
perature. Sections were viewed without counterstaining using differen-
tial interference contrast microscopy. The images presented were
digitized with a Hamamatsu CCD camera and assembled in Microsoft
PowerPoint.
Dot-blot AssayNitrocellulose membrane (Nitro Plus, 0.45 m, Mi-
cron Separations, Inc., Westboro, MA) was prewet with 10 mM Tris-
buffered saline, pH 7.2 (TBS), and CEP-HSA (50 l/well, 2 g) was
applied by gravity to membrane wells in a dot-blot microfiltration
apparatus (Bio-Rad). Blocking solution (1% ovalbumin in TBS, 300 l)
was then applied to each well, followed by 0.5% Tween 20 in TBS
(TBST, 2 300 l), and a vacuum was used to pull the solutions
through the membrane. Plasma (100 l of 1:10 dilution in TBS contain-
ing 0.2% ovalbumin) was then applied by gravity to each well followed
by TBST (3 300 l). Secondary antibody (horseradish peroxidase-
conjugated anti-human Ig, 100 l, 1:1000 dilution TBS containing 1%
ovalbumin) was then applied by gravity to each well followed by TBST
(3 300 l). The membrane was then removed from the microfiltration
apparatus; chemiluminescence reagent was applied (ECL Plus, Amer-
sham Biosciences), and the membrane was blotted dry with filter paper
and exposed to x-ray film (Hyperfilm, Amersham Biosciences) for 30
min. The film was scanned with a densitometer (Bio-Rad GS-710 Cal-
ibrated Imaging Densitometer), and the signal was quantified using
PDQuest image analysis software (Bio-Rad).
StatisticsFor statistical analysis of plasma levels of CEP immuno-
reactivity and autoantibodies, p values were calculated by independent
CEP Biomarkers for AMD 42029

