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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:778-785.)
© 1997 American Heart Association, Inc.

Articles

The Effect of Oxidizing Cholesterol on Gastrointestinal Absorption, Plasma Clearance, Tissue Distribution, and Processing by Endothelial Cells

Louis H. Krut; Joseph W. Yang; Gustav Schonfeld; ; Richard E. Ostlund, Jr

From the Metabolism Division (L.H.K., J.W.Y., R.E.O.) and the Division of Atherosclerosis, Nutrition, and Lipid Research (L.H.K., G.S.), Washington University School of Medicine, St Louis, Mo.

Correspondence to Dr Louis H. Krut, St Mary's Health Center, Department of Internal Medicine, 6420 Clayton Rd, St Louis, MO 63117.

	Abstract

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Abstract Little is known about the absorption or metabolism of oxysterols. Toward better appreciating the metabolic consequences of oxidizing cholesterol, we compared labeled cholesterol with the labeled oxysterols 7-hydroxycholesterol, 7ß-hydroxycholesterol, and 7-ketocholesterol prepared from [4-¹⁴C]cholesterol, [26,26,26,27,27,27-²H₆]cholesterol, and [23,24,25,26,27-¹³C₅]cholesterol. Gastrointestinal absorption of oxysterols in rats was 91.5±0.3% compared with 75±1.1% for cholesterol, determined by fecal collection (P<.001). When injected intravenously and followed by gas chromatography/mass spectrometry, 7-hydroxycholesterol was cleared at 23 times the rate of cholesterol. After 5 minutes, only 1.2±0.2% of 7-hydroxycholesterol remained in the plasma, whereas 28.0±1.7% of cholesterol and 40.0±2.5% of a triglyceride emulsion injected simultaneously were still present. [¹⁴C]7-Hydroxycholesterol injected intravenously was also rapidly cleared from plasma, was widely distributed in tissues and organs, and showed evidence of extensive metabolism at 5 minutes. The fractional rate of uptake of radiolabeled oxysterols by cultured endothelial cells was 15.7 times that of cholesterol (P<.001), and the fractional rate of efflux was 3.4 times that of cholesterol (P<.001). Oxysterols passed through endothelial cells grown on transwell membranes at a rate 4.3 times that of cholesterol (P<.001). Fractional oxysterol transport across the endothelial cell monolayer was increased 62±17% when HDL was added to the medium in the lower chamber (P=.003). Oxysterols were extensively metabolized to even more polar metabolites during endothelial cell transit. These properties of oxysterols potentially provide a mechanism for enhancing transport of cholesterol through tissues and preventing accumulation of cholesterol in those cells that can oxidize it.

Key Words: cholesterol • cholesterol absorption • oxysterols • endothelial cells • kinetics

	Introduction

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Oxidation products of cholesterol (oxysterols) have potent biological actions, but the role of oxysterols in physiology is still controversial. It has been reported that oxysterols themselves cause atherosclerosis and are directly angiotoxic,^{1 2 3} but other investigators have found no evidence of toxicity and, quite to the contrary, that they prevent or retard atherogenesis.^{4 5 6} Oxysterols act synergistically with phosphatidylcholine to enhance the solubility of cholesterol in vitro,⁷ effect the clearance of tabletted cholesterol implanted subcutaneously,⁸ and inhibit hydroxymethylglutaryl–coenzyme A reductase,⁹ all properties that are potentially beneficial. Thus, oxysterols are likely to have multiple biological effects.

The net effect of oxysterols is of direct interest since they are both synthesized by the body and eaten in foods. Oxysterols are critical intermediates in the synthesis of bile acids by the liver¹⁰ and can be measured in plasma.¹¹ Plasma levels appear to be regulated, as suggested by the observation that bile acid sequestrant treatment elevates plasma 7-hydroxycholesterol in humans up to 25-fold.^{12 13} Despite modern technology in food processing, oxysterols are still found in many foods.^{14 15 16}

It is plain that whether we ingest them or synthesize them, oxysterols are an inescapable part of our biological milieu, yet knowledge of their actions is sketchy and unclear. Toward understanding the metabolic consequences of oxidizing cholesterol, we measured oxysterol absorption and clearance from plasma in rats, the distribution in organs and tissues, and the kinetics of oxysterol uptake and release in cultured endothelial cells. These results show that the metabolism of cholesterol is significantly modified when it is oxidized.

