Generation of Cholesterol Carboxyaldehyde by the Reaction of Singlet

Chem. Res. Toxicol. 2009, 22, 875–884

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Generation of Cholesterol Carboxyaldehyde by the Reaction of Singlet Molecular Oxygen [O2 (1∆g)] as Well as Ozone with Cholesterol Miriam Uemi, Graziella E. Ronsein, Sayuri Miyamoto, Marisa H. G. Medeiros, and Paolo Di Mascio* Departamento de Bioquímica, Instituto de Química, UniVersidade de Saõ Paulo, CP26077, CEP 05513-970, Saõ Paulo, SP, Brazil ReceiVed NoVember 24, 2008

A few years ago, it was reported that ozone is produced in human atherosclerotic arteries, on the basis of the identification of 3β-hydroxy-5-oxo-5,6-secocholestan-6-al and 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (ChAld) as their 2,4-dinitrophenylhydrazones. The formation of endogenous ozone was attributed to water oxidation catalyzed by antibodies, with the formation of dihydrogen trioxide as a key intermediate. We now report that ChAld is also generated by the reaction of cholesterol with singlet molecular oxygen [O2 (1∆g)] that is produced by photodynamic action or by the thermodecomposition of 1,4-dimethylnaphthalene endoperoxide, a defined pure chemical source of O2 (1∆g). On the basis of 18O-labeled ChAld mass spectrometry, NMR, light emission measurements, and derivatization studies, we propose that the mechanism of ChAld generation involves the formation of the well-known cholesterol 5R-hydroperoxide (5R-OOH) (the major product of O2 (1∆g)-oxidation of cholesterol) and/or a 1,2-dioxetane intermediate formed by O2 (1∆g) attack at the ∆5 position. The Hock cleavage of 5ROOH (the major pathway) or unstable cholesterol dioxetane decomposition (a minor pathway, traces) gives a 5,6-secosterol intermediate, which undergoes intramolecular aldolization to yield ChAld. These results show clearly and unequivocally that ChAld is generated upon the reaction of cholesterol with O2 (1∆g) and raises questions about the role of ozone in biological processes. Introduction 1

Cholesterol (cholest-5-en-3β-ol; Ch ) is a neutral lipid found in all membrane compartments of mammalian cells. Like all unsaturated lipids, cholesterol is susceptible to oxidation in the presence of reactive oxygen species (ROS), giving rise to a variety of potentially mutagenic and cytotoxic species (1-4). Lipid peroxidation (5, 6) is a good example of ROS-induced oxidative damage to unsaturated membrane lipids, which has been linked to neurodegenerative disease, cardiovascular diseases, and aging (7-12). These modifications may be triggered by free radical species such as peroxyl, oxyl, or hydroxyl radicals (13) or by nonradical species such as singlet molecular oxygen [O2 (1∆g)], ozone (O3), and peroxynitrite (13-15). Ozonization of cholesterol has been shown to form several products, including 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (CSec) (the major product) and 3β-hydroxy-5β-hydroxy-Bnorcholestane-6β-carboxaldehyde (ChAld) (16, 17). In 1992, Pryor et al. (18) identified these cholesterol oxidation products in rat lung tissue after exposing the animals to O3 by using a derivatization reaction with 2,4-dinitrophenylhydrazine (DNPH). Since then, these hydrazone derivatives have been used as biomarkers for O3 (19, 20). Babior et al. (21) proposed that neutrophils generate ozone by catalytic conversion of singlet molecular oxygen to ozone. They suggested that this toxic ROS contributes to the antimi* To whom correspondence should be addressed. Tel: (55) (11) 30913815 (ext. 224). Fax: (55) (11) 38155579. E-mail: [email protected]. 1 Abbreviations: O2 (1∆g), singlet molecular oxygen; Ch, cholesterol; CSec, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al; ChAld, 3β-hydroxy-5βhydroxy-B-norcholestane-6β-carboxaldehyde.

