©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Use of an O Inhalation Technique and Mass Isotopomer Distribution Analysis to Study Oxygenation of Cholesterol in Rat
EVIDENCE FOR IN VIVO FORMATION OF 7-OXO-, 7beta-HYDROXY-, 24-HYDROXY-, AND 25-HYDROXYCHOLESTEROL (*)

(Received for publication, March 6, 1995; and in revised form, June 12, 1995)

Olof Breuer (§) Ingemar Björkhem

From the Karolinska Institutet, Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Huddinge University Hospital, S-141 86 Huddinge, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cholesterol oxidation products (oxysterols) have been detected in many different tissues, often at concentrations 10^3 to 10^4 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 ^18O(2) 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 ^18O(2) instead of O(2). Control rats were kept in O(2)-containing atmosphere throughout the experiment. The ^18O 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 ^18O, was established for cholest-5-ene-3beta,7alpha-diol, cholest-5-ene-3beta,7beta-diol, 7-oxocholesterol, cholest-5-ene-3beta,24-diol, cholest-5-ene-3beta,25-diol, and cholest-5-ene-3beta,27-diol. Additionally, it seems likely that cholest-5-ene-3beta,4beta-diol is formed in vivo. The ^18O labeling pattern suggests that there is incomplete equilibration between the liver and plasma pools of cholest-5-ene-3beta,27-diol. No evidence for the in vivo formation of 5,6-oxygenated oxysterols was obtained.


INTRODUCTION

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 10^3 to 10^4 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-^14C]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-3beta,7beta-diol, cholestane-3beta,5alpha,6beta-triol, 5alpha,6alpha-epoxycholestan-3beta-ol, 5beta,6beta-epoxycholestan-3beta-ol, and cholest-5-ene-3beta,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-3beta,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-3beta,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-3beta,25-diol stimulates calcifying osteoblast-like vascular cells in the arterial wall(13) .

Presented here is an ^18O(2) 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 ^18O in oxysterols. The abundance of ^18O-enriched compounds in liver and plasma is determined by gas/liquid chromatography-mass spectrometry and mass isotopomer distribution analysis.


MATERIALS AND METHODS

Chemicals

^18O(2) (95% isotopic and 98% chemical purity) was obtained from Cambridge Isotope Laboratories, Andover, MA, in 1-liter break-seal ampoules of atmospheric pressure. The isotopic purity was checked by gas/liquid chromatography-mass spectrometry. O(2) (98% chemical purity) was purchased from AGA AB, Sweden. Antifoam M (dimethylpolysiloxane/silic acid colloid; 94:6, w/w) was obtained from Draco Läkemedel AB, Lund, Sweden. Hypnorm® (mixture of 10 mg of fluanisone and 0.315 mg of phentanyl citrate in 1 ml of water) was purchased from Janssen Pharmaceutica, Belgium. Tween® 80 (polyoxyethylenesorbitan monooleate) was purchased from Kebo Lab, Stockholm, Sweden.

Equipment for ^18OInhalation Experiments

Fig. 1shows a schematic diagram of the equipment used for ^18O(2) inhalation experiments. An airtight cage (inner measurements 6.9 6.9 24.7 cm) was constructed by gluing together sheets of polyacrylamide. The CO(2)-removing apparatus consisted of an aquatic pump (Type Schego Prima, Schemel & Goetz GmbH and Co. KG, Offenbach am Main, Germany; 660 ml/min flow rate, modified in order to obtain an inlet pipe and to seal off surrounding air), a glass tube containing 10 ml of silica gel and a 100-ml round flask containing a slurry of 90 ml of water, 2.5 g of Ca(OH)(2), 200 mg of Antifoam M, and 5 mg of thymolphthalein. The latter compound, being a pH indicator, was used for signaling impending saturation of the CO(2) absorber. Oxygen was delivered to the cage through a small water seal in order to prevent nitrogen diffusion from the cage to the oxygen container. A physiological composition of the cage atmosphere (i.e. 80% nitrogen) was thus maintained throughout the experiment. Plastic bags (Uribag, Swedish Hospital Supply SHS AB, Sweden) were used as oxygen containers in order to keep the system under an atmospheric pressure. However, a minor fall in cage pressure (about 1 mm of water) was unavoidable due to the water seal. The transfer of ^18O(2) from the glass ampoules to the plastic bags was accomplished by means of a vacuum desiccator and a nonreturn valve (obtained from Cykelbjörk, Sweden).


Figure 1: Schematic diagram of the equipment used for ^18O(2) inhalation.



