Cholesterol Photooxidation Research Articles

Features of the supercritical CO2-assisted immobilization of fluorinated tetraphenylporphyrins into tetrafluoroethylene copolymers

The immobilization of fluorinated tetraphenylporphyrins (FTPPs) into tetrafluoroethylene copolymers (fluoroplast F-42 and MF-4SK, a perfluorinated sulfonic acid cation exchanger in H+-form) is conducted in supercritical CO2 (scCO2). The effects the conditions of immobilization (the temperature and pressure of scCO2, reaction time, and the addition of cosolvents) and the structure of the carrier polymer have on the content of porphyrin in these polymers is studied. The porphyrin-loaded polymer systems are shown to exhibit photosensitizing activity in anthracene and cholesterol oxidation in scCO2. Under conditions of photocatalysis, chemical and functional stability is a feature of only MF-4SK polymer systems; this is attributed to the formation of protonated forms of the porphyrins and their interaction with SO3 −-groups of the polymer (an ion exchange process), which prevents leaching of the FTPP from the polymer matrix. The photocatalytic process actually occurs inside the matrix of the perfluorinated copolymer, with the protonated form of the porphyrin acting as a photosensitizer. The rate constant of anthracene photooxidation in the presence of FTPP-loaded MF-4SK films in scCO2 is found to pass through a maximum as a function of the porphyrin content and the polymer film thickness. The use of such catalytic systems for cholesterol photooxidation in scCO2 is shown to produce a virtual monoproduct (yield, ~10%): 6-formyl-B-norcholestane-3,5-diol, a compound with high biological activity.

Cholesterol photo-oxidation: A chemical reaction network for kinetic modeling

In this work we studied the effect of polyunsaturated fatty acids (PUFAs) methyl esters on cholesterol photo-induced oxidation. The oxidative routes were modeled with a chemical reaction network (CRN), which represents the first application of CRN to the oxidative degradation of a food-related lipid matrix. Docosahexaenoic acid (DHA, T-I), eicosapentaenoic acid (EPA, T-II) and a mixture of both (T-III) were added to cholesterol using hematoporphyrin as sensitizer, and were exposed to a fluorescent lamp for 48h. High amounts of Type I cholesterol oxidation products (COPs) were recovered (epimers 7α- and 7β-OH, 7-keto and 25-OH), as well as 5β,6β-epoxy. Fitting the experimental data with the CRN allowed characterizing the associated kinetics. DHA and EPA exerted different effects on the oxidative process. DHA showed a protective effect to 7-hydroxy derivatives, whereas EPA enhanced side-chain oxidation and 7β-OH kinetic rates. The mixture of PUFAs increased the kinetic rates several fold, particularly for 25-OH. With respect to the control, the formation of β-epoxy was reduced, suggesting potential inhibition in the presence of PUFAs.

Kinetics of 25‐hydroperoxycholesterol formation during photo‐oxidation of crystalline cholesterol

25-Hydroxycholesterol (25-OH), a side-chain product of cholesterol oxidation, has emerged as one of the important issues in food chemistry and biochemistry, because of its involvement in several human pathologies. This oxysterol is derived from both enzymatic and non-enzymatic pathways. However, the latter mechanism has been scarcely studied in either food or model systems. In this work, a kinetic model was developed to evaluate the formation of 25-OH and its precursor 25-hydroperoxycholesterol (25-OOH) during photo-oxidation of cholesterol for 28 days under fluorescent light. 25-OOH was estimated by an indirect method, using thin-layer chromatography coupled with gas chromatography-mass spectrometry. Peroxide value (POV) and cholesterol oxidation products (COPs) were determined. POV showed a hyperbolic behavior, typical of a crystalline system in which the availability of cholesterol is the limiting factor. Further reactions of hydroperoxides were followed; in particular, after photo-oxidation, 25-OOH (0.55 mg g(-1) ) and 25-OH (0.08 mg g(-1) ) were found in cholesterol, as well as seven other oxysterols, including 7-hydroxy and 5,6-epoxy derivatives. The application of kinetic models to the data showed good correlation with theoretical values, allowing derivation of the kinetic parameters for each oxidation route. The results of this work confirm that cholesterol in the crystalline state involves different oxidation patterns as compared to cholesterol in solution. Moreover, the numerical fit proved that hydroperoxidation is the rate-limiting step in 25-OH formation.