crobial and inflammatory actions of these cells based on evidence of chemistry of ozone reacting with indigo blue carmine to produce insatin sulfonic acid (22). Later, Wentworth et al., using mass spectrometry measurements, inferred the formation of ozone in atherosclerotic plaques by the presence of the Csec and ChAld derivatives as their 2,4-dinitrophenylhydrazones (20). Recently, these compounds were also related to Alzheimer’s disease since they were found in human brain tissue and were shown to initiate protein misfolding of amyloid β peptide in Vitro (23, 24). Csec and ChAld can also accelerate R-synuclein fibrilization, which has been associated with Parkinson’s disease and Lewy body dementia (25). Smith (26) proposed that CSec and ChAld can be generated by O2 (1∆g) attack on cholesterol via decomposition of the 1,2dioxetane intermediate formed at the ∆5 bond. Thus, the reaction of O2 (1∆g) as well as O3 with cholesterol might give these products. Our goal in this study was to determine whether CSec and ChAld can, in fact, be generated by O2 (1∆g) attack on cholesterol. O2 (1∆g) is commonly produced by dye-sensitized photooxidation reactions (27, 28). Type I (free radical-mediated) and type II (singlet dioxygen-mediated) photooxidation of cholesterol have been well documented, and specific cholesterol hydroperoxide intermediates have been identified as primary products in each case (1, 28, 29) (Figure 1). In type I photooxidation reactions, the epimeric pair 3β-hydroxycholest5-ene-7R-hydroperoxide (7R-OOH) and 3β-hydroxycholest-5ene-7β-hydroperoxide (7β-OOH) is generally the most prominent hydroperoxide product, with minor amounts of the dihydroxy derivatives 7R-OH and 7β-OH being formed, along with 7-ketone (7-one), and epimeric 5,6-epoxides (1, 28, 30). In type II photooxidation reactions, only three primary hydro-

10.1021/tx800447b CCC: $40.75  2009 American Chemical Society Published on Web 04/10/2009

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Figure 1. Primary products of cholesterol photooxidation.

Scheme 1. Thermal Decomposition of 1,4-Dimethylnaphthalene Endoperoxide (DMNO2) to O2 (1∆g) and the Corresponding 1,4-Dimethylnaphthalene (DMN)

peroxides of cholesterol have been identified: 3β-5R-cholest6-ene-5-hydroperoxide (5R-OOH), 3β-hydroxycholest-4-ene-6Rhydroperoxide (6R-OOH), and 3β-hydroxycholest-4-ene-6βhydroperoxide (6β-OOH), with the yield of 5R-OOH typically being the highest (28, 29, 31). Although O2 (1∆g) does not produce 7R-OOH and 7β-OOH, this pair can arise via allylic rearrangement of 5R-OOH (32-34) (Figure 1). In addition to these products, we have now established that ChAld is formed during photooxidation reactions with different photosensitizers and also by reaction of cholesterol with a defined pure chemical source of O2 (1∆g), which is generated by thermal decomposition of 1,4-dimethylnaphthalene endoperoxide (DMNO2) (35, 36) (Scheme 1).

Materials and Methods Materials. Cholesterol (cholest-5-en-3β-ol) was obtained from Sigma (St. Louis, MO). Silica gel 60 (230-400 mesh), 2,4dinitrophenylhydrazine (DNPH), methylene blue, rose bengal, and 5,10,15,20-tetra-(4-pyridyl)-21H,23H-porphine (porphyrin) were purchased from Aldrich (Steinheim, Germany). Deuterium chloroform (CDCl3) and deuterium benzene (C6D6) were supplied from Cambridge Isotope Laboratories (Rio de Janeiro, Brazil). Methanol and all other solvents were of HPLC grade and were acquired from Merck (Rio de Janeiro, Brazil). The 18O2 gas cylinder (99% 18O) came from Isotec-Sigma (St. Louis, MO). The water used in the