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

1
6
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

t test (two sample assuming equal variances) using Excel 98 for Macin-
tosh. For correlation of CEP immunoreactivity and autoantibody titer
with AMD, a logistic regression model was fit with both variables as
predictors using SAS/STAT software, Version 8.2 from SAS Institute
Inc., Cary, NC. A predicted score was calculated for each plasma donor
and the c statistic, which is a measure of how well the predictor
variables can distinguish AMD cases from controls, was determined.
RESULTS
We produced CEP-adducted proteins using Paal-Knorr con-
densation of DOHA with the -amino group of lysyl residues.
The unambiguous synthesis of DOHA, DOHA-phosphatidyl-
choline (DOHA-PC), CEP, and CEP-PC adducted dipeptide and
verifications by NMR are documented in the Supplemental
Material. DOHA was then used to chemically modify KLH,
BSA, HSA, and GPDH to yield CEP-adducted antigens and
reference proteins.
Characterization of DOHA Modified HSATo probe for the
presence of CEP adducts in HSA following modification with
DOHA, the protein was digested with trypsin and analyzed by
mass spectrometry (Fig. 2). Two HSA peptides containing ap-
parent lysyl CEP adducts were identified by MALDI-TOF mass
spectrometry at m/z 1141 and m/z 2021 (Fig. 2A). Tandem
nanoelectrospray mass spectrometric analysis of m/z 1141 re-
vealed HSA peptide
234
AFK*WAVAR
241
and unambiguously
confirmed a CEP adduct on lysyl residue Lys
236
(Fig. 2B).
Electrospray MS/MS analysis of m/z 2021 revealed HSA pep-
tide
170
HPYFYAPELLFFAK*R
184
and a CEP adduct on lysyl
residue Lys
183
(Fig. 2C). The pyrrole content in this prepara-
tion of CEP-HSA was estimated using the Ehrlich assay to be
1.51.6 mol of pyrrole per mol of protein. Complete structural
analysis of the DOHA-modified albumin was not pursued, and
other residues containing CEP adducts could be present.
Anti-CEP Antibody SpecificityTo evaluate the binding
specificities of anti-CEP rabbit polyclonal antibodies raised to
DOHA-modified KLH, competitive ELISAs were performed
with HSA adducted with CEP, CPP, CHP, or PP-ACA, and the
results (Fig. 3 and Table I) were compared with that of anti-
CPP antibodies (7). The CEP and CPP adducts differ by only a
single CH
2
group; nevertheless, inhibition of anti-CEP anti-
body binding by CPP-HSA is remarkably weak, i.e. 0.1% cross-
reactivity. Inhibition of anti-CPP binding by CEP-HSA is some-
what greater; however, anti-CPP also exhibits high structural
specificity, i.e. only 1.3% cross-reactivity. Neither antibody ex-
hibits significant cross-reactivity with CHP-HSA, which con-
tains a much larger carboxyalkyl group than the haptens
against which anti-CEP or anti-CPP were raised. Another pyr-
rolated species, PP-6-ACA-HSA, which contains an n-pentyl
rather than a carboxyalkyl side chain, showed no cross-reac-
tivity with either of the antibodies. Similar structural selectiv-
ity of anti-CEP antibody was observed when a different coating
agent, CEP-GPDH, was used. As expected, unmodified HSA or
GPDH were not recognized by anti-CEP antibody.
CEP Immunoreactivity fromHOHA-PCModification of HSA
Recently we showed that 2-(4-hydroxy-7-oxohept-5-enoyl)phos-
phatidylcholine (HOHA-PC), a minor oxidative product from
DHA-PC, can react with the -amino group of a lysine-
containing dipeptide to form CEP adducts (12). HOHA-PC will
also modify HSA resulting in anti-CEP immunoreactivity as
shown in Fig. 4A; however, the antibody inhibition curves do
not parallel that for the CEP-HSA generated by DOHA modi-
fication of HSA. Presumably this is because the CEP epitopes
generated with HOHA-PC are mostly in the form of PC esters
rather than the free acids against which the antibodies were
raised. Treatment of the reaction product mixture with KOH
converted the CEP-PC esters into the corresponding free acids
whose inhibition curves parallel that for CEP-HSA (Fig. 4B).