With this equipment, the ^18O(2) fraction of total oxygen (l) in the cage atmosphere increased according to as a function of consumed oxygen (V(O)(2)). In this equation, l(max) denotes the isotopic purity of the delivered ^18O(2) and k is a constant that is defined by the cage volume. When the supply of ^18O(2) was depleted, O(2) was reconnected. The ^18O(2) fraction thereafter decreased according to , where l(0) denotes the ^18O(2) fraction of total oxygen at the point O(2) was reconnected.

^18O(2) Inhalation Experiments

Rats (130 g body weight) of an outbred Sprague-Dawley strain were given free access to a standard chow and water before the investigation started. Two experiments (A and B) were conducted. In each experiment, one control rat and one ^18O(2)-exposed rat were used. A central venous catheter was introduced into the vena cava via the jugular vein under neuroleptic anesthesia (Hypnorm®, 0.5 ml/kg body weight intramuscular injection). Further anesthesia was thereafter given intravenously (Hypnorm® diluted 1:10 with sterile water, in a dose of 0.5 ml/kg body mass every 30 min). The animals were placed in the airtight cages (see above) and O(2) was delivered during a 15-min equilibration period. ^18O(2) was then given to the test rat, while the control continued receiving O(2). During the ^18O(2) exposure, several samples of the cage atmosphere were taken with 1-ml syringes and the fraction of ^18O(2) of total oxygen was determined by GLC-MS (^1)(cf.Fig. 2).


Figure 2: The fraction of ^18O(2) 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 ^18O(2) was depleted after 178 min, O(2) 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 ^18O(2) exposure, O(2) 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.

Gas/Liquid Chromatography-Mass Spectrometry

Oxysterols were isolated from thawed liver homogenates and plasma samples after mild alkaline hydrolysis essentially as described previously(16) . The oxysterols were then converted to TBDMSi ethers by evaporating the solvent under a stream of argon and subsequently treating the residue with 100 µl of tert-butyldimethylsilylimidazole/dimethylformamide (from Supelco, Inc., Bellefonte, PA) at 22 °C overnight. Thereafter, 1 ml of water was added and the mixture was extracted twice with ethyl acetate. After evaporation under a stream of argon, the residue was dissolved in 50 µl of hexane. Of this mixture, 2 µl were subjected to GLC-MS in selective ion monitoring mode on an HP 5890 gas/liquid chromatograph equipped with a 25-m HP Ultra-1 fused silica column (0.2 mm inner diameter, 0.33 µm film thickness) coupled to an HP 5970 quadrupole type mass spectrometer (Hewlett-Packard, Palo Alto, CA). Helium was used as carrier gas with a column pressure of 75 kiloPascal. Samples (2 µl) dissolved in hexane were injected in splinterless mode. Electron impact ionization at 70 eV was applied. The column temperature was kept at 180 °C for 1 min, subsequently raised at a rate of 35 °C/min to 270 °C, and finally increased 20 °C/min to 310 °C.

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-3beta,5alpha,6beta-triol and cholest-5-ene-3beta,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-3beta,25-diol, loss of one tert-butyl group) and M - 57 - 18 (cholestane-3beta,5alpha,6beta-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-3beta,25-diol and 7-oxocholesterol coeluted under these conditions (Table 1), cholest-5-ene-3beta,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.

Mass Isotopomer Distribution Analysis

Oxysterols were analyzed by GLC-MS in selective ion monitoring mode. The m/z corresponding to fragment ions composed of the lightest natural occurring isotopes (m), as well as the isotopomers at m+2 and m+4 were monitored. The natural mass isotopomer distributions of the fragment ions were determined by analyzing reference samples, i.e. either liver samples from a control rat, or plasma samples collected at the beginning of an ^18O(2) inhalation experiment (cf.Fig. 3). These distributions were found to be close to those calculated theoretically from the elementary compositions of the fragment ions (cf.Table 2).


Figure 3: Ion fragmentograms of cholest-5-ene-3beta,7beta-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 ^18O(2)-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 ^18O atom were subtracted from the ion intensity at m+4. The molar distribution of oxysterols containing zero, one, and two ^18O 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 ^18O is very low. Therefore, there is no sizable skew of natural enrichment of these clusters during progressive ^18O enrichment(17) .

Copper Oxidation of Cholesterol

One ml of the incubation buffer (10 mM sodium phosphate, 5 µM copper sulfate, and 160 mM sodium chloride in aqueous solution, pH 7.4) was added to 100 µg of cholesterol dissolved in 2 mg of Tween. The mixture was incubated for 12 h at 37 °C under an ^18O(2)/argon (80:20, v/v) atmosphere and magnetic stirring. The experiment was repeated with O(2) instead of ^18O(2) in order to obtain a reference sample. Mass isotopomer distribution analysis of oxysterols isolated from the reaction mixture was performed as described above.


