(Received for publication, March 6, 1995; and in revised form, June 12, 1995)
From the
Cholesterol oxidation products (oxysterols) have been detected
in many different tissues, often at concentrations 10 to
10
times lower than cholesterol. This constitutes a
considerable risk of quantitation errors, since even a minor oxidation
of cholesterol during sample processing would yield a substantial
increase of oxysterol levels. It has therefore been suggested that some
of the oxysterols do not occur in vivo and their detection in
tissues merely are artifacts produced in vitro.
In the
present work, an O
inhalation technique was
developed in order to clarify which oxysterols are produced in
vivo. Rats were exposed for 3 h to an atmosphere with a
composition similar to normal air, except that it contained
O
instead of
O
.
Control rats were kept in
O
-containing
atmosphere throughout the experiment. The
O enrichment of
oxysterols in plasma and liver was determined by gas/liquid
chromatography-mass spectrometry and mass isotopomer distribution
analysis.
In vivo formation of oxysterols, indicated by
enrichment in O, was established for
cholest-5-ene-3
,7
-diol, cholest-5-ene-3
,7
-diol,
7-oxocholesterol, cholest-5-ene-3
,24-diol,
cholest-5-ene-3
,25-diol, and cholest-5-ene-3
,27-diol.
Additionally, it seems likely that cholest-5-ene-3
,4
-diol is
formed in vivo. The
O labeling pattern suggests
that there is incomplete equilibration between the liver and plasma
pools of cholest-5-ene-3
,27-diol. No evidence for the in vivo formation of 5,6-oxygenated oxysterols was obtained.
Oxysterols are compounds that are formed as a result of cholesterol oxidation. Such reactions occur either as a part of enzymatic cholesterol metabolism or due to autoxidation. It has been shown that many oxysterols exert various biological effects(1, 2) , including inhibition (3) or stimulation (4, 5) of important enzymes in cholesterol metabolism.
During the last decades many attempts have been made to
quantify oxysterols in various tissues and
foodstuffs(1, 6) . However, the results of several of
these investigations have been ambiguous or even contradictory.
Cholesterol is present in close proximity to oxysterols, often at
concentrations 10 to 10
times higher than those
of oxysterols. This constitutes a considerable risk of quantitation
artifacts, as even a minor oxidation of cholesterol during sample
preparation would yield a substantial elevation of oxysterol levels.
In all investigations of oxysterols in biological systems, the possibility of oxysterol formation in vitro can hardly be excluded, despite all antioxidative measures taken. Thus, an overriding problem in quantitative analyses is to estimate the contribution from formation in vitroversus presence in vivo. One way of solving this is to trace the in vitro oxidation with radiolabeled cholesterol(7) . However, such methods call for an exceptional purity of the labeled cholesterol. Furthermore, autoxidation of highly purified labeled cholesterol might occur before it is added to samples.
In a carefully performed study by Kudo et al.(7) , oxysterols were assayed after addition of
highly purified [4-C]cholesterol in order to
detect in vitro oxidation during sample processing. After
correction for such in vitro formation, plasma samples from
two human subjects were reported to contain very little or none of
cholest-5-ene-3
,7
-diol,
cholestane-3
,5
,6
-triol,
5
,6
-epoxycholestan-3
-ol,
5
,6
-epoxycholestan-3
-ol, and
cholest-5-ene-3
,25-diol(7) .
The findings by Kudo et al.(7) raise the question whether the above
oxysterols are formed at all in vivo. The answer is important
in view of the fact that cholest-5-ene-3,25-diol is known as one
of the most potent inhibitors of 3-hydroxy-3-methylglutaryl-CoA
reductase, the rate-limiting enzyme of cholesterol
synthesis(8, 9, 10) . In addition,
cholest-5-ene-3
,25-diol down-regulates the synthesis of
low-density lipoprotein receptors(11) , stimulates
acyl-CoA:cholesterol O-acyltransferase(4, 5) , and inhibits
3-hydroxy-3-methylglutaryl-CoA synthase(12) . It has also been
suggested that cholest-5-ene-3
,25-diol stimulates calcifying
osteoblast-like vascular cells in the arterial wall(13) .
