Conversion of 7-ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells

Article abstract of DOI:10.1194/jlr.M014217

Of the different oxygenated cholesterol metabolites, 7-ketocholesterol (7KCh) is considered a noxious oxysterol implicated in the development of certain pathologies, including those found in the eye. Here we elucidated whether sterol 27-hydroxylase cytochrome P450 27A1 (CYP27A1) is involved in elimination of 7KCh from the posterior part of the eye: the neural retina and underlying retinal pigment epithelium (RPE). We first established that the affinities of purified recombinant CYP27A1 for 7KCh and its endogenous substrate cholesterol are similar, yet 7KCh is metabolized at a 4-fold higher rate than cholesterol in the reconstituted system in vitro. Lipid extracts from bovine neural retina and RPE were then analyzed by isotope dilution GC-MS for the presence of the 7KCh-derived oxysterols. Two metabolites, 3β,27-dihydroxy- 5-cholesten-7-one (7KCh-27OH) and 3β-hydroxy-5-cholesten-7-one-26-oic acid (7KCh-27COOH), were detected in the RPE but not in the neural retina. 7KCh-27OH was also formed when RPE homogenates were supplemented with NADPH and the mitochondrial redox system. Quantifications in human RPE showed that CYP27A1 is indeed expressed in the RPE at 2-4-fold higher levels than in the neural retina. The data obtained represent evidence for the role of CYP27A1 in retinal metabolism of 7KCh and suggest that, in addition to cholesterol removal, the functions of this enzyme could also include elimination of toxic endogenous compounds.

Full text of DOI:10.1194/jlr.M014217

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Conversion of 7-ketocholesterol to oxysterol
metabolites by recombinant CYP27A1 and retinal
pigment epithelial cells
†
1,§
§,
Gun-Young Heo,* Ilya Bederman, Natalia Mast,* Wei-Li Liao, Illarion V. Turko, **
2
,
and Irina A. Pikuleva *
†
Departments of Ophthalmology and Visual Sciences,* and Pediatrics, Case Western Reserve University,
§
Cleveland, OH; Institute for Bioscience and Biotechnology Research, Rockville, MD; and
Division Analytical Chemistry,** National Institute of Standards and Technology, Gaithersburg, MD
Abstract Of the different oxygenated cholesterol metabo-
Supplementary key words 3␤,27-dihydroxy-5-cholesten-7-one • 3␤-
hydroxy-5-cholesten-7-one-26-oic acid• isotope dilution gas chromatog-
lites, 7-ketocholesterol (7KCh) is considered a noxious oxy-
sterol implicated in the development of certain pathologies,
including those found in the eye. Here we elucidated whether
sterol 27-hydroxylase cytochrome P450 27A1 (CYP27A1) is
involved in elimination of 7KCh from the posterior part of
the eye: the neural retina and underlying retinal pigment
epithelium (RPE). We first established that the affinities of
purified recombinant CYP27A1 for 7KCh and its endoge-
nous substrate cholesterol are similar, yet 7KCh is metabolized
at a 4-fold higher rate than cholesterol in the reconstituted
system in vitro. Lipid extracts from bovine neural retina and
RPE were then analyzed by isotope dilution GC-MS for the
presence of the 7KCh-derived oxysterols. Two metabolites,
raphy-mass spectrometry
cytochrome P450 27A1
• age-related macular degeneration •
7-Ketocholesterol (3␤-hydroxy-5-cholesten-7-one or
7KCh), a product of cholesterol auto-oxidation and an oxy-
sterol, attracts constant attention because of its deleterious
pro-inflammatory and pro-apoptotic effects in vitro (re-
viewed in Refs. 1–4). However, it still remains to be proven
whether these in vitro properties are of physiological rele-
vance. 7KCh is the most abundant oxysterol of nonenzy-
matic origin in atherosclerotic plaques, indicating that it
may play a role in the initiation and/or development of
atherosclerosis (2, 5). In the eye, 7KCh was found in hu-
man cataracts, but it was not detected in clear lenses (6).
Experimental data suggest that 7KCh disrupts the highly
regulated differentiation program of the lens epithelial
cells, thus compromising lens growth and transparency
3␤,27-dihydroxy-5-cholesten-7-one (7KCh-27OH) and 3␤-
hydroxy-5-cholesten-7-one-26-oic acid (7KCh-27COOH),
were detected in the RPE but not in the neural retina. 7KCh-
2
7OH was also formed when RPE homogenates were sup-
plemented with NADPH and the mitochondrial redox
system. QuantificationsinhumanRPEshowedthatCYP27A1
is indeed expressed in the RPE at 2-4-fold higher levels than
in the neural retina. The data obtained represent evidence
for the role of CYP27A1 in retinal metabolism of 7KCh and
suggest that, in addition to cholesterol removal, the func-
tions of this enzyme could also include elimination of toxic
endogenous compounds.—Heo, G-Y., I. Bederman, N. Mast,
W-L. Liao, I. V. Turko, and I. A. Pikuleva. Conversion of
(
7). Studies on monkeys demonstrate that 7KCh is also
present at very low levels in the neural retina and at
3-5-times higher levels in the retinal pigment epithelium
(RPE)-choriocapillaris region beneath the neural retina
(8). Similarly, low levels of 7KCh were found in the retina
of untreated rats, but exposure to intense, constant light
increased the levels of 7KCh ف6-fold (9). In light-exposed
7
-ketocholesterol to oxysterol metabolites by recombinant
CYP27A1 and retinal pigment epithelial cells. J. Lipid Res.
011. 52: 1117–1127.
2
This work was supported by National Institutes of Health Grants EY-018383
I.A.P.) and AG-024336 (I.A.P.). Its contents are solely the responsibility of the
Abbreviations: 27OH, 27-hydroxycholesterol; 7KCh, 7-ketocholes-
terol;7KCh-27OH,3␤,27-dihydroxy-5-cholesten-7-one;7KCh-27COOH,
3␤-hydroxy-5-cholesten-7-one-26-oic acid; Adr, adrenodoxin reductase;
(
authors and do not necessarily represent the official views of the National Insti-
tutes of Health. I.A.P. is recipient of a Jules and Doris Stein Professorship from
the Research to Prevent Blindness Foundation (New York). Certain commercial
materials, instruments, and equipment are identified in this article to specify the
experimental procedure as completely as possible. In no case does such identifica-
tion imply a recommendation or endorsement by the National Institute of Stan-
dards and Technology (NIST), nor does it imply that the materials, instruments,
or equipment identified are necessarily the best available for the purpose.
