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

Article abstract of DOI:10.1002/anie.201914637

In homogenous solution, cholesterol autoxidation leads to a mixture of epimers of 5 primary products, whose concentrations vary in the presence/absence of antioxidants, such as vitamin E. Two of the products (5α-OOH and 6β-OOH) undergo Hock fragmentation

Full text of DOI:10.1002/anie.201914637

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Accepted Article
Title: On the Products of Cholesterol Autoxidation in Phospholipid
Bilayers and the Formation of Secosterols Derived Therefrom
Authors: Emily Schaefer, Nadia Zopyrus, Zosia Zielinski, Glenn Facey,
and Derek Andrew Pratt
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201914637
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On the Products of Cholesterol Autoxidation in Phospholipid
Bilayers and the Formation of Secosterols Derived Therefrom
Emily L. Schaefer, Nadia Zopyrus, Zosia A. M. Zielinski, Glenn A. Facey, and Derek A. Pratt*[a]
Dedicated to Dr. Keith U. Ingold, in recognition of his lifetime contributions to chemistry, on the occasion of his 90th birthday.
Abstract: In homogenous organic solution, cholesterol autoxidation
leads to a mixture of epimers of 5 primary products, whose
concentrations vary in the presence/absence of antioxidants, such as
Vitamin E. Two of the products (5a-OOH and 6b-OOH) undergo Hock
fragmentation to yield electrophilic secosterols implicated in disease.
Herein, we show that the product distribution is similar in phospholipid
bilayers, in that the 7-OOHs are the major products, but the presence
of Vitamin E has no effect on the product distribution. Cholesterol 7a-
OOH, but not 7b-OOH, undergoes Hock fragmentation to yield a
mixture of unprecedented A-ring cleavage products and 6,7-epoxides.
When subjected to typical derivatization conditions, 7a-OOH yields
products with essentially indistinguishable chromatographic and
spectroscopic features from the previously identified secosterols,
casting further doubt on their controversial origin from endogenous O3.
Cholesterol (chol) is among the most abundant of lipids in
mammalian phospholipid bilayers[1] and elevated levels thereof,
as part of circulating low-density lipoproteins, is a key risk factor
for cardiovascular disease.[2,3] In organic solution, chol
autoxidizes to a variety 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] Cholesterol undergoes a variety of
other oxidative transformations leading to so-called oxysterols,[6–
8]
several of which have been implicated in disease
pathogenesis.[9–11] Among the more intriguing[12–14] oxidation
products are the electrophilic secosterols A and B (secA and
Scheme 1. Products of cholesterol autoxidation via H-atom abstraction and
addition pathways (A) and demonstrated routes to secosterols A and B (B).
secB).[15,16] These compounds, which were allegedly identified in
diseased heart and brain tissue,[17,18] were proposed to arise from
the oxidation of chol by ozone formed from the antibody-catalyzed
oxidation of water.[19,20] In previous work, we demonstrated
5a-OOH and 6b-OOH are formed from chol autoxidation in
phospholipid bilayers. Moreover, it was hitherto unknown if the
alternative pathways for their formation (Scheme 1B). Specifically,
we showed that chol 5a-OOH, (the major product of the reaction
of 1O2 with cholesterol and a minor product of chol autoxidation in
the presence of Vitamin E), undergoes acid-catalyzed (Hock)
fragmentation to yield secA and secB.[21,22] Similarly, chol 6b-OOH,
epimers of chol 7-OOH undergo Hock fragmentation to give
secosterol products. Herein we address these outstanding points.
The product 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]
The compounds 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+-H2O] ® [M+H+-2H2O] MS/MS transition (385.4 ®
367.4). The 5,6-epoxides (the b-epimer of which unfortunately co-
elutes with chol 4a-OH), were identified by the [M+H+] ®
[M+H+-H2O] MS/MS transition (403.4 ® 385.4).[26] Using this
approach (see Supporting Information),[27] we investigated the
product distribution in azo-initiated (MeOAMVN) autoxidations of
cholesterol-embedded unilamellar liposomes prepared from
either egg phosphatidylcholine (PC) or soy lecithin (43.5 mol%
chol, ~130 nm diameter). The results are shown alongside those
previously obtained in PhCl in Figure 1.
