Mechanisms of Singlet Oxygen-Dependent Formation of Ozone, Bioactive Lipid Aldehydes, and Amide-Type Aldehyde Adducts in Biological Systems

Abstract

The cholesterol secosterol aldehydes, namely 3β-hydroxy-5-oxo-5, 6-secocholestan-6-al (secosterol A) and its aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (secosterol B) are highly bioactive compounds. They have been detected in human tissues and may significantly contribute to the pathophysiology of conditions such as diabetes, certain cancers, atherosclerosis and Alzheimer’s disease. Previously, they were considered unique products of cholesterol ozonolysis. Hence they were used as indicators for formation of ozone endogenously. However, the formation of ozone in biological systems has been questioned partly because of inadequate understanding of the mechanisms of its formation. The original mechanism proposes that antibodies or amino acids catalyze the oxidation of water with singlet oxygen to form dihydrogen trioxide (HOOOH). Then HOOOH decomposes to form ozone (O3) and hydrogen peroxide (H2O2). However, in aqueous solutions, HOOOH was found to decompose readily to singlet oxygen and water rather than ozone and hydrogen peroxide. Alternatively it has been suggested that ozone can be formed by first oxidation of amino acids with singlet oxygen. Then by a further reaction of the amino acid oxidation products with singlet oxygen to form zwitterionic polyoxidic species that decompose to form ozone and amino acids or amino acid oxidation products. Because of previous doubts on the occurrence of biological ozone, an alternative mechanism for the formation of the secosterol aldehydes has been proposed. It involves oxidation of cholesterol by singlet oxygen to form cholesterol-5α-hydroperoxide, followed by acid-catalyzed decomposition (Hock cleavage) of the cholesterol-5α-hydroperoxide to secosterol A and subsequent conversion of secosterol A to secosterol B. However, Hock cleavage of cholesterol-5α-hydroperoxide results in the formation of mainly secosterol B and negligible amounts of secosterol A. The secosterol A compound is implicated as the major secosterol in atherosclerotic tissues. Additionally, it was postulated that primary amines such as lysine may catalyze the conversion of cholesterol-5α-hydroperoxide (Ch-5α-OOH), to the secosterol aldehydes. However, no experimental evidence was provided. Therefore, this study tested the hypotheses that (i) reaction of singlet oxygen with amino acids and their oxidation products yields ozone and (ii) amines react with cholesterol-5α-hydroperoxide to form secosterol aldehydes. The first hypothesis was tested by exposing methionine (C5H11NO2S) and methionine sulfoxide (C5H11NO3S) to a singlet oxygen-generating system consisting of myeloperoxidase-hydrogen peroxide-halide system in the presence of the ozone ‘indicator’ molecules, indigo carmine and vinyl benzoic acid. The finding that methionine sulfoxide was more efficient than methionine in converting vinyl benzoic acid and indigo carmine to 4-carboxybenzaldehyde and isatin sulfonate, respectively, supported conversion of methionine sulfoxide to trioxidic anionic species RS+(OOO-)CH3 as a precursor of ozone or ozone-like oxidants. The second hypothesis was tested by generating cholesterol-5α-hydroperoxide by the photosensitized oxidation of cholesterol. Then exposed the hydroperoxides to lysine in the presence of 2,6-ditertiary butyl- 4-hydroxytoluene (BHT) to limit free radical reactions. Analysis of the reaction mixtures by electrospray ionization mass spectrometry revealed the formation of the secosterol aldehydes as well as various types of secosterol-amine adducts including carbinolamines, Schiff’s bases and amide-type adducts. The amide-type adducts in vitro and in vivo contribute to pathophysiological processes such as hexanoyl-lysine. They are also considered biomarkers of lipid oxidation in foods. Their mechanism of formation however is not well understood. Recently it was postulated that such adducts may be formed by the reaction of aldehydes with amines to form Schiff’s bases, followed by reaction of the Schiff’s bases with hydroperoxides to form unstable peroxide intermediates that rearrange to amide-type adducts and alcohols. However the peroxide intermediate was not detected by liquid chromatography-electrospray ionization mass spectrometry LC-ESI-MS as a direct evidence for this mechanism in this study. Thus, an alternative mechanism was proposed, involving the oxidation of carbinolamine adducts by singlet oxygen. In this case, dioxetane derivatives of cholesterol decompose into triplet carbonyls which transfer some of their energy to triplet oxygen to generate singlet oxygen. Apart from the amine-mediated decomposition of cholesterol hydroperoxide, the analogous amine-mediated decomposition of linoleic acid hydroperoxide was also investigated. Analysis of the products by GC-MS revealed the formation of hexanal, 2-pentyfuran and 2-nonenal. Detection of 2-pentylfuran signified the formation of 4-hydroperoxy-2-nonenal. This is a key precursor of the 4-hydroxy-2-nonenal, a major cytotoxic product of linoleic acid oxidation, and whose mechanisms of formation is of great interest. Another objective of this study was to determine the effect of uric acid on the conversion of linoleic acid hydroperoxides to aldehydic products. Thus, the aldehyde forming reactions were done in the presence of uric acid. Interestingly, uric acid, even without the amines, was found to promote conversion of the hydroperoxides to aldehydes. Thus, the present study obtained evidence for the hypotheses that some amino acids react with singlet oxygen to form ozone and that amines such as lysine mediate the decomposition of cholesterol-5α-hydroperoxide to form secosterol aldehydes, and the analogous conversion of linoleic acid hydroperoxide to hexanal and 4-hydroxy-2-nonenal. Based on identification cholesterol-secosterol aldehyde adducts by ESI-MS spectrometry, a new mechanism for the formation of amide-type aldehydes was proposed. It was also found that uric acid promotes the conversion of lipid hydroperoxides to toxic aldehydes, and this may explain the paradoxical association of hyperuricemia with various physiological disorders, despite its known antioxidant activities.