The yield of CEP epitope from modification of HSA with
HOHA-PC after 24 h was estimated to be 0.5% relative to the
phospholipid fragment.
Carboxyalkylpyrrole Immunoreactivity from Oxidation of
PUFA or DHA-PCIn vitro free radical-initiated oxidations of
arachidonic acid, linoleic acid, or DHA for 72 h in the presence
of HSA generated carboxyalkyl pyrrole immunoreactivity. Pro-
tein-bound pyrroles were detected by ELISA Method B using
the appropriate BSA-bound pyrrole as coating agent and the
corresponding HSA-bound pyrrole as standard. For CEP and
CPP, barely detectable levels of immunoreactivity were ob-
served, namely 8 mol of CEP/mol of HSA, 7 10
5
% yield
relative to DHA and 5 mol of CPP/mol of HSA, 40 10
5
%
yield relative to arachidonate. A significantly greater level
of CHP immunoreactivity was observed, namely 2.8 mmol of
CHP/mol of HSA, 0.02% yield relative to linoleate. Oxidation
of DHA-PC liposomes for 24 h in the presence of HSA also
generated barely detectable CEP immunoreactivity, but follow-
ing hydrolysis of the PC esters to the corresponding free acids
with 0.2 N KOH, CEP immunoreactivity increased to 50 mol/
mol HSA or 4 10
4
% yield relative to DHA-PC. We speculate
that the increase in CEP immunoreactivity following saponifi-
cation is because of the importance of strong interactions of the
free carboxyl group with the anti-CEP antibody, interactions
that are not possible when the carboxyl group is esterified to
2-lyso-phosphatidylcholine. A previous study of CHP immuno-
reactivity generated upon oxidation of low density lipoprotein
in vitro also found a large increase consequent to saponification
of the oxidized low density lipoprotein (7). The sensitivity of
CEP yield to its phospholipid origin is presumably a conse-
quence of interference by the free carboxyl group in HOHA with
pyrrole formation. In HOHA-PC, the carboxyl group is masked
by esterification. CHP yield from HODA is presumably much
greater than CEP yield from HOHA because the carboxyl group
in HODA does not interfere with CHP formation because of the
longer chain separating it from the -hydroxy-,-unsaturated
aldehyde functionality in HODA (7 carbons) compared with
HOHA (2 carbons).
CEP Immunoreactivity in RetinaImmunocytochemical
analysis of mouse retina demonstrated intense CEP immuno-
reactivity in the photoreceptor outer segments and RPE and
lighter immunoreactivity in the inner plexiform layer (Fig. 5A).
In contrast, when the anti-CEP antibody was preincubated
with CEP-protein antigen, all labeling was eliminated. The
distribution of CEP immunoreactivity in the RPE and photo-
receptor outer segments was confirmed using 9 eyes from three
mice with tissue fixation performed shortly after death (2 h).
Mouse retinal tissues were consistently labeled over the pho-
toreceptors and RPE over antibody dilution ranges of 1:100 to
1:10,000. Comparison of anti-CEP immunostaining of human
AMD retina with healthy retina (Fig. 5, B and C) show that for
the two samples analyzed, more CEP immunoreactivity was
present in the AMD tissue than the normal retina. This is
concordant with our previous detection of more CEP immuno-
reactivity by Western analyses of proteins obtained from RPE/
Bruchs membrane/choroid tissue from 11 AMD donors com-
pared with 11 normal controls (6).
CEP Immunoreactivity in Human PlasmaThe levels of
CEP immunoreactivity were measured by ELISA in human
plasma from (i) 19 donors with diagnosed AMD and of average
age 82 years, (ii) 19 normal donors of average age 83 years
without AMD, and (iii) 9 young healthy donors of average age
27 years. Protease inhibitors, a metal chelator, and an antiox-
idant were added to human plasma to limit in vitro oxidation/
degradation, and all samples were quench-frozen immediately
in liquid nitrogen after plasma isolation (21). A comparison of
CEP immunoreactivity detected in each donor group is pre-
CEP Biomarkers for AMD 42030

