Free radicals are believed to be involved in leukocyte induced tissue injury. The present studies were performed to determine whether low density lipoprotein (LDL) might serve as a mediator of tissue injury after leukocyte induced free radical oxidation of LDL. Our results show that incubation of LDL with monocytes or polymorphonuclear leukocytes (PMN) leads to oxidation of the lipoprotein rendering it toxic to proliferating fibroblasts. Monocyte activation enhances these effects. Butylated hydroxytoluene (BHT), vitamin E (vit E) and glutathione (GSH) virtually prevent the oxidation of LDL and the formation of cytotoxic LDL, indicating that these alterations are mediated by leukocyte-derived free radicals. This is the first demonstration that short-lived free radicals emanating from phagocytic cells could mediate cell injury through the action of a stable cytotoxin formed by the oxidation of LDL. The fact that lipoproteins can transfer a cytotoxic effect from leukocytes to proliferating cells reveals a pathway for cell destruction which may have implications in atherosclerotic plaque progression, macrophage mediated toxicity to tumor cells and tissue injury by inflammatory processes.
Previous studies have shown that Blueberries possess anti-angiogenic, anti-infective, anti-inflammatory, anti-carcinogenic, and anti-Alzheimer's properties (Bagchi et al. 2004, Joseph et al. 2003). Whole Blueberry Extract (WBE) was prepared from Vaccinium corymbosum L Bluecrop, by macerating the whole crushed blueberries in 80% acetone/water. The above WBE fraction was lyophilized and tested using the Enzyme Linked Immuno-Sorbent Assay (ELISA) for Vascular Endothelial Growth Factor (VEGF) in U937 and Eahy926 cell lines. The WBE fraction of Vaccinium corymbosum L bluecrop was found to inhibit VEGF in both the cell lines and the inhibitory activity was proportional to its concentration. These observations indicate that Whole Blueberry Extract possesses potent inhibitory activity on VEGF that results in anti-angiogenic property which helps in the protective action against certain cancers. Cytotoxicity studies were also performed on these cell lines for the effective inhibitory concentrations of the WBE fraction and were found to be non toxic. The above WBE fraction was further fractionated into different polyphenolic fractions and the highly active fraction was determined.
Our purpose was to determine whether the action of oxidative free radicals released by endothelial cells and vascular smooth muscle cells grown in culture could be responsible for certain modifications to low density lipoprotein (LDL). In these experiments we showed that after a 48-hour incubation with human umbilical vein endothelial cells or bovine aortic smooth muscle cells, human LDL: 1) became oxidized, as evidenced by reactivity to thiobarbituric acid; 2) lost variable amounts of sterol relative to protein (up to 20%); 3) had an increased relative electrophoretic mobility (by 30% to 70%); and 4) became toxic to proliferating fibroblasts. None of these changes occurred after a 48-hour incubation with confluent fibroblasts or bovine aortic endothelial cells, and all could be virtually prevented by the presence of butylated hydroxytoluene or other free radical scavengers. The results suggest that cells modifying LDL do so in part by an oxidation of LDL subsequent to cellular generation of free radicals.
In presence of oleate and taurocholate, differentiated CaCo-2 cell monolayers on membranes were able to assemble and secrete chylomicrons. Under these conditions, both cellular uptake and secretion into chylomicrons of β-carotene (β-C) were curvilinear, time-dependent (2–16 h), saturable, and concentration-dependent (apparent Km of 7–10 μM) processes. Under linear concentration conditions at 16 h incubation, the extent of absorption of all-trans β-C was 11% (80% in chylomicrons), while those of 9-cis- and 13-cis-β-C were significantly lower (2–3%). The preferential uptake of the all-trans isomer was also shown in hepatic stellate HSC-T6 cells and in a cell-free system from rat liver (microsomes), but not in endothelial EAHY cells or U937 monocyte-macrophages. Moreover, extents of absorption of α-carotene (α-C), lutein (LUT), and lycopene (LYC) in CaCo-2 cells were 10%, 7%, and 2.5%, respectively. Marked carotenoid interactions were observed between LYC/β-C and β-C/α-C.The present results indicate that β-C conformation plays a major role in its intestinal absorption and that cis isomer discrimination is at the levels of cellular uptake and incorporation into chylomicrons. Moreover, the kinetics of cellular uptake and secretion of β-C, the inhibition of the intestinal absorption of one carotenoid by another, and the cellular specificity of isomer discrimination all suggest that carotenoid uptake by intestinal cells is a facilitated process.