Adx, adrenodoxin; CYP27A1, cytochrome P450 27A1; KP , potassium
i
phosphate buffer; PL, phospholipid; RPE, retinal pigment epithelium;
SIM, selective ion monitoring.
1
Present affiliation of W-L. Liao: Expression Pathology, Inc., Rock-
ville, MD 20850.
2
To whom correspondence should be addressed.
Manuscript received 20 January 2011 and in revised form 11 March 2011.
e-mail iap8@case.edu
Published, JLR Papers in Press, March 16, 2011
DOI 10.1194/jlr.M014217
The online version of this article (available at http://www.jlr.org)
contains supplementary data in the form of one figure.
This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 52, 2011
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rats, 7KCh was observed in both the RPE-choriocapillaris
region and neural retina, and in the latter, 7KCh was im-
munolocalized to the regions with high mitochondrial
content, the ganglion cell layer and photoreceptor inner
segments (9). 7KCh is studied in the retina because of the
possible connection to age-related macular degeneration
by CYP27A1 in the reconstituted system in vitro and pres-
ent evidence that this P450 also oxidized 7KCh in vivo in
bovine RPE. Our results suggest that cytochrome P450-
mediated metabolism could be at least one of the mecha-
nisms whereby RPE removes toxic 7KCh, thus expanding our
understanding of the retinal significance of CYP27A1.
(
4), a major cause of blindness in the elderly in industrial-
ized countries (10). Loss of vision in age-related macular
degeneration is caused by degeneration of the neural ret-
ina and RPE in the macula (11), a region near the center
of the retina.
MATERIALS AND METHODS
Materials
Cholesterol and 7KCh were purchased from Steraloids, Inc.
7
KCh is putatively a toxic compound, yet the mecha-
2
(
Newport, RI); [25,26,26,26,27,27,27- H ]cholesterol (choles-
7
nisms whereby it is formed and eliminated in vivo are not
fully understood. Evidence was obtained indicating that
iron is likely involved in generation of 7KCh in the neural
retina (9). The source of iron was not determined, but fer-
ritin and/or cytochrome c are suspected (4). The routes
of 7KCh elimination from the retina are also under inves-
tigation. Four pathways are considered: i) enzymatic oxi-
dation by cholesterol 25-hydroxylase and cytochrome P450
enzymes 27A1 and 46A1 (CYP27A1 and CYP46A1); ii) re-
duction by 11␤-hydroxysteroid dehydrogenase type 1; iii)
efflux involving transporters such as ABCA1 and ABCG5;
and iv) sulfonation by sulfotransferases (reviewed in Ref.
2
terol-D ) and [26,26,26,27,27- H ]27-hydroxycholesterol (27OH-
7
5
D ) were from Medical Isotopes (Pelham, NH); [25,26,26,
5
2
2
6,27,27,27- H ]7KCh (7KCh-D ) was from CDN Isotopes (Que-
7 7
3 3
bec, Canada); [ H]7KCh and [ H]cholesterol were from Ameri-
can Radiolabeled Chemicals, Inc. (St. Louis, MO); 27OH,
7
KCh-27OH and 7KCh-27COOH were from Avanti (Alabaster,
AL). Human recombinant CYP27A1, mitochondrial redox part-
ners adrenodoxin (Adx) and adrenodoxin reductase (Adr), and
microsomal P450 oxidoreductase were expressed and purified
as described (18–21). Bovine eyes were obtained from a local
slaughterhouse 3–5 h after animals were sacrificed. The anterior
segment was removed, and the vitreous humor was poured out to
expose the retina. The neural retina was carefully separated from
the RPE, placed in a tube, and washed one time (by centrifuga-
tion at 2,000 g) with PBS. The remaining eye cup was incubated
4
). Of these four possible pathways, we favor enzymatic
oxidation of 7KCh by CYP27A1, a ubiquitous sterol 27-
hydroxylase with relatively broad substrate specificity and
multiple physiological roles. Indeed, CYP27A1 is involved
in cholesterol elimination from extrahepatic tissues, me-
tabolizes bile acid intermediates in the liver, and activates
2
+
on ice with 5 ml of PBS containing 1 mM EDTA to chelate Ca
and facilitate dissociation of the RPE from the basement mem-
brane (22). In 30 min, the RPE was gently brushed off, aspirated,
and subjected to repeated washes by differential centrifugation
(
23) until the supernatant was clear and colorless and the pel-
vitamin D in the kidney (12–14). CYP27A1 is also a mito-
3
leted RPE cells were essentially free of the contaminating non-
melanotic cells, erythrocytes, and rod outer segments as assessed
by brightfield microscopy. Fluorescent microscopy (excitation at
488 and emission at 515 nm) did not detect autofluorescent rod
outer segments, erythrocytes, or macrophages, thus confirming
the analysis done by light microscopy. The isolated RPE was ei-
ther used immediately or flash-frozen in liquid nitrogen and
stored at Ϫ80°C. Cadaveric human eyes were processed previ-
ously (24). The RPE was gently scraped under the dissecting mi-
croscope with a crescent knife, aspirated with a glass microcapillary
tube, and frozen. Assessment of the purity of these preparations
by brightfield and fluorescent microscopies did not reveal signifi-
cant contaminations: only few nonmelanotic nonautofluorescent
cells of undetermined origin were present. Human tissue use
conformed to the Declaration of Helsinki and institutional re-
view at Case Western Reserve University. Eye specimens were ob-
tained from de-identified donors following informed consent
of their families.
chondrial protein whose immunolocalization in the retina
overlaps with that of 7KCh: photoreceptor inner segments,
ganglion cell layer, and RPE (15). Furthermore, 7KCh was
found to be 27-hydroxylated in human macrophages and
HepG2 cells (16, 17); it was suggested to be the substrate
for CYP27A1 based on the site of hydroxylation, reduction
of its metabolism by the cells treated with several CYP27A1
inhibitors, and absence of 27-hydroxylated 7KCh metabo-
lite in the medium when CYP27A1-deficient human mac-
rophages were incubated with exogenous radioactive
7
KCh (16). These studies, however, did not assess whether
enzymes other than CYP27A1 were also inhibited and
whether CYP27A1 deficiency affected the uptake of exog-
enous radioactive 7KCh by the cultured cells.