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

1
6
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

FIG. 2. Localization of CEP adducts in DOHA-modified HSA. A, MALDI-TOF mass spectrum of a tryptic digest of CEP-HSA. Arrows denote
internal standards; dots indicate tryptic peptides from HSA; and asterisks highlight possible CEP-modified peptides based on a mass addition of
122.0366 for the CEP adduct. B, nanoelectrospray MS/MS spectrum of the doubly charged ion m/z 571 (singly charged m/z 1141.6302 in A) from
HSA residues 234241 supporting CEP modification of Lys
236
. C, nanoelectrospray MS/MS spectrum of the doubly charged ion m/z 1101 (singly
charged m/z 2021.0678 in A) from HSA residues 170184 supporting CEP modification of Lys
183
.
CEP Biomarkers for AMD 42031

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

1
6
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

sented in Fig. 6. Mean CEP immunoreactivity ( S.D.) ob-
served in the plasma of AMD donors (15.9 7.4 pmol/ml) was
significantly higher than in the plasma of younger donors
(10.5 3.7 pmol/ml, p 0.05) and also 1.5-fold higher than in
the plasma from older healthy donors (10.5 2.8 pmol/ml, p
0.004). The mean levels of anti-CEP immunoreactivity meas-
ured in triplicate revealed 411% intra-assay variability and
37% inter-assay variability. The plasma level of CEP adducts
is lower than that of CHP adducts (175275 pmol/ml) (7) and
reflects the lower plasma lipid concentration of DHA compared
with linoleate, e.g. low density lipoprotein particles contain
about 29 molecules of DHA versus about 1100 molecules of
linoleate (22).
Anti-CEP Autoantibodies in Human PlasmaAutoantibody
titers against CEP protein adducts were measured by ELISA
Method C in human plasma from 19 donors with AMD and 19
older normal donors (Fig. 7). Plasma from the AMD donors
exhibited a 2-fold higher average CEP autoantibody titer (3.4
3.1 S.D.) than plasma from age-matched normal controls (1.5
0.4 S.D.), and the difference was statistically significant (p
0.02). Titer here is defined as the ratio of binding to CEP-
modified BSA (A) versus unmodified BSA (A
o
) and high titer as
any A/A
o
value greater than 2 S.D. above the average titer of
the controls (namely for these data sets, A/A
o
2.3). The
prevalence of high anti-CEP autoantibody titers from AMD
donors was 53% (10:19) compared with 21% (4:19) of the age-
matched controls.
A dot-blot assay was also employed to probe for anti-CEP
autoantibodies in which CEP-HSA was immobilized on nitro-
cellulose membrane, reacted with human plasma and then
secondary antibody, and the membrane scanned for spot optical
density. Results from the dot blot assay were in good agree-
ment with the ELISA data in Fig. 7 and revealed a mean
optical density value (0.15 0.05) for AMD plasma that was
2.5-fold higher (p 0.01) than the mean optical density value
(0.06 0.04) obtained for the age-matched control group (not
shown). AMD and age-matched normal plasma exhibiting high
levels of anti-CEP antibodies in both assays are highlighted
with stars in Fig. 7.
DISCUSSION
The cellular, biochemical, and molecular events contributing
to the etiology of AMD remain poorly understood. AMD is
usually defined as either dry or wet and is a slow, progres-
sive degenerative disease with both genetic and environmental
risk factors (1, 3). Advanced stage AMD, also known as the wet,
exudative, or neovascular form of the disease, affects only
10% of those with AMD and is characterized by abnormal
blood vessel growth from the choriocapillaris through the RPE
(neovascularization), resulting in possible hemorrhage, exuda-
tion, scarring, and/or serous retinal detachment (23). The vast
majority (90%) of all AMD is the non-neovascular or dry
form of AMD and is characterized by numerous large macular
drusen, atrophy of the RPE, and loss of macular photoreceptors
(23). Oxidative damage appears to contribute to the pathogen-
esis of AMD (4) based on epidemiological studies showing that
smoking significantly increases the risk of AMD (1, 24). The
molecular mechanism for how smoking enhances the risk for
AMD is not known. We speculate that reactive oxygen and
nitrogen species derived from tobacco smoke in the lungs leads
to oxidative protein modifications in the blood that contribute
to drusen formation and choroidal neovascularization. Results
from a recent clinical trial (5) also demonstrate that the pro-
gression of AMD can be slowed in some individuals by high
daily doses of antioxidant vitamins and zinc. Direct evidence of
oxidative damage in AMD donor eye tissues include elevated
levels of CEP adducts uniquely derived from the oxidative
fragmentation of DHA (6).
As an approach to better understanding the role of DHA-
derived oxidative damage in AMD, we have prepared CEP-
modified proteins and anti-CEP antibodies and initiated com-
FIG. 4. Antibody inhibition before and after saponification. A,
anti-CEP binding to CEP-BSA in the presence of CEP-HSA () or
products from reaction of HOHA-PC with HSA for 1 (), 8 (), and 24 h
(f) at 37 C before saponification. B, anti-CEP binding to CEP-BSA in
the presence of CEP-HSA () or products from reaction of HOHA-PC
with HSA for 1 (), 8 (), and 24 h () at 37 C after treatment with 0.2
N KOH.
FIG. 3. Antibody specificity. A, anti-CEP binding to CEP-BSA in
the presence of CEP-HSA (E) or anti-CEP binding to CEP-GPDH in the
presence of CEP-HSA (), CPP-HSA (), CHP-HSA (f), or PP-ACA-
HSA (). B, anti-CEP binding to CEP-BSA in the presence of CEP-HSA
(), CPP-HSA (), CHP-HSA (), or PP-ACA-HSA (f). C, anti-CPP
binding to CPP-BSA in the presence of CPP-HSA (), CEP-HSA (), or
CHP-HSA ().
TABLE I
Selectivity of rabbit polyclonal anti-CEP and anti-CPP antibodies
CEP Biomarkers for AMD 42032