Oxysterols, oxidized derivatives of cholesterol, may enter the circulation as contaminants of cholesterol-containing food, arise through peroxidation of lipoproteins, or be generated by enzymatic reactions. They are found in serum associated either with lipoproteins or with albumin. In these studies, 25-hydroxycholesterol (25OHC) was used as a model oxysterol to investigate the effect of esterification on the association of oxysterols with serum components and their delivery to cultured cells. 25OHC added in vitro to fresh human serum was readily esterified during incubation at 37 degrees C, most likely by serum lecithin:cholesterol acyltransferase (LCAT) as it was blocked by known inhibitors of LCAT. The 25OHC-esters formed were identified as monoesters by comparing their elution on high performance liquid chromatography and thin-layer chromatography with that of chemically synthesized 25OHC mono- and diesters. Esterification doubled the percentage of 25OHC associated with lipoproteins, concomitantly decreasing the amount associated with albumin. Whereas unesterified 25OHC readily transferred between isolated lipoproteins, 25OHC-esters remained associated with donor lipoproteins unless human lipoprotein-deficient serum was added. That cholesteryl ester transfer protein (CETP) mediated transfer of 25OHC-esters was demonstrated by the ineffectiveness of rat lipoprotein-deficient serum as well as by the ability of IC-4, an anti-CETP monoclonal antibody, to suppress the transfer. Esterification of 25OHC in serum limited its entry into human erythrocytes and fibroblasts (GM 3468A cells) in vitro. Up-regulation of fibroblast low density lipoprotein (LDL)-receptors enhanced the uptake of esterified 25OHC from medium, but did little to enhance the total uptake of 25OHC. Thus, esterification of oxysterols in serum shifts their distribution away from albumin into LDL and high density lipoprotein (HDL) and limits their availability to cells in culture.
The effect of oxysterols on efflux of cholesterol from mouse L-cell fibroblasts, rat Fu5AH hepatoma cells, J774 macrophages, and human EA.hy 926 endothelial cells was studied. Cells were preincubated with 25-hydroxycholesterol (25-OHC) either during labeling of the cells with [3H]cholesterol or during equilibration after labeling. Subsequently, the release of [3H]cholesterol into medium containing 0.2 mg HDL3/ml was measured and the fractional release of cellular [3H]cholesterol was calculated. Pretreatment with 25-OHC (1 microgram/ml) caused a 30% reduction in [3H]cholesterol efflux from L-cells during 8 h of incubation with HDL3. 25-OHC also inhibited cholesterol efflux from Fu5AH and J774 cells, but the effect was less marked. There was only a small, nonsignificant reduction of efflux from EA.hy 926 cells. The mechanisms of 25-OHC-induced inhibition of cellular cholesterol efflux was further studied in L-cells, because of their sensitivity to 25-OHC treatment. The effect of 25-OHC on cholesterol efflux was dose-dependent, with significant effects seen at 25-OHC concentrations as low as 50 ng/ml. The half-time for cholesterol efflux from 25-OHC-treated cells (5 micrograms/ml) was 13.0 +/- 3.3 h compared to 5.7 +/- 1.0 in control cells, corresponding to a 55% reduction in the rate of cholesterol release. Other oxysterols, including 7-ketocholesterol, 7 alpha- and 7 beta-hydroxycholesterol, and 22(S)-hydroxycholesterol also inhibited [3H]cholesterol efflux from L-cells significantly, but to a lesser degree. 