H-Atom Abstraction vs Addition: Accounting for the Diverse Product Distribution in the Autoxidation of Cholesterol and Its Esters

Abstract

We recently communicated that the free-radical-mediated oxidation (autoxidation) of cholesterol yields a more complex mixture of hydroperoxide products than previously appreciated. In addition to the epimers of the major product, cholesterol 7-hydroperoxide, the epimers of each of the regioisomeric 4- and 6-hydroperoxides are formed as is the 5α-hydroperoxide in the presence of a good H-atom donor. Herein, we complete the story by reporting the products resulting from competing peroxyl radical addition to cholesterol, the stereoisomeric cholesterol-5,6-epoxides, which account for 12% of the oxidation products, as well as electrophilic dehydration products of the cholesterol hydroperoxides, 4-, 6-, and 7-ketocholesterol. Moreover, we interrogate how their distributionand abundance relative to the H-atom abstraction productschanges in the presence of good H-atom donors, which has serious implications for how these oxysterols are used as biomarkers. The resolution and quantification of all autoxidation products by LC-MS/MS was greatly enabled by the synthesis of a new isotopically labeled cholesterol standard and corresponding selected autoxidation products. The autoxidation of cholesteryl acetate was also investigated as a model for the cholesterol esters which abound in vivo. Although esterification of cholesterol imparts measurable stereoelectronic effects, most importantly reflected in the fact that it autoxidizes at 4 times the rate of unesterified cholesterol, the product distribution is largely similar to that of cholesterol. Deuteration of the allylic positions in cholesterol suppresses autoxidation by H-atom transfer (HAT) in favor of addition, such that the epoxides are the major products. The corresponding kinetic isotope effect (k H/k D ∼ 20) indicates that tunneling underlies the preference for the HAT pathway.

On the Products of Cholesterol Autoxidation in Phospholipid Bilayers and the Formation of Secosterols Derived Therefrom

“…Cholesterol (chol) is among the most abundant of lipids in mammalian phospholipid bilayers and elevated levels thereof, as part of circulating low‐density lipoproteins, is a key risk factor for cardiovascular disease . In solution, chol autoxidizes to a variety of products (Scheme A), of which the epimers of the 7‐hydroperoxide (7‐OOH) and 5,6‐epoxide (5,6‐epoxy) are predominant . Chol undergoes a variety of other oxidative transformations leading to so‐called oxysterols, several of which have been implicated in disease pathogenesis .…”

“…The product distribution shown in Scheme A was established from analyses of samples of autoxidations of chol in chlorobenzene (PhCl) by normal phase HPLC‐APCI + ‐MS/MS . The compounds were identified by direct comparison to authentic standards obtained by chemical synthesis and quantified against deuterated internal standards.…”

“…[31] Fully consistent with the results in solution, no chol 5a-OOH was observed in phospholipids.T his was expected based upon the rapid rate of b-fragmentation of the 5aperoxyl radical (k b = 3.8 10 5 s À1 in chlorobenzene). [5] In our previous studies,w es howed that good H-atom donors (for example,p entamethylchromanol (PMC), at runcated analogue of a-tocopherol (a-TOH), the most potent form of vitamin E), can effectively compete with b-fragmentation, trapping the 5a-peroxyl radical and leading to the observation of the 5a-OOH ( Figure 2A). In stark contrast, no chol 5a-OOH was observed in liposomal autoxidations loaded with a-TOH( Figure 2B).…”

“…[2,3] In solution, chol autoxidizes to av ariety of products (Scheme 1A), of which the epimers of the 7-hydroperoxide (7-OOH) and 5,6-epoxide (5,6-epoxy) are predominant. [4,5] Chol undergoes av ariety of other oxidative transformations leading to so-called oxysterols, [6][7][8] several of which have been implicated in disease pathogenesis. [9][10][11] Among the more intriguing [12][13][14] oxidation products are the electrophilic secosterols Aa nd B (secA and secB).…”

“…Thep roduct distribution shown in Scheme 1A was established from analyses of samples of autoxidations of chol in chlorobenzene (PhCl) by normal phase HPLC-APCI + -MS/MS. [5] Thec ompounds were identified by direct comparison to authentic standards obtained by chemical synthesis and quantified against deuterated internal standards.The hydroperoxides were identified following reduction to their corresponding alcohols by the [M+ +H + ÀH 2 [26] Using this approach (see the Supporting Information), [27] we investigated the product distribution in azo-initiated (MeOAMVN) autoxidations of chol-embedded unilamellar liposomes prepared from either egg phosphatidylcholine (PC) or soy lecithin (43.5 mol %c hol, approximately 130 nm diameter). Ther esults are shown alongside those previously obtained in PhCl in Figure 1.…”