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

1
6
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

parative analysis of CEP immunoreactivity and autoantibody
titers in human plasma. Paal-Knorr condensation of DOHA
with the -amino group of lysyl residues was used to prepare
CEP-adducted peptides and proteins. The unambiguous syn-
thesis of DOHA and its phosphatidylcholine ester (DOHA-PC)
was confirmed by NMR as was the unambiguous production of
CEP and CEP-PC-modified dipeptide (Supplemental Material).
DOHA was then used to chemically modify KLH, BSA, HSA,
and GPDH to yield CEP-adducted antigen and/or reference
proteins containing from 1 to 1.6 mol of pyrrole per mol of
protein. To confirm further the presence of CEP adducts in
DOHA-modified HSA, the modified protein was digested with
trypsin, and CEP adducts were localized to HSA residues
Lys
183
and Lys
236
by MALDI-TOF and tandem electrospray
mass spectrometric analyses.
CEP Antibody SelectivityThe utility of anti-CEP antibod-
ies for the detection of DHA-derived CEP adducts in biological
samples depends on their ability to discriminate between CEP
and similar 2-(-carboxyalkyl)pyrrole modifications generated
from other lipid oxidation products such as CPP and CHP
adducts (Fig. 1). We found that our polyclonal anti-CEP and
anti-CPP antibodies recognize their respective epitopes with
excellent discrimination despite the fact that they differ by only
a single CH
2
group. This remarkable structural selectivity
persisted when another protein (i.e. CEP-GPDH) was used to
coat ELISA wells instead of CEP-BSA. To provide the observed
discrimination between these very similar epitopes, the fit
must be tight and include strong, geometrically rigorous inter-
actions of the carboxyl groups in the CEP and CPP structures.
In view of this exquisite selectivity, it is not surprising that
both antibodies cross-react very weakly (0.050.24%) with
CHP-HSA that contains a much larger carboxyalkyl group
than either CEP or CPP. Neither antibody cross-reacts with
PP-ACA-HSA that lacks a carboxyl (Table I). The utility of the
anti-CEP antibody in Western blot analyses was previously
demonstrated at an antibody concentration of 1 g/ml (6).
Immmunocytochemical Localization of CEP in RetinaDHA
is not uniformly distributed throughout the retina but, rather,
is concentrated in the photoreceptor rod outer segments and
FIG. 5. Localization of CEP immunoreactivity in retina. Immu-
nohistochemical analysis of retinal sections with anti-CEP antibody is
shown in the left panels and control tissue sections detected with
non-immune sera in the right panels. A, mouse retina shows prominent
CEP staining in the photoreceptor outer segments and RPE. Less
intense staining is evident in the inner plexiform layer (IPL), and little
if any staining is seen in the outer limiting membrane (OLM), outer
nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer
(INL), or ganglion cell layer (GCL). B, sections from normal human
retina; C, AMD macula and peripheral photoreceptors immunostained
with anti-CEP antibody. More CEP immunoreactivity can be seen in
the AMD sample.
FIG. 6. CEP immunoreactivity in human plasma from young,
old, and AMD donors. CEP immunoreactivity was detected by ELISA
in human plasma from AMD donors (, n 19, average age 83 years),
and from healthy older donors (, n 19, average age 82 years) or
healthy younger donors (f, n 9, average age 27 years). Individual and
mean levels detected (E) plus standard deviation are shown for each
data set.
FIG. 7. Anti-CEP autoantibodies in plasma from AMD and nor-
mal donors. Anti-CEP autoantibodies were detected by ELISA in
human plasma from the AMD donors (n 19, average age 83 years) and
older healthy donors (n 19, average age 82 years) depicted in Fig. 6.
Each bar represents the mean S.D. of at least three ELISA. The
normal healthy donor mean and mean plus 1 S.D. are indicated by solid
and dashed lines, respectively. Stars indicate plasma that exhibit anti-
CEP IgG reactivity greater than 1 S.D. above the normal mean in both
ELISA and dot-blot analyses. The mean CEP autoantibody titer is
higher in the AMD samples than in the normal samples.
CEP Biomarkers for AMD 42033