25-Hydroxycholesterol (5 micrograms/ml) reduced efflux from both normal and cholesterol-enriched cells by 31 and 14%, respectively. Inhibition of efflux was similar when reconstituted HDL3-apolipoprotein/phosphatidylcholine particles or small unilamellar phosphatidylcholine vesicles were used as cholesterol acceptors instead of HDL3. The content of phospholipids, cholesterol and the FC/PL ratio of intact cells and from isolated plasma membrane vesicles were the same for control and 25-OHC-treated cells. Efflux of [3H]cholesterol from plasma membranes isolated from 25-OHC-treated cells was 20% less than efflux from membranes from control cells. The difference in efflux observed in intact cells is partially explained by the reduction in efflux from the plasma membrane. In conclusion, our studies suggest that oxysterols, especially 25-hydroxycholesterol, can reduce cellular cholesterol efflux in vitro. Therefore oxysterols, either endogenous or derived from the diet, may influence cellular cholesterol efflux in vivo, the first step in reverse cholesterol transport.
Carotenoids and α-tocopherol are dietary, lipophilic antioxidants that may protect plasma lipoproteins from oxidation, a process believed to contribute to atherogenesis. Previous work demonstrated that after the Cu(II)-initiated oxidation of human low density lipoprotein (LDL) in vitro, carotenoids and α-tocopherol were destroyed before significant lipid peroxidation took place, and that α-tocopherol was destroyed at a much faster rate than were the carotenoids. Additionally, in vitro enrichment of LDL with β-carotene, but not with lutein or lycopene, inhibited LDL oxidation. In the present studies the impact of LDL carotenoid and α-tocopherol content on LDL oxidation by human endothelial cells (EaHy-1) in culture was assessed. LDL isolated from 11 individual donors was incubated at 0.25 mg protein/mL with EaHy-1 cells in Ham's F-10 medium for up to 48 h. Formation of lipid hydroperoxides was assessed by chemical analysis and the contents of lutein, β-cryptoxanthin, lycopene, β-carotene and α-tocopherol were determined by high performance liquid chromatography. The extent of lipid peroxidation correlated with the endogenous α-tocopherol content of the LDL but not with its content of carotenoids. As in the Cu(II)-initiated system, carotenoids and α-tocopherol were destroyed before significant peroxidation took place, but, in the cell-mediated system, α-tocopherol and the carotenoids were destroyed at comparable rates. Also, like the Cu(II)-initiated oxidation, enrichment of the LDL with β-carotene protected it from oxidation by the endothelial cells. However, enrichment with either lutein or lycopene actually enhanced the cell-mediated oxidation of the LDL. Thus, the specific content of carotenoids in low density lipoprotein (LDL) clearly modulates its susceptibility to oxidation, but individual carotenoids may either inhibit or promote LDL oxidation.— Dugas, T. R., D. W. Morel, and E. H. Harrison. Impact of LDL carotenoid and α-tocopherol content on LDL oxidation by endothelial cells in culture. J. Lipid Res. 1998. 39: 999–1007. Carotenoids and α-tocopherol are dietary, lipophilic antioxidants that may protect plasma lipoproteins from oxidation, a process believed to contribute to atherogenesis. Previous work demonstrated that after the Cu(II)-initiated oxidation of human low density lipoprotein (LDL) in vitro, carotenoids and α-tocopherol were destroyed before significant lipid peroxidation took place, and that α-tocopherol was destroyed at a much faster rate than were the carotenoids. Additionally, in vitro enrichment of LDL with β-carotene, but not with lutein or lycopene, inhibited LDL oxidation. In the present studies the impact of LDL carotenoid and α-tocopherol content on LDL oxidation by human endothelial cells (EaHy-1) in culture was assessed. LDL isolated from 11 individual donors was incubated at 0.25 mg protein/mL with EaHy-1 cells in Ham's F-10 medium for up to 48 h. Formation of lipid hydroperoxides was assessed by chemical analysis and the contents of lutein, β-cryptoxanthin, lycopene, β-carotene and α-tocopherol were determined by high performance liquid chromatography. The extent of lipid peroxidation correlated with the endogenous α-tocopherol content of the LDL but not with its content of carotenoids. As in the Cu(II)-initiated system, carotenoids and α-tocopherol were destroyed before significant peroxidation took place, but, in the cell-mediated system, α-tocopherol and the carotenoids were destroyed at comparable rates. Also, like the Cu(II)-initiated oxidation, enrichment of the LDL with β-carotene protected it from oxidation by the endothelial cells. However, enrichment with either lutein or lycopene actually enhanced the cell-mediated oxidation of the LDL. Thus, the specific content of carotenoids in low density lipoprotein (LDL) clearly modulates its susceptibility to oxidation, but individual carotenoids may either inhibit or promote LDL oxidation.