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• W1994559481 abstract "The aim of this study was to determine in humans whether oxidized cholesterol in the diet is absorbed and contributes to the pool of oxidized lipids in circulating lipoproteins. When a meal containing 400 mg cholestan-5α,6α-epoxy-3β-ol (α-epoxy cholesterol) was fed to six controls and three subjects with Type III hyperlipoproteinemia, α-epoxy cholesterol in serum was found in chylomicron/chylomicron remnants (CM/RM) and endogenous (VLDL, LDL, and HDL) lipoproteins. In controls, α-epoxy cholesterol in CM/RM was decreased by 10 h, whereas in endogenous lipoproteins it remained in the circulation for 72 h. In subjects with Type III hyperlipoproteinemia, α-epoxy cholesterol was mainly in CM/RM. In vitro incubation of the CM/RM fraction containing α-epoxy cholesterol with human LDL and HDL that did not contain α-epoxy cholesterol resulted in a rapid transfer of oxidized cholesterol from CM/RM to both LDL and HDL. In contrast, no transfer was observed when human serum was substituted with rat serum, suggesting that cholesteryl ester transfer protein is mediating the transfer. Thus, α-epoxy cholesterol in the diet is incorporated into the CM/RM fraction and then transferred to LDL and HDL, contributing to lipoprotein oxidation. Moreover, LDL containing α-epoxy cholesterol displayed increased susceptibility to further copper oxidation in vitro.It is possible that oxidized cholesterol in the diet accelerates atherosclerosis by increasing oxidized cholesterol levels in circulating LDL and chylomicron remnants. The aim of this study was to determine in humans whether oxidized cholesterol in the diet is absorbed and contributes to the pool of oxidized lipids in circulating lipoproteins. When a meal containing 400 mg cholestan-5α,6α-epoxy-3β-ol (α-epoxy cholesterol) was fed to six controls and three subjects with Type III hyperlipoproteinemia, α-epoxy cholesterol in serum was found in chylomicron/chylomicron remnants (CM/RM) and endogenous (VLDL, LDL, and HDL) lipoproteins. In controls, α-epoxy cholesterol in CM/RM was decreased by 10 h, whereas in endogenous lipoproteins it remained in the circulation for 72 h. In subjects with Type III hyperlipoproteinemia, α-epoxy cholesterol was mainly in CM/RM. In vitro incubation of the CM/RM fraction containing α-epoxy cholesterol with human LDL and HDL that did not contain α-epoxy cholesterol resulted in a rapid transfer of oxidized cholesterol from CM/RM to both LDL and HDL. In contrast, no transfer was observed when human serum was substituted with rat serum, suggesting that cholesteryl ester transfer protein is mediating the transfer. Thus, α-epoxy cholesterol in the diet is incorporated into the CM/RM fraction and then transferred to LDL and HDL, contributing to lipoprotein oxidation. Moreover, LDL containing α-epoxy cholesterol displayed increased susceptibility to further copper oxidation in vitro. It is possible that oxidized cholesterol in the diet accelerates atherosclerosis by increasing oxidized cholesterol levels in circulating LDL and chylomicron remnants. A large body of evidence supports the hypothesis that oxidized lipoproteins, particularly oxidized LDL, play a pathogenic role in atherosclerosis (1Steinberg D. Oxidative modification of LDL and atherogenesis.Circulation. 1997; 95: 1062-1071Google Scholar, 2Chisolm G.M. Penn M.S. Oxidized lipoproteins and atherosclerosis.in: Fuster V. Ross R. Topol E.J. Atherosclerosis and Coronary Heart Disease. Raven Press, NY1996: 129-149Google Scholar). Oxidized LDL has numerous atherogenic properties with a variety of cell types in culture, including induction of inflammatory genes, stimulation of monocyte chemotactic factor production, the potentiation of monocyte-endothelial cell adhesion, accelerated deposition of lipids in macrophages, cytotoxicity with endothelial cells, and modulation of growth factors. Additionally, oxidized LDL-like particles and lipid peroxidation products are present in atherosclerotic lesions (3Palinski W. Rosenfeld M.E. Yla-Herttuala S. Gurtner G.C. Socher S.S. Butler S.W. Parthasarathy S. Carew T.E. Steinberg D. Witztum J.L. Low density lipoprotein undergoes oxidative modification in vivo.Proc. Natl. Acad. Sci. USA. 1989; 86: 1372-1376Google Scholar, 4Yla-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. Evidence for the presence of oxidatively modified LDL in atherosclerotic lesions of rabbit and man.J. Clin. Invest. 