CYP27A1 is a strong candidate for being the major en-
zyme eliminating 7KCh in the retina. Yet, in previous stud-
ies by others, hydroxylated products of 7KCh were not
detected either in monkey retina or photodamaged rat
retina when retinal lipid extracts were analyzed by high
performance liquid chromatography (HPLC) coupled to
mass spectrometry (MS) based on atmospheric pressure
chemical ionization (8, 9). Therefore, in the present work,
we investigated retinal metabolism of 7KCh by a highly
sensitive analytical tool, isotope dilution gas chromatogra-
phy-mass spectrometry (GC-MS) with selected ion moni-
toring (SIM) following in vitro studies evaluating 7KCh as
a substrate for purified recombinant CYP27A1. We show
here that 7KCh was efficiently hydroxylated several times
Isolation of mitochondrial phospholipids (PL) from
bovine neural retina and RPE and preparation of PL
vesicles for the enzyme assay
Each tissue (ف5 g) was homogenized with Teflon grinder in 10
ml of 50 mM Tris-HCl (pH 7.4), containing 250 mM sucrose, 5
mM MgCl , 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiotre-
2
itol, 100 ␮g/ml of butylated hydroxytoluene (BHT), and a cock-
tail of protease inhibitors (Roche Complete EDTA-free, 1
tablet/50 ml buffer). Tissue homogenates were subjected to cen-
trifugation at 1,000 g for 20 min to remove unbroken cell, nuclei,
and cell debris followed by centrifugation of the resultant super-
natant at 9,000 g for 20 min to pellet the mitochondria. Mito-
chondria were resuspended in 3 ml of the homogenization buffer
1
118
Journal of Lipid Research Volume 52, 2011

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(
1
ف5 mg protein/ml) with the Tris-HCl concentration reduced to
0 mM, and then mixed with 6 ml of the Folch reagent (chloro-
addition of the NADPH-regenerating system to initiate the en-
zyme reaction, which was carried out for 2 min.
form-methanol, 2:1, V/V) (25) by vigorous vortexing for 30 s.
Phase separation was facilitated by centrifugation at 2,000 g for
Spectral binding assay with purified recombinant human
CYP27A1
10 min; the lower organic phase was removed, placed in a glass
tube, and dried under the nitrogen gas. Subsequent separation
of nonphosphorous lipids and PLs was as described (26). Briefly,
lipid extract was dissolved in 100 ␮l of chloroform and applied
on a dry 690 mg Sep-Pak Silica Cartridge (Waters Corporation,
Milford, MA). The cartridge was first washed with 20 ml of chlo-
roform to elute nonphosphorous lipids and then with 30 ml of
methanol to elute PLs. Both nonphosphorous lipids and PLs
were dried under the nitrogen gas and redissolved in 2 ml of
chloroform. Absence of cholesterol and other nonphosphorous
lipids in the PL fraction was confirmed by thin layer chromatog-
raphy (TLC) on plates with silica gel using hexane/diethyl ether/
methanol/acetic acid (90/20/5/2, V/V/V/V) as resolving mo-
bile phase; bands were detected by iodine. The PL concentration
was measured using ammonium ferrothiocyanate as described
Titrations with cholesterol and 7KCh were carried out at 30°C
as described (28). Sterols were added from 1 mM stock in 4.5%
aqueous 2-hydroxypropyl-␤-cyclodextrin to a 1-ml solution of 50
mM KP , pH 7.2, containing 0.4 ␮M CYP27A1, 1 mM EDTA, 0.5
i
M NaCl, and 10% glycerol. The K values were calculated by non-
d
linear least-squares fitting using the quadratic form of the single-
site binding equation (29):
2
'
A = 0.5 'Amax K +[E]+[S]ꢂ (K +[E]+[S]) ꢂ 4[E][S]
ꢁ (Eq. 1)
ꢀ
d
d
where ⌬A is the spectral response at different substrate con-
centrations [S], ⌬Amax is the maximal amplitude of the spectral
response, and [E] is the enzyme concentration.
(
27). The calibration curve was generated using known amounts
Quantification of sterols in bovine tissues
Fresh neural retina or RPE was weighed and homogenized in
of 1-palmitoyl-2-oleoyl-sn-glycero-3-[phosphor-rac-1(1-glycerol)].
Stock solution of PL vesicles for reconstitution with CYP27A1 was
prepared by evaporating 1 mg of PL solution in chloroform in a
glass tube, then adding 1 ml of 40 mM potassium phosphate buffer
10 vol (w/v) of 40 mM KP , pH 7.2, containing 1 mM EDTA and
i
100 µg/ml BHT. Tissue homogenization was followed by centrif-
ugation at 1,500 g for 15 min to remove cell debris. Supernatant
aliquots (ف15 mg of total protein per tube) were used either for
lipid extraction or enzyme assays. For the former, two tubes were
used, each supplemented with the same amounts of cholesterol-
D (500 nmol) and 27OH-D (1 nmol) but different amounts of
(KPi), pH7.2, containing 1 mM EDTA, vortexing, and placing
the glass tube in an ice-water bath that was sonicated for 15 min
with a Digital Sonifier (Branson, Danbury, CT) at a 50% amplitude.
Enzyme assays with purified recombinant human
CYP27A1
7
5
7KCh-D7 (0.2 or 0.02 nmol) for quantification of 7KCh and
In studies assessing the ability to hydroxylate 7KCh, 0.02 ␮M
or 0.2 ␮M CYP27A1 was reconstituted with 2 ␮M 7KCh, 100,000
cpm of [ H]7KCh, Adx and Adr used at 30- and 4-fold molar
excesses over CYP27A1, respectively, and NADPH-regenerating
system (1 mM NADPH, 1 U of isocitrate dehydrogenase, and
7KCh-27OH, respectively. Lipids were extracted from tissue ho-
mogenates five times by vortexing with 2.5 vol of chloroform/
methanol (2:1, V/V) for 1 min. The chloroform phases were
combined and evaporated to dryness at room temperature under
vacuum using a Savant SC2104 SpeedVac Concentrator (Thermo
Scientific, Ashville, NC). Oxysterols were separated from choles-
terol by solid phase extraction as described (30). Briefly, the
dried lipid extract was dissolved in 10 ml of 70% ethanol and
applied to a Varian C18 SPE column (1000 mg, Varian Inc., Lake
Forest, CA) equilibrated with 30 ml of 70% ethanol. The flow-
through fraction was collected and combined with the eluate
from the subsequent column wash with 40 ml of 70% ethanol.