	Methods

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Cholesterol Oxidation
Cholesterol and cholesterol tracers were subjected to oxidation by the method of Bergstrom and Wintersteiner¹⁷ as modified for microgram and milligram amounts of material.¹⁸ This gives the oxidation products 7-ketocholesterol and 7- and 7ß-hydroxycholesterol and leaves a substantial amount of unaltered cholesterol. Oxidation was performed on natural cholesterol (Sigma), [4-¹⁴C]cholesterol (51.3 mCi/mmol, DuPont New England Nuclear), and also on the stable isotopic tracers [26,26,26,27,27,27-²H₆]cholesterol and [23,24,25,26,27-¹³C₅]cholesterol previously described.¹⁹ Oxysterols were separated by TLC on 2.5x7.5-cm microplates coated with silica gel 60 (Alltech) in the solvent system ethyl acetate:heptane (3:1, vol/vol) and eluted with ethanol. For some experiments, [4-¹⁴C]7-hydroxycholesterol and [4-¹⁴C]7-ketocholesterol were purified by HPLC on normal-phase silica using 90:10 hexane:isopropanol as mobile phase.²⁰ 1,2-[³H]Cholesterol (50.3 Ci/mmol) was purchased from ARC.

GC/MS
Plasma lipids were extracted and saponified, and the sterols were recovered.²¹ Microgram amounts of the sterols to be derivatized were dried under nitrogen and heated in polytetrafluoroethylene-lined screwcapped glass vials for 2 hours at 80°C in 0.5 mL dry pyridine with methoxyamine hydrochloride (Sigma Chemical Co; 20 mg/mL) and then at 65°C for 30 minutes after adding 1.0 mL of 1/1 Regisil RC-3 (Regis) in dry pyridine. The sterols were chromatographed on a 2-mmx137-cm column packed with 3% OV-17 on Gas Chrom Q at 250°C with helium flow at 30 mL/min and passed into a Finnigan 3200 electron ionization mass spectrometer with a quadrupole mass analyzer. The oxysterols eluted in the order 7-hydroxycholesterol (14.8 minutes), cholesterol (18.1 minutes), 7ß-hydroxycholesterol (21.9 minutes), and 7-ketocholesterol (44.7 minutes). The principal ions in these peaks obtained with natural materials were m/z 456, 368, 456, and 470, respectively. These ions were increased by 5 mass units for the ¹³C preparation and by 6 units for the ²H preparation. The major ion of 7-ketocholesterol gave too small a peak for reliable quantitation in plasma. Because the principal ion of natural cholesterol, m/z 368, was so intense, m/z 371 (from natural cholesterol) was monitored instead, since the ratio of m/z 371/368 is constant. The plasma concentration of labeled cholesterol or oxysterols was calculated by dividing the peak area of interest by the area of natural plasma cholesterol at m/z 371 after correcting for any spillover¹⁹ and normalizing with similar ratios obtained from in vitro mixture of the infused tracers with natural cholesterol. This method of computation corrects for differences in ion intensities of the various compounds and for any plasma volume changes that could have been substantial and variable. Recoveries of 7-hydroxycholesterol and 7ß-hydroxycholesterol added directly to plasma were 103±9.0% (mean±SEM) and 87.9±6.1%, respectively.

Animal Studies
Study 1
This experiment was designed to compare absorption of cholesterol with that of oxysterols by the method of Zilversmit and Hughes,²² modified for use with stable isotopes.¹⁹ The oxidized preparations of [²H]cholesterol and [¹³C]cholesterol, each containing both cholesterol and oxysterols, were taken up in sterile ethanol at a concentration of 10 mg/mL, warmed to 37°C, and mixed with a phospholipid emulsion containing 10 g triglyceride per 100 mL (Intralipid, Kabi Pharmacia) in the ratio 1:9. One milliliter of the ²H mixture, containing 1.0 mg total sterols (453 µg cholesterol, 116 µg 7-hydroxycholesterol, 136 µg 7ß-hydroxycholesterol, and 298 µg 7-ketocholesterol) and 90 mg triglyceride, was given by gavage, and 1.0 mL of the ¹³C mixture of the same composition was given by intraperitoneal injection to four Sprague-Dawley male rats with body weight in the range of 250 to 300 g. Each rat was fitted with an Elizabethan collar to prevent coprophagy, housed in an individual cage, and given a standard laboratory diet and water ad libitum. Blood was sampled from a tail vein before and at 1, 2, 3, and 5 days after administration. Intraperitoneal injection of cholesterol tracer in rats gives cholesterol pool size and kinetics expected from intravenous injection.²³

Study 2
The clearance from plasma of deuterated cholesterol and oxysterols was studied in four male rats. The rats were anesthetized with intraperitoneal sodium pentobarbital (40 mg/kg) and anesthesia maintained with inhaled ether as required. Each rat received 1.0 mL of the deuterated cholesterol/oxysterol mixture in Intralipid given as a bolus into the dorsal vein of the penis. This route of administration allows ready recognition of extravasation, which would exclude the animal from study. About 1 mL of blood was sampled from the retro-orbital plexus before the intravenous bolus (zero time) and at 5, 30, 90, 150, and 180 minutes after the injection. The sample was transferred to a microfuge tube containing enough heparin to prevent clotting. Each rat was given 2.0 mL of 0.9% NaCl subcutaneously after bleeding at 30, 90, and 150 minutes to replace plasma volume. Plasma volume was estimated as 8% of body weightx0.6. Plasma triglycerides were measured by Lipid Research Clinics methods²⁴ after subtracting a blank to account for glycerol present in Intralipid.