Uemi et al. experiments was treated with the Nanopure Water System (Barnstead, Dubuque, IA). Synthesis of 3β-Hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (ChAld) by the Photooxidation of Cholesterol. ChAld was synthesized by the photooxidation of cholesterol using different sensitizers. Cholesterol (200 mg) was dissolved in 20 mL of chloroform using a 100 mL round-botton flask, and 250 µL of methylene blue solution (10 mM in methanol) was added. Under continuous stirring and in an oxygen-saturated atmosphere, the solution was cooled at 4 °C and irradiated using two tungsten lamps (500 and 300 W) for 2.5 h. The ChAld sample was purified from cholesterol and its hydroperoxides by flash column chromatography, using silica gel 60 (230-400 mesh). The column was equilibrated with hexane, and then a gradient of hexane and ethyl ether was used. Preparative thin-layer chromatography (TLC) was performed on ChAld using Merck 0.25 mm coated silica gel Kieselgel 60 F254 plates; elution was performed with a solution of ethyl acetate and isooctane (1:1). The same conditions were used for two others sensitizers: rose bengal (final concentration of 100 µM) and porphyrin (final concentration of 350 µM). Photooxidation of Cholesterol in an 18O2-saturated Atmosphere. Briefly, the same procedure described above was followed, although the oxygen in the system was removed by successive freezing and thawing under vacuum. This procedure was repeated at least five times to ensure the complete removal of 16O2. Thereafter, the whole system was connected to an 18O2 gas cylinder under 0.5 atm. Ozonization of Cholesterol. The ozonization of cholesterol was carried out using an Aquazone instrument plus (Red Sea Fish Pharm Ltd., Eilat, Israel) to generate ozone in a 10 mM solution of cholesterol in CHCl3 under a flow of 10 mL/min for 10 min without reduction with zinc. Reaction of Cholesterol with O2 (1∆g) Generated by 1,4-Dimethylnaphthalene Endoperoxide. For this study, 19 mg of DMNO2 endoperoxide and 98 mg of cholesterol were dissolved in 600 µL of CCl4 (final concentrations of 0.5 and 0.2 M, respectively) in a 7 mm NMR tube closed under constant stirring at 50 °C for 4 h. Aliquots of 150 µL were removed after 2 and 4 h of reaction. CCl4 was removed under argon flow and dissolved in EtOH (final concentrations approximately 5 and 2 mM of cholesterol and DMNO2, respectively) to be used in derivatization reactions using DNPH. NMR Spectroscopy Analysis of ChAld. The isolated ChAld was analyzed by NMR using a DRX500 instrument, Avance series (Bruker-Biospin, Rheinstetten, Germany), operating at 11.7 T. The instrument was equipped either with a 5-mm direct detection probe (13C, HETCOR spectra) or a 5-mm trinuclear, inverse detection probe with z-gradient (TXI) (1H and 2D spectra). Temperature was controlled by a BVT2000 accessory. All chemical shifts were expressed in ppm relative to the deuterium solvent or TMS, and data were acquired and processed using TOPSIN 1.3 (BrukerBiospin, Rheinstetten, Germany). 1H NMR (500.13 MHz) and 13C NMR (125.77 MHz) spectra were recorded at 7 and 25 °C, using CDCl3 or C6D6. The 1H NMR spectrum was acquired with a spectral width of 6830.6 Hz (≈13.6 ppm) and 64 K data points, providing a digital resolution of 0.10 Hz. For 13C NMR spectra, a spectral width of 32679.74 Hz (≈260 ppm) and 64 K data points were set, providing a digital resolution of 0.50 Hz (see Supporting Information). Mass Spectrometry Analysis of ChAld. ChAld was analyzed by mass spectrometry using an Esquire Plus 3000 (Bruker Daltonik Inc., Billerica, MA) in the positive electrospray ionization mode. For the analysis, a 100 µM solution of ChAld in methanol and water (9:1, v/v) was injected directly through the instrument at a flow rate of 160 µL/h. The flow rate of drying gas was kept at 4.0 L/min, and the nebulizing gas pressure was maintained at 82.7 kPa. The capillary potential was set to 4.5 kV, the source temperature was set to 250 °C, and the capillary current was set to 4.9 nA. Comparative HPLC/MS/MS analyses of unlabeled and 18Olabeled ChAld were carried out in a Shimadzu HPLC system (Tokyo, Japan) coupled to a Quattro II mass spectrometer (Micro-