RESULTS AND DISCUSSION

^18OInhalation Experiments and Methodological Considerations

In order to test the hypothesis that all oxysterols of non-enzymatic origin are artifacts, rats were subjected to ^18O(2) inhalation experiments. If this hypothesis is valid, one would find that only oxysterols of enzymatic origin would have enrichment in ^18O at other positions than C-3. The incorporation of ^18O at C-3 merely reflects the de novo cholesterol synthesis during the period of ^18O(2) exposure.

In experiment A, three blood samples were taken: (i) at time 0 as a reference, (ii) at the point when the ^18O 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 ^18O 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 ^18O(2) 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, ^18O(2) 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 ^18O(2) 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 ^18O(2) exposure) were lower than in the reference sample (a plasma sample obtained at time 0 or a liver sample from an O(2)-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 ^18O(2) inhalation experiments, oxysterols should always be compared with cholesterol. In order to signify ^18O incorporation at other positions than C-3, and hence in vivo oxidation, the ^18O incorporation in an oxysterol has to be considerably larger than that of cholesterol. It should be noted that ^18O enrichment does not exclude the possibility that the compound is to some extent of dietary origin or formed in vitro.

Analysis of Hydroxylations at Position C-7

Cholesterol oxidation by copper in an ^18O(2) atmosphere yielded oxysterols with O at the C-3 position and ^18O at other positions such as C-25 or C-7. The major ^18O-labeled compounds were cholest-5-ene-3beta,7alpha-diol and cholest-5-ene-3beta,7beta-diol (Table 5), as well as 5alpha,6alpha-epoxycholestan-3beta-ol, 5beta,6beta-epoxycholestan-3beta-ol and 7-oxocholesterol (data not shown). These oxysterols were subjected to mass spectrometric analysis as TMSi-derivatives. As shown in Table 5, most of the ^18O at position C-7 in cholest-5-ene-3beta,7alpha-diol and cholest-5-ene-3beta,7beta-diol was retained after loss of one trimethylsiloxy group yielding the M - 90 fragment ion. Thus, for the 7-hydroxylated diols it is possible to discriminate between incorporation of ^18O at C-3 and C-7 by comparing the ^18O enrichment of the molecular ion with that of the M - 90 fragment ion.



^18O Enrichment of Cholesterol and Its Precursor Lathosterol

As presented in Table 3and Table 4, a considerable ^18O enrichment of lathosterol, a well known cholesterol precursor, was observed in plasma as well as liver. In contrast, the ^18O enrichment of cholesterol in samples from plasma and liver barely exceeded the background noise.

Cholest-5-ene-3beta,7alpha-diol

Cholest-5-ene-3beta,7alpha-diol is a known product of the key enzyme in bile acid synthesis, cholesterol 7alpha-hydroxylase. This enzyme is a cytochrome P-450-dependent monooxygenase, i.e. it utilizes molecular oxygen for the stereospecific addition of one oxygen atom to the substrate. It has been established that the cholest-5-ene-3beta,7alpha-diol found in blood is predominantly of hepatic origin and that the serum concentration of this compound covaries with the activity of hepatic cholesterol 7alpha-hydroxylase(18) . In accordance with this, a marked enrichment of cholest-5-ene-3beta,7alpha-diol in ^18O was observed in samples from plasma as well as liver (cf.Table 3and Table 4).

Analysis of TMSi-derivatized liver preparations showed that a large fraction of the M - 90 fragment ion was ^18O-labeled (cf. Table 5). Hence, most of the ^18O was incorporated at position C-7.

Cholest-5-ene-3beta,7beta-diol

It is generally believed that cholest-5-ene-3beta,7beta-diol is predominantly of non-enzymatic origin. According to Kudo et al., most, if not all, of the cholest-5-ene-3beta,7beta-diol found in human blood samples is formed during sample processing(7) . In our experiments, there was an appreciable ^18O enrichment of this compound in plasma and liver samples. In contrast to all the other oxysterols analyzed, the di-^18O incorporation in cholest-5-ene-3beta,7beta-diol in liver (experiment B) was nearly as high as the mono-^18O incorporation. As shown in Table 5, analysis of the M - 90 fragment ion of TMSi-derivatized cholest-5-ene-3beta,7beta-diol from a liver preparation confirmed the incorporation of ^18O at C-7. Altogether, these results indicate the presence of molecules with a considerable di-^18O incorporation at both positions C-3 and C-7, as well as molecules with high mono-^18O incorporation at either position C-3 or C-7. In view of the very high enrichment of lathosterol in ^18O, it is tempting to suggest that cholest-5-ene-3beta,7beta-diol is predominantly formed from newly synthesized cholesterol, or a cholesterol precursor, by enzymatic reactions in vivo.