Synthesis of 7-Hydroperoxycholesterol and Its Separation, Identification, and Quantification in Cholesterol Heated Model Systems

7-Hydroperoxycholesterol is considered to be an intermediate compound of the cholesterol oxidation path as the first product formed when cholesterol is oxidized by triplet oxygen. However, there is a limitation on cholesterol mechanism studies because of the lack of 7-hydroperoxycholesterol analytical standard due to its low stability. To verify the formation of hydroperoxides in cholesterol model systems heated at 140, 180, and 220 °C, 7α-hydroperoxycholesterol was synthesized by cholesterol photooxidation followed by rearrangement at room temperature in chloroform. Its structure was confirmed on the basis of 13C NMR and mass spectra obtained by APCI-LC-MS. The synthesized compound was also used as standard for the quantification of 7-hydroperoxycholesterol as the sum of 7α- and 7β-hydroperoxycholesterol. The results demonstrated that 7-hydroperoxycholesterol is the first compound formed when the temperature is lower (140 °C). However, the concentration of the 7-hydroperoxycholesterol depends on the temperature and time of exposure: the higher the time, the higher the amount of 7-hydroperoxycholesterol at lower temperatures, and the lower the time, the lower the amount of 7-hydroperoxycholesterol at higher temperatures (180 and 220 °C). By the formation of 7-hydroperoxycholesterol, the known cholesterol oxidation mechanism in three phases (initiation, propagation, and termination) could be confirmed; once at lower temperatures, the stage of cholesterol oxidation is at initiation, at which hydroperoxide formation predominates.

The retinal oxysterol pathway: a unifying hypothesis for the cause of age-related macular degeneration

To summarize recent findings implicating toxic agents resulting in photooxidation of cholesterol in the etiology of age-related macular degeneration. Understanding the role of these agents and the existing pathways for their neutralization may lead to novel therapeutic approaches. The human eye is now known to produce significant quantities of 7-ketocholesterol and related substances as a direct result of photoreceptor function. These substances are highly toxic to retinal cells and the eye has been shown to be unique among human organs in expressing three separate enzymatic pathways that neutralize these agents. Drusen are recently shown to contain significant accumulations of 7-ketocholesterol, likely as a result of failure of these neutralization pathways. In addition to its direct tissue toxicity, which may trigger death of retinal pigment epithelium and photoreceptor cells, ketocholesterol is a potent attractor of macrophages and induces macrophages to express both vascular endothelial growth factor F and metalloproteinases. The role of the former in neovascularization is well understood, whereas the latter is capable of directly inducing breaks in Bruch's membrane. The toxic role of 7-ketocholesterol and existing pathways for its neutralization may point the way to a unified theory that explains the cause of age-related macular degeneration and points towards novel therapeutic interventions.

Effect of the nature of polymer matrix on the process of cholesterol photooxidation in the presence of immobilized tetraphenylporphyrins

It was shown that carrying out the reaction of cholesterol photosensitized oxidation in the presence of porphyrins immobilized on the hydrolyzed copolymer of tetrafluorethylene and H+ form of perfluoro-3,6-dioxo-5-methyl-6-sulfonylfluorideoctene-1 leads to the formation of not previously described new products: 6β-phormyl-B-norcholestan-3β,5β-diol, 6β-chlorocholestan-3β,5α-diol, cholestan-3β,5α,6β-triol, and 5α-chlorocholestan-3β,6β-diol.

Cholesterol photooxidation as affected by combination of riboflavin and fatty acid methyl esters

The effect of combination of riboflavin and fatty acid methyl esters on cholesterol photooxidation was studied. Samples containing both riboflavin and fatty acid methyl esters were exposed to light as 25 °C for 28 days. The various cholesterol oxidation products (COPs) were analyzed by gas chromatography–mass spectrometry (GC–MS), while riboflavin was determined by high-performance liquid chromatography (HPLC). Results showed that both riboflavin and fatty acid methyl esters could promote COPs formation and cholesterol degradation, and the degradation fits a first-order model. Riboflavin had a more pronounced effect than fatty acid methyl esters. 7-Keto was generated in highest amount, followed by 5,6β-EP, 3,5- cholestadien-7-one, 5,6α-EP, 7α-OH and 7β-OH. The formation of 3,5-cholestadien-7-one was probably due to dehydration of 7-keto through energy liberated during illumination.

Photodynamically generated 3-beta-hydroxy-5 alpha-cholest-6-ene-5- hydroperoxide: toxic reactivity in membranes and susceptibility to enzymatic detoxification.