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

1
6
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

the RPE (25, 26). Notably this DHA-rich region of the retina is
exposed to high levels of oxygen and is responsible for captur-
ing photons (i.e. phototransduction). Immunocytochemistry
demonstrated that these cell layers exhibited intense CEP im-
munoreactivity in mouse retina (Fig. 5). Normal human retina
also exhibited CEP immunoreactivity in photoreceptor cells,
but the intensity was much greater in AMD retina. Each RPE
cell interacts with the tips of about 50 photoreceptor outer
segments, and 10% of each outer segment is shed daily and
phagocytized by the RPE. This process is thought to be in part
a repair mechanism for the high photooxidative stress prevail-
ing in the retina and certainly introduces significant amounts
of oxidatively modified proteins and DHA-containing lipids
from the photoreceptor cells into the RPE. The RPE forms an
integral part of the blood-retinal barrier and is responsible for
vectorial transport of nutrients to the photoreceptor cells and
waste products to the blood. We hypothesize that normal pro-
tective mechanisms against oxidative stress are compromised
in AMD, resulting in oxidative damage that impairs the ability
of the RPE to process and degrade waste products from the
photoreceptors. A consequence of this impairment appears to
be increased deposition of CEP-modified proteins in the adja-
cent Bruchs membrane (6) and circulatory system (Fig. 6).
CEP Adducts Are Generated in Low YieldAfter 2472 h in
vitro oxidation of DHA or DHA-PC with HSA, we found the
yield of CEP adduct to be low, namely in the range of 0.74
ppm relative to the DHA or lipid and 850 ppm relative to
HSA. Saponification was required for detection of the higher
levels from DHA-PC. These very low levels of CEP modifica-
tions are due in part to the complicated array of DHA oxidation
products that compete with formation of the HOHA and
HOHA-PC fragmentation products that produce CEP adducts
(12). Reactions with other DHA oxidation products can result
in alternative protein modifications such as Michael adducts,
Schiff bases, or protein cross-links (6, 12). In vitro reaction of
HOHA-PC with HSA for 24 h yielded significantly greater
levels of CEP adducts (5000 ppm or 0.5% yield relative to the
lipid fragment); however, this yield is still relatively low and
most likely reflects the susceptibility of HOHA-PC itself to
further oxidation and rearrangements (12). In contrast, the
yield of CHP-HSA adducts (200 ppm) from linoleate (and
HODA) is much higher than CEP. This is due in part to fewer
double bonds in linoleate than DHA and consequently fewer
possible alternative oxidation products. There is also less pos-
sible interference by the carboxyl group with pyrrole genera-
tion for HODA due to the greater distance between the incipi-
ent pyrrole ring and the carboxyl group compared with HOHA.
The absence of carboxyl group interception of intermediates in
the pyrrole-forming process may also explain the higher appar-
ent yields of CEP-HSA obtained from oxidation of the phospho-
lipid ester (i.e. DHA-PC) followed by saponification than pro-
duced by oxidation of the free acid DHA. These in vitro studies
show that CEP adducts are generated from DHA in low yield
over a few days. Analyses of human plasma (Figs. 6 and 7) and
human ocular tissues (Fig. 5) demonstrate CEP adducts accu-
mulate in vivo and are more abundant in tissues from AMD
donors than from normal tissues (6).