1989; 84: 1086-1095Google Scholar). Moreover, it has been shown that antioxidants in the diet can slow the progression of atheroscerosis in animal models (5Carew T.E. Schwenke D.C. Steinberg D. Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit LDL degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit.Proc. Natl. Acad. Sci. USA. 1987; 84: 7725-7729Google Scholar, 6Kita T. Nagano Y. Yokode M. Ishii K. Kume N. Ooshima A. Yoshida H. Kawai C. Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia.Proc. Natl. Acad. Sci. USA. 1987; 84: 5928-5931Google Scholar, 7Parker A.R. Sabrah T. Cap M. Gill B.T. Relation of vascular oxidative stress, α-tocopherol, and hypercholesterolemia to early atherosclerosis in hamsters.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 349-358Google Scholar). Thus, there is strong evidence that oxidized lipoproteins play a key role in atherogenesis, but the site and mechanisms by which lipoproteins are oxidized is far from resolved. It is not clear whether oxidized lipoproteins form locally in the artery wall, as suggested by several investigators (1Steinberg D. Oxidative modification of LDL and atherogenesis.Circulation. 1997; 95: 1062-1071Google Scholar, 2Chisolm G.M. Penn M.S. Oxidized lipoproteins and atherosclerosis.in: Fuster V. Ross R. Topol E.J. Atherosclerosis and Coronary Heart Disease. Raven Press, NY1996: 129-149Google Scholar), or are sequestered in atherosclerotic lesions following the uptake of circulating oxidized lipoproteins. We have been focusing our studies on the role of diet and have demonstrated that potentially atherogenic oxidized lipoproteins in the circulation are at least partially derived from oxidized lipids in food and contribute to atherosclerosis in animal models. The American diet contains large quantities of oxidized fatty acids (8Addis P.B. Hassel C.A. Safety issues with antioxidants in foods.in: Finley J.W. Robinson S. Armstrong D.J. Food Safety Assessment. American Chemists Society, Washington, DC1992: 346-376Google Scholar, 9Yagi K. Kiuchi K. Saito Y. Miike A. Kayahara N. Tatano T. Ohishi N. Use of a new methylene blue derivative for determination of lipid peroxides in foods.Biochem. Int. 1986; 12: 367-371Google Scholar) and oxidized cholesterol (10Addis P.B. Park P.W. Guardiola F. Codony R. Analysis and health effects of cholesterol oxides.in: McDonald R.E. Min D.B. Food Lipids and Health. Marcel Dekker, Inc., NY1996: 199-240Google Scholar, 11Van de Bovenkamp P. Kosmeijer-Schuil T.G. Katan M.B. Quantification of oxysterols in Dutch foods: egg products and mixes diets.Lipids. 1988; 23: 1079-1085Google Scholar, 12Addis P.B. Park P.W. Cholesterol oxide content in food.in: Peng S.K. Morin R.J. Biological effects of cholesterol oxides. CRC press, Boca Raton, FL.1992: 71-88Google Scholar, 13Angulo A.J. Romera J.M. Ramirez M. Gil A. Determination of cholesterol oxides in dairy products. Effects of storage conditions.J. Agric. Food Chem. 1997; 45: 4318-4323Google Scholar) due to the fact that a large portion of the fat and cholesterol in the diet is often prepared in a fried, heated, or processed form. Studies in our laboratory have demonstrated that oxidized fatty acids in the diet result in oxidized lipids in serum lipoproteins in rodents and rabbits. In rats, the levels of oxidized fatty acids in the serum are proportional to the quantity of oxidized fatty acids in the diet, with increased dietary oxidized fatty acids leading to increased exogenous and endogenous serum oxidized lipoprotein levels (14Staprans I. Rapp J.H. Pan X-M. Feingold K.R. The effect of oxidized lipid in the diet on serum lipoprotein peroxides in control and diabetic rats.J. Clin. Invest. 1993; 92: 638-643Google Scholar). The quantity of oxidized fatty acids in chylomicrons isolated from the mesenteric lymph drainage of the small intestine and postprandial serum also correlates directly with the amount of oxidized fatty acids administered to the animal (15Staprans I. Pan X-M. Miller M. Rapp J.H. Effect of dietary lipid peroxides on metabolism of serum chylomicrons in rats.Am. J. Physiol. 