— Dugas, T. R., D. W. Morel, and E. H. Harrison. Impact of LDL carotenoid and α-tocopherol content on LDL oxidation by endothelial cells in culture. J. Lipid Res. 1998. 39: 999–1007. The carotenoids are a group of plant pigments obtained in the diet from consumption of a variety of fruits and vegetables. These lipophilic compounds circulate in human plasma predominately in low density lipoprotein (LDL) (1Romanchik J. Morel D. Harrison E. Distribution of carotenoids and α-tocopherol among lipoproteins does not change when human plasma is incubated in vitro.J. Nutr. 1995; 125: 2610-2617Google Scholar, 2Krinsky N. Cornwell D. Oncley J. The transport of vitamin A and carotenoids in human plasma.Arch. Biochem. Biophys. 1958; 73: 233-246Google Scholar, 3Mathews-Roth M. Gulbrandsen C. Transport of β-carotene in serum of individuals with carotenemia.Clin. Chem. 1974; 20: 1578-1579Google Scholar, 4Bjornson L. Kayden H. Miller E. Moshell A. The transport of α-tocopherol and β-carotene in human blood.J. Lipid Res. 1976; 17: 343-352Google Scholar). These micronutrients, some of which have pro-vitamin A activity, are not essential in the diets of humans. Since the finding that carotenoids efficiently quench singlet oxygen (5Sundquist A. Briviba K. Sies H. Singlet oxygen quenching of carotenoids.Methods Enzymol. 1994; 234: 384-388Google Scholar), however, much research has focussed on their possible antioxidant function in diseases such as cancer and atherosclerosis (6Singh V.N. Role of β-carotene in disease prevention with special reference to cancer.in: Ong A.S.H. Packer L. Lipid-Soluble Antioxidants: Biochemistry and Clinical Applications. Birkhauser Verlag, Basel, Switzerland1992: 208-227Google Scholar, 7Sies H. Stahl W. Sundquist A. Antioxidant functions of vitamins.Ann. NY Acad. Sci. 1992; 669: 7-20Google Scholar, 8Gaziano J. Hennekens C. Antioxidant vitamins in the prevention of coronary artery disease.Cont. Int. Med. 1995; 7: 9-14Google Scholar) whose etiologies are believed to involve free radical lipid oxidation (9Berliner J. Heinecke J. The role of oxidized lipoproteins in atherogenesis.Free Radical Biol. Med. 1996; 20: 707-727Google Scholar). Over 100 epidemiological studies have demonstrated a lower risk of both cancer and atherosclerosis associated with the intake of fruits and vegetables high in carotenoids and other antioxidant vitamins (10Diaz M. Frei B. Vita J. Keaney J. Antioxidants and atherosclerotic heart disease.N. Engl. J. Med. 1997; 337: 408-416Google Scholar, 11Garewal H. Schantz S. Emerging role of β-carotene and antioxidant nutrients in prevention of oral cancer.Arch. Otolaryngol. Head Neck Surg. 1995; 121: 141-144Google Scholar, 12Kim S. Lee-Kim Y. Suh M. Chung E. Cho S. Cho B. Suh I. Serum levels of antioxidant vitamins in relation to coronary artery disease: a case control study of Koreans.Biomed. Environ. Sci. 1996; 9: 229-235Google Scholar, 13Marchand L.L. Intake of specific carotenoids and lung cancer risk.Cancer Epidemiol Biomarkers Prev. 1993; 2: 183-187Google Scholar). One large intervention study in Linxian, China, showed that supplementation with β-carotene and α-tocopherol reduced the risk of cancer by 13% (14Blot W. Li J. Taylor P. Guo W. Dawsey S. Wang G. Yang C. Zheng S. Gail M. Li G. Nutrition intervention trials in Linxian, China: supplementation with specific vitamin, mineral combinations, cancer incidence and disease-specific mortality in the general population.J. Natl. Cancer Inst. 1993; 85: 1483-1492Google Scholar). Though these effects were positive, other intervention trials have shown different results. In one, supplementation with β-carotene had no effect on cancer or coronary heart disease (Physician's Health Study) (15Hennekens