Antioxidant Enzymes in the
Aging Human Retinal Pigment
Mark R.
Epithelium
Liles; David A. Newsome, MD; Peter D. Oliver, PhD
\s=b\ The antioxidant enzymes catalase
and superoxide dismutase have integral
roles in controlling reactive oxygen radicals that can harm cells. In the present
study, we quantitated catalase activity in
retinal pigment epithelium, retina, iris,
and vitreous from human donors. To our
knowledge, our results represent the first
quantitation of catalase activity in human
retinal pigment epithelium and show six\x=req-\
fold greater catalase activity in retinal
pigment epithelium than in other ocular
tissues analyzed (P<.0001). To investigate whether aging or macular degeneration affects retinal pigment epithelium
catalase or superoxide dismutase activities, we measured enzyme levels in retinal pigment epithelium from donors 50 to
90 years of age with and without evidence of macular degeneration. Superoxide dismutase activity showed no significant correlations with aging or
macular degeneration, while catalase activity decreased with age (P<.02) and
macular degeneration (P<.05) in both
macular and peripheral retinal pigment
epithelium.
(Arch Ophthalmol. 1991;109:1285-1288)
'" he functional integrity of the retina
depends on the maintenance activi¬
ties of the retinal pigment epithelium
(RPE).1 The RPE cells ingest and de-
Accepted for publication May 16, 1991.
From the Sensory and Electrophysiology Research Unit, Touro Infirmary (Mr Liles and Drs
Newsome and Oliver), and the Departments of
Ophthalmology (Dr Newsome) and Anatomy (Dr
Oliver), Tulane University School of Medicine,
New Orleans, La.
Reprint requests to Sensory and Electrophysiology Research Unit, Touro Infirmary, 1401
Foucher St, New Orleans, LA 70115 (Dr
Newsome).
grade
numerous
(ROS) every day,
rod outer segments
a process that re¬
the photoreceptors.2 However,
phagocytosis initiates the production
of Superoxide anions (02~),3 a reactive
news
form of oxygen that
damage directly
or
cellular
conversion to
can cause
by
more reactive and damaging hydroxyl radical (OH·)4"8:
202+2H+-»H202+02
Fe2++H202-»Fe3*+.OH+OH-.
Oxygen radical exposure to the RPE
can also result from photic reactions910
and from high Po2 values due to the
diffusion of oxygen through the RPE
cell layer from the adjacent choroidal
circulation.1112 Thus, the unique func¬
the
tions and location of the RPE cause its
environment to be inundated with oxy¬
gen radicals that cause cellular
damage.
The manifestations of aging and
macular degeneration (MD) in the
RPE include the deposition of drusen,
neovascularization, the thickening of
Bruch's membrane, and the accumula¬
tion of the aging pigment, lipofus¬
cin.1314 Oxygen radical damage may
accelerate
these
age-related
changes.15'1'' For example, lipofuscin is
thought to consist partially of lipids
from ingested ROS that have been
oxidatively modified into a nondegradable form.1'"19 To our knowledge, a
functional role for Superoxide during
RPE phagocytosis is unknown, but
Superoxide (or the action of hydroxyl
radicals) can inhibit the lysosomal en¬
zymes that normally degrade ROS.20,21
The accumulation of lipofuscin would
probably accelerate as a result of in¬
complete lipid degradation and in¬
creased lipid peroxidation.2223 Dorey
and colleagues24 have documented a
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positive correlation between lipofuscin
accumulation and photoreceptor loss in
the human macula.
Antioxidant enzymes have the vital
role of protecting the RPE from oxy¬
gen radical damage.1 The two species
of Superoxide dismutase (SOD) (cop¬
per-zinc and manganous) dismutate
Superoxide anions, forming hydrogen
peroxide (H,02) as a product.20 As pre¬
viously mentioned, H202 can give rise
to reactive hydroxyl radicals; thus,
SOD acts in concert with two other
enzymes, catalase and glutathione peroxidase, both of which convert H202 to
the nontoxic products water and mo¬
lecular oxygen. The combined action of
these three enzymes forms one meta¬
bolic pathway for protection against
oxidative assault.26'27
The activity of catalase is closely
linked to the mechanisms of ROS deg¬
radation. Catalase is localized within
the peroxisomes of the RPE,2829 and,
after the fusion of the peroxisome with
the phagosome, catalase is also present
within secondary lysosomes, which
contain ROS in varying stages of deg¬
radation.30 Phagocytosis of ROS has
been reported to cause a mobilization
of peroxisomes in vivo (frog) and to
increase catalase activity in cultured
human RPE cells.Z9,31 The most impor¬
tant role of catalase activity in the
RPE may be the prevention of lipid
peroxidation and lysosomal enzyme in¬
hibition by removing H202 from the
phagosome.