To compare the initial distribution of cholesterol with oxysterols, three male Sprague-Dawley rats weighing 290 to 390 g were anesthetized with 90 mg/kg ketamine and 13 mg/kg xylazine. Intralipid (0.5 mL) containing 0.25 µCi [4-¹⁴C]7-hydroxycholesterol isolated by HPLC and 3.5 µCi [1,2-³H]cholesterol in 50 µL ethanol was injected into the dorsal vein of the penis. Blood was collected in a heparinized syringe from the abdominal aorta at 5 minutes and the animal was rapidly exsanguinated. Plasma was separated by centrifugation and counted. Red cells were washed four times with 0.15 mol/L NaCl containing 25 mmol/L phosphate, pH 7.4. Organs, tissues, and red cells were extracted by the method of Bligh and Dyer²⁵ and counted. Total fat mass was assumed to be 16% of body weight,²⁶ and total muscle mass was taken as 38% of body weight.²⁷

Study 3
Absorption of cholesterol and oxysterols was determined by fecal recovery of orally administered tracers. [¹⁴C]Oxysterols (60.2% 7-ketocholesterol, 17.1% 7ß-hydroxycholesterol, and 22.7% 7-hydroxycholesterol) were purified by TLC.¹⁸ Radiosterols for each rat were taken up in 100 µL ethanol and added to 900 µL Intralipid. Two rats received [³H]cholesterol:[¹⁴C]cholesterol (dpm of ³H:¹⁴C, 1.48x10⁶:8.88x10⁴) by gavage. Four rats received [³H]cholesterol:[¹⁴C]oxysterols (dpm of ³H:¹⁴C, 1.18x10⁶:8.0x10⁴) by gavage. The animals were fitted with Elizabethan collars, housed individually in metabolic cages, and given a standard laboratory diet and water ad libitum. Daily stool from each of the six rats was collected for 4 consecutive days, weighed, homogenized with water (1:2, wt/vol), extracted by the method of Bligh and Dyer,²⁵ and saponified, and the sterols were recovered and counted.²¹

Cell-Culture Studies
Fetal bovine pulmonary artery endothelial cells (CCL 209, American Type Culture Collection) were grown in minimum essential medium containing 10% fetal bovine serum, 0.1 mmol/L nonessential amino acids, and 1 mmol/L sodium pyruvate on collagen-coated surfaces in a humidified incubator under 5% CO₂ in air. Oxysterols and cholesterol in ethanol were each added to 49 volumes of Puck's saline G²⁸ containing a 1:100 dilution of Intralipid. This solution was then further diluted 1:20 into a BSA medium prepared by substituting 0.1% BSA for fetal bovine serum, such that the final concentrations obtained were 0.75 µmol/L total oxysterols or 0.78 µmol/L cholesterol, 0.1 vol% ethanol, and 0.005 g triglyceride per 100 mL.

For uptake and release studies, cells were plated in 12-well clusters at 30x10³ cells per square centimeter and grown for 5 days or until confluent. Growth medium was changed every second day, and 24 hours before study the cells were washed twice with saline G and the medium was replaced with BSA medium. For uptake experiments, the cells were rapidly washed three times with 1.0 mL cold saline G, and 1.0 mL BSA medium containing the isotopic tracers was introduced into the wells. The clusters were then returned to the incubator and placed on a horizontally rotating surface for up to 24 hours. At the end of the period, the cells were washed rapidly three times with 1.0 mL cold saline G and allowed to dry. Ethanol (1.0 mL) was added to each well, the cluster was covered, and sterols were extracted at room temperature for 20 minutes with occasional mixing. Aliquots of the ethanolic extract were counted. This extraction method gave results equal to a more extensive procedure in which cells were grown on glass coverslips and extracted three times with 0.5 mL ethanol and twice with 1 mL 2:1 chloroform:methanol followed by drying of the extracts and counting. For studies of radiolabeled sterol release, cells were exposed to tracers for 24 hours simultaneously with those done for the uptake studies. They were then rapidly washed three times with cold saline G, and fresh BSA medium containing 100 µg protein per milliliter human HDL, prepared by ultracentrifugation,²⁹ was added and radioactivity in the medium was measured for up to 24 hours. The release with time was based on the radioactivity found in aliquots removed, allowing for volume changes, and expressed as a percentage of the total radioactivity that had accumulated in cells after 24 hours of uptake.