1993; 264: G561-G568Google Scholar, 16Staprans I. Rapp J.H. Pan X-M Feingold K.R. Oxidized lipids in the diet are incorporated by the liver into VLDL in rats.J. Lipid Res. 1996; 37: 420-430Google Scholar). Thus, oxidized fatty acids in the diet are absorbed by the small intestine, transported in chylomicrons to the liver, and utilized to form VLDL that are secreted into the circulation (16Staprans I. Rapp J.H. Pan X-M Feingold K.R. Oxidized lipids in the diet are incorporated by the liver into VLDL in rats.J. Lipid Res. 1996; 37: 420-430Google Scholar). Most importantly, we have demonstrated that oxidized fatty acids in the diet are proatherogenic and accelerate fatty streak formation in the aorta of cholesterol-fed rabbits (17Staprans I. Rapp J.H. Pan X-M. Hardman D.A. Feingold K.R. Oxidized lipids in the diet accelerate lipid deposition in the arteries of cholesterol-fed rabbits.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 533-538Google Scholar). It has also been shown in several laboratories that, similarly to fatty acids, oxidized cholesterol in the diet is incorporated into serum lipoproteins of rats (18Bascoul J. Domerque N. Mourot J. Derby G. Crastes de Paulet A. Intestinal absorption and fecal excretion of 5,6α-epoxy-5α-cholesta-3β-ol by male Wistar rat.Lipids. 1986; 21: 744-747Google Scholar, 19Osada K. Sasaki E. Sugano M. Lymphatic absorption of oxidized cholesterol in rats.Lipids. 1994; 29: 555-559Google Scholar) and rabbits (20Peng S.K. Phillips G.A. Xia G-Z. Morin R.J. Transport of cholesterol autoxidation products in rabbit lipoproteins.Atherosclerosis. 1987; 64: 1-6Google Scholar, 21Vine D.F. Mamo J.C.L. Beilin L.J. Mori T. Croft K.D. Dietary oxysterols are incorporated in plasma triglyceride-rich lipoproteins, increase their susceptibility to oxidation and increase aortic cholesterol concentration in rabbits.J. Lipid Res. 1998; 39: 1995-2004Google Scholar, 22Staprans I. Pan X-M. Rapp J.H. Feingold K.R. Oxidized cholesterol in the diet accelerates the development of aortic atherosclerosis in cholesterol-fed rabbits.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 977-983Google Scholar). Recently, we have demonstrated that oxidized cholesterol in the diet also accelerates fatty streak lesion formation in aortas of cholesterol-fed rabbits (22Staprans I. Pan X-M. Rapp J.H. Feingold K.R. Oxidized cholesterol in the diet accelerates the development of aortic atherosclerosis in cholesterol-fed rabbits.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 977-983Google Scholar). In LDL receptor-deficient and apolipoprotein E (apoE)-deficient mice (murine models in which atherosclerosis more closely resemble human lesions), again the feeding of oxidized cholesterol resulted in a significant increase in aortic fatty streak lesions (23Staprans I. Pan X-M. Rapp J.H. Grunfeld C. Feingold K.R. Oxidized cholesterol in the diet accelerate the development of atherosclerosis in LDL-receptor- and apolipoprotein E-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 708-714Google Scholar). Thus, our observations in animals clearly demonstrate that diets containing oxidized lipids, either oxidized fatty acids or cholesterol, increase the development of atherosclerosis. In studies in humans, we have shown that the quantity of oxidized fatty acids in the diet also correlates with the levels of oxidized lipids in postprandial serum chylomicrons/chylomicron remnants (CM/RM) (24Staprans I. Rapp J.H. Pan X-M. Kim K.Y. Feingold K.R. Oxidized lipids in the diet are a source of oxidized lipid in chylomicrons of human serum.Arterioscler. Thromb. 