in pyridine, conditions where 4a is completely stable (ESI,w
Table S10, Fig. S7-S8)). A comparison of this IDGCMS assay
with the 2,4-DNP hydrazine method in a plasma analysis of
1b-derivatives in the apoEÀ/À mouse model of atherosclerosis
reveals that the measured levels are not significantly different
[GCMS method = 7.5 Æ 1.3 nM (n = 4); 2,4-DNP hydrazine
method = 9.3 Æ 2.4 nM (n = 6); p 4 0.05], suggesting that 4a
is not contributing significantly to the measured amounts of 2b
in this system (ESI,w Fig. S9).
Analysis of human arterial plaque extracts by IDGCMS
reveals a mean [1a(b)] of 32 Æ 15 pmol mgÀ1 of tissue which is
similar to levels we previously reported for 2b in plaque
extracts2 (ESI,wFig. S10–S12). Interestingly, treatment of these
arterial plaque extracts with Zn/acetic acid prior to lipid
extraction results in an increase in the measured 1a(1b) levels,
suggesting that either 4a or cholesterol ozonides are present in
these atheroma samples and are being converted into 1a and
1b during tissue processing. We are currently investigating this
finding (ESI,w Fig. S11 and S12).
We are also interested in understanding to what extent the
acid-catalyzed rearrangement of 4a or a chemical oxidant with
the chemical signature of ozone may be contributing to
cholesterol secosterol biogenesis in vivo. As described vide
supra authentic 4a is stable at physiological pH for 420 h
and 5a-hydroperoxide 4a has been shown to be isolable from
biological samples by a number of groups.4,10 However, one
cannot discount that the formation of 1b, which is ubiquitous
in all biological samples we have tested thus far, does not, at
least in part, arise from this route.
Pratt and co-workers1 have shown that Hock-cleavage of
authentic 4a in organic solvents leads to levels of 1bc1a and
we herein have shown that 4a, generated by chemical- and
biological-oxidations of cholesterol in aqueous buffers,
generates 2b (with no measurable 2a) upon 2,4-DNP hydrazine
derivatization. Ozonolysis of 3 in aqueous buffers generates
1ac1b (at all physiologically-relevant pHs either in the
presence or absence of added chemical reductant). We have
also studied the ozone-mediated oxidation of hLDL-bound
cholesterol in aqueous buffer over a range of pH values,
temperatures and durations of reaction and found that the
ratio 1a : 1b varies from B15 : 1 to B4 : 1, but in all cases, the
level of atheronal-A is greater than atheronal-B (ESI,w
Table S12). An analysis of our original report of the levels of
cholesterol secosterols (measured as their hydrazones 2a
and 2b) in human arterial plaque samples reveals that the
ratio of 2a : 2b varies greatly (61.3 : 1 to 0.06 : 1, n = 28)
(ESI,w Table S11). However, the ratio of 2a : 2b is 41 in 20 of
the 28 samples and 410 in 10 of the 28 samples. Therefore, the
dominant form of cholesterol 5,6-secosterols in arterial plaque
extracts, where aldolization is minimal, is 1a.2 Based upon our
new understanding of the ozone and singlet oxygen pathways
from 3 into 2a and 2b (after 2,4-DNP hydrazine derivatization),
this clinical data offers support for an oxidant with the
chemical signature of ozone playing a composite role in
arterial plaque cholesterol secosterol 1a biogenesis.
While the debate as to the relevance of ozone generation in
biological systems continues,11 we continue to search for more
direct markers of this oxidant in biological extracts. The
terminal products from cholesterol ozonolysis, 1a and 1b,
may not discriminate the pathways to their formation under
all conditions, however, a ratio of 1a to 1b of 41 is strong
support for oxidation of 3 by an oxidant with the chemical
signature of ozone. Our current search is aimed at quantifying
more direct species that originate as a result of the primary
chemical reaction of ozone with biological molecules, such as
1,2,4-trioxolanes.
Financial support from the Skaggs Institute for Chemical
Biology and the NIH (AG028300).
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This journal is The Royal Society of Chemistry 2009
3100 | Chem. Commun., 2009, 3098–3100