The combined eluates represented the oxysterol fraction. The
cholesterol fraction was eluted next by washing the column with
20 ml of ethanol. The oxysterol and cholesterol fractions were
evaporated to dryness in a SpeedVac Concentrator and methyl-
ated with 3 ml of freshly prepared ethereal diazomethane at
room temperature for 30 min. After methylation, the fractions
were dried under nitrogen, derivatized with 60 ␮l of bis-
(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (TMS)
at 60°C for 1 h, and subjected to GC-MS.
3
5
mM trisodium isocitrate). Enzymatic reactions proceeded
at 37°C in 1 ml of 40 mM KP buffer, pH 7.4, containing 1 mM
i
EDTA, and were terminated by addition of 5 ml of methanol:
chloroform (2:1, V/V). The two phases were separated by a
1
0 min-centrifugation at 2,000 g; the lower phase was isolated
and dried under nitrogen (25). The dried extract was then dis-
solved in 100 ␮l of acetonitrile:methanol:H O (50:20:30, V/V)
2
and subjected to HPLC using a Shimadzu HPLC system
(
Tokyo, Japan) with a Nova-Pak C18 column (3.9 × 150 mm,
Waters, Millford, MA) connected to a ␤-RAM radioactivity
detector (INUS Systems Inc., Tampa, FL). The flow rate (1 ml/
min) was kept at 100% solvent A (acetonitrile:methanol:water,
5
0:20:30, V/V) for 5 min, then a linear gradient between sol-
vent A and solvent B (100% methanol) over 15 min was used,
after which the flow was kept at 100% solvent B for another
5
min. Peak areas corresponding to 7KCh and its metabo-
lites were integrated and used to calculate the substrate
metabolism.
Saponification was performed on a dried lipid extract as de-
scribed (31). Briefly, 2 ml of 20% KOH in methanol was added
and vortexed to dissolve lipids. Then 2 ml of diethyl ether was
added, followed by vortexing and flushing the solution with ni-
trogen to remove air. The solution was vigorously shaken on ice
for 3 h in a capped glass tube, then the reaction was stopped by
20% acetic acid (2 ml). Hexane (2.5 ml) was added, and the up-
per phase was isolated and dried. The dried extract was dissolved
in 10 ml of 70% ethanol and used for separation on a Varian C18
SPE column as described above.
To identify metabolites of 7KCh, a separate enzyme assay was
carried out, in which only unlabeled 7KCh was used. The concen-
trations of CYP27A1 and 7KCh were 0.2 ␮M and 2 ␮M, respec-
tively, and those of Adx and Adr were 6 ␮M and 0.8 ␮M,
respectively; the reaction time was 5 min. The products were iso-
lated by HPLC, followed by analysis by GC-MS.
To determine the rates of cholesterol and 7KCh hydroxyla-
tion, a third set of experiments was conducted in which 0.05 ␮M
CYP27A1 was added to 1 ml of 40 mM KP , pH 7.4, containing 1
i
GC-MS analysis
mM EDTA, 20 ␮g of PLs, 30 ␮M cold sterol substrate, ف1 nM
3
[
H]labeled sterol substrate (100,000 cpm), 1.5 ␮M Adx, and 0.2
The derivatized samples were analyzed using an Agilent 5973N-
MSD mass spectrometer equipped with an Agilent 6890 gas chro-
matograph. A DB-35ms (Agilent Technologies, Santa Clara, CA)
␮
M Adr. The assay mixture was incubated at room temperature
for 30 min and then at 37°C for additional 10 min followed by the
Metabolism of 7-ketocholesterol by CYP27A1
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capillary column (30 m × 0.25 mm × 0.25 mm) was used for all
analyses. The mass spectrometer was operated in the electron im-
pact (EI) ionization mode. Gas chromatographic conditions
were as follows: 2 µl sample was injected in splitless mode (inlet
was kept at 270°C with the helium flow at 1.0 ml/min) at the
initial 150°C. The oven was first kept at 150°C for 2 min, ramped
at 10°C/min to 310°C, and held for 20 min isothermally. The ion
source filament was operated at 70 eV. The mass detector was set
at 310°C. We utilized both scan (total ion monitoring, m/z range
of 100-700 Da) and SIM modes and the optimal dwell time (10
msec per ion). The following ions (m/z) were monitored in the
This result is consistent with previous studies by us and
others showing multiple oxidations of cholesterol and 5␤-
cholestane-3␣,7␣,12␣-triol by CYP27A1 to yield C27 alco-
hol, C27 aldehyde, and C27 acid and dependence of the
product profile on the enzyme concentration and reac-
tion time (32, 33).
To confirm that 7KCh in the enzyme assay was oxidized
by CYP27A1 at C27, we used GC-MS analyses. An equi-
molar mixture of authentic 7KCh, 7KCh-27OH, 7KCh-
27COOH as well as cholesterol, 27OH, and 5-cholestenoic
acid (27-COOH) was prepared. The sterols were subjected
to methylation and trimethylsilylation, and column and
chromatography conditions were identified that enabled
good separation of all six sterols and high sensitivity of de-
tection (supplemental Fig. IA). The limit of detection of
SIM mode: 368 (cholesterol); 375 (cholesterol-D ); 417 (27OH),
7
4
22 (27OH-D ), and 472 (7KCh); 479 (7KCh-D ); 455, 470, 545,
5
7
and 560 (7KCh-27OH); and 411, 426, 501, and 516 (7KCh-
7COOH). For quantification, calibration curves were generated
2
using a fixed concentration of the internal standard, deuterated
sterol analog, and varying concentrations of the corresponding
unlabeled sterol.
7
KCh-27OH in this study was ف1 pmol/injection in SIM
mode (supplemental Fig. IA, inset), which is higher than
15 pmol of 7KCh-27OH/injection, reported previously
(16).
Following optimization of the conditions of GC-MS,
CYP27A1 was again incubated with 7KCh under condi-
tions that produced comparable amounts of the two me-
tabolites (0.2 µM CYP27A1, 5 min reaction time, Fig. 1D).
The metabolites were isolated by HPLC and analyzed indi-
vidually by GC-MS after methylation and trimethylsilylation.
The HPLC peak putatively corresponding to 7KCh-27OH
Incubations with RPE homogenates
RPE homogenates (ف15 mg of total protein) were incubated
either with 10 mM NADPH or with 10 mM NADPH plus 5 ␮M
Adr and 15 ␮M Adx or with 10 mM NADPH plus 5 ␮M P450 oxi-
doreductase. Incubations were carried out for 60 min at 37°C
with constant shaking. Extraction of lipids and GC-MS analysis
were as described above.