1994; 14: 1900-1905Google Scholar, 25Staprans I. Hardman D.A. Pan X-M. Feingold K.R. Effect of oxidized lipids in the diet on oxidized lipid levels in serum chylomicrons of diabetic patients.Diabetes Care. 1999; 22: 300-306Google Scholar). Oxidized fatty acids in the diet are absorbed by the small intestine, incorporated into chylomicrons and chylomicron remnants, and appear in the bloodstream where they contribute to the total body pool of oxidized lipid. Moreover, these postprandial lipoproteins containing diet-derived oxidized fatty acids were more susceptible to oxidation in vitro. The purpose of the present study was 1) to determine whether in humans oxidized cholesterol in the diet is absorbed by the small intestine and contributes to the pool of oxidized lipids, 2) to determine the distribution of dietary oxidized cholesterol among all serum lipoproteins, including CM/RM, VLDL, HDL, and LDL, and 3) to determine whether α-epoxy cholesterol containing LDL is more susceptible to further oxidation. This study was performed on six control subjects (four males and two females) and three male subjects with familial Type III hyperlipoproteinemia (apolipoprotein phenotype E2/E2 and elevated lipids) (26Mahley R.W. Alterations in plasma lipoproteins induced by cholesterol feeding in animals including man.in: Dietschy J.M. Gotto A.M. Ontko J.A. Disturbances in Lipid and Lipoprotein Metabolism. American Physiological Society, Bethesda, MD1987: 181-197Google Scholar). All control subjects were selected from volunteers employed at the Veterans Affairs Medical Center, San Francisco. Blood was drawn from each subject after a 12 h fast (0 time) for measurement of fasting serum triglycerides, cholesterol, and α-epoxy cholesterol levels. All subjects were nonsmokers, were moderately active, and consumed a typical American diet. None of the subjects was on vitamin or antioxidant therapy. Control subjects had normal serum triglyceride (<2.3 mmol/l) and cholesterol (<5.2 mmol/ dl) levels. None of the subjects was obese (BMI < 30), had diabetes, congestive heart failure, or gastrointestinal disorders. Control subjects were not taking any lipid-lowering medication. Subjects with familial Type III hyperlipoproteinemia were recruited from the Lipid Clinic at University of California, San Francisco, and all their medications were discontinued for at least 3 weeks prior to the experiment. This study was approved by the Committee on Human Research at University of California, San Francisco, and written consent was obtained from all experimental subjects. Cholestan-5α,6α-epoxy-3β-ol (α-epoxy cholesterol) was used as a source of oxidized cholesterol in a test meal. It was purchased from Steraloids Inc. (Newport, RI), and was selected because it is one of the major oxidized cholesterol components in heated or stored foods (10Addis P.B. Park P.W. Guardiola F. Codony R. Analysis and health effects of cholesterol oxides.in: McDonald R.E. Min D.B. Food Lipids and Health. Marcel Dekker, Inc., NY1996: 199-240Google Scholar, 11Van de Bovenkamp P. Kosmeijer-Schuil T.G. Katan M.B. Quantification of oxysterols in Dutch foods: egg products and mixes diets.Lipids. 1988; 23: 1079-1085Google Scholar, 12Addis P.B. Park P.W. Cholesterol oxide content in food.in: Peng S.K. Morin R.J. Biological effects of cholesterol oxides. CRC press, Boca Raton, FL.1992: 71-88Google Scholar, 13Angulo A.J. Romera J.M. Ramirez M. Gil A. Determination of cholesterol oxides in dairy products. Effects of storage conditions.J. Agric. Food Chem. 1997; 45: 4318-4323Google Scholar). It has also been detected in human serum (27Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry.Anal. Biochem. 1995; 225: 73-80Google Scholar, 28Sevanian A. Hodis H.N. Hwang J. Mcleod L.L. Peterson H. Characterization of endothelial cell injury by cholesterol oxidation products found in oxidized LDL.J. Lipid Res. 1995; 36: 1971-1986Google Scholar, 29Murakami H. Tamasawa N. Matsui J. Yasujima M. Suda T. Plasma oxysterols and tocopherol in patients with diabetes mellitus and hyperlipidemia.Lipids. 2000; 35: 333-338Google Scholar) and atherosclerotic lesions (30Vaya J. Aviram M. Mahmood S. Hayek T. Grenadier E. Selective distribution of oxysterols in atherosclerotic lesions and in human plasma lipoproteins.Free Rad. Res. 2000; 34: 485-497Google Scholar, 31Breuer O. Dzeletovic S. Lund E. Diczfalusy U. The oxysterols cholest-5ene-3β,4α-diol, cholest-5-ene-3β,4β-diol and cholestane-3β,5α,6α-triol are formed during the in vitro oxidation of low density lipoprotein, and are present in human atherosclerotic plaques.Biochim. Biophys. Acta. 1996; 1302: 145-152Google Scholar). It is efficiently absorbed as indicated by studies with experimental animals (19Osada K. Sasaki E. Sugano M. Lymphatic absorption of oxidized cholesterol in rats.Lipids. 