Presented here is an O
inhalation technique
that was developed in order to clarify which oxysterols are produced in vivo in rat. A similar methodology has been used previously
to study the metabolism of monoamines (14) and the synthesis of
bile acids(15) . With the experimental model, an in vivo cholesterol oxidation results in an incorporation of
O in oxysterols. The abundance of
O-enriched
compounds in liver and plasma is determined by gas/liquid
chromatography-mass spectrometry and mass isotopomer distribution
analysis.
Figure 1:
Schematic diagram of the equipment
used for O
inhalation.
With this equipment, the O
fraction of
total oxygen (l) in the cage atmosphere increased according to as a function of consumed oxygen (V
). In this equation, l
denotes the isotopic purity of the delivered
O
and k is a constant that is defined
by the cage volume. When the supply of
O
was
depleted,
O
was reconnected. The
O
fraction thereafter decreased according to , where l
denotes the
O
fraction of total oxygen at the point
O
was reconnected.
Figure 2:
The fraction of O
of total oxygen in the cage atmosphere. The points of blood
sampling are indicated by arrows.
In experiment A, blood samples were
drawn at 0, 178, and 205 min from the test rat and at 0 and 190 min
from the control rat. On each occasion a 2-ml sample of blood was
collected in a glass tube (containing 0.12 ml of 0.34 M potassium EDTA) and stored on ice. Subsequently, 2 ml of 0.9%
saline was infused via the catheter. When the supply of O
was depleted after 178 min,
O
was delivered to the cage for 25 min. The
final blood samples were then drawn, and the animals were killed by
cervical dislocation. The livers were immediately isolated and
homogenized in 0.1 M potassium phosphate buffer (containing 2
mM EDTA and 11 µg/ml butylated hydroxytoluene, pH 7.4, 9
ml of buffer/g of liver) using a Potter-Elvehjem type homogenizer.
Plasma was obtained by low speed centrifugation of the blood samples in
the cold. The liver homogenates and plasma samples were stored for 20
days at -20 °C.
In experiment B, blood samples, 2
ml, were drawn at 0 and 210 min from the test rat as well as the
control animal. On each occasion, 2 ml of 0.9% saline was infused
subsequently. After 183 min of O
exposure,
O
was delivered to the cage for 22 min. The
cage was then opened wide at 205 min to allow equilibration with room
air for 5 min. The final blood samples were thereafter drawn, the
animals were killed, and the livers isolated. Liver homogenates and
plasma samples were prepared and stored as in experiment A.
TBDMSi derivatives seemed to improve the mass spectrometric sensitivity as compared to TMSi derivatives. This was probably due to a lower degree of fragmentation and more efficient gas/liquid chromatographic separation from interfering compounds. The loss of one tert-butyl group from the molecular ion (M) yielded an intense fragment ion (M - 57), which was selected for mass isotopomer distribution analysis of most oxysterols.
However, cholestane-3,5
,6
-triol and
cholest-5-ene-3
,25-diol did not elute from the GLC column,
probably due to underivatized hydroxyl groups at the sterically
hindered C-6 and C-25 positions, respectively. As TMSi ethers are less
bulky than TBDMSi ethers, the above positions were successfully
TMSi-derivatized after TBDMSi-derivatization by evaporating the solvent
and treating the residue with 100 µl of
pyridine/hexamethyldisilazane/trimethylchlorosilane (3:2:1, v/v/v) at
60 °C for 30 min. The preparations were then reanalyzed while the
fragment ions at M - 57 (cholest-5-ene-3
,25-diol, loss of
one tert-butyl group) and M - 57 - 18
(cholestane-3
,5
,6
-triol, loss of tert-butyl and
hydroxyl groups) were monitored. The derivatives analyzed, retention
times, and m/z monitored are shown in Table 1. Although
cholest-5-ene-3
,25-diol and 7-oxocholesterol coeluted under these
conditions (Table 1), cholest-5-ene-3
,25-diol was easily
resolved by its higher m/z (531 versus 457).