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 ^18O incorporation at the C-3 position of cholest-5-ene-3beta,7beta-diol was observed in the present investigation, it is not likely that this compound is produced secondary to lipid peroxidation. Neither does this ^18O labeling pattern support formation by epimerization of cholest-5-ene-3beta,7alpha-diol or reduction of 7-oxocholesterol.

7-Oxocholesterol

The formation of 7-oxocholesterol has been observed secondary to lipid peroxidation (21) and lipoxygenase-catalyzed reactions(22) . 7-Oxocholesterol has also been detected in large quantities in air-aged cholesterol(23) . It is known that 7-oxocholesterol is readily formed from cholesterol unless anti-oxidative precautions are taken(1) . Furthermore, copper-catalyzed cholesterol oxidation under an ^18O atmosphere yielded a substantial amount of ^18O-enriched 7-oxocholesterol (data not shown). In the present investigation, an appreciable monoincorporation of ^18O in 7-oxocholesterol was observed in preparations from plasma as well as liver (cf. Tables III and IV).

It is noteworthy that the measured fraction of ^18O-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 ^18O incorporation in 7-oxocholesterol in vivo is even higher than observed in the present investigation.

5,6-Oxygenated Oxysterols

Cholestane-3beta,5alpha,6beta-triol is known as the hydrolysis product of 5alpha,6alpha-epoxycholestan-3beta-ol and 5beta,6beta-epoxycholestan-3beta-ol(1, 6) . As cholestane-3beta,5alpha,6beta-triol was difficult to detect due to low abundance, mass isotopomer analysis was only possible in one sample from a liver preparation (cf.Table 3). In this single observation, no ^18O enrichment of cholestane-3beta,5alpha,6beta-triol was found.

In the present investigation, no enrichment of 5alpha,6alpha-epoxycholestan-3beta-ol in ^18O 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 5alpha,6alpha-epoxycholestan-3beta-ol and cholestane-3beta,5alpha,6beta-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 ^18O. Thus, the absence of ^18O enrichment does not per se exclude formation in vivo.

With the conditions employed for gas/liquid chromatography, 5beta,6beta-epoxycholestan-3beta-ol partially coeluted with interfering compounds. As a consequence, mass isotopomer distribution analysis of 5beta,6beta-epoxycholestan-3beta-ol was hampered.

Cholest-5-ene-3beta,4beta-diol

We have recently identified cholest-5-ene-3beta,4beta-diol as one of the predominating oxysterols in human plasma and rat liver. (^2)The mass isotopomer distribution of this compound was not analyzed in plasma from experiment A, since the occurrence of cholest-5-ene-3beta,4beta-diol was not known at the time the experiment was conducted. As the fraction of mono-^18O-labeled cholest-5-ene-3beta,4beta-diol in liver samples and a single plasma sample (from experiment B) was about 2-3 times higher than the fraction of ^18O-labeled cholesterol, an in vivo formation of cholest-5-ene-3beta,4beta-diol seems likely.

Cholest-5-ene-3beta,24-diol

It was not possible to determine the mass isotopomer distribution of cholest-5-ene-3beta,24-diol in liver preparations, since the concentration of this compound was too low. The analysis of plasma preparations showed a mono-^18O enrichment of cholest-5-ene-3beta,24-diol that was markedly higher than that of cholesterol. It has been shown previously that a purified preparation of sterol 27-hydroxylase from porcine liver is able to catalyze 24-hydroxylation of cholesterol, although the reaction rate is much lower as compared to 27-hydroxylation(24) . Altogether, an enzymatic origin of cholest-5-ene-3beta,24-diol has to be considered.

Cholest-5-ene-3beta,25-diol

In the present investigation, cholest-5-ene-3beta,25-diol was appreciably mono-^18O-labeled in preparations from plasma as well as liver (cf. Tables III and IV). Incorporation of ^18O at C-25 was confirmed by analysis of TMSi-derivatized oxysterols in a liver preparation from an ^18O(2)-exposed rat. The mono-^18O-labeled fraction (42%) of the molecular ion was essentially as large as the ^18O-labeled fraction (43%) of a C-25-containing fragment ion (M - 415), whereas the di-^18O-labeled fraction (4%) of the molecular ion was small. These findings are consistent with a mono-labeling at C-25. Thus, it is evident that cholest-5-ene-3beta,25-diol is produced in vivo.