Cholesterol Oxidation by O2 (1∆g) mass, Manchester, UK) with a Z-spray source. The system was equipped with a 250 × 4.60 mm (particle size 5 µm) C18 reversephase column (Phenomenex). The separation was carried out using isocratic elution with 2% formic acid (0.1%) in acetonitrile at a flow rate of 1.0 mL/min. The eluant was monitored at 210 nm. A small fraction of the eluant was directed into the mass spectrometer at a flow rate of 150 µL/min. MS/MS analyses were performed with the atmospheric pressure chemical ionization (APcI) source in the positive ion mode. The source and APcI probe temperatures were maintained at 150 and 500 °C, respectively. The optimal flow rates of the drying and nebulizing gases were found to be 300 L/h and 15 L/h, respectively. The corona voltage was adjusted to 4 kV. The cone voltage was set at 25 V and the collision energy at 10 eV. Full-scan data were acquired over a mass range of 100-500 m/z. A relative quantification was performed with ChAld photooxidized with 18O atoms using multiple reaction monitoring (MRM) analyses. For this purpose, unlabeled, mono- and double-labeled ChAld were estimated using their specific fragmentation transitions: 419 to 383 (unlabeled), 421 to 383 (labeled with one 18O atom), and 423 to 385 (labeled with two 18O atoms). Derivatization Using DNPH. For the isolated ChAld, 100 µL of a solution of ChAld (5 mM in EtOH) was introduced into an amber glass bottle. Then, 800 µL of EtOH and 100 µL of 5 mM DNPH solution previously prepared in 1 M HCl were added. The solution was bubbled under argon flow for 5 min, and the reaction was conducted at 37 °C for 2.5 h. The products of cholesterol photooxidation generated in the presence of different sensitizers (methylene blue, rose bengal, and porphyrin) were also analyzed. After each photooxidation reaction (100 mg of cholesterol), CHCl3 was removed under reduced pressure, and the residue was dissolved in 10 mL of hexane and ethyl ether (1: 1, v/v). A 100 µL aliquot of each solution was removed and diluted in 2 mL of hexane and ethyl ether (1: 1, v/v), and a small amount of silica gel previously dried at 120 °C for 1 h was added to remove the sensitizer. The solution was filtered, the solvent was removed under nitrogen flow, the solid was dissolved in 900 µL of EtOH in an amber glass bottle, and 100 µL of 5 mM solution of DNPH (previously prepared in 1 M HCl) was added. The solution was bubbled under argon flow for 5 min, and the reaction was conducted at 37 °C for 2.5 h. After the ozonization reaction, an aliquot of 100 µL was removed and transferred into an amber glass bottle. CHCl3 was removed under nitrogen flow, and the residue was dissolved in 900 µL of EtOH. Later, this solution was reacted with 100 µL of 5 mM solution of DNPH as described previously. For the reaction products of the cholesterol with DMNO2, an aliquot of 100 µL (final concentrations of cholesterol and DMNO2 were 5 and 1 mM, respectively) was removed and transferred to an amber glass bottle. The CCl4 of the solution was removed under nitrogen flow. The residue was dissolved in 900 µL of EtOH, and 100 µL of 5 mM DNPH solution was added as described above. HPLC/MS Analysis of 2,4-Dinitrophenylhydrazones of Cholesterol Oxidation Products. The derivatization reaction products were analyzed by the Shimadzu HPLC system coupled to a Quattro II mass spectrometer. For analysis, 30 µL of derivatization reaction products were injected through a C18 reversed-phase column (250 × 4.6 mm, 5 µm, Shimadzu, Tokyo, Japan), thermostatized at 25 °C, and eluted in the isocratic mode. The mobile phase consisted of 90% acetonitrile and 10% of a solution composed of 90% acetonitrile, 8% water, and 2% methanol at a flow of 1 mL/min. The source and desolvation temperatures of the mass spectrometer were kept at 150 and 200 °C, respectively. The cone voltage was set at 35 V, and the flow rates of the drying and nebulizing gases were 350 L/h and 15 L/h, respectively. The data were acquired in the negative electrospray ionization mode, and the UV-vis detector was set at 360 nm. Photooxidation of Cholesterol at Low Temperature for Chemiluminescence Studies. A 25 mM solution of cholesterol in CHCl3 was prepared with 0.16 mM methylene blue in a 100 mL round-bottom flask. Under continuous stirring and in an oxygensaturated atmosphere, the solution was cooled at -60 °C in a dry

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Figure 2. Mass spectrum of ChAld acquired in the positive electrospray ionization mode by direct infusion. A 100 µM solution of ChAld in methanol and water (9:1, v/v) was injected directly through the instrument at a flow rate of 160 µL/h.

ice and ethanol bath and irradiated using two tungsten lamps (500 and 300 W) for 2 h and 50 min. The chemiluminescence assay was conducted using a photocounter model FACT 50 MK III (Thorn Emi Electron Tubes inc., Rockway, NJ). The sample was placed in a quartz cuvette inside a dark box. Light emission was detected by a photomultiplier (model 9658B, Electron Tubes Ltd., Ruislip, UK) sensitive in the red region (300-800 nm), which was cooled thermoelectrically to -20 °C. For the assay, 3 mL of a 5 mM solution of 9,10-dibromoanthracene (DBA) in toluene was thermostatted at 70 °C for 10 min inside the cuvette. After that, 200 µL of cholesterol-photosensitized solution at -60 °C was injected into the cuvette, and light emission was recorded.

Results Cholesterol Photooxidation and ChAld Characterization. Cholesterol was subjected to photooxidation using methylene blue as a photosensitizer. The reaction products were purified by flash chromatography and analyzed by TLC as described in previous studies (29, 37). Two main fractions were isolated and analyzed by HPLC-MS/MS and NMR spectroscopy. The fraction eluted with a mixture of hexane/diethylether (50:50) was a white solid, and analysis by HPLC showed that it contained a mixture of hydroperoxides of cholesterol, which were identified as 7R-OOH, 6β-OOH, and 5R-OOH by NMR analysis. The second fraction, which was eluted with hexane/ diethylether (60:40), contained a transparent oil. The 1H NMR analysis showed a double peak at 9.87 ppm (J ) 2.9 Hz), characteristic of aldehyde groups [see Supporting Information, Figure S1]. The presence of the aldehyde group was confirmed by 13C NMR, DEPT135, and by two-dimensional COSY, HETCOR, and HMBC experiments [see Supporting Information, Figures S2, S3, S4, S5, respectively, and Table S1]. In addition to the NMR data, we also confirmed the presence of the aldehyde group by infrared absorption at 2867 cm-1 [see Supporting Information, Figure S6]. The complete analysis of the NMR data led to the identification of a new product in photooxidation reactions, identified as ChAld, an aldehyde formed in the ozonization reaction of cholesterol (16, 17). Analysis of this product by HPLC-MS/ MS showed a molecular ion at m/z 419, which is in agreement with the structure proposed. Besides this main peak, another two peaks at m/z 401 and 383 were detected, corresponding to the loss of one and two water molecules, respectively. Sodium and methanol adducts were also detected and correspond to m/z 441 and 473, respectively (Figure 2). Complementary elemental analysis of the isolated ChAld was done, confirming the carbon and hydrogen percentage of the compound [see Supporting Information, Table S2].