Studies to compare the rates at which cholesterol and oxysterols traverse endothelial cells were done with Costar 6-well clusters, each well fitted with a transwell insert. The bottom of the insert is a 0.4-µm-pore-size membrane (24 mm diameter) on which cells are plated and which is inserted into the wells of the cluster. The endothelial cells at confluence separate the culture medium into two reservoirs and form a barrier to diffusion. Collagen-coated transwell membranes were primed by incubation in growth medium for 48 hours and then seeded at 74.5x10³ cells per square centimeter. The upper reservoir was filled with 1.5 mL medium and the lower with 2.6 mL to prevent volume changes between them. The cells became confluent within 5 days. The cells were then preincubated for 24 hours with BSA medium in the top and bottom reservoirs. Monitoring of sterol transport across endothelial cells was done by adding radiolabeled sterols to the upper reservoirs and counting aliquots taken from the bottom reservoirs at the times given in "Results." The integrity of the endothelial cell monolayer was assessed by microscopic visualization and confirmed by the consistency of the findings between wells.

Means±SEMs are presented for triplicate samples. The significance of differences between treatments over multiple time periods was determined by ANOVA using the general linear model of the Statistical Analysis System software (SAS Institute, Cary, NC). Rate constants were calculated using Simulation, Analysis, and Modeling (SAAM),³⁰ which was obtained from the Resource Facility for Kinetic Analysis, University of Washington. Single-compartment models were used.

	Results

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In Vivo Studies
Study 1
After administration of labeled cholesterol and oxysterol mixtures by gavage and by intraperitoneal injection, [²H]cholesterol and [¹³C]cholesterol were readily detectable by GC/MS in all rat plasmas obtained at 1 to 5 days. However, oxysterols similarly labeled and given in similar amounts at the same time could not be detected in any of the samples. Oxysterols added directly to rat plasma gave the expected peak areas, ruling out an artifactual loss of oxysterols during sample preparation. Thus, it was not possible to measure oxysterol absorption with these tracers by the Zilversmit and Hughes method²² using a standard plasma sampling protocol lasting several days.

Study 2
The kinetics of [²H]cholesterol and [²H]oxysterols injected intravenously into rats is shown in Fig 1. After 5 minutes, 28.0±1.7% of the native cholesterol was still present in the plasma compartment. However, only 1.2±0.2% of the initial 7-hydroxycholesterol and 8.0±1.0% of the 7ß-hydroxycholesterol remained (both P<.001 with respect to cholesterol). Thus, the oxysterols were cleared 3.5 to 23 times as fast as cholesterol in the first 5 minutes. The difference between 7-hydroxycholesterol and 7ß-hydroxycholesterol was statistically significant (P=.006). After the initial rapid clearance, the small amount of residual oxysterol decreased slowly in plasma with a single exponential decay of T½=86.0±8.0 minutes, possibly reflecting recycling between tissues and plasma. The initial clearance of oxysterols was also faster than that of Intralipid triglycerides injected simultaneously (Fig 1).

View larger version (23K): [in this window] [in a new window]   Figure 1. Clearance of sterols from plasma. The fraction of [2H]cholesterol, [2H]7- and [2H]7ß-hydroxycholesterol, and triglycerides present in the plasma compartment at various times after intravenous injection was determined as described in "Methods." Where standard error bars are absent, they are within the symbol. Values are from four rats.

To determine the distribution in tissues, [4-¹⁴C]7-hydroxycholesterol and [³H]cholesterol complexed with Intralipid were injected intravenously. The Table shows the distribution of radioactivity found in plasma after 5 minutes. Radioactivity from ¹⁴C reflecting 7-hydroxycholesterol was widely disbursed at this time and varied in different tissues compared with the ³H radioactivity from cholesterol. While 22.7% of the total ¹⁴C radioactivity was still present at 5 minutes, a lipid extract of plasma²⁵ fractionated by HPLC in 90:10 hexane:isopropanol showed that only 19% of the recovered ¹⁴C radioactivity comigrated with [4-¹⁴C]7-hydroxycholesterol. Thus, only 4.3% of the injected oxysterol was still in plasma, indicating that extensive metabolism had occurred. It is likely that this small amount of residual oxysterol is an overestimate, because the [4-¹⁴C]7-hydroxycholesterol radioactivity HPLC peak was broad and asymmetrical compared with the narrow peak given by the starting material, suggesting that coeluting compounds were contained in it. Thus, the clearance from plasma of both deuterated and [¹⁴C]7-hydroxycholesterol tracers is in agreement. In contrast, the recovery in plasma of ³H radioactivity at 5 minutes was 49.9%, and of this 89% comigrated with cholesterol (including 8% present in cholesterol ester), indicating that 44.4% of the injected [³H]cholesterol was still present in plasma.