Catalase activity has not been quantitated previously in human RPE cells.
In the present study, we compared
catalase activity in the RPE with that
of the retina, iris, and vitreous humor.
We examined SOD and catalase activi¬
ties in human RPE (macular and pe-
2. The peripheral region was defined as
the RPE within the anterior third of the
globe. The RPE (macular and peripheral),
retina, iris, and vitreous samples were
placed in separate polypropylene tubes in a
40-mmol/L TRIS hydrochloride buffer (pH
7.8) with 0.2 mmol/L of pentetic acid (diethylenetriamine-pentaacetic acid), and were
stored at 20°C for no more than 7 days.
Catalase Activity in Human
Ocular Tissue
Tissue Type (n)
Retinal pigment
epithelium (7)
(7)
Erythrocytes (3)
centrifugation (Eppendorf Centrifuge 5415,
Brinkmann Instruments Co, Westbury,
NY) at 13 000 rpm for 10 minutes and the
supernatants containing soluble cellular
Fig 1. —Photomicrograph of the posterior
pole of an eye from a 79-year-old patient with
known macular degeneration. The retina has
been partially removed (arrowheads indicate
cut edge) to reveal the drusen (arrows) in the
central macular region. ON indicates optic
nerve
(original magnification
13).
ripheral) from donors with and without
evidence of MD who ranged in age
from 50 to 90 years.
MATERIALS AND METHODS
Tissue
Human donor eyes were obtained from
the National Disease Research Interchange
(Philadelphia, Pa) within 24 hours of death.
The chorioretinal appearance was assessed
using
stereoscopic dissecting microscope
magnification ( 10). Eyes with dru¬
sen and pigment changes (Fig 1) in the
macular region of the RPE were said to
have MD. Eyes free of macular drusen and
with a normal-appearing fundus and no
known history of ocular disease were classi¬
fied as normal. Each sample was given an
identification number, and the investigator
performing the enzyme determinations was
a
at low
masked
as
to the characteristics of each
sample.
RPE Isolation
After removal of the corneas, lenses,
vitreous, and retinas, the RPE cells were
isolated by scraping with a small spatula
(Moria, Paris, France). Macular and periph¬
eral RPE were isolated separately, with
anatomical distinctions as follows.
1. By our definition, the macular region
was defined as all RPE cells within a circu¬
lar area having a radius from the fovea to
the optic nerve, an area including both the
macula and the surrounding perimacular
RPE. Inclusion of perimacular RPE was
necessary to acquire sufficient protein foienzyme determinations, particularly in
cases of MD.
collected and stored in ali¬
quote of 20 µ at -80°C. Protein content of
the supernatants was determined using a
microassay (Bio-Rad, Richmond, Calif)
with bovine
standard.
serum
albumin (BSA)
as
a
Assay for Catalase Activity
Catalase activity (EC 1.11.1.6) was mea¬
sured by the method of Cohen and col¬
leagues3 with some modifications. This
method depends on the first-order decom¬
position of H202 by catalase and the subse¬
quent measurement of residual H202. The
decomposition of H202 by catalase in our
samples was stopped at exactly 3.0 minutes
after the initiation of the reaction for each
sample by the addition of concentrated sulfuric acid. The concentration of residual
H202 was established by subsequent reac¬
tion with an excess of potassium permanga¬
nate and by measurement of potassium
permanganate concentration at 480 nm on a
spectrophotometer (DU-64, Beckman In¬
struments, Norcross, Ga). To reduce the
quantity of cellular protein needed for cata¬
lase determination, all volumes were re¬
duced by a factor of 25 from Cohen's
procedure.
Samples at a concentration of 25 µg cellu¬
lar protein per milliliter were analyzed in
duplicate, with heat-inactivated catalase
samples (100°C, 5 minutes) as controls.