1
a minor product of the reaction of O2 with chol as well as chol
autoxidation, yields secA and secB.[4] The acid-mediated
reactions of these hydroperoxides are relevant given the
presence of acidic microenvironments within the cell[23–25] and that
the alleged identification of secA and secB was made following
acid-catalyzed derivatization to their corresponding 2,4-
dinitrophenylhydrazones. However, it was hitherto unknown if
[a]
Prof. Dr. D. A. Pratt, E. L. Schaefer, N. Zopyrus, Z. A. M. Zielinski,
G. A. Facey
Department of Chemistry and Biomolecular Sciences
University of Ottawa
10 Marie Cure Pvt., Ottawa, Ontario, CANADA K1N 6N5
E-mail: dpratt@uottawa.ca
Supporting information for this article is given via a link at the end of
the document.
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Figure 1. Representative chromatograms of chol autoxidation products in unilamellar liposomes of egg phosphatidylcholine. Hydroperoxide species were reduced
to their corresponding alcohols for detection by APCI+-MS/MS (A). Product distributions from MeOAMVN-initiated (20 mM) autoxidations of chol in PhCl at 37°C for
16 h (as previously reported)[5] compared to product distributions of MeOAMVN-initiated (1 mM) autoxidations of chol in liposomal egg phosphatidylcholine or soy
lecithin at 37°C after 16 h; inset: distributions of minor products (B).
Incorporation of chol into liposomes did not afford a
substantial difference in the product distribution. The epimers of
chol 7-OOH were still the predominant products, although their
relative abundance in egg PC relative to PhCl was bolstered
somewhat at the expense of the second most abundant 5,6-epoxy
chol products. Interestingly, more epoxide was observed in the
more highly unsaturated soy lecithin liposomes (ca. 64%
polyunsaturated fatty acid content compared to 15% in egg PC),
suggesting that a more fluid membrane facilitates peroxyl radical
addition over H-atom abstraction. Interestingly, the minor 4-OOH
and 6-OOH products were also observed in liposomes.[28]
Although 4-OOH have been indirectly observed in tissue samples
(as 4α/β-OH presumably derived from 4-OOH),[29,30] to the best of
our knowledge, no mention of 6-OOH derived from autoxidation
has been made. Instead, observation of 6-OOH has long been
considered a biomarker for 1O2.[31]
medium impacts the product distribution directly (chol 7-
OOH/epoxide ratio) and indirectly (whether chol 5-OOH is formed
or not). Moreover, it suggests that under physiological conditions,
where a-TOH is the most reactive lipid-soluble antioxidant, 5a-
OOH is unlikely to form by chol autoxidation.
Given the overwhelming abundance of 7-OOH in chol
autoxidations in solution and phospholipid bilayers, we sought to
investigate its propensity to undergo the Hock fragmentation
reactions that are so facile for 5a-OOH and 6b-OOH (Figure 3A).
Initial treatment of chol autoxidation samples with acid revealed
that while the 7a-OOH was readily consumed, 7b-OOH persisted
(Figure 3B). As such, treatment of chol autoxidation mixtures or
authentic chol 7α-OOH[34] with ethanolic HCl in the presence of
2,4-dinitrophenylhydrazine (DNPH) to trap the carbonyls formed
upon Hock fragmentation yielded very similar HPLC/UV-Vis/MS
profiles. Representative results are shown in Figure 4A, in which
four prominent product peaks are evident in the chromatogram.
Interestingly, the first two peaks co-eluted with the hydrazones of
secA and secB, respectively, and the latest eluting peak
corresponded to the hydrazine derivative of 7-ketocholesterol, the
dehydration/oxidation product of 7-OOH.