C. Buring J. Manson J. Stampfer M. Rosner B. Belanger C. LaMotte F. Gaziano J. Ridker P. Willet W. Peto R. Lack of effect of long-term supplementation with beta-carotene on the incidence of malignant neoplasms and cardiovascular disease.N. Engl. J. Med. 1996; 334: 1145-1149Google Scholar), while in another, the α-Tocopherol, β-Carotene Cancer Prevention Study, supplementation with β-carotene in male Finnish smokers resulted in an 18% increase in lung cancer, but had no effect on coronary heart disease (16α-Tocopherol, β-Carotene Prevention Study Group The effect of vitamin E and β-carotene on the incidence of lung cancer and other cancers in male smokers.N. Engl. J. Med. 1994; 330: 1029-1035Google Scholar). In addition, in one intervention trial in the U.S., the Carotene and Retinol Efficacy Trials (CARET), supplementation of male and female smokers with both β-carotene and retinyl palmitate resulted in a 28% increase in lung cancer (17Ommen G. Goodman G. Thornquist M. Balmes J. Cullen M. Glass A. Keogh J. Meyskens F. Valanis B. Williams J. Barnhart S. Hammar S. Effects of a combination of β-carotene and vitamin A on lung cancer and cardiovascular disease.N. Engl. J. Med. 1996; 334: 1150-1155Google Scholar). More recently, carotenoids other than β-carotene have gained attention. In particular, lycopene is being investigated for its possible activity in preventing prostate, stomach, and pancreatic cancer (18Franceshi S. Tomatoes and risk of digestive tract cancers.Int. J. Cancer. 1994; 59: 181-184Google Scholar, 19Clinton S. Emenhiser C. Schwartz S. Bostwick D. Williams A. Moore B. Erdman J.W. Cis-trans lycopene isomers, carotenoids, and retinol in the human prostate.Cancer Epidemiol. Biomarkers Prev. 1996; 5: 823-833Google Scholar, 20Gerster H. The potential role for lycopene for human health.J. Am. Coll. Nutr. 1997; 16: 109-126Google Scholar) and myocardial infarction (21Kohlmeier L. Kark J.D. Gomez-Gracia E. Martin B.C. Steck S.E. Kardineal A.F.M. Ringstad J. Tham M. Masaev V. Riemersma R. Martinmoreno J.M. Huttunen J.K. Kok F.J. Lycopene and myocardial infarction risk in Euramic Study.Am. J. Epidem. 1997; 146: 618-626Google Scholar). Before intervention trials are attempted, however, much more investigation of these carotenoids needs to be performed. A major goal of the present investigation was to study the potential roles of all of the major carotenoids in human plasma on endothelial cell-mediated oxidation of LDL, which is implicated in atherogenesis (9Berliner J. Heinecke J. The role of oxidized lipoproteins in atherogenesis.Free Radical Biol. Med. 1996; 20: 707-727Google Scholar). Previous investigations by our laboratory and others have focussed on chemically-initiated oxidation of LDL (9Berliner J. Heinecke J. The role of oxidized lipoproteins in atherogenesis.Free Radical Biol. Med. 1996; 20: 707-727Google Scholar, 21Kohlmeier L. Kark J.D. Gomez-Gracia E. Martin B.C. Steck S.E. Kardineal A.F.M. Ringstad J. Tham M. Masaev V. Riemersma R. Martinmoreno J.M. Huttunen J.K. Kok F.J. Lycopene and myocardial infarction risk in Euramic Study.Am. J. Epidem. 1997; 146: 618-626Google Scholar, 22Romanchik J. Harrison E. Morel D. Addition of lutein, lycopene or β-carotene to LDL or serum in vitro: effects on carotenoid distribution, LDL composition and LDL oxidation.J. Nutr. Biochem. 1997; 8: 681-688Google Scholar, 23Reaven P. Khouw A. Beltz W. Parthasarathy S. Witztum J. Effect of dietary antioxidant combinations in humans: protection of LDL by vitamin E but not by b-carotene.Arterioscler. Thromb. 1993; 13: 590-600Google Scholar). After Cu(II)-initiated oxidation of LDL, it was shown that α-tocopherol and the carotenoids were destroyed before significant oxidation took place, with α-tocopherol being destroyed at a faster rate than the carotenoids (21Kohlmeier L. Kark J.D. Gomez-Gracia E. Martin B.C. Steck S.E. Kardineal A.F.M. Ringstad J. Tham M. Masaev V. Riemersma R. Martinmoreno J.M. Huttunen J.K. Kok F.J. Lycopene and myocardial infarction risk in Euramic Study.Am. J. Epidem. 1997; 146: 618-626Google Scholar). Additionally, in vitro enrichment with β-carotene, but not with lutein or lycopene, inhibited LDL oxidation (21Kohlmeier L. Kark J.D. Gomez-Gracia E. Martin B.C. Steck S.E. Kardineal A.F.M. Ringstad J. Tham M. Masaev V. Riemersma R. Martinmoreno J.M. Huttunen J.K. Kok F.J. Lycopene and myocardial infarction risk in Euramic Study.Am. J. Epidem. 