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Figure 3. Mass spectra of ChAld acquired in the positive electrospray ionization mode. (A) Mass spectrum of unlabeled ChAld. (B) Mass spectrum of 18O-labeled ChAld.

Figure 4. MS/MS analyses of ChAld obtained in the positive electrospray ionization mode. (A) Fragment ions of unlabeled ChAld. (B) Fragment ions of ChAld labeled with one 18O atom. (C) Fragment ions of ChAld labeled with two 18O atoms. Inset: relative amount of unlabeled, mono, and double-labeled ChAld quantified using their specific fragmentation transitions 419 to 383 (unlabeled), 421 to 383 (labeled with one 18O atom), and 423 to 385 (labeled with two 18O atoms). The results were expressed as the relative amount of each specific transition compared to the sum of the areas of the three transitions.

In order to gain further information about the structure and mechanism of formation of the photoproduct, the photodynamic process was carried out in an 18O2-saturated atmosphere. This approach was used to generate 18O2 (1∆g), and consequently, ChAld was labeled as a photoproduct. Figure 3 compares the mass spectra of cholesterol photosensitized in 16O2- and 18O2saturated atmospheres. Under a 16O2-saturated atmosphere, ChAld displayed a molecular ion at m/z 419 and the loss of three water molecules (ions at m/z 401, 383, and 365) (Figure 3A). These data were supported by the results of 18O-labeled

ChAld. Labeled ChAld exhibited mainly a molecular ion at m/z 421 (Figure 4B), consistent with an increase of two atomic mass units (amu) relative to the unlabeled molecule. These results indicate that one atom of 18O2 was incorporated into the ChAld. MS/MS analysis of the labeled ChAld (fragment ions of m/z 421) confirmed the previously attributed fragmentation, showing fragment ions at 401, 383, and 365 relative to H218O and 2 H216O (Figure 4B). In 18O-labeled ChAld, it is also possible to see a small molecular ion signal at m/z 423. This increase by 4 amu over the unlabeled ChAld molecule is consistent with a small

Cholesterol Oxidation by O2 (1∆g)


Figure 5. Scheme of CSec and ChAld derivatization reactions with DNPH, with the formation of corresponding hydrazones DNPH-Csec and DNPH-ChAld.

incorporation of two atoms of 18O2 (Figure 3B). Fragmentation of m/z 423 is also in agreement with the incorporation of two 18 O atoms in the cholesterol molecule (Figure 4C). A relative quantification was performed on ChAld photooxidized with 18O atoms. For this purpose, unlabeled, mono- and double-labeled ChAld were estimated using their specific fragmentation transitions: 419 to 383 (unlabeled), 421 to 383 (labeled with one 18O atom), and 423 to 385 (labeled with two 18O atoms). The results were expressed as the relative amount of each specific transition compared to the sum of the areas of the three transitions and are depicted in the inset of Figure 4. Comparing the transitions, it is possible to see that the majority of ChAld generated has just one 18O atom (85% of the total amount of ChAld). A small portion of ChAld has two 18O atoms (approximately 10%). Also, it is possible to see the unlabeled Chald (5%). This small portion of unlabeled molecule could be due to contamination with 16O atoms during the photooxidation process. ChAld Analysis by Derivatization with DNPH. In order to compare the aldehydes of cholesterol formed in photooxidation with those formed in ozonization, we conducted experiments using DNPH to detect the corresponding 2,4dinitrophenylhydrazone of 3β-hydroxy-5-oxo-5,6-secocholestan6-al (DNPH-CSec) and 2,4-dinitrophenylhydrazone of 3,5dihydroxy-B-norcholestan-6β-carboxaldehyde (DNPH-ChAld) (19) (Figure 5). The reaction of pure isolated ChAld with DNPH showed a predominant peak at the retention time of 31.4 min (Figure 6A), which was identified as DNPH-ChAld. The results obtained from the derivatization of cholesterol ozonization products showed two predominant peaks as described previously (19).

Figure 6. Liquid chromatography-mass spectrometry analysis of 2,4dinitrophenylhydrazone derivatives generated from different oxidizing systems using DNPH. Mass chromatograms were obtained by selecting the ion at m/z 597. (A) Isolated ChAld, (B) ozonization products, (C) photodynamic process using methylene blue (MB), and (D) clean pure source of O2 (1∆g) DMNO2.