Plasma CEP Immunoreactivity and CEP Autoantibody Titer
Correlate with AMDWe found the mean levels of CEP immu-
noreactivity and autoantibody titer in AMD plasma statisti-
cally to be significantly higher than in controls. In addition, the
variability of CEP immunoreactivity and autoantibody was
about 23 times higher in plasma from AMD donors than from
the older controls. This higher variability likely reflects the
complex genetic basis of AMD susceptibility and possible envi-
ronmental differences among the donors. Interestingly, the
variability of CEP immunoreactivity was about 30% greater in
the plasma from younger controls than from older normal do-
nors. The basis of this higher apparent variability is presently
not known. However, AMD is a progressive disease, and we
speculate that oxidative modifications such as CEP adducts
will gradually increase in the plasma of those susceptible to
developing the disease and be detectable before the manifesta-
tion of retinal pathology. Our analyses of human plasma show
that a large fraction of the AMD donors exhibited both elevated
CEP immunoreactivity and high anti-CEP autoantibody titer
(Fig. 8). Specifically, 12 of the 19 AMD donors (63%) exhibited
both high immunoreactivity and high autoantibody titer com-
pared with only 1 of the 19 age-matched controls (5%). How-
ever, high CEP immunoreactivity was not always associated
with high anti-CEP autoantibody titer. Select plasma from
both AMD or control donors exhibited higher levels of either
CEP immunoreactivity or autoantibody titer but not both. Nev-
ertheless, 92% of the individuals exhibiting both antigen and
autoantibody levels above the mean for non-AMD controls had
AMD (region IV in Fig. 8). Logistic regression also revealed
larger probabilities of AMD associated with increased levels of
CEP immunoreactivity and autoantibodies. The logistic regres-
sion model obtained using the available data as predictors of
AMD fit the equation: ln(probability of AMD/(1 probability of
AMD)) 4.80 0.26(CEP immunoreactivity) 0.82(CEP
autoantibody titer). The c statistic based on this preliminary
model was 0.80 (95% confidence interval), indicating that by
using both variables there is an 80% likelihood that a randomly
selected person with AMD is likely to receive a higher predicted
probability of having AMD than is a person without AMD. This
probability was greater than using either CEP immunoreactiv-
ity (0.77) or CEP autoantibody titer (0.73) alone. Given the
apparently low incidence of false positives, a combination of
these two parameters, namely plasma CEP immunoreactivity
and autoantibody titer, may have diagnostic utility. A much
larger clinical investigation is now warranted to test whether
this approach could be useful in predicting AMD susceptibility.
Finally, the presence of elevated levels of CEP autoantibod-
ies in AMD plasma supports suggestions by others that im-
mune-mediated events may be associated with the pathogene-
sis of AMD (2729). Over time, immune mediated events may
FIG. 8. Correlation between CEP immunoreactivity and anti-
CEP autoantibody titer in plasma. The horizontal and vertical
dashed lines indicate the mean values of CEP autoantibody titer and
CEP immunoreactivity, respectively, for healthy donors of average age
82 years (from Figs. 6 and 7). Solid symbols represent AMD donors, and
open symbols denote normal donors. Over 60% (12:19) of the AMD
donors cluster in region IV; 92% (12:13) of the data in region IV is from
AMD donors.
CEP Biomarkers for AMD 42034