RESULTS
(
retention time 13 min, Fig. 1A, B) eluted as one peak
Enzyme assays with purified recombinant human
CYP27A1 and identification of 7KCh metabolites
during GC (Fig. 2A) at the retention time of the authentic
7KCh-27OH (supplemental Fig. I) with a fragmentation
pattern very similar to that of the authentic standard (sup-
plemental Fig. IB): a prominent molecular ion at m/z 560
(M) and fragment ions at 545 (M-15), 470 (M-90), and 455
(M-90-15) (Fig. 2C). The HPLC peak putatively corre-
sponding to 7KCh-27COOH (retention time 8.5 min, Fig.
1A, B) eluted as three peaks during GC, one major and
two smaller peaks (Fig. 2B) with the retention time of the
major peak (34.8 min) corresponding to that of the au-
thentic 7KCh-27COOH (supplemental Fig. I) and mass
spectrum similar to that of authentic 7KCh-27COOH (sup-
plemental Fig. IB): a prominent molecular ion at m/z 516
and fragment ions at 501 (M-15), 426 (M-90), and 411
(M-90-15) (Fig. 2D). We did not identify the smaller peaks
in Fig. 2B because they were nonsterol compounds. Thus,
similar to endogenous substrates cholesterol and 5␤-
cholestane-3␣,7␣,12␣-triol,7KChcouldbeoxidizedCYP27A1
multiple times, yielding hydroxylated and carboxylated
products. Identification of 7KCh-27COOH and its MS
characterization enabled future search of this metabolite
in biological samples.
As it had not previously been shown that purified
CYP27A1 metabolizes 7KCh, two enzyme concentrations
(
2
0.02 µM and 0.2 µM) and different reaction times (up to
h) were tested in the in vitro reconstituted system, and
then the product formation was assessed by HPLC. At 0.02
µM CYP27A1 and 5 min reaction time, only one product
peak, putatively 7KCh-27OH, was seen during HPLC sepa-
ration (Fig. 1A). Yet two product peaks were observed
when the enzyme reaction proceeded for 2 h (Fig. 1B).
The retention time of the second product peak was earlier
than that of the putative 7KCh-27OH, indicating that it is
the more polar metabolite. Consequently, by analogy with
our previous studies investigating cholesterol as a substrate
for CYP27A1 (32), we hypothesized that the second prod-
uct was 7KCh-27COOH. The kinetics of the formation of
the putative 7KCh-27OH and 7KCh-27COOH in the en-
zyme assay were different (Fig. 1C, D). At low CYP27A1
concentration (0.02 µM), the 7KCh-27OH content in-
creased up to 30 min of reaction time but then started to
decline. In contrast, the amount of 7KCh-27COOH was
constantly increasing and reached the levels of 7KCh-
27OH after 120 min of enzymatic reactions (Fig. 1C). At
10-fold higher CYP27A1 concentration (0.2 µM), 7KCh-
27COOH became a predominant metabolite after only 15
Comparison of 7KCh and cholesterol as substrates for
CYP27A1
Next we determined affinities of purified recombinant
min of the enzymatic reaction. After 60 min, almost all
KCh was converted to 7KCh-27COOH (Fig. 1D). Thus,
CYP27A1 can metabolize 7KCh in vitro and yield different
products depending on the conditions of the enzyme as-
say: higher enzyme concentration and longer reaction
time lead to the formation of the more polar metabolite.
CYP27A1 for 7KCh and cholesterol (Table 1). The K values
d
7
were found to be similar (0.07 ␮M for 7KCh and 0.14 ␮M
for cholesterol), indicating that in the inner mitochon-
drial membrane where CYP27A1 resides and cholesterol
content is low (34), 7KCh could compete with choles-
terol for the CYP27A1 active site. Then we determined the
1
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Fig. 1. Product formation in the incubations of CYP27A1 with 7KCh. A, B: Chromatograms of HPLC sepa-
ration of the products formed after 5 min and 120 min of enzymatic reaction, respectively, when the concen-
tration of CYP27A1 in the enzyme assay was 0.02 ␮M. C, D: Kinetics of the product formation with 0.02 and
0
.2 ␮M CYP27A1, respectively. In all assays, the concentration of 7KCh was 2 ␮M, and the reaction volume
was 1 ml. The results represent the average of triplicate measurements ± SD. The error bars are not seen
because they are smaller than the symbol size.
rate of 7KCh and cholesterol hydroxylation in the in vitro
enzyme assay. Mitochondrial PLs were isolated from bo-
vine neural retina and RPE and used for reconstitution
with CYP27A1 in the presence of saturating substrate and
redox partner concentrations. The conditions of the assay
were optimized for the formation of only one metabolite,
27-alcohol, with the product formation linear with time
and enzyme concentration. Under these steady-state con-
ditions, the rate of 7KCh hydroxylation was about 4-fold
higher than that of cholesterol in both retinal and RPE
Fig. 2. Total ion chromatograms (A and B, black traces) and mass spectra (C and D) of the 7KCh metabo-
lites (after methylation and trimethylsilylation) formed in the enzyme assay (5 min at 37°C) with 0.2 nmol of
purified recombinant CYP27A1 and 2 nmol of 7KCh. The products were isolated by HPLC, and each was
analyzed individually by GC-MS. Red traces show the negative control, in which CYP27A1 was boiled prior to
initiation of the enzyme reaction.
Metabolism of 7-ketocholesterol by CYP27A1
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TABLE 1. Spectral binding properties and enzymatic activity of CYP27A1 against cholesterol and 7KCh
Ϫ1
c
Turnover Number (min
)
a
b
K
d
(␮M)
⌬Amax
Retinal PL
RPE PL
Cholesterol
0.14 ± 0.03
0.07 ± 0.01
0.05 ± 0.01
0.12 ± 0.01
10.56 ± 0.37
40.40 ± 2.51
9.28 ± 0.61
36.28 ± 1.48
7
KCh
a
b
Calculated on the basis of the spectral binding.
Maximal amplitude of the substrate-induced spectral response per nanomol of CYP27A1.
Assessed by enzyme activity assay as described in Material and Methods. The results represent the average of
c
triplicate measurements ± SD.