1997; 146: 618-626Google Scholar). Though much work has been reported for chemically initiated oxidation of LDL, little work has been reported for the presumably more pathophysiological initiation of oxidation by vascular cells. We present here the results of studies where LDL oxidation was initiated using EaHy-1 cells, a human endothelial cell line. Results presented here show that as in the Cu(II)-initiated system, α-tocopherol and the carotenoids were destroyed before significant oxidation took place, but in this case, α-tocopherol and the carotenoids were destroyed at comparable rates. As with the Cu(II)-initiated system, β-carotene enrichment protected LDL from oxidation, but contrary to what was expected, enrichment with lutein and lycopene enhanced the oxidation. Finally, further analysis of the data shows that there was no correlation between the extent of oxidation and endogenous carotenoid levels, however, there was a strong correlation between oxidation and endogenous α-tocopherol. All carotenoids used in this study, either for HPLC quantitation or for in vitro enrichment, were selected for their high purity and quality. Lycopene and lutein were gifts from Dr. Gary Beecher of the USDA (Beltsville, MD). Lutein was also purchased from Kemin Industries (Des Moines, IA). β-Carotene was obtained from Fluka, and β-cryptoxanthin was a gift from Hoffman-LaRoche (Basel, Switzerland). A human endothelial cell line, EaHy-1, was a gift from Dr. Mahamad Navab at UCLA. Cell culture media, Dulbecco's modified Eagle's Medium (DMEM) and Ham's F-10 medium, trypsin·EDTA and heat-inactivated fetal bovine serum (FBS) were obtained from Bio-Whittaker (Walkersville, MD). HAT (5 mm hypoxanthine, 0.02 mm aminopterin, 0.8 mm thymidine) media supplement (Hybri-Max®, 50×) for hybridoma cells and gentamicin solution (10 mg/mL) were purchased from Sigma (St. Louis, MO). Reagents used for protein or oxidation assays, including Folin and Ciocalteu's Phenol Reagent, lauryl sulfate (sodium dodecyl sulfate), xylenol orange (3,3′-bis[N, N-di(carboxymethyl)-aminomethyl]-o-cresol-sulfonephthalein), butylated hydroxytoluene (2,6-di-t-butyl-p-cresol), and 2-thiobarbituric acid (4,6-dihydroxyprimidine-2-thiol), were obtained from Sigma. Malonaldehyde bis(dimethyl acetal), or 1,1,3,3-tetramethoxypropane, 99%, was purchased from Aldrich. The tetrahydrofuran (THF) used in these experiments contained no BHT (butylated hydroxytoluene), so it was stored under N2 and was pretested before each use with starch iodide test papers (Fisher) to insure that the THF was peroxide-free. All other incidental reagents or solvents were of high purity and were used as received. Plasma was collected from eleven different fasted donors (7 males and 4 females), denoted here as donors A–K. The donors were all healthy individuals between the ages of 22 and 45 years, who were not currently taking medication or vitamin supplements. After collection, plasma was either stored at -20°C or was used immediately. Lipoproteins were isolated by the sequential density gradient ultracentrifugation method described by Hatch and Lees (24Hatch F. Lees R. Practical methods for plasma lipoprotein analysis.Adv. Lipid Res. 1968; 6: 1-68Google Scholar). Low-density lipoprotein (LDL) was identified as the fraction with a density of 1.019–1.063 g/mL. Once the LDL was isolated, it was used immediately with no storage. Before incubation with cells, the LDL was first desalted by gel filtration (Pharmacia Biotech Sephadex® G-25 M (PD-10) gel filtration columns). Protein was determined by the modified Lowry protein assay (25Markwell M. Haas S. Bieber L. Tolbert N. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.Anal. Biochem. 