Cholesterol and lathosterol were analyzed as TMSi ethers under essentially similar GLC conditions as above, except that the final oven temperature was 300 °C.
The identities of the derivatized sterols were confirmed by comparing their retention times with those of high-purity references.
Figure 3:
Ion fragmentograms of
cholest-5-ene-3,7
-diol in plasma obtained in experiment B
(see ``Materials and Methods''). The m+0, m+2, and
m+4 isotopomers were monitored at m/z 573, 575, and 577,
respectively. Fragmentograms from a sample collected at 0 (leftpanel) and 210 min (rightpanel) are
shown.
In analysis of oxysterols from O
-exposed animals, the contribution from the
natural isotopomer cluster of unlabeled molecules was subtracted from
the monitored intensity at m+2. Subsequently, the contributions
from the isotope clusters of molecules with zero or one
O
atom were subtracted from the ion intensity at m+4. The molar
distribution of oxysterols containing zero, one, and two
O
atoms was then calculated from the cluster-corrected ion intensities.
This mode of correction for isotope clusters is legitimate because
natural enrichment of oxygen in
O is very low. Therefore,
there is no sizable skew of natural enrichment of these clusters during
progressive
O enrichment(17) .
In experiment
A, three blood samples were taken: (i) at time 0 as a reference, (ii)
at the point when the O labeled fraction of total oxygen
in the cage air had reached its maximum, and (iii) at the point when
there was about a 50% decrease in the labeled fraction of oxygen. This
sampling scheme would determine whether any oxysterols are produced in vivo. If this is the case, the difference in
O
enrichment of oxysterols between the last two plasma samples would give
an indication of the biological half-life. However, with such a
sampling scheme there is a theoretical risk that
O
dissolved in plasma or bound to hemoglobin might oxidize
cholesterol during sample processing. To overcome this, conditions were
modified in experiment B. As shown in Fig. 2, the final
samples in this experiment were taken after exposure to normal air for
5 min. During this period of time,
O
should
have been cleared from the tissues to be sampled while compounds with a
short half-life would still be detectable.
The results from the O
exposure experiments are presented in Table 3and Table 4. Negative values of isotopomer
distribution were obtained when the measured
[m+2]/[m] or
[m+4]/[m] ion intensity ratios in the test
sample (a sample obtained after
O
exposure)
were lower than in the reference sample (a plasma sample obtained at
time 0 or a liver sample from an
O
-exposed
control rat). Such negative values should be regarded as a part of the
background noise, which had an amplitude of approximately ±2%
isotope distribution.
In the evaluation of results from O
inhalation experiments, oxysterols should
always be compared with cholesterol. In order to signify
O
incorporation at other positions than C-3, and hence in vivo oxidation, the
O incorporation in an oxysterol has to
be considerably larger than that of cholesterol. It should be noted
that
O enrichment does not exclude the possibility that
the compound is to some extent of dietary origin or formed in
vitro.
Analysis of TMSi-derivatized
liver preparations showed that a large fraction of the M - 90
fragment ion was O-labeled (cf. Table 5).
Hence, most of the
O was incorporated at position C-7.
The formation of both epimeric
7-hydroxycholesterols from cholesterol during NADPH-dependent enzymatic
lipid peroxidation has been observed in liver microsomes(19) .
These enzymatic reactions reduce Fe to
Fe
(20) , which in turn nonspecifically catalyzes
cholesterol autoxidation. Since a substantial
O
incorporation at the C-3 position of cholest-5-ene-3
,7
-diol
was observed in the present investigation, it is not likely that this
compound is produced secondary to lipid peroxidation. Neither does this
O labeling pattern support formation by epimerization of
cholest-5-ene-3
,7
-diol or reduction of 7-oxocholesterol.