PLs (Table 1). Along with the binding data, this result sug-
gested that 7KCh has a potential to be a substrate for
CYP27A1 in vivo, and it justified our subsequent search of
lack of a good internal standard. Saponification of the
RPE lipid extract led to significant reduction of the 7KCh-
27OH peak and the disappearance of the 7KCh-27COOH
peak, indicating decomposition of these sterols even
though they were hydrolyzed on ice. Hence, all subsequent
studies have been carried out only on nonsaponified ex-
tracts of RPE.
7
KCh metabolites in bovine neural retina and RPE.
Analysis of retinal and RPE extracts for the presence of
7
KCh and its metabolites
Several precautions were taken to avoid artificial auto-
Three preparations of the RPE were additionally iso-
lated, and all three showed similar but ف5-fold lower con-
tent of 7KCh (ف8 pmol/mg protein) compared with the
first preparation (Fig. 4). Also, no ion peaks correspond-
ing to 7KCh-27OH and 7KCh-27COOH were observed in
these preparations. Therefore, we used these samples and
reconstituted them with NADPH and either mitochon-
drial Adr and Adx, which transfer electrons from NADPH
to all mitochondrial P450s including CYP27A1, or the mi-
crosomal P450 oxidoreductase, which reduces all mi-
crosomal P450s. These experiments were carried out
because tissue disruption and homogenization usually
leads to quick oxidation of endogenous NADPH and, con-
sequently, terminate the P450-mediated enzymatic reac-
tions. These reactions, however, could be reinitiated if the
homogenate is supplemented with NADPH and the P450
redox partners that may be at limiting amounts in a bio-
logical sample.
oxidation of cholesterol to 7KCh during the workup of
biological samples (35). Only tissues immediately isolated
from fresh bovine eyes (3-5 h postmortem according to
the slaughterhouse) were used, and BHT and EDTA were
always included in the buffer for tissue homogenization.
Lipid extracts were always evaporated at room tempera-
ture under vacuum, followed by the rapid separation of
the oxysterol fraction from cholesterol by using solid-
phase extraction. To ensure high accuracy of the measure-
ments, the best possible internal standard, 7KCh-D , was
7
used because its extraction and derivatization efficiency is
very similar to that of endogenous 7KCh. Also, to deter-
mine interpreparation variability, pooled samples of the
neural retina and RPE were prepared four times, each
time from a group of animals with different demographics
(
age, breed, and ratio of bulls to cows), fed either grass or
grain, and sacrificed at two different slaughterhouses.
The first preparation of the neural retina and RPE was
analyzed for free (unconjugated) and total (conjugated)
forms of sterols. The detector was programmed in a SIM
Incubations with RPE homogenates
The ion peaks characteristic for 7KCh-27OH were only
observed in the ion chromatograms of the extracts from
the incubations with NADPH and a mitochondrial re-
dox system (Fig. 4). The 7KCh-27OH concentration (ف1
pmol/mg protein) was similar in all three preparations.
No ion peaks characteristic for 7KCh-27OH were detected
in the extracts from control incubations lacking NAPDH
and a redox system or from incubations with NADPH and
a microsomal redox partner P450 oxidoreductase (Fig. 4).
Thus, the RPE can metabolize 7KCh to 7KCh-27OH, and
this enzymatic reaction is specific to a mitochondrial P450.
To confirm that CYP27A1 is active under the experimental
conditions used, we measured its cholesterol hydroxylase
activity and the levels of 27OH formed from cholesterol.
The concentration of 27OH increased ف260-fold after ini-
tiation of the enzymatic reaction with NADPH and Adr
and Adx (2 versus 521 pmol/mg protein), and to a much
lesser extent, ف5-fold and 3-fold, respectively, in the pres-
ence of NADPH and NADPH plus the microsomal redox
partner P450 oxidoreductase (Fig. 4). Thus, even when
the concentration of cholesterol in the RPE homogenate
was four orders of magnitude higher than that of 7KCh,
mode for the ions characteristic for 7KCh, 7KCh-D , 7KCh-
7
2
7OH, and 7KCh-27COOH. In the neural retina, only mo-
lecular ions for 7KCh-D (m/z 479) and 7KCh (m/z 472)
7
were observed (Fig. 3A), which eluted as two major peaks
at the retention time of the authentic 7KCh-D and 7KCh,
7
respectively. In this preparation, the concentration of free
7
KCh in the neural retina was 25 pmol/mg protein, and it
remained the same after cold saponification, indicating
lack of esterification. Ion peaks for free 7KCh were also
present in the RPE (Fig. 3B) but at a concentration higher
than that in the neural retina (41 pmol/mg), with saponi-
fication leading to further increase in the level of the
sterol to 51 pmol/mg. Unlike the neural retina, the nonsa-
ponified extract of RPE also contained four ion peaks
characteristic for 7KCh-27OH (m/z 455, 470, 545, and 560)
and 7KCh-27COOH (m/z 411, 426, 501, and 516), which
coeluted for each metabolite at the retention times of the
authentic standards (Fig. 3B). The content of 7KCh-27OH
was estimated based on the calibration curve using 7KCh-
D as internal standard and found to be 1 pmol/mg pro-
7
tein. We did not quantify 7KCh-27COOH because of the
1
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Fig. 3. SIM chromatograms of the oxysterol frac-
tions (after methylation and trimethylsilylation) iso-
lated from the neural retina (A) and RPE (B). Each
ion trace is shown in different color: m/z 479
(
black), m/z 472 (red), m/z 560 (blue), m/z 545 (ma-
genta), m/z 470 (orange), m/z 455 (gray), m/z 516
green), m/z 411 (cyan), m/z 426 (violet), and m/z
01 (brown). The insets show enlarged views of the
(
5
chromatograms at the retention times corresponding
to the elution of 7KCh-27OH and 7KCh-27COOH.
CYP27A1 was able to metabolize 7KCh, yielding 7KCh-
7OH. The latter is likely because mitochondria were not
significantly disrupted during homogenization and the ra-
tio between cholesterol and 7KCh in the inner mitochon-
drial membrane was significantly different from that in the
homogenate. Incubations with the homogenates provide
additional evidence for the involvement of CYP27A1 in
the metabolism of 7KCh in the RPE.
2
Fig. 4. Changes in sterol levels after bovine RPE homogenate was incubated with the components of the
cytochrome P450 redox systems. Vertical bars (open, gray, black, and checkered) show the concentrations of
cholesterol (Chol), 27OH, 7KCh, and 7KCh-27OH. The results represent the average of the triplicate mea-
surements of each of the three RPE preparations ± SD. Where not shown, error bars are smaller than symbol
size. The inset shows SIM chromatogram confirming the formation of 7KCh-27OH in the incubations with
the mitochondrial redox system (5 ␮M adrenodoxin reductase, 15 ␮M adrenodoxin, and 10 mM NADPH, 1
h, 37°C). Coloring of the traces is the same as in Fig. 3.