View this table: [in this window] [in a new window]   Table 1. Tissue Distribution of [4-14C]7-Hydroxycholesterol and [1,2-3H]Cholesterol Radioactivity 5 Minutes After Intravenous Injection

Study 3
The absorption of radiolabeled cholesterol and oxysterols was determined by fecal recovery after administration by gavage. Fig 2 shows that at the end of 4 days, the cumulative recovery of [³H]cholesterol was 25.0±1.1% compared with 8.5±0.3% for [¹⁴C]oxysterols (P=.0009). This result corresponds to absorption of 75% for cholesterol and 91.5% for oxysterols. Fig 3 shows that the ratio of oxysterols recovered to cholesterol recovered fell steeply on day 1 and continued to decline over the next 3 days (solid symbols). When [¹⁴C]cholesterol and [³H]cholesterol were gavaged together, the ratio of isotopes in stool was constant and close to unity, as expected (open symbols). These data imply that, relative to cholesterol, absorption of oxysterols was both more rapid and more complete.

View larger version (16K): [in this window] [in a new window]   Figure 2. Cumulative recovery of [3H]cholesterol and [14C]oxysterols in feces after simultaneous administration to four rats by gavage.

View larger version (11K): [in this window] [in a new window]   Figure 3. Isotope ratios of sterols in stool from the experiment of Fig 2 () and from two rats given pure [14C]cholesterol and pure [3H]cholesterol by gavage (). The 14C/3H index is the ratio of isotopes found in stool divided by the ratio administered.

Possible selective degradation of cholesterol and oxysterols by bowel bacteria was examined by incubating feces with radioactive sterols. Feces from normal Sprague-Dawley male rats never exposed to radioisotopes were mixed with 6 vol 0.15 mol/L NaCl (wt/vol) to give a homogeneous slurry in which the solids did not sediment on standing, and small volumes of [³H]cholesterol and [¹⁴C]oxysterol in ethanol were added. Four aliquots were taken into separate containers before and after anaerobic incubation (BBL GasPak Anaerobic System, Becton Dickinson and Co) for 22 hours at 37°C, and lipids were extracted²⁵ immediately and counted. The [¹⁴C]oxysterol/[³H]cholesterol ratio was 0.043±0.003 before and 0.048±0.001 after incubation, P=.18. A similar preparation incubated aerobically using [¹⁴C]cholesterol and [¹⁴C]oxysterol with separation by TLC before counting showed a [¹⁴C]oxysterol/[¹⁴C]cholesterol ratio of 0.61±0.03 before incubation and 0.57±0.02 after incubation, P=.24.

In Vitro Studies
The fractional uptake of radiolabeled cholesterol into endothelial cells in standard monolayer culture was compared with uptake of the mixture of radiolabeled oxysterols (see "Methods"). A marked difference between cholesterol and oxysterols was found over 24 hours (Fig 4). The calculated T½ for uptake was 0.65±0.05 hours for oxysterols and 10.2±1.0 hours for cholesterol, a 15.7-fold difference (P<.001). The fractional rate of release of labeled sterols from endothelial cells that had first been loaded for 24 hours is shown in Fig 5. Oxysterol efflux was significantly more rapid than that of cholesterol P<.001 by ANOVA). At 5 hours, the release of oxysterols was 51.4±2.2% of the initial value compared with 14.9±0.2% for cholesterol, a 3.4-fold difference (P<.001).

View larger version (12K): [in this window] [in a new window]   Figure 4. Uptake of [14C]cholesterol and [14C]oxysterols by endothelial cells. Cells were incubated with solubilized cholesterol or oxysterols for the indicated times and then washed, and the sterols were extracted with ethanol and counted. Each point represents triplicate wells. Where standard error bars are missing, they are within the symbol.

View larger version (12K): [in this window] [in a new window]   Figure 5. Release of [14C]cholesterol and [14C]oxysterols from endothelial cells in culture. Cells were loaded with either labeled cholesterol or oxysterols for 24 hours and then washed, and the medium was sampled for 5 hours, as described in "Methods." Each point depicts the percent of dpm initially present that had been released as estimated from triplicate wells.

We studied whether oxysterols pass through endothelial cells by using a transwell system in which the lower wells contained 100 µg/mL HDL. Fig 6 shows the appearance over time in the lower wells of radioactivity from oxysterols and cholesterol that had been added to the upper wells. The transfer of radioactivity from cholesterol was much slower than that from oxysterols, such that at 24 hours only 7.1±1.1% of cholesterol had been transferred (solid bars) compared with 30.8±3.3% of oxysterols (open bars), a 4.3-fold difference (P<.001).

View larger version (27K): [in this window] [in a new window]   Figure 6. The appearance in the bottom wells of transwell culture dishes of [14C]cholesterol and [14C]oxysterol radioactivity expressed as a percentage of the amount introduced into the top wells at zero time. Intralipid (50 µg/mL) or HDL (100 µg/mL) were added to the bottom wells of the indicated experiments.