Fully consistent with the results in solution, no 5a-OOH was
observed in phospholipids. This was expected based upon the
rapid rate of b-fragmentation of the 5a-peroxyl radical (kb = 3.8 ´
105 s-1 in chlorobenzene).[4] In our previous studies, we showed
that good H-atom donors (e.g. pentamethylchromanol,
a
truncated analog 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
5a-OOH (Figure 2A). In stark contrast, no 5a-OOH was observed
in liposomal autoxidations loaded with a-TOH (Figure 2B). We
surmised this may be due to the dramatically lower H-atom
transfer reactivity exhibited by a-TOH in phospholipids (kinh = 4.7
´ 103 M-1s-1 in egg PC compared to 3.6 ´ 106 M-1s-1 in PhCl).[32]
This difference has been attributed to the strong H-bonding
interaction between a-TOH and the phospholipid phosphate
diester moiety, which renders it unreactive to H-atom transfer
(Figure 2C).[33] Consistent with this view, when autoxidations
were carried out on chol-loaded liposomes supplemented with the
more reactive, less strongly H-bonding antioxidant, phenoxazine
(kinh = 2.3 ´ 105 in egg PC),[32] 5a-OOH was observed and its
Although it was entirely reasonable that secA and secB
would appear in chol autoxidation samples that had undergone
derivatization (since they contain 6b-OOH which we have shown
to yield secA and secB under these conditions)[4], we were
surprised to see these products derived from derivatization of
authentic chol 7a-OOH. To confirm the identity of these products
– and ascertain the identity of the compound in the peak eluting
at 21 minutes – we carried out a preparative-scale experiment and
isolated the compounds for characterization. Doing so revealed
that the compounds eluting at 12.5 and 19 minutes were neither
the DNPH derivatives of secA and secB, respectively, nor the
expected Hock fragmentation products of chol 7a-OOH shown in
Figure 3A. Although we were unable to confidently assign a
structure to the compound co-eluting with the hydrazone of secA,
the compound co-eluting with the hydrazone of secB at 19
minutes, as well as that contained in the following peak at 21
minutes, were confidently assigned to the unprecedented A-ring
cleavage products shown in Figure 4B.
concentration
increased
with
increasing
phenoxazine
concentration (Figure 2D). Thus, it is clear that the exact same
mechanistic scheme operates in phospholipid bilayers as in
solution. However, these results also demonstrate that the
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Figure 2. Influence of radical-trapping antioxidant (RTA) on the product distribution of chol autoxidation (A). The chol 5α-OOH to chol 7-OOH ratio in chol
autoxidations as a function of both media and identity of RTA (B).[5] The potency of α-TOH is diminished in phospholipid bilayers relative to organic solution (e.g.
PhCl) due to formation of strong H-bonds with the phosphate diester moiety (C), resulting in a significant decrease in reactivity to peroxyl radicals (kinh) (D).[32,35,36]
These structural assignments were enabled by the results
of parallel experiments carried out in the absence of DNPH (see
Supporting Information). These revealed that the two primary
reaction paths which are followed in the acid-mediated
decomposition of chol 7a-OOH are distinct from that expected
based on the reactivity of chol 5α-OOH and chol 6β-OOH (Figure
3A). Instead, the major product is not an aldehyde at all, but the
a-hydroxyepoxides shown in Scheme 2A (or a-alkoxyepoxides
when the reactions were carried out in alcohol). These products
are, of course, ‘invisible’ in the above derivatizations because they
lack carbonyls with which DNPH can react.[37] Unambiguous
elucidation of the structures of the pair of epimers was made
possible by single crystal x-ray crystallographic characterization
of the b-epimer of both the a-hydroxy- and a-methoxyepoxides
(see Supporting Information). The majority of the remaining mass
balance consisted of the A-ring cleavage product also shown in
Scheme 2A. To the best of our knowledge, this is the first
occurrence of an A-ring cleavage product derived from chol
oxidation. Thus, in addition to our exhaustive characterization
efforts (see Supporting Information), we prepared 2,2,4,4-d4-chol
7a-OOH and subjected it to the same reaction conditions to
support the assignment of the exomethylene protons and the
protons adjacent the aldehydic center as originating from C2 and
C4, respectively (see Supporting Information). Furthermore, we
subjected 7a-OOH in which the 3-OH was acetylated to the same
conditions, to find that A-ring cleavage was significantly
suppressed in favour of the a-hydroxyepoxides (see Supporting
Information).
fully consistent with the fact that only chol 7a-OOH underwent
fragmentation; the p orbital of the C5-C6 double bond and the sO-
Figure 3. Expected products of acid-catalyzed (Hock) fragmentation of chol 7-
OOH based on previous observations with chol 5α-OOH and chol 6β-OOH (A)
and the comparative consumption of the 7α-OOH versus 7β-OOH isomer under
acid-catalyzed (Hock) fragmentation conditions (B).
A mixture of chol
It should be noted that this is the most compelling evidence
to date supporting the intermediacy of an epoxycarbenium ion
intermediate in the mechanism of Hock fragmentation,[38] and is
autoxidation products (obtained from MeOAMVN-initiated autoxidation of chol
in chlorobenzene) was subjected to acidic conditions (0.01 M HCl in acetone).