The first one eluted at 18.9 min and corresponds to DNPHCSec, while the second one had the same retention time (31.4 min) as that of DNPH-ChAld observed in chromatogram A (Figure 6B). To investigate the involvement of O2 (1∆g) as the

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Figure 7. Five light emission in the visible region. (A) Light emission generated upon injection of cholesterol photooxidized at -60 °C (final concentration of 1.7 mM) into a solution containing 5 mM 9,10- dibromoanthracene (DBA) (a) and control 5 mM DBA (b). (B) Proposed scheme for dioxetane formation and cleavage, yielding excited species, which could then transfer energy to DBA.

oxidant responsible for the formation of ChAld, we used a defined pure chemical source of O2 (1∆g), generated by thermodecomposition of 1,4-dimethylnaphthalene endoperoxide (DMNO2) (35, 36). The same DNPH-ChAld peak was observed in both the cholesterol photooxidation using methylene blue as a photosensitizer (Figure 6C) and in the reaction of cholesterol with DMNO2 (Figure 6D). The formation of DNPH-ChAld was also observed using different photosensitizers in the photooxidation of cholesterol [see Supporting Information, Figure S7]. Mass spectrometry analyses of these peaks acquired in negative electrospray mode as described by Wentworth et al. (20) showed the same m/z 597 for both peaks [see Supporting Information, Figure S8]. These results, in addition to the previously documented formation of ChAld by ozonization, indicate that both photooxidation reactions and the thermodecomposition of DMNO2 lead to the formation of ChAld. Chemiluminescence Measurement after Photooxidation of Cholesterol at Low Temperature. In order to elucidate the mechanism of formation of ChAld containing both 18O atoms, we investigated the involvement of a cholesterol dioxetane intermediate during photooxidation reactions. To determine whether cholesterol photooxidation can result in dioxetaneassociated chemiluminescence, light emission intensity in the presence of 9,10-dibromoanthracene (DBA) was measured. For this assay, DBA, a well-known triplet carbonyl energy acceptor, was used (38, 39). The light emission signal observed after injection of photooxidized cholesterol solution in the cuvette containing DBA (Figure 7 A, a) at 70 °C can be explained by the formation of a dioxetane in the ∆5 double bond of the cholesterol molecule. No chemiluminescence was observed with DBA alone (Figure 7 A, b). The observed light emission is therefore assigned as arising from a triplet carbonyl species produced by the thermolysis of a dioxetane intermediate (Figure 7B). The thermal cleavage of a dioxetane intermediate generates an excited triplet carbonyl species, which can transfer energy to DBA. The excited-state of DBA (DBA*) is responsible for the flash of light and the slow decay of signal observed in the chemiluminescent measurement (38-40) (Figure 7 A, a and B).

Discussion Free radical-mediated reaction by a strong oxidant such as HO• or radical-mediated propagation reactions can occur directly

by hydrogen abstraction at the C7 of cholesterol. As a result, cholesterol hydroperoxides that include the epimeric pair of 7ROOH and 7β-OOH are produced (41) (Figure 1). Other nonhydroperoxide compounds correspond to the diol derivatives (7R-OH, 7β-OH), the 7-one, and the epimeric 5,6-epoxides (42) (Figure 1). Singlet dioxygen reactions with cholesterol are characterized by the ene-addition of O2 (1∆g), leading to a different peroxidation pattern compared to that observed in radical reactions (Vide supra). The peroxides thus generated are the 5R-OOH, 6R-OOH, and 6β-OOH (29) (Figure 1). 5R-OOH is considered to be a definitive fingerprint for O2 (1∆g) (28, 43). In fact, 5R-OOH has been used as an O2 (1∆g) reporter for isolated membrane and cellular systems (28, 31, 44). However, the use of this biological trap is limited because of its low reactivity and the fact that 5R-OOH is not particularly stable and can undergo rapid allylic 1,3-rearrangement to a mixture ofcholesterol7-hydroperoxidesundersomeconditions(32,34,45,46). Nevertheless, the presence of 5R-OOH appears to be diagnostic for O2 (1∆g) generation. Irradiation of cholesterol-containing liposomes in the presence of a phthalocyanine derivative showed 40-fold more 5R-OOH than 7R-OOH/7β-OOH (31). 5R-OOH was detected in the skin of rats exposed to intense visible light after ingesting sensitizing compounds (47), providing good evidence for the occurrence of an O2 (1∆g) reaction in ViVo. Besides the well-documented generation of hydroperoxides, our study shows the formation of ChAld in the reaction of cholesterol with O2 (1∆g). The ChAld generated in the photooxidation reaction of cholesterol was identified by NMR, infrared, HPLC/MS/MS, and derivatization analyses. Moreover, the involvement of O2 (1∆g) as the oxidant responsible for the formation of ChAld was also confirmed by using a defined pure chemical source of O2 (1∆g) that was generated by the thermodecomposition of DMNO2, followed by a derivatization reaction using DNPH (Scheme 1 and Figure 6). Two mechanisms may be responsible for the generation of ChAld in the reaction of cholesterol with O2 (1∆g). One is the process known as Hock fragmentation, which can occur when ene-hydroperoxides are treated with Lewis acids (48). In this mechanism, allylic hydroperoxides may undergo acid-catalyzed heterolysis of the peroxide bond generating a positive oxygen fragment (Figure 8). The instability of the positive oxygen derivative with respect to a carbonium ion would promote the migration of groups to the electron-deficient oxygen in concert