Metabolism of 7-ketocholesterol by CYP27A1
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Concentrations of CYP27A1 in the RPE
lizes 7KCh and oxidizes this sterol ف4-fold more efficiently
than cholesterol, despite a similar affinity for 7KCh and
cholesterol. These data served as the biochemical basis for
the search of the C27-hydroxylated metabolites of 7KCh in
the retina.
Quantifications were carried out as described (24) using
multiple reaction monitoring and individual samples from
four human donors. These samples were isolated during
the course of our previous studies in which we measured
CYP27A1 expression in the neural retina; in the present
work, we determined the CYP27A1 levels in the RPE. De-
pending on the donor, the enzyme concentration per mil-
ligram of total protein was 2- to 4-fold higher in the RPE
than in the neural retina (Table 2), thus confirming that
CYP27A1 is indeed expressed in the RPE and providing an
explanation as to why we did not detect metabolites of
From previous studies by others (8, 9) we knew that
7
KCh is present in the retina but its hydroxylated deriva-
tives were not detected by HPLC-MS with atmospheric
pressure chemical ionization mode. We also knew that
7
KCh is one of the most difficult oxysterols to measure
because even minor auto-oxidation of highly abundant
cholesterol during sample isolation and workup will lead
to falsely high levels of 7KCh and overestimation of its con-
tent by as much as a 1,000-fold (35). We were also aware
that to accurately quantify 7KCh, as well as any other ste-
rol, its deuterated analog should be added to a biological
sample prior to the beginning of any manipulations. There-
fore, to assay bovine neural retina and RPE for 7KCh and
its C27-oxygenated metabolites, we employed a highly sen-
sitive and accurate GC-MS methodology with SIM detection,
used deuterated 7KCh as internal standard, and main-
tained anaerobic conditions during sample processing.
The measured levels of free 7KCh were in a low picomo-
lar range; they were 1.6-fold higher in the RPE (41 pmol/
mg protein) than in the retina (25 pmol/mg of protein)
as assessed by the measurements in one preparation. Simi-
lar to cholesterol and oxysterols formed enzymatically
7
KCh in the neural retina. For each GC-MS injection, the
same amount of total protein was used to extract lipids ei-
ther from the neural retina or RPE. In the RPE, the levels
of 7KCh metabolites were close to the limits of detection.
Hence, in the neural retina, where both 7KCh and
CYP27A1 are present at concentrations lower than in the
RPE (per mg of protein), less 7KCh metabolites are likely
generated, and these amounts may be below the limits of
detection of the utilized protocol. Thus, our study does
not exclude metabolism of 7KCh by CYP27A1 in the neu-
ral retina. Instead, it suggests that a higher sensitivity is
required to investigate metabolism of 7KCh in this ana-
tomical element. Note that normalizations based on total
protein do not reflect local concentrations of CYP27A1 in
the neural retina and RPE. The neural retina is a multilay-
ered and multicellular organ with uneven expression of
CYP27A1 as assessed by immunohistochemistry (15),
whereas the RPE represents one layer of the same cell
type. Therefore, concentrations of CYP27A1 in some reti-
nal cells could be comparable to those in the RPE.
(
7
36), saponification did not significantly increase the
KCh content either in the neural retina or RPE, indicat-
ing that sulfonation or other types of esterification are un-
likely to be the major mechanisms by which retinal cells
eliminate or reduce the toxicity of this metabolite. These
data are consistent with undetectable retinal levels of
mRNA for sulfotransferase 2B1b (4), one of the enzymes
that could sulfonate 7KCh (37).
DISCUSSION
A notable difference between oxysterols generated en-
zymatically (36) and the nonenzymatically formed 7KCh
was a significant interpreparation variability in the levels of
the latter. Of the four preparations tested, a 40 picomo-
lar/mg protein concentration of 7KCh was detected only
in one preparation of the RPE, whereas the other three
preparations contained similar and much lower levels of
7KCh (ف8 pmol/mg protein). A 5-fold variation in the
content of 7KCh in the RPE could be due to differences in
animal housing, feeding, demographics, and duration of
This study was undertaken to further elucidate choles-
terol metabolism in the retina and the role of CYP27A1 in
this process. Previously we measured retinal and the RPE
content of cholesterol and sterol metabolites generated
enzymatically (36). In the present work, we focused on
7
KCh, the nonenzymatic product of cholesterol oxidation,
and investigated its possible elimination by CYP27A1. By
using in vitro assays, we demonstrated for the first time
that purified recombinant CYP27A1 binds and metabo-
TABLE 2. Quantification of CYP27A1 in human RPE
a
Donor (pmol/mg tissue protein)
CYP27A1 peptide
#12
#13
#17
#20
RPE
LYPVVPTNSR
VVLAPETGELK
EIEVDGFLFPK
Consensus
1.45 ± 0.12
1.30 ± 0.16
1.65 ± 0.32
1.46 ± 0.24
2.06 ± 0.12
1.90 ± 0.23
2.08 ± 0.20
2.01 ± 0.18
1.11 ± 0.33
1.15 ± 0.11
1.26 ± 0.12
1.17 ± 0.20
1.61 ± 0.23
1.39 ± 0.10
1.72 ± 0.05
1.57 ± 0.19
b
Neural retina
Consensus
0.46 ± 0.04
0.51 ± 0.05
0.57 ± 0.05
0.53 ± 0.06
a
The concentration was calculated for three experimental replicates by monitoring three transitions per
individual peptide and presented as mean ± SD. For the consensus, the data for three peptides from CYP27A1 were
combined and presented as mean ± SD. The monitored transitions are the same as in Ref. 24.
b
Taken from Ref. 24.
1
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exposure to sunlight. Studies of the photodamaged rat
retinas demonstrated that the levels of 7KCh are increased
compared with the 27COOH/cholesterol ratio may reflect
either more efficient metabolism of 7KCh relative to cho-
lesterol or less efficient elimination of 7KCh-27OH rela-
tive to 27COOH. The former would be in agreement with
the results of our in vitro studies evaluating cholesterol
and 7KCh as the substrates for CYP27A1, as well with prior
investigations by others showing cytotoxic, pro-inflamma-
tory, and apoptotic effects of 7KCh on the RPE-derived
cells (4, 15, 44). 27-Hydroxylation seems to reduce toxicity
of 7KCh as demonstrated by survival of the RPE cells
treated with 7KCh-27OH at concentrations and time in
which 7KCh essentially killed the entire culture (15). Thus,
high 7KCh-27OH/7KCh ratio in a cell would be consistent
with attenuated toxicity of 7KCh-27OH relative to 7KCh.