In the same experiment (Fig 6), to determine whether the presence of HDL in the lower well influenced oxysterol accumulation, we also measured transfer of radioactivity in the absence of HDL and in the presence of 50 µg/mL Intralipid as a nonspecific lipid acceptor. The difference between these last two conditions was not statistically significant by ANOVA. In the absence of HDL, the percent of oxysterols transferred was reduced to 61.9±6.6% of that observed in the presence of HDL (P=.0007).

To determine whether oxysterols are altered during transit through endothelial cells, the medium from the bottom well of a transwell culture dish was collected after 24 hours' incubation with [¹⁴C]oxysterols and compared with the original oxysterols by TLC. Radioactivity in the starting material (Fig 7, top) was principally in the expected defined oxysterols that migrate well in the TLC system. However, after passage through the endothelial cells, the distribution of radioactivity was markedly different (Fig 7, bottom), with most remaining at the origin, suggesting that the oxysterols had been modified to become even more polar. The recovery of unaltered oxysterols incubated in culture medium for 24 hours in the absence of cells was 95%.

View larger version (24K): [in this window] [in a new window]   Figure 7. The distribution of radioactivity in oxysterols before addition to endothelial cells (above) and after passage through the cells into the bottom of a transwell system (below). The percent of counts recovered after TLC is given. O indicates the origin; O-, the region between the origin and 7-hydroxycholesterol; 7, 7-hydroxycholesterol; 7ß, 7ß-hydroxycholesterol; 7K, 7-ketocholesterol; and K-C, the region between 7-ketocholesterol and the solvent front.

Despite the absence of morphological changes in cells cultured with oxysterols, the possibility that endothelial cell barrier function might be altered by oxysterols^{31 32} was studied specifically. Nonlabeled oxysterols were either omitted or introduced at 0.75 µmol/L or 7.5 µmol/L to test their effect on both uptake and release of [4-¹⁴C]cholesterol. Fig 8 shows that oxysterols added even at 10 times the concentration used for the studies of Figs 4 through 6 had no effect on the uptake or release of [4-¹⁴C]cholesterol. Similar results were found with mouse J774 macrophages (data not shown).

View larger version (28K): [in this window] [in a new window]   Figure 8. Effect of 7.5 µmol/L unlabeled oxysterols on uptake (A) and release (B) of radiolabeled cholesterol in endothelial cells. The values shown are the means from three wells at each time point.

To determine whether there was a difference in the distribution of cholesterol and oxysterols with respect to Intralipid and albumin in the culture medium, the following experiment was performed. Intralipid was initially clarified by ultracentrifugation for 30 minutes at 36 000 rpm in a Beckman SW41 rotor, the infranatant was discarded, and the Intralipid pellicle was brought to the original volume in saline G. [³H]Cholesterol and [¹⁴C]7-ketocholesterol in ethanol were added to Intralipid, as described in "Methods" and then to minimum essential medium, buffered with 25 mmol/L HEPES, pH 7.4, ±0.1% bovine albumin, to yield a final concentration of 7.5 nmol/L [³H]cholesterol and 0.75 mmol/L [¹⁴C]7-ketocholesterol. After incubation for 4 hours at 37°C, triplicate aliquots were centrifuged and the infranatants counted. Without albumin, 27.1±2.0% of the cholesterol and 29.6±2.0% of the oxysterol were recovered in the infranatant. When albumin was present, these amounts increased to 46.2±3.6% and 39.9±3.7%, respectively. These differences in distribution of oxysterols and cholesterol, both with and without albumin, were not statistically significant.

	Discussion

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The findings in the in vivo studies show clearly that compared with cholesterol, oxidized cholesterol is absorbed more rapidly and more completely from the bowel (Figs 2 and 3) and cleared far more rapidly from plasma (Fig 1). Oxysterol is rapidly taken up in tissues and organs (Table). The in vitro studies support and extend some of the in vivo findings.

GC/MS was used to follow plasma clearance of oxysterols because it allowed us to be certain of the chemical identity of the materials being analyzed by means of selected ion monitoring. This method has advantages over radioisotopic tracers, which cannot distinguish oxysterols from their metabolites without prior separation. The pertinence of this point is borne out by our studies with [¹⁴C]7-hydroxycholesterol, in which only 19% of the radioactivity remaining in rat plasma at 5 minutes was actually 7-hydroxycholesterol as determined by HPLC (Table). The notably rapid rate at which oxysterols are cleared from plasma is thought to reflect avid uptake by the endothelial cell, an interpretation supported by the in vitro studies (Fig 4) and the widespread distribution of ¹⁴C from oxysterol 5 minutes after intravenous injection (Table). This need not mean that this property is peculiar to the endothelial cell but rather that this cell and blood cells are the first cells with which compounds introduced into plasma come in contact. The avidity of oxysterols for cell membrane is demonstrated by the marked selectivity in uptake by red cells relative to cholesterol (Table), and this may be related to the interaction of oxysterols with the lipids in cell membranes.⁷