Singlet oxygen (1O2)-mediated photooxidation of cholesterol gives three hydroperoxide products: 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 alpha-hydroperoxide (6 alpha-OOH) and 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH). These species have been compared with respect to photogeneration rate on the one hand and susceptibility to enzymatic reduction/detoxification on the other, using the erythrocyte ghost as a cholesterol-containing test membrane and chloroaluminum phthalocyanine tetrasulfonate (AlPcS4) as a 1O2 sensitizer. Peroxide analysis was accomplished by high-performance liquid chromatography with mercury cathode electrochemical detection (HPLC-EC[Hg]). The initial rate of 5 alpha-OOH accumulation in AlPcS4/light-treated ghosts was found to be about three times greater than that of 6 alpha-OOH or 6 beta-OOH. Membranes irradiated in the presence of ascorbate and ferric-8-hydroxyquinoline (Fe[HQ]2, a lipophilic iron complex) accumulated lesser amounts of 5 alpha-OOH, 6 alpha-OOH and 6 beta-OOH but relatively large amounts of another peroxide pair, 3 beta-hydroxycholest-5-ene-7 alpha- and 7 beta-hydroperoxide (7 alpha, 7 beta-OOH), suggestive of iron-mediated free radical peroxidation. When photoperoxidized membranes containing 5 alpha-OOH, 6 alpha,6 beta-OOH and 7 alpha,7 beta-OOH (arising from 5 alpha-OOH rearrangement) were incubated with glutathione (GSH) and phospholipid hydroperoxide glutathione peroxidase (PHGPX), all hydroperoxide species underwent HPLC-EC(Hg)-detectable reduction to alcohols, the relative first order rate constants being as follows: 1.0 (5 alpha-OOH), 2.0 (7 alpha,7 beta-OOH), 2.4 (6 alpha-OOH) and 3.2 (6 beta-OOH).(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid peroxidation and loss of potassium from red blood cells produced by phototoxic quinolones

Alterations of the cationic permeability of red blood cell membranes induced by the photosensitiser nalidixic acid were demonstrated by evaluating the potassium loss from intact erythrocytes. The results show that an increase in intracellular potassium efflux, preceeds the photohemolysis induced by nalidixic acid. The addition of a nonpermeable osmotic solute, such as sucrose, inhibited photohemolysis but not the potassium loss, indicating a colloid osmotic lysis. Lipid peroxidation induced by nalidixic acid and other photosensitiser quinolones (oxolinic acid and rosoxacin) was time irradiation-dependent. Although rosoxacin was the most photoperoxidative, none of the three quinolones studied produced significant lipid peroxidation. However, of the three quinolones studied, only rosoxacin considerably diminished the percentage of the cholesterol extracted from red blood cell membranes. It is postulated that the increased cation permeability induced by nalidixic and oxolinic acids cannot be attributed to cholesterol oxidation nor to lipid peroxidation; a more probable mechanism is photo-oxidation of amino acid residues of the membrane proteins. However, the lysis induced by rosoxacin is caused by photo-oxidation of cholesterol, not excluding other cellular targets.

Photooxidation of Cholesterol in Butter

ABSTRACTThe effect of exposure to fluorescent light on the oxidative stability of cholesterol in unpackaged butter was investigated. Oxidation of cholesterol in butter occurred, although the oxidation products were detectable only after prolonged exposure to light. Cholesterol oxides were more concentrated at the surface than throughout the entire butter block and were detectable after 8 days of exposure to light. 5‐Cholesten‐313, 7α‐diol and its 7β‐epimer, products of free radical attack on cholesterol, were detected among the oxidation products, along with a compound suspected to be 6‐cholesten‐3β, 5α‐diol. This latter compound is only known to form following singlet oxygen attack on cholesterol.

Reactivity of singlet molecular oxygen with cholesterol in a phospholipid membrane matrix. A model for oxidative damage of membranes

Photooxidation of cholesterol in liposomes with hematoporphyrin sensitization has been studied. With liposomal samples in which the hematoporphyrin is incorporated in the membrane, the yield of the characteristic singlet oxygen product, 3β-hydroxycholest-6-ene 5α-hydroperoxide, was approximately 6 times greater than that observed in the samples in which the hematoporphyrin was outside the membrane. Small amounts of 3β-hydroxycholest-5-ene 7α- and 7β-hydroperoxides, radical autoxidation products, were formed in both samples. Photolysis of a dispersion of cholesterol in an aqueous solution of hematoporphyrin gave no singlet oxygen products. It is concluded from these results that endogenous singlet oxygen when formed in the phospho-lipid membrane has a sufficiently long lifetime to effect oxygenation of cholesterol; whereas exogenous singlet oxygen generated outside the membrane is quenched by solvent before appreciable diffusion into the membrane can occur.

Cholesterol hydroperoxide formation in red cell membranes and photohemolysis in erythropoietic protoporphyria.

3beta-Hydroxy-5alpha-hydroperoxy-Delta(6)-cholestene is produced in protoporphyrin-containing red blood cell ghosts irradiated with approximately 400-nanometer light in the presence of oxygen. Incorporation of this cholesterol photooxidation product into normal red blood cells leads to increased osmotic fragility and eventual hemolysis. These results may be relevant to photohemolysis associated with erythropoietic protoporphyria.