It is
noteworthy that the measured fraction of O-enriched
7-oxocholesterol decreased with harsh conditions for hydrolysis (data
not shown). This could be explained by an enhanced oxygen exchange
between water and the 7-ketone due to a strong alkaline environment. It
is therefore possible that the
O incorporation in
7-oxocholesterol in vivo is even higher than observed in the
present investigation.
In the present
investigation, no enrichment of 5,6
-epoxycholestan-3
-ol
in
O was observed in plasma or liver. Also, we have
experienced that quantitations of 5,6-oxygenated cholesterol in various
tissues are less reproducible than measurements of monohydroxylated
cholesterol species.
Altogether, it seems plausible that a
substantial part of 5,6
-epoxycholestan-3
-ol and
cholestane-3
,5
,6
-triol is of dietary origin or, as
suggested by Kudo et al.(7) , formed during sample
processing. However, another possibility is that the 5,6-oxygen
functions originate from water which has a negligible incorporation of
O. Thus, the absence of
O enrichment does not per se exclude formation in vivo.
With the
conditions employed for gas/liquid chromatography,
5,6
-epoxycholestan-3
-ol partially coeluted with
interfering compounds. As a consequence, mass isotopomer distribution
analysis of 5
,6
-epoxycholestan-3
-ol was hampered.
This is also in line with the recent findings of Johnson et
al.(25) . Rats were given deuterium oxide
(DO), and the subsequent incorporation of deuterium in
oxysterols was determined (25) . A considerable incorporation
of deuterium in cholest-5-ene-3
,25-diol in liver was observed,
whereas no deuterium incorporation in cholest-5-ene-3
,7
-diol
was found (25) . Johnson et al. therefore concluded
that no autoxidation had occurred and that cholest-5-ene-3
,25-diol
was formed in vivo. However, it should be noted that the
absence of deuterium incorporation in cholest-5-ene-3
,7
-diol
does not necessarily indicate that cholesterol oxidation at C-25 occurs in vivo.
Cytochrome P-450-dependent 25-hydroxylation of cholesterol has been observed in rat liver mitochondria along with 27-hydroxylation (26) . It is still uncertain whether there are two separate systems for 25- and 27-hydroxylation or only one 27-hydroxylase with broad substrate specificity.
Interestingly, the O enrichment of
7-oxygenated sterols from plasma and liver was lower in experiment A
than in experiment B. Since the 7-oxygenated oxysterols are all common
autoxidation products, this finding might suggest that the samples in
experiment A were autoxidized in vitro to some extent.
In
experiment B, cholest-5-ene-3,7
-diol,
cholest-5-ene-3
,7
-diol, cholest-5-ene-3
,25-diol, and
cholest-5-ene-3
,27-diol had all a substantial enrichment in
O, close to the suggested maximum, and it is therefore
evident that these compounds were predominantly formed in
vivo. A lower degree of
O incorporation was observed
for cholest-5-ene-3
,4
-diol, cholest-5-ene-3
,24-diol, and
7-oxocholesterol. This may be due to a longer biological half-life or in vitro oxidation. Some of the
O in
7-oxocholesterol might also have been lost due to equilibration between
the 7-keto group and water.
To summarize, in vivo formation
of oxysterols has been established, not only for compounds generally
known to be products of enzymatic reactions
(cholest-5-ene-3,7
-diol and cholest-5-ene-3
,27-diol),
but also for cholest-5-ene-3
,7
-diol,
cholest-5-ene-3
,24-diol, cholest-5-ene-3
,25-diol, and
7-oxocholesterol. It seems likely that also
cholest-5-ene-3
,4
-diol is formed in vivo.
As no
evidence for in vivo formation of
5,6
-epoxycholestan-3
-ol or
cholestane-3
,5
,6
-triol could be obtained with the
present technique, it is possible that these compounds mainly are of
dietary origin or artifacts produced in vitro.