Consumption of chol 7α- and 7β-OOH was monitored by HPLC-APCI+-MS/MS.
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Figure 4. Chromatographic analysis of products observed when chol 7α-OOH is treated with DNPH (200 µM) in ethanolic HCl (0.1 M) for 1 hour (A, grey trace).
Products were analysed by reverse phase HPLC-ESI--MS (4.6 × 150 mm Atlantis C18 HPLC column, flow rate = 1 mL/min, 75:20:5 ACN:MeOH:H2O) with
simultaneous UV detection (360 nm). Representative chromatograms for authentic DNPH hydrazone products derived from secA and B (A, teal traces) are shown
along with the corresponding mass spectra. DNPH-derived hydrazones derived from chol 7α-OOH are indicated along with their corresponding mass spectra (B).
O* are not properly aligned for fragmentation to occur in the 7b-
OOH, as illustrated in Scheme 2B. The conformational change
required to achieve the epoxycarbenium ion from protonated 7b-
OOH forces both the A- and B-rings into sub-optimal half-chair
conformations, as opposed to the more favourable A-ring chair
and B-ring twist-boat arising from protonation of chol 7a-OOH.
DNPH derivatization of the isolated A-ring cleavage
products yielded the corresponding hydrazones. The epoxide and
diol coincided with the mixture of compounds eluting at 19
minutes and the monoethylated product coincided with the
compound eluting at 21 minutes. Interconversion of the epoxide
and diol was noted in both the DNPH derivatized and
underivatized samples, leading to differing ratios as a function of
derivatization conditions and time. Related to this point, it should
be noted that the ionization for the molecular ion of the derivatized
diol (m/z = 597) was not particularly strong by ESI; instead, m/z =
579 and 615 were prominent peaks, implying facile dehydration
to the epoxide (or a ketone) and hydration of the imine in the
source. Nevertheless, carrying out selected ion monitoring of
derivatized samples of either chol autoxidations or simply chol 7a-
OOH at m/z = 597 yielded chromatograms where these products
and secB were essentially indistinguishable.
In conclusion, consistent with earlier reports,[39,40] the
autoxidation of cholesterol in phospholipid bilayers yields
primarily chol 7-OOH and chol 5,6-epoxide. Chol 6-OOHs and 4-
OOHs are also observed, but to a significantly lower extent, and
importantly, we find that the ratio of the major products is
influenced by the level of unsaturation in the bilayer. Chol 5α-
OOH is not observed – even in the presence of Vitamin E, which
is capable of trapping chol 5α-OO• in solution, but not in
phospholipid bilayers where it is less effective due to H-bonding
to the phosphate diester moiety. Thus, it appears likely that chol
6b-OOH is the source of electrophilic secA and secB in tissue
samples subjected to acid-catalyzed derivatization. Alternatively,
given the predominance of chol 7-OOH and the foregoing results
that demonstrate fragmentation of the 7a-OOH epimer under
derivatization conditions yields distinct products that have similar
chromatographic and spectroscopic properties to secA and secB,
it is possible that they were misidentified.[41]
Scheme 2. Chol 7α-OOH undergoes Hock fragmentation via an α-epoxycarbenium ion intermediate to generate α-hydroxy/alkoxyepoxide and A-ring cleavage
products (A). Chol 7β-OOH does not undergo Hock fragmentation due to the poor overlap between the p-MO of the C5-C6 double bond and the sO-O* MO of the
protonated hydroperoxide moiety. Driving the protonated hydroperoxide along this reaction coordinate forces the A- and B-rings into half-chair conformations when
starting from the β-epimer vs an A-ring chair and B-ring twist-boat in the case of the α-epimer, the former being 4 kcal/mol lower in energy than the latter (B).
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[26]
[27]
7-Ketocholesterol, a dehydration product of chol 7-OOH, is detected
by UV at 234 nm.
Acknowledgements
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This work was supported by grants from the Natural Sciences and
Engineering Research Council of Canada and the Canada
Foundation for Innovation. ELS and ZAMZ thank the goverments
of Ontario and Canada, respectively, for OGS and NSERC post-
graduate scholarships.
[28]
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Emily L. Schaefer, Nadia Zopyrus, Zosia
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On the Products of Cholesterol
Autoxidation in Phospholipid Bilayers
and the Formation of Secosterols
Derived Therefrom
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