1978; 87: 206-210Google Scholar). The LDL or plasma sample was first divided into two parts. One was enriched with carotenoid in vitro and the other, which served as an unenriched control, was stored untreated at 4°C. To achieve in vitro carotenoid enrichment, stock solutions of either 2 mm lutein in ethanol, 3 mm lycopene in THF, or 8 mm β-carotene in THF were first prepared. These stock solutions were checked for purity by HPLC before use and discarded if there was any evidence of degradation of the carotenoids. Plasma or LDL was then incubated at 4°C in the dark on a gentle rotary (blood) mixer with 2% v/v of the appropriate carotenoid stock solution for 16 h (overnight). The resulting concentrations of carotenoid incubated in the presence of plasma or LDL were thus either 40 μm lutein, 60 μm lycopene, or 160 μm β-carotene. The plasma or LDL was filtered using a 0.45-μm syringe filter to remove the majority of unincorporated carotenoid. The lipoproteins were then (re)isolated by the sequential density gradient ultracentrifugation method mentioned earlier, further reducing the amount of unincorporated carotenoid. Lipoprotein integrity was compared for the unenriched and enriched samples by measuring the electrophoretic mobility on an agarose gel (Beckman Paragon® lipoprotein (LIPO) agarose gel electrophoresis kit), as well as the cholesterol-to-protein and triglyceride-to-protein ratios for each of the two samples. Results of these measurements showed that there was no effect of in vitro enrichment on either LDL mobility on an agarose gel or on cholesterol- or triglyceride-to-protein ratios, suggesting that there was no change in physical properties or composition of the lipoprotein. We analyzed the lipoprotein carotenoid content before and after enrichment with each carotenoid. Importantly, the enrichment of LDL with specific carotenoids (i.e., lutein, lycopene, β-carotene) had no significant effect on the levels of the other endogenous carotenoids. Moreover, after enrichment there was no indication by HPLC analysis of any “new” carotenoid peaks or breakdown products. Thus, the enrichment of LDL with pure solution of a given carotenoid increased only that carotenoid in the lipoprotein. The EaHy-1 cells used in these experiments, human aortic endothelial cells immortalized by hybridization to cells from a human adenoma, were shown previously to maintain their characteristic cobblestone morphology, presence of Factor VIII-related antigen, and ability to take up acetylated LDL (26Navab M. Hough G. Stevenson L. Drinkwater D. Laks H. Fogelman A. Monocyte migration into the subendothelial space of a coculture of adult human aortic endothelial and smooth muscle cells.J. Clin. Invest. 1988; 82: 1853-1863Google Scholar, 27Navab M. Imes S. Hama S. Hough G. Ross L. Bork R. Valente A. Berliner J. Drinkwater D. Laks H. Fogelman A. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein.J. Clin. Invest. 1991; 88: 2039-2046Google Scholar, 28Reaven P. Ferguson E. Navab M. Powell F. Susceptibility of human LDL to oxidative modification. Effects of variations in β-carotene concentration and oxygen tension.Arterioscler. Thromb. 1994; 14: 1162-1169Google Scholar). These cells were grown here in DMEM medium containing 10% FBS, 0.125 mg/mL gentamycin, 4.5 mg/mL glutamine, and HAT medium supplement (50×, dissolved in 10 mL deionized water and diluted 1:50 in the DMEM). Cells were plated into 6-well plates 3–4 days before the start of the oxidation experiments. Once cells were grown to confluence, the DMEM was removed and the cells were washed with sterile phosphate buffered saline (PBS). The cells were then incubated at 37°C with 2 mL per well of 0.25 mg/mL LDL in Ham's F-10 medium for 0–48 h. As a control for the spontaneous oxidation of the LDL that might result from incubation at 37°C in the absence of cells, the 0.25 mg/mL LDL was also incubated in wells without cells at 37°C for 0–48 h. After various lengths of incubation, the LDL-containing medium was removed and aliquots of a stock solution of BHT in ethanol were added such that the final concentration was 25 μm BHT. This was done to prevent further oxidation of the samples both during storage and during the oxidation assays. Aliquots of medium containing BHT were taken for measurements of lipid oxidation and stored prior to assay at -20°C for less than 1 week. The cells were again washed with PBS, and the amount of remaining cell protein per well was measured using the modified Lowry protein assay (25Markwell M. Haas S. Bieber L. Tolbert N. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.Anal. Biochem. 