The initial dissemination of oxysterol to peripheral tissues, with rapid change of the [¹⁴C]7-hydroxycholesterol in plasma, suggests that there is partial metabolism in many tissues followed by recycling through plasma to the liver for conversion into bile acids. This possibility is supported by the work of Fredrickson and Ono,³³ who reported in rats nearly quantitative recovery of intravenously injected [4-¹⁴C]25- and [4-¹⁴C]26-hydroxycholesterol radioactivity in biliary acidic steroids within 4 hours. We found on analysis of liver that ¹⁴C radioactivity overestimated 7-hydroxycholesterol relative to the ³H radioactivity of cholesterol. While the normalized ¹⁴C/³H ratio recovered in liver was similar to that injected, fractionation of a liver extract showed only 10.3% of the ¹⁴C radioactivity eluted as expected for 7-hydroxycholesterol, indicating rapid metabolism of this sterol. At the same time, 89% of the ³H radioactivity was still cholesterol. While we have not identified the metabolic sites accounting for the extensively altered [¹⁴C]7-hydroxycholesterol in plasma, it seems unlikely that it could all be attributed to recycling of those metabolites from the liver to plasma over 5 minutes.

Support for the view that oxysterols are cleared rapidly from plasma is consistent with the work of others. Bascoul et al³⁴ studied the absorption of oxysterols with ¹⁴C and ³H preparations of an epoxide of cholesterol in rats by the method of Zilversmit and Hughes.²² In experiments in which very good absorption was found, isotopes in the plasma of the animals on day 2 are seen to give counts about 0.01% to 0.02% of those administered. The daily feeding to rats of 3 µCi of ¹⁴C in a mixture of labeled oxysterols for 6 consecutive days left insufficient radioactivity in the plasma on day 7 to measure the distribution in lipoproteins. Peng et al³⁵ fed squirrel monkeys a single dose of 150 µCi [25-³H]hydroxycholesterol and reported on its distribution in lipoproteins after 24 hours, but the residual radioactivity is again seen to be a very small fraction of that given. It seems to us that the most notable feature of the behavior of oxysterols in this context is the rapidity with which they are cleared from plasma, in our data up to 99% in 5 minutes (Fig 1). Such rapid clearance is not unique, since 92% of oxidized LDL was cleared from rat plasma in 2 minutes.³⁶ Although different oxysterols are cleared at different rates (Fig 1), rapid clearance is common to many oxysterols.

Our conclusion from study 3 that absorption of oxysterols is 91.5±0.3% (significantly greater than that of cholesterol) concurs with the findings of Bascoul et al³⁴ in which the absorption of cholesterol-5,6--epoxide in rats was 93±22%. From the report by Fredrickson and Ono³³ and our own data showing gross alteration of 7-hydroxycholesterol in liver within 5 minutes, it seems likely that the isotope identifying oxysterols we recovered in feces, even from day l, reflects to a considerable degree if not entirely metabolic products of the oxysterols administered. Our measure of oxysterol absorption could therefore be an underestimate. Endogenous cholesterol present in the rat intestine in our experiments is unlikely to be of a level to cause saturation of percent cholesterol absorption, which could confound the interpretation of the data. Zilversmit and Hughes²² found in rats that oral unlabeled cholesterol at 2 or 4 mg/d per 100 g animal weight, or allowing coprophagy, had no effect on measurement of percent cholesterol absorption using radioisotopes, and similar results have been reported over a range of cholesterol intake from 190 to 820 mg/d in humans.³⁷

Consideration was also given as to whether different pool sizes for cholesterol and oxysterols might have confounded the interpretation of our in vitro data. For uptake studies, cholesterol and oxysterol tracers at the same concentration (0.78 and 0.75 µmol/L, respectively) presented an equal probability for uptake of either class of sterol. There is clearly selectivity in the uptake of oxysterols (Fig 4). In studies of release (Fig 5) and transit of radioactivity (Fig 6), the fractional rates of transfer for oxysterols analyzed by first-order tracer kinetics, which are independent of pool size,³⁸ exceeded those for cholesterol. The release of cholesterol mass from macrophages is first order over widely varying cholesterol pool size.³⁹ Since tracers move in both directions in our systems, the absolute movement in any one direction might be underestimated, and this underestimate would be greater with the faster-moving compounds. The transfer of oxysterols across endothelial cells (Fig 6) shows that what we term uptake and release of oxysterols is not necessarily confined to movement between the cell membrane and the medium bathing it.

Although it has been reported previously that endothelial cells in culture oxidize cholesterol,^{40 41} it was not anticipated that oxysterols would be altered in their passage through endothelial cells, yet extensive metabolism to more polar compounds was found (Fig 7). It is now evident that metabolic alteration of oxysterols is not exclusive to the hepatocyte. Both the identity of the compounds formed and the significance of this observation remain to be defined. Our finding that [4-¹⁴C]7-hydroxycholesterol in plasma is rapidly altered suggests that this may also occur in vivo.