Figure 8. Proposed reaction mechanisms between cholesterol (Ch) and singlet molecular oxygen (O2, 1∆g) generating the 5R-OOH or a dioxetane intermediate, which is subsequently cleaved to generate CSec that in turn undergoes intramolecular aldolization to the final ChAld.

with the rearrangement of the carbon skeleton. The resulting oxycarbonium ion is nucleophilically attacked by H2O, leading to the corresponding hemiacetal, which cleaves to two carbonyl fragments, a process called Hock-cleavage (48). It should be noted that such cleavages were reported to occur also in the absence of any added acid (49). A second plausible mechanism to explain ChAld generation involves the formation of a 1,2-dioxetane at the ∆5 bond of cholesterol. Subsequent corresponding sterol dioxetane decomposition would then yield 5,6-secosterol aldehyde without O3 participation. Several authors have proposed the formation of electronically excited carbonyls in lipoperoxidation via the thermal cleavage of dioxetane intermediates (supposedly by (2 + 2) cycloaddition of O2 (1∆g) to polyunsaturated fatty acids (PUFA)) (50-52). Because the thermolysis of dioxetanes can produce a high yield of triplet carbonyls (38), it has been suggested that the cycloaddition of O2 (1∆g) to PUFA, producing dioxetanes, could provide a plausible mechanism for low-level chemiluminescence. Nonetheless, it has been established that the reaction of O2 (1∆g) with linoleic acid yields almost exclusively the corresponding hydroperoxides and 1,4-endoperoxides, instead of dioxetanes (1,2-cycloaddition derivatives) (53). It has been shown that 1,2-addition of O2 (1∆g) to alkenes yields dioxetanes only in the case of electron-rich olefins lacking R-hydrogens or rigid olefins (54). Another possible mechanism for cholesterol dioxetane formation is a peroxycyclization route, involving the cyclization of the cholesterol hydroperoxide 5ROOH to the corresponding dioxetane. In fact, it has been suggested that triplet-excited species can be generated from the

cyclization of the alkylperoxyl radical to a dioxetane intermediate followed by its thermolysis (55). Recently, Brinkhorst et al. (56) have shown that 5R-OOH, the product of O2 (1∆g) oxidation of cholesterol, undergoes acidcatalyzed (Hock) cleavage of the C5-C6 bond. We performed the photooxidation of cholesterol in an 18O2-saturated atmosphere, generating 18O2 (1∆g) and obtaining double-labeled hydroperoxides (including 5R-18O18OH) and also ChAld. However, the majority of ChAld obtained contained just one atom of 18O2 (see the inset of Figure 4). This provides evidence for the involvement of 5R-18O18OH in 18O-labeled ChAld formation and suggests that a Hock-cleavage mechanism is a major pathway (Figure 8). During this mechanism, one 18O atom of ChAld is lost, and a 16O atom from a water molecule is added to the molecule. Mass spectrometry analyses of 18O-labeled ChAld show mainly a molecular ion at m/z 421, which corresponds to the incorporation of one atom of 18O. This result is in agreement with Hock cleavage since ChAld has one 18O atom, (probably derived from the 5R-OOH moiety) and one 16O atom (probably derived from the water molecule involved in the mechanism) (Figure 8). Interestingly, a small portion of the generated ChAld exhibited a molecular ion at m/z 423, consistent with an increase by four atomic units relative to the unlabeled molecule (m/z 419). This result indicates that two atoms of 18O2 were incorporated into the ChAld, probably through the formation and cleavage of an intermediate dioxetane. A second mechanism involved in the generation of ChAld was investigated with a chemiluminescence assay using DBA.