This is also in line with the recent findings of Johnson et al.(25) . Rats were given deuterium oxide (D(2)O), and the subsequent incorporation of deuterium in oxysterols was determined (25) . A considerable incorporation of deuterium in cholest-5-ene-3beta,25-diol in liver was observed, whereas no deuterium incorporation in cholest-5-ene-3beta,7beta-diol was found (25) . Johnson et al. therefore concluded that no autoxidation had occurred and that cholest-5-ene-3beta,25-diol was formed in vivo. However, it should be noted that the absence of deuterium incorporation in cholest-5-ene-3beta,7beta-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.

Cholest-5-ene-3beta,27-diol

It has been shown previously that a cytochrome P-450-dependent cholesterol 27-hydroxylase is present in liver(26) , as well as many other organs and tissues(27, 28) . As expected, a high degree of mono-^18O incorporation of cholest-5-ene-3beta,27-diol was observed in plasma preparations (cf.Table 3and Table 4). In contrast, the ^18O-enriched fraction of cholest-5-ene-3beta,27-diol isolated from liver preparations was rather small. It has been suggested that 27-hydroxylation and secretion of cholest-5-ene-3beta,27-diol might be an important mechanism for the elimination of redundant intracellular cholesterol from endothelial cells (29) and macrophages(30) . Furthermore, recent findings suggest that there is a continuous flux of cholest-5-ene-3beta,27-diol from peripheral tissues to the human liver. (^3)After uptake by the liver, 27-oxygenated sterols are converted into bile acids and excreted(27) . In the present investigation, the low degree of ^18O incorporation in cholest-5-ene-3beta,27-diol in liver preparations suggests an incomplete equilibration between the liver and the plasma pools of cholest-5-ene-3beta,27-diol.

Concluding Remarks

With the ^18O(2) inhalation technique, the extent of ^18O incorporation in an oxysterol depends on the biological half-life of the sterol, the equilibration time of the system, and the extent of in vitro autoxidation. In the present investigation, rats were exposed to about 80% ^18O(2) (fraction of total oxygen) for more than 90 min, as shown in Fig. 2. Theoretically, one would therefore expect a maximum of 80% mono-^18O incorporation in an oxysterol with a very short half-life. The enrichment of lathosterol in ^18O, a steroid that is not formed by in vitro oxidation and with a fast turnover, was about 55%. It is therefore tempting to suggest that the maximum obtainable mono-^18O incorporation in oxysterols is close to 55% under the conditions employed.

Interestingly, the ^18O 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-3beta,7alpha-diol, cholest-5-ene-3beta,7beta-diol, cholest-5-ene-3beta,25-diol, and cholest-5-ene-3beta,27-diol had all a substantial enrichment in ^18O, close to the suggested maximum, and it is therefore evident that these compounds were predominantly formed in vivo. A lower degree of ^18O incorporation was observed for cholest-5-ene-3beta,4beta-diol, cholest-5-ene-3beta,24-diol, and 7-oxocholesterol. This may be due to a longer biological half-life or in vitro oxidation. Some of the ^18O 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-3beta,7alpha-diol and cholest-5-ene-3beta,27-diol), but also for cholest-5-ene-3beta,7beta-diol, cholest-5-ene-3beta,24-diol, cholest-5-ene-3beta,25-diol, and 7-oxocholesterol. It seems likely that also cholest-5-ene-3beta,4beta-diol is formed in vivo.

As no evidence for in vivo formation of 5alpha,6alpha-epoxycholestan-3beta-ol or cholestane-3beta,5alpha,6beta-triol could be obtained with the present technique, it is possible that these compounds mainly are of dietary origin or artifacts produced in vitro.


FOOTNOTES

*
This work was supported by grants from the Swedish Medical Research Council, the Marianne and Marcus Wallenberg Foundation, the Swedish Society for Medical Research, the Swedish Society of Medicine and ``Förenade Liv'' Mutual Group Life Insurance Company, Stockholm, Sweden. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 46-8-7461072; Fax: 46-8-7748393.

(^1)
The abbreviations used are: GLC-MS, gas/liquid chromatography-mass spectrometry; TBDMSi, tert-butyldimethylsilyl; TMSi, trimethylsilyl.

(^2)
O. Breuer, unpublished observation.

(^3)
E. Lund, personal communication.


ACKNOWLEDGEMENTS

We acknowledge Dr. Erik Lund for presenting valuable ideas on derivatization techniques for gas/liquid chromatography and John Öhman for assisting in the assembly of the ^18O(2) inhalation equipment.


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