Indeed, if a metabolite is as toxic as the parent compound,
it will not be accumulated at comparable levels in a cell.
The ability of the RPE to metabolize 7KCh was also con-
firmed in the incubations with the homogenates from the
preparations where the 7KCh content was low (ف8 pmol/
mg protein) and its metabolites could not be detected by
GC-MS. 7KCh-27OH was formed only when the homoge-
nates were supplemented with the mitochondrial P450 re-
dox system. In these experiments, ف1/5 of 7KCh was
converted to 7KCh-27OH, but only ف1/200 of cholesterol
was converted to 27OH. More efficient metabolism of
7KCh relative to cholesterol suggested that CYP27A1
should be seriously considered as a contributor to 7KCh
elimination from the RPE. Yet studies of other possible
elimination pathways are required to assess quantitative
significance of 27-hydroxylation in the overall metabolism
of 7KCh in the RPE. Also, it is important to identify the
carriers of 7KCh-derived oxysterols inside and outside the
cell. Oxysterol-binding protein was found to interact with
7KCh and 27OH (45, 46), and oxysterol-binding protein-
related protein 2 was found to bind 7KCh (47). 27OH and
27COOH were shown to spontaneously diffuse across
plasma membranes and become associated outside the
cell either with HDL (27OH) or albumin (27COOH) (48,
49). Conceivably, upon exit from the mitochondrion,
7KCh-27OH could interact with one of the oxysterol trans-
port proteins and be delivered to the plasma membrane,
which it quickly traverses, followed by complex formation
with either HDL or albumin. This mechanism would be
concordant with the finding that the neural retina and
RPE express many proteins necessary for HDL lipid trans-
port, for example, apolipoprotein A1 (50), the major com-
ponent of HDL. Immunohistochemistry localization on
monkey retina found apolipoprotein A1 in Bruch’s mem-
brane, which represents the interface between the RPE
and choroid, as well as on the apical side of the RPE (this
side faces the interphotoreceptor matrix and neural ret-
ina) and in the interphotoreceptor matrix surrounding
the photoreceptor outer segments (50). Thus, 7KCh me-
tabolites can be accepted by HDL on either side of the
RPE. Consequently, they can diffuse from the RPE through
either apical or basal sides. Yet passive diffusion usually re-
quires a concentration gradient between the donor cell
membrane and the extracellular acceptor. Because of the
intensity of the choroidal blood flow, the HDL-7KCh me-
6
-fold when animals are exposed to intense, constant light
(
9). The first preparation of the neural retina and RPE was
the only one obtained in late spring, whereas the other
three preparations were isolated throughout autumn. De-
spite interpreparation variability, a range of the 7KCh con-
centrations in our study was still significantly lower than
that reported in monkey and rat tissues: 0.21 pmol/nmol
of free cholesterol in bovine neural retina versus 1-1.6
pmol/nmol of free cholesterol in untreated monkey and
rat retinas; and 0.07-0.36 pmol/nmol of free cholesterol in
bovine RPE versus 5-8 pmol/nmol of free cholesterol in
monkey RPE-choriocapillaris (8, 9). These differences
could represent interspecies variations and be pertinent to
the way the quantifications were carried out. In our study,
sterol concentrations were determined by plotting the ra-
tio of the ion peak areas (endogenous sterol/deuterated
internal standard) against the calibration generated sepa-
rately for each sterol of interest. In sterol quantifications
in monkeys and rats, the concentrations were derived from
the peak areas specific to cholesterol and 7KCh after nor-
malization to the peak area of the internal standard
␤-sitosterol (8, 9). In any case, the range of the 7KCh con-
centrations in bovine neural retina and RPE (0.07-0.36
pmol/nmol cholesterol) was more comparable to the
7
KCh levels in hamster brain (ف0.05 pmol/nmol choles-
terol) (38) with higher concentrations in the neural retina
possibly reflecting its unique oxidative environment (re-
viewed in Ref. 4).
Increased levels of 7KCh in the first preparation of bo-
vine RPE enabled the detection of its metabolites, 7KCh-
2
7OH and 7KCh-27COOH, whose identification was based
on the coelution of four fragment ions specific for 7KCh-
7OH and 7KCh-27COOH at the retention times of the
2
authentic standards. The sites of oxygenation in 7KCh as
well as expression in the RPE strongly suggest that 7KCh-
2
7OH and 7KCh-27COOH are the enzymatic products of
CYP27A1, consistent with our in vitro studies using puri-
fied recombinant enzyme. Furthermore, it is conceivable
that 7KCh could be formed in the mitochondrion because
the mitochondrial respiratory chain is known to produce
reactive oxygen species, which in turn can oxidize choles-
terol to yield 7KCh and other products (39, 40). Recent
studies also suggest that in the retina cholesterol oxidation
could involve ferritin, which colocalizes with 7KCh (9).
Ferritin was shown to release bound iron upon light expo-
sure (41) and induce lipid peroxidation in photoreceptor
outer segments (42). This mechanism is supported by
identification of a new form of ferritin, mitochondrial fer-
ritin, which localizes in the mitochondrial matrix (43).
The steady-state levels of 7KCh-27OH in bovine RPE were
ف1/40 of those of 7KCh, and the metabolite to substrate
ratio would presumably have been even higher if we could
have quantified 7KCh-27COOH. But even when assessed
based on 7KCh-27OH, this ratio is more than two orders of
magnitude higher than the ratio of the other CYP27A1
metabolite and substrate pair, 27COOH and cholesterol
(
ف1/14,000) (38). Such high 7KCh-27OH/7KCh ratio
Metabolism of 7-ketocholesterol by CYP27A1
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tabolite complex will be removed more quickly from the
basal side of the RPE than from the apical. Therefore, we
hypothesize that the majority of 7KCh metabolites will dif-
fuse through the basal side of the RPE. With respect to
physiological relevance, if CYP27A1 is indeed important
for elimination of toxic 7KCh from the RPE, CYP27A1 de-
ficiency should lead to accumulation of 7KCh and may af-
fect normal retinal function. Experiments are underway in
this laboratory to investigate whether accumulation of
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