1994; 29: 555-559Google Scholar). Moreover, it has been demonstrated that α-epoxy cholesterol is not formed enzymatically during cholesterol transport in vivo (32Kudo K. Emmons G.T. Casserly E.W. Vis D.P. Smith L.C. St Pyrek J. Schroepfer G.J. Inhibitors of sterol synthesis. Chromatography of acetate derivatives of oxygenated sterols.J. Lipid Res. 1989; 30: 1097-1111Google Scholar). After a 12 h fast, six control subjects and three subjects with familial Type III hyperlipoproteinemia were given a dose of 400 mg α-epoxy cholesterol dissolved in olive oil (0.5 ml/kg body weight) and added to 100 g of carbohydrate (mashed potatoes). For control purposes, in another experiment the same subjects were given a similar dose of nonoxidized cholesterol as a test meal and serum and lipoprotein fractions were tested for α-epoxy cholesterol generated during the isolation procedure. The subjects tolerated the test meal well, and none had gastrointestinal symptoms. At 2 h, 4 h, 6 h, 8 h, and 10 h after the consumption of the test meal, 50 ml blood samples were obtained for the determination of serum triglycerides, cholesterol, and α-epoxy cholesterol levels. In three control subjects, serum samples were also obtained at 24 h, 48 h, and 72 h. All serum samples were stored in ice and contained 10 μM EDTA and 5 μM BHT throughout the sample processing. The α-epoxy cholesterol levels were measured in serum and the amount of α-epoxy cholesterol was expressed as micrograms of oxidized cholesterol per decaliters of serum. The subjects were not permitted to consume any food for the 10 h test period. Water was allowed ad libidum. To determine the dose response, three control subjects, in addition to ingesting the 400 mg dose, were given decreasing amounts of α-epoxy cholesterol (200 mg, 100 mg, and 50 mg), and serum was collected at 6 h for the determination of the oxidized cholesterol levels. In each individual, there was at least a 2 week time period between the consumption of any of the test meals. Initially, serum samples collected at 2 h time intervals after the administration of the test meal were separated into fractions using apoB-100 immunoaffinity columns (the column does not bind apoB-48) as described by Schneeman et al. (33Schneeman B.O. Kotite L. Todd K.M. Havel R.J. Relationship between the response of triglyceride-rich lipoproteins in blood plasma containing apolipoproteins B-48 and B-100 to a fat-containing meal in normolipemic humans.Proc. Natl. Acad. Sci. USA. 1993; 90: 2069-2073Google Scholar): 1) apoB-100 containing lipoproteins (VLDL, IDL, LDL) and 2) CM/RM and HDL lipoprotein fractions. Thus, a highly enriched diet-derived apoB-48 fraction could be separated from most apoB-100-containing serum lipoproteins (34Campos E. Kotite L. Blanche P. Yasushi M. Frost P.H. Masharani U. Krauss R.M. Havel R.J. Properties of triglyceride-rich and cholesterol-rich lipoproteins in the remnant-like particle fraction of human blood plasma.J. Lipid Res. 2002; 43: 365-374Google Scholar). Serum (4 ml) was applied on columns and the unbound fraction containing apoB-48 CM/RM and HDL were collected to be further separated by density centrifugation (35Havel R.J. Eder H. Bragdon J. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Google Scholar) into CM/RM fraction (d < 1.019), and HDL fraction (d = 1.063–1.225). The bound fraction was eluted from the column, collected as described (33Schneeman B.O. Kotite L. Todd K.M. Havel R.J. Relationship between the response of triglyceride-rich lipoproteins in blood plasma containing apolipoproteins B-48 and B-100 to a fat-containing meal in normolipemic humans.Proc. Natl. Acad. Sci. USA. 1993; 90: 2069-2073Google Scholar, 34Campos E. Kotite L. Blanche P. Yasushi M. Frost P.H. Masharani U. Krauss R.M. Havel R.J. Properties of triglyceride-rich and cholesterol-rich lipoproteins in the remnant-like particle fraction of human blood plasma.J. Lipid Res. 2002; 43: 365-374Google Scholar), then further fractionated into VLDL (d < 1.006), IDL (d = 1.006–1.019), and LDL (d = 1.019–0.063) by ultracentrifugation (35Havel R.J. Eder H. Bragdon J. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Google Scholar). All lipoproteins from all time points examined were characterized by determining triglyceride, cholesterol, and α-epoxy cholesterol content. The recovery of α-epoxy cholesterol was 85–95% when the sum of α-epoxy cholesterol in each lipoprotein was compared with α-epoxy cholesterol in serum. The transfer of α-epoxy cholesterol from CM/RM fraction to serum-endogenous lipoproteins was determined by incubating diet-derived α-epoxy cholesterol containing CM/RM fraction with fasting serum that was free of α-epoxy cholesterol. Three normal subjects were fed an α-epoxy cholesterol-containing meal as described above to obtain CM/RM fraction at 2 h, 4 h, 6 h, and 8 h after the consumption of the test meal. Postprandial serum samples from each time point were collected and applied to a apoB-100 affinity column to obtain the unbound fraction for CM/RM isolation as described above. Four milliliters of fasting serum (d > 1.006) were incubated at 37°C for 12 h (36Guerin M. Dolphin P.J. Chapman M.J. A new in vitro method for simultaneous evaluation of cholesteryl ester exchange and mass transfer between HDL and apoB-containing lipoprotein subspecies.Arterioscl. Thromb. 1994; 14: 199-206Google Scholar) with the CM/RM fraction that was derived from 4 ml serum. After a 12 h incubation, the CM/RM, LDL, and HDL fractions were isolated from the incubation mixture by sequential centrifugation, and the amounts of α-epoxy cholesterol in these fractions were determined by gas-liquid chromatography (GLC) as described below. In these experiments, α-epoxy cholesterol in the CM/RM fraction was ∼2% of the total cholesterol mass. The transfer was expressed as a percentage of α-epoxy cholesterol distribution among LDL, HDL, and the remaining CM/RM. The original CM/RM fraction was designated as 100%. To eliminate the possibility that oxidized cholesterol in endogenous lipoproteins was generated during the experimental procedure, in a separate experiment, incubation was also carried out in the presence of CM/RM fraction that did not contain oxidized cholesterol. To determine the rate of transfer during the incubation over a 12 h time period, α-epoxy cholesterol containing purified CM/RM fraction (isolated from serum 6 h after the consumption of the test meal) was incubated at 37°C with fasting serum and the transfer was monitored for 12 h. Samples were removed and examined for α-epoxy cholesterol in CM/RM fraction and in endogenous LDL and HDL. For control purposes, the CM/RM fraction was incubated with a similar amount of rat serum that does not contain cholesteryl ester transfer protein (CETP). Susceptibility of LDL to oxidation was performed using the procedure of Esterbauer et al. (37Esterbauer H. Striegl G. Puhl H. Rotheneder M. Continuous monitoring of in vitro oxidation of human low density lipoprotein.Free Rad. Res. Comm. 1989; 6: 67-75Google Scholar). Freshly isolated, antioxidant-free LDL from four control subjects was incubated (200 μg cholesterol/ml incubation mixture) with CuSO4 (final concentration 1.5 μmol/l) at 37°C, and conjugated dienes were measured at 234 nm every 15 min for 5 h. Susceptibility to oxidation was expressed as the “lag time,” and was determined from the intercept of lines drawn through the linear portion of the lag and propagation phases for each sample as described by us previously (24Staprans I. Rapp J.H. Pan X-M. Kim K.Y. Feingold K.R. Oxidized lipids in the diet are a source of oxidized lipid in chylomicrons of human serum.Arterioscler. Thromb. 1994; 14: 1900-1905Google Scholar). The lag time was compared for LDL isolated from serum samples obtained at 0 time and 8 h after the consumption of the test meal containing 400 mg α-epoxy cholesterol. Total cholesterol and triglyceride concentration in serum and lipoproteins was measured using Sigma (St. Louis, MO) kits #352-20 and #339-20, respectively. α-Epoxy cholesterol in serum and serum-lipoprotein fractions and free and esterified cholesterol in LDL were determined by GLC using the procedure described by Hughes et al. (38Hughes H. Mathews B. Lenz M.L. Guyton J.R. Cytotoxicity of oxidized LDL to porcine aortic smooth muscle cells is associated with oxysterols 7-ketocholesterol and 7-hydroxycholesterol.Arterioscler. Thromb. 