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

1
6
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

be contributory, for example, to the formation of AMD drusen;
however, it is not yet clear that such events are causative. We
hypothesize that protein modifications derived from the oxida-
tion of lipids and carbohydrates may be primary catalysts in
AMD pathology and likely play significant roles in both drusen
biogenesis and choroidal neovascularization (6). Immune re-
sponses triggered by oxidative lipid-derived protein modifica-
tion may play a more dominant role in autoimmune pathologies
than generally recognized. Plasma analyses for oxidative pro-
tein modifications such as CEP and the associated autoanti-
bodies may provide an early warning system for predicting
those at risk of developing AMD and facilitate timely therapeu-
tic intervention preventing retinal degeneration and vision
loss.
AcknowledgmentsWe thank Henry F. Hoff for helpful discussions
and James Bean and Ed Maschau in the Department of Biostatistics
and Epidemiology, Cleveland Clinic Foundation, for statistical
analyses.
REFERENCES
1. Evans, J. R. (2001) Prog. Retin. Eye Res. 20, 227253
2. Klein, R., Klein, B. E., and Linton, K. L. (1992) Ophthalmology 99, 933943
3. Stone, E. M., Sheffield, V. C., and Hageman, G. S. (2001) Hum. Mol. Genet. 10,
22852292
4. Beatty, S., Koh, H., Phil, M., Henson, D., and Boulton, M. (2000) Surv.
Ophthalmol. 45, 115134
5. AREDS Research Group (2001) Arch. Ophthalmol. 119, 14171436
6. Crabb, J. W., Miyagi, M., Gu, X., Shadrach, K., West, K. A., Sakaguchi, H.,
Kamei, M., Hasan, A., Yan, L., Rayborn, M. E., Salomon, R. G., and
Hollyfield, J. G. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 168214687
7. Kaur, K., Salomon, R. G., ONeil, J., and Hoff, H. F. (1997) Chem. Res. Toxicol.
10, 13871396
8. Sun, M., Deng, Y., Batyreva, E., Sha, W., and Salomon, R. G. (2002) J. Org.
Chem. 67, 35753584
9. Podrez, E. A., Poliakov, E., Shen, Z., Zhang, R., Deng, Y., Sun, M., Finton, P. J.,
Shan, L., Gugiu, B., Fox, P. L., Hoff, H. F., Salomon, R. G., and Hazen, S. L.
(2002) J. Biol. Chem. 277, 3850338516
10. Podrez, E. A., Poliakov, E., Shen, Z., Zhang, R., Deng, Y., Sun, M., Finton, P. J.,
Shan, L., Febbraio, M., Hajjar, D. P., Silverstein, R. L., Hoff, H. F., Salomon,
R. G., and Hazen, S. L. (2002) J. Biol. Chem. 277, 3851738523
11. Subbanagounder, G., Deng, Y., Borromeo, C., Dooley, A. N., Berliner, J. A., and
Salomon, R. G. (2002) Vascul. Pharmacol. 38, 201209
12. Gu, X., Sun, M., Gugiu, B., Hazen, S., Crabb, J. W., and Salomon, R. G. (2003)
J. Org. Chem 68, 37493761
13. Fliesler, S. J., and Anderson, R. E. (1983) Prog. Lipid Res. 22, 79131
14. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H.,
Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk,
D. C. (1985) Anal. Biochem. 150, 7685
15. DeCaprio, A. P., Jackowshi, S. J., and Regan, K. A. (1987) Mol. Pharmacol. 32,
542548
16. Crabb, J. W., Nie, Z., Chen, Y., Hulmes, J. D., West, K. A., Kapron, J. T.,
Ruuska, S. E., Noy, N., and Saari, J. C. (1998) J. Biol. Chem. 273,
2071220720
17. West, K. A., Yan, L., Miyagi, M., Crabb, J. S., Marmorstein, A. D.,
Marmorstein, L., and Crabb, J. W. (2001) Exp. Eye Res. 73, 479491
18. Miyagi, M., Sakaguchi, H., Darrow, R. M., Yan, L., West, K. A., Aulak, K. S.,
Stuehr, D. J., Hollyfield, J. G., Organisciak, D. T., and Crabb, J. W. (2002)
Mol. Cell. Proteomics 1, 293303
19. Sayre, L. M., Sha, W., Xu, G., Kaur, K., Nadkarni, D., Subbanagounder, G.,
and Salomon, R. G. (1996) Chem. Res. Toxicol. 9, 11941201
20. MacDonald, R. C., MacDonald, R. I., Menco, B. P., Takeshita, K., Subbarao,
N. K., and Hu, L. R. (1991) Biochim. Biophys. Acta 1061, 297303
21. Salomon, R. G., Batyreva, E., Kaur, K., Sprecher, D. L., Schreiber, M. J.,
Crabb, J. W., Penn, M. S., DiCorletoe, A. M., Hazen, S. L., and Podrez, E. A.
(2000) Biochim. Biophys. Acta 1485, 225235
22. Steinbrecher, U. P. (1987) J. Biol. Chem. 262, 36033608
23. Abdelsalam, A., Del Priore, L., and Zarbin, M. A. (1999) Surv. Ophthalmol. 44,
128
24. Seddon, J. M., Willett, W. C., Speizer, F. E., and Hankinson, S. E. (1996) J. Am.
Med. Assoc. 276, 11411146
25. Alvarez, R. A., Aguirre, G. D., Acland, G. M., and Anderson, R. E. (1994)
Investig. Ophthalmol. Vis. Sci. 35, 402408
26. Wang, N., and Anderson, R. E. (1992) Curr. Eye Res. 11, 783791
27. Hageman, G. S., Luthert, P. J., Chong, N. H. V., Johnson, L. V., Anderson,
D. H., and Mullins, R. F. (2001) Prog. Retin. Eye Res. 20, 705732
28. Johnson, L. V., Ozaki, S., Staples, M. L., Erickson, P. A., and Anderson, D. H.
(2000) Exp. Eye Res. 70, 441449
29. Penfold, P. L., Madigan, M. C., Gilles, M. C., and Provis, J. M. (2001) Prog.
Retin. Eye Res. 20, 385414
CEP Biomarkers for AMD 42035

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

1
6
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m