Fig 6 shows that oxysterols pass through endothelial cells even when the acceptor medium is free of HDL. However, the presence of HDL in the medium markedly promotes this transfer of oxysterols, as has been found for cholesterol.⁴² It is possible that HDL could have a role in reverse oxysterol transport, just as it is thought to have in reverse cholesterol transport.^{42 43} The demonstrated rapid uptake and rapid release by cells of oxysterols is in keeping with the rapidity with which they reach the liver from extrahepatic sites and the speed at which they are metabolized.^{33 36} When cholesterol is mixed with oxysterols and implanted subcutaneously, both are rapidly solubilized and rapidly cleared, mainly via incorporation into HDL. This process is unlike the fate of cholesterol, which is not cleared at all when it is implanted alone.⁸

We found no effect of oxysterols on transfer of cholesterol. Previous work has shown that oxysterols decrease endothelial cell barrier function to albumin,^{31 32} but this occurs only at concentrations of 50 µmol/L or higher. We used a concentration of 0.75 µmol/L and found no effect on cholesterol transfer, even at 10 times that level (Fig 8).

Oxysterol metabolism may be especially important with respect to hepatic and endothelial cells. Only these cells and blood cells are normally exposed to LDL at the concentration found in plasma. The liver has 0.1 µm fenestrae in liver sinusoids, which allow LDL to bypass endothelium,⁴⁴ and this could account for absence of selectivity in the initial uptake of oxysterol and cholesterol by the liver (Table). Neither hepatocytes nor endothelial cells ordinarily accumulate cholesterol pathologically. Both can oxidize cholesterol, the liver through synthesis of 7-hydroxycholesterol and bile acids and the endothelial cell, at least in culture, through oxidation of LDL cholesterol taken up from the medium.^{40 41} Cholesterol oxidation is therefore a pathway that might allow these cells to cope with the entry of amounts of lipoprotein cholesterol in excess of their needs without becoming loaded with cholesterol. It has also been suggested that oxidation of cholesterol by macrophages might provide a means for clearing their excess cholesterol.⁴⁵ Although the liver produces bile acids, the final cholesterol oxidation products, it is not necessarily the source of all the precursor sterols. The present data indicate that oxysterols produced peripherally or derived from the diet could be transported rapidly to the liver for further metabolism.

The effects of oxysterols and the cellular enzymes that produce and metabolize them are sometimes difficult to predict. Although oxysterols inhibit LDL receptor activity in extrahepatic cells,⁴⁶ transfection of nonliver cells with the enzyme 7-hydroxylase actually increases LDL receptor activity and confers a resistance to downregulation by cholesterol that is typical of liver.⁴⁷ The proposed mechanism is that the enzyme preferentially inactivates an oxysterol repressor, presumably by further oxidation. Likewise, although oxysterols repress LDL receptors in vitro, plasma levels of 7-hydroxycholesterol are increased manyfold in individuals taking bile acid sequestrants,^{12 13} in whom LDL receptor activity is actually increased. Some of these paradoxical effects may be due to differences between oxysterols. We report here that oxysterols, including 7-hydroxycholesterol, traversing endothelial cells are greatly altered, for the most part being converted to more polar compound(s) (Fig 7). This may be noteworthy, because it has been reported that the effect of oxysterols on cholesterol homeostasis is related more to their polarity than to their oxygen function.⁴⁸

If endogenously generated oxysterols have a physiological function, it seems possible that oxysterols in the diet, which are likely to be well absorbed, might also have such a role. It has been reported that cholestane-3ß,5,6ß-triol prevents hypercholesterolemia and atheroma in animals fed atherogenic diets.⁵ It has also been postulated that the virtual elimination of spontaneously generated oxysterols from the diet in the developed countries has direct relevance to the evolution of CHD in those countries during this century.⁸

The current results show that cholesterol oxidation markedly alters the properties of the parent compound such that some of the resulting properties could have physiological benefits. Further work needs to be done to clarify the role of oxysterols in atherosclerosis.

	Selected Abbreviations and Acronyms

 

dpm = disintegrations per minute GC/MS = gas chromatography/mass spectrometry HPLC = high-performance liquid chromatography TLC = thin-layer chromatography

	Acknowledgments

 
This study was supported in part by NIH grant R01-50420 (to Dr Ostlund), the Washington University Mass Spectrometry Resource (RR-00954), the Washington University Diabetes Center (DK 20579), and the Division of Atherosclerosis, Nutrition, and Lipid Research. Part of the work was done during sabbatical leave of Dr Krut from the Department of Medicine, Baragwanath Hospital and University of Witwatersrand, Johannesburg, South Africa.

Received January 30, 1996; accepted July 23, 1996.

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