Serial dilutions of purified catalase of
known activity were used to generate a
standard curve of catalase activity (data not
shown). One unit of catalase activity decom¬
of H202 per minute (pH 7.0,
poses 1.0 µ
25°C). Catalase activity in experimental
samples was based on this standard curve
and expressed in units per milligram of
protein. As a reference point, we used this
method to determine catalase activity in
human erythrocytes and found 163 ±7.5
U/mg of protein.
Assay for SOD Activity
Superoxide dismutase (EC 1.15.1.1) ac¬
tivity was determined using the procedure
of Bensinger and Johnson,1 with lumines¬
cence quantitated via the photon monitor of
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'
13.83 ± 0.69
11.42 ± 0.93
1.11 ± 0.45
162.5 ± 7.45
Retina (7)
Vitreous (7)
Preparation of Intracellular Protein
All chemicals were obtained from Sigma
Chemical Co, St Louis, Mo, unless other¬
wise specified. Tissue samples were soni¬
cated (VirSonic 50, Virtis Co Ine, Gardiner,
NY) at 30% power for 30 seconds in 100 µ ,
of ice-cold 40 mmol/L TRIS hydrochloride
buffer (pH 7.8) with 0.2 mmol/L of pentetic
acid. The tissue sonicates were subjected to
were
82.63 ± 5.93
Iris
-
proteins
Catalase Activity,
U/mg of Protein
liquid scintillation counter (LS5000TD,
Beckman Instruments). This procedure de¬
pends on the ability of SOD to inhibit
Superoxide luminol-generated chemiluminescence. Superoxide radicals were gener¬
ated by the enzymatic reaction of xanthine
oxidase (1.6x10" U/mL) acting on 1.0
mmol/L of hypoxanthine. To initiate the
production of chemiluminescence, xanthine
oxidase was added to a solution of 0.1
mmol/L of luminol in 20 mmol/L of TRIS
hydrochloride buffer (pH 7.8) containing
0.1 mmol/L of pentetic acid and 1 mg/mL of
bovine serum albumin. Purified SOD or
cellular proteins were added to the solution,
and the reduction of measured photon emis¬
sion was expressed as percent inhibition of
luminol chemiluminescence. One unit of
SOD activity was defined as the quantity of
cellular protein or purified enzyme produc¬
ing 50% inhibition of photon emission.
a
Statistical
Analysis
Statistical analyses were performed us¬
ing a correlation analysis on the SAS pro¬
gram (SAS Institute Ine, Cary, NC). Data
on enzyme activity correlated with the de¬
cade of donor age were analyzed using a
paired Student's t test on the SAS program.
RESULTS
Analysis of the catalase activity of
peripheral RPE, retina, iris, and vitre¬
ous from seven nondiseased subjects
revealed that the peripheral RPE had
six times more catalase activity than
any other ocular tissue analyzed (Ta¬
ble). There were statistically signifi¬
cant differences between the levels of
catalase activity in the RPE and in the
iris, retina, and vitreous (P<.0001 for
all comparisons).
There was a consistent decrease by
decade in the catalase activity of both
macular and peripheral RPE (Fig 2).
From the 50- to 60-year age group to
the 80- to 90-year age group, there was
a 57% decrease in macular RPE
(P<.05) and a 38% decrease in periph¬
eral RPE (P<.02). This represents a
1.5-fold greater decrease in macular
RPE catalase activity than in periph¬
eral RPE.
Catalase activity in macular and pe¬
ripheral
RPE was plotted against the
age of each subject (Fig 3). Curves
were generated using a linear regres¬
sion analysis, representing average
Fig 3.—Correlation of catalase activity in macular (left) and peripheral (right) retinal pigment
epithelium (RPE) with donor age. The lines represent the linear regression analysis of normal
(solid lines) and diseased (dotted lines) subjects. Ten of the subjects were normal (circles); 14
had disease (triangles): six, macular degeneration and eight, peripheral disease.
Fig 2.—Catalase activity in macular (shaded
bars) and peripheral (hatched bars) retinal
pigment epithelium (RPE) correlated with do¬
nor age grouped by decade.
values of normal and diseased eyes.