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The chemiluminescence associated with the photooxygenated cholesterol solution in the presence of DBA may be due to the generation of excited triplet carbonyls. These excited species can emit phosphorescence in the visible region. The weak direct light emission from these excited species can be enhanced by the addition of fluorescent compounds, such as DBA (40). We propose thermolysis of dioxetane intermediates as a source of triplet carbonyls during cholesterol photooxydation, the dioxetanes being formed by (2 + 2) cycloaddition of singlet dioxygen to the ∆5 double bond of cholesterol or by a peroxycyclization of 5R-OOH (Figure 8). The dioxetane decomposes yielding 5,6-secosterol and finally ChAld as the end product of intramolecular aldolization (Figure 8). Judging from the experimental data and the literature, dioxetane is likely present only in trace amounts, and thus, the last mechanism is a minor pathway for the formation of the 5,6-secosterol aldehyde. Wentworth et al. reported the generation of O3 in biological systems based on two reactions they believe to yield specific products upon ozone oxidation: one is the oxidation of indigo carmine by ozone yielding isatin sulfonic acid, and the second is the oxidation of cholesterol yielding CSec and ChAld (20, 22). However, many researchers have expressed reservations about the biomarkers used to infer the presence of ozone. Concerned about the first specific biomarker, Kettle et al. reexamined the process whereby neutrophils bleach indigo carmine. They concluded that, in addition to O3, the superoxide anion can also convert indigo carmine to isatin sulfonic acid, indicating that this compound is not a specific marker for O3 (57). Also, Smith (26) questioned whether the second reaction proposed by Wentworth, namely, the oxidation of cholesterol yielding CSec and ChAld, was specific for ozone oxidation and indicated that the misidentification of specific oxysterols in tissues can lead to an improper conclusion of steroid biochemistry. Because of these concerns, the characterization of CSec and ChAld from their chromatographic properties and from a single mass spectral negative ion m/z 597 (formulated as [M H]- ion of the hydrazone derivatives) has been considered by many researchers to be inadequate evidence of O3 as the oxidant (15, 26). Additionally, other unidentified oxysterols with the same m/z ion could exist. For example, the reaction of 3β-5hydroxy-5R-cholestan-6-one (an autoxidation product of cholesterol) with DNPH could also generate the same m/z 597. Moreover, the possibility of 1,2-dioxetane formation through O2 (1∆g) attack at the ∆5 bond of cholesterol cannot be completely discounted since subsequent sterol dioxetane decomposition would yield CSec and ChAld without O3 participation. Sies (58) was also concerned with the endogenous production of ozone in the vasculature and suggested that a systematic examination of various oxidants (in terms of their capability to generate CSec and ChAld) is necessary to corroborate their use as signature products for the endogenous formation of ozone. Pryor et al. have addressed this issue in a review dealing with gases affecting oxidative stress (15). They also suggest that ozone may not be the only oxidant capable of oxidizing cholesterol to CSec and ChAld since appropriate controls to prove that this reaction is a unique signature for ozone were not published. Furthermore, although this reaction does occur in Vitro and could occur in ViVo, the complexity of the biological milieu, both with regard to competing reactions and substrate concentrations and locations, raises questions about the biological relevance of ozone oxidation of cholesterol and about this reaction’s involvement in disease progression. Production of

Uemi et al.

ozone by cells would require substantial energy, and once generated near membranes, ozone would be expected to oxidize polyunsaturated lipids faster than lipids such as cholesterol. Taking into account these observations and the results obtained in this article, cholesterol oxidation with consequent formation of ChAld and CSec may be the result of a complex process, where other oxidants, including O2 (1∆g), can take part. In conclusion, our observations show clearly and unequivocally that ChAld is generated upon the reaction of cholesterol with O2 (1∆g), and they support a new pathway that might explain the presence of significant amounts of ChAld and CSec in neurodegenerative and cardiovascular diseases. As is well known, O2 (1∆g) can be generated in many different biologically relevant systems, including enzymatic reactions (59-61), the reaction of hypochlorite with hydrogen peroxide (as in activated phagocytes) (62), and Russell-type (63) decomposition of peroxides such as lipid hydroperoxides (64-67). In addition, it is important to note that the ozonization of many compounds can also give rise to O2 (1∆g) (68-72), which could then react with cholesterol to give ChAld, as demonstrated in this work. Acknowledgment. We thank Professor A.W. Girotti (Medical College of Wisconsin) and Professor J. W. Baader (Universidade de Saõ Paulo) for helpful discussions, and Dr. M. A. Oliveira (Universidade de Saõ Paulo) for assistance with the chemiluminescence assay. This work was supported by FAPESP (Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo), CNPq (Conselho Nacional para o Desenvolvimento Cientı´fico e Tecnolo´gico), Instituto do Mileˆnio: Redoxoma, INCT de Processos Redox em Biomedicina-Redoxoma, and by Fundo Bunka de Pesquisa - Banco Sumitomo Mitsui (M.U., Fellow). G.E.R. is recipient of a FAPESP fellowship. Supporting Information Available: NMR, infrared, and elemental analysis, and liquid chromatography-mass spectrometry of 2,4-dinitrophenylhydrazones derivatives generated from different systems using DNPH. This material is available free of charge via the Internet at http://pubs.acs.org.

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TX800447B

Generation of Cholesterol Carboxyaldehyde by the Reaction of Singlet

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