1994; 14: 1177-1185Google Scholar) and previously by us (22Staprans I. Pan X-M. Rapp J.H. Feingold K.R. Oxidized cholesterol in the diet accelerates the development of aortic atherosclerosis in cholesterol-fed rabbits.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 977-983Google Scholar, 23Staprans I. Pan X-M. Rapp J.H. Grunfeld C. Feingold K.R. Oxidized cholesterol in the diet accelerate the development of atherosclerosis in LDL-receptor- and apolipoprotein E-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 708-714Google Scholar). Lipid samples were derivatized with Sylon BFT and injected into gas chromatograph (Hewlett-Packard 5890, Palo Alto, CA) fitted with DB-1 column (J&W Scientific, Folsom, CA). Free and esterified α-epoxy cholesterol and cholesterol in lipoproteins was determined by measuring the difference between the amount before (free cholesterol) and after saponification (total cholesterol). Fatty acid composition in LDL was determined as described by us previously for lymph chylomicrons (24Staprans I. Rapp J.H. Pan X-M. Kim K.Y. Feingold K.R. Oxidized lipids in the diet are a source of oxidized lipid in chylomicrons of human serum.Arterioscler. Thromb. 1994; 14: 1900-1905Google Scholar). Triglycerides were isolated by TCL, hydrolyzed, and fatty acids were transmethylated with boron trifluoride methanol. Vitamin E in LDL was measured as described previously (24Staprans I. Rapp J.H. Pan X-M. Kim K.Y. Feingold K.R. Oxidized lipids in the diet are a source of oxidized lipid in chylomicrons of human serum.Arterioscler. Thromb. 1994; 14: 1900-1905Google Scholar). Standard α-epoxy cholesterol was obtained from Steraloids Inc. (Newport, RI), and a mixture of fatty acid standards was obtained from Matreya, Inc (Pleasant Gap, PA). Data are presented as mean ± SEM. The mean differences between groups were assessed with Student’s t-test. The mean lag time for LDL susceptibility to oxidation was determined using paired Student’s t-test. Significance was expressed as P < 0.05. Table 1 summarizes the age, BMI, and lipid values of the control and Type III hyperlipoproteinemic subjects. As expected, in the hyperlipoproteinemic subjects, serum lipid values are elevated because of a decrease in the clearance of serum chylomicron and VLDL remnants (IDL).TABLE 1Characteristics of study subjectsControl Subjects n = 6Subjects with Hyperlipoproteinemia n = 3Age (years)52.9 ± 3.060.0 ± 2.5BMI25.8 ± 0.727.3 ± 0.7Triglycerideammol/ dl.0.90 ± 0.194.0 ± 0.24 (P < 0.001)Cholesterolammol/ dl.5.31 ± 0.298.20 ± 1.18 (P < 0.02)HDL cholesterolammol/ dl.1.54 ± 0.170.99 ± 0.10LDL cholesterolammol/ dl.2.67 ± 0.221.82 ± 0.35α-Epoxy cholesterol/cholesterol (μg/mg)bα-Epoxy cholesterol-cholesterol ratio in postprandial dietary chylomicrons/chylomicron remnants (CM/RM) fraction collected 6 h after the consumption of the test meal.22.20 ± 4.901.32 ± 0.21 (P < 0.05)α-Epoxy cholesterol, cholestan-5α,6α-epoxy-3β-ol. All values are given as means ± SEM.a mmol/ dl.b α-Epoxy cholesterol-cholesterol ratio in postprandial dietary chylomicrons/chylomicron remnants (CM/RM) fraction collected 6 h after the consumption of the test meal. Open table in a new tab α-Epoxy cholesterol, cholestan-5α,6α-epoxy-3β-ol. All values are given as means ± SEM. In our initial studies, we determined the effect of different α-epoxy cholesterol quantities in the test meal on α-epoxy cholesterol levels in postprandial serum. We found that the serum levels of α-epoxy cholesterol strongly correlated (r = 0.997; P < 0.001; n = 3) with the amount of α-epoxy cholesterol in the test meal (50–400 mg). To ensure detection and to obtain accurate measurements, in these studies the quantity of α-epoxy cholesterol administered in the test meal was relatively large (400 mg) because the sensitivity of GLC for detecting small quantities of α-epoxy cholesterol is limited. At baseline before the feeding of the test meal, we did" @default.
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• W1994559481 date "2003-04-01" @default.
• W1994559481 modified "2023-10-02" @default.
• W1994559481 title "Oxidized cholesterol in the diet is a source of oxidized lipoproteins in human serum" @default.
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