Note that all of the eyes with MD fell
below this normal curve. The slope of
the normal curve is steeper for macu¬
lar (-0.42) than for peripheral RPE
( 0.13), but our data do not indicate a
statistically significant difference in
the rate of decrease depending on ana¬
tomical location.
The difference in catalase activity
between diseased and nondiseased
RPE is depicted in Fig 4. A 32%
reduction in catalase activity was ob¬
served in both macular and peripheral
RPE cells from subjects with MD rela¬
tive to normal donor subjects (macu¬
—
lar, P<.05; peripheral, P<.005).
Cellular proteins from the same do¬
nors analyzed for catalase activity
were used for analysis of SOD activity.
No statistically significant differences
in SOD activity were correlated with
disease status in macular or peripheral
RPE (Fig 5). Neither was there a
correlation between SOD levels and
the age of individual donors (data not
shown).
COMMENT
study repre¬
knowledge,
quantitative analysis of
catalase activity in human RPE cells.
The high levels of catalase activity
To
our
this
sents the first
observed in the RPE relative to the
retina, iris, and vitreous suggest that
catalase is an important component of
the RPE antioxidant system (Table).
This observation coincides with re¬
ports of substantial immunoreactive
catalase activity in rat and bovine
RPE.34
In this study, we found no statisti¬
cally significant differences in total
SOD activity with respect to aging
between the sixth and ninth decades,
MD, or anatomical location. However,
we did not distinguish between the
copper-zinc and manganous forms of
Fig 4.—Catalase activity in macular (shaded
bars) and peripheral (hatched bars) retinal
pigment epithelium (RPE) demonstrating a
decrease in activity correlated with disease
Fig5.—Superoxide dismutase (SOD) activity
(shaded bars) and peripheral
(hatched bars) retinal pigment epithelium
(RPE) divided on the basis of disease status.
in macular
status.
owing to a restriction on the
quantity of protein available for en¬
zyme determinations. The degree of
SOD
variation among individual donors was
much greater for SOD activity than for
catalase activity (compare Figs 4 and
5). It may be significant that when
RPE cells are cultured there is much
less variation among donors in SOD
activities, probably because cell cul¬
ture conditions represent a more uni¬
form environment (unpublished obser¬
vations). This suggests that the
observed variation in SOD activities
was primarily a function of individual
differences and not a technical problem
SOD
method
for
with
the
determination.
Oxidative damage to ocular tissues
from H202 has been shown to be re¬
duced by catalase activity. Bhuyan and
Bhuyan35 induced cataracts in the rab¬
bit by inhibiting catalase activity with
3-aminotriazole, which resulted in a
twofold to threefold increase in H202 in
the aqueous and vitreous humor rela-
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tive to controls. In another study using
rabbits, the effects of treatment with
3-aminotriazole followed by exposure
to H202 produced pathological effects
that were most pronounced in older
animals.36 Furthermore, this greater
susceptibility to H202-induced damage
in older animals was correlated with an
age-related reduction in catalase activ¬
ity in the iris and corneal endothelial
cells (50% and 35%, respectively).
The decrease in catalase activity ob¬
served in RPE cells from the sixth to
the ninth decades of human life is
likely influenced by various factors. An
age-related decrease in quantitated
catalase activity could result from a
decrease in expression of catalase gene
products,87 formation of inactive cata¬
lase complexes,32 or deficiencies in es¬
sential metal cations (eg, iron, zinc,
and copper).38"40 The observed decrease
in catalase activity may also be indica¬
tive of a decrease in the phagocytosis
and/or degradation of ROS in the aging
human RPE.
Our data demonstrate an age-relat¬
ed decrease in catalase activity. This
decrease appears more pronounced in
eyes with MD and occurs within the
same time frame as other age-related
changes in the RPE, such as the accu¬
mulation of lipofuscin. Further studies
are necessary to ascertain the signifi¬
cance of catalase in RPE function and
to determine whether the age-related
reduction of its antioxidant activity
contributes to the clinical manifesta¬
tions of MD.
This study was supported by grant EY-006677
from the National Institutes of Health, Bethesda,
Md (Dr Newsome). Support of the J. Willard
Marriott Foundation, Washington, DC, is also
acknowledged.
We are grateful to Clay Hinrichs for his techni¬
cal expertise in performing statistical analyses.
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