1978; 87: 206-210Google Scholar). In brief, 1 mL reagent A (containing SDS and described in the modified Lowry assay) was placed in each well and was incubated for 1 h with gentle shaking of the plate. From each well, 100 μL was then taken for the assay. The amount of lipid oxidation that had taken place in each LDL sample was assessed using the TBARS (thiobarbituric acid-reactive substances) (29Morel D. Chisolm G. Antioxidant treatment of diabetic rats inhibits lipoprotein oxidation and cytotoxicity.J. Lipid Res. 1989; 30: 1827-1834Google Scholar) and/or the FOX (ferrous oxidation/xylenol orange) (30Jiang Z. Hunt J. Wolff S. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low-density lipoprotein.Anal. Biochem. 1992; 202: 384-389Google Scholar) assays. Both the samples of LDL incubated in the presence of endothelial cells and the LDL incubated cell-free were assayed for lipid oxidation. For the TBARS assay, 400 μL of each LDL sample was assayed as described previously (29Morel D. Chisolm G. Antioxidant treatment of diabetic rats inhibits lipoprotein oxidation and cytotoxicity.J. Lipid Res. 1989; 30: 1827-1834Google Scholar). As a standard for the quantitation of TBARS, the assay was also performed using several dilutions of a known amount of malonaldehyde bis(dimethyl acetal) (Aldrich). Before the FOX assay could be performed, the lipid hydroperoxides were first extracted from 100 μL of the LDL samples using the methanol extraction procedure described by Jiang, Hunt, and Wolff (30Jiang Z. Hunt J. Wolff S. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low-density lipoprotein.Anal. Biochem. 1992; 202: 384-389Google Scholar). The extracts were then dried under nitrogen and redissolved in 100 μL methanol–water 9:1 (v/v) solution. The FOX assay (30Jiang Z. Hunt J. Wolff S. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low-density lipoprotein.Anal. Biochem. 1992; 202: 384-389Google Scholar) was performed on the samples, as well as on dilutions of 30% hydrogen peroxide used as a standard. The cholesterol contents of the plasma or lipoproteins were determined using the Total Cholesterol 50 assay kit (enzymatic, colorimetric, from Sigma). Likewise, triglycerides were determined using the Triglyceride (GPO-Trinder) 50 assay kit (enzymatic, colorimetric, from Sigma). To determine the carotenoid content of the LDL, the carotenoids were first extracted from 2-mL aliquots of the 0.25 mg/mL LDL samples using the method described by Barua et al. (31Barua A. Batres R. Furr H. Olson J. Analysis of carotenoids in human serum.J. Micronutr. Anal. 1989; 5: 291-302Google Scholar). The extracts were evaporated with a gentle stream of N2 and were redissolved in 200 μL acetonitrile–isopropanol 1:1 solution. The carotenoid and α-tocopherol contents were then determined by the HPLC method reported originally by Barua and Furr (32Barua A. Furr H. Extraction and analysis by high-performance liquid chromatography of carotenoids in human serum.Methods Enzymol. 1992; 213: 273-281Google Scholar). The analysis was performed using a Waters 600E HPLC pump, a 717 plus autosampler, and a 996 photodiode array detector. Conditions for the separation included a 25 cm, 4.6 ID Supercosil® (Supelco) C18 reverse-phase column, acetonitrile–dichloromethane–methanol–octanol 90:15:10:0.1 (v/v/v/v) containing 0.01% t-butylamine mobile phase, and a flow rate of 1 mL/min. Absorbance was monitored between 250 and 600 nm, and the UV spectrum was sampled at 2-sec intervals. Elution and absorption of the α-tocopherol and the carotenoids were determined on separate channels using Millenium software (Waters) version 2.10. For the α-tocopherol channel, the chromatogram was extracted at 290 nm, while for the carotenoid channel, at 450 nm. Quantitation and identification of the α-tocopherol or individual carotenoids were done by comparison to standards. The individual carotenoids and α-tocopherol were identified by comparison of both retention times and UV spectra. Carotenoid and α-tocopherol compositions of the various LDL samples were compiled and are listed in Table 1.TABLE 1.Endogenous carotenoid composition of LDL samples from the various donorsDonorLuteinβ-CryptoxanthinLycopeneβ-Caroteneα-Tocopherolnmol/mg proteinA0.070.160.240.156.87B (I)0.090.080.360.120.68B (II)0.050.171.230.678.14C0.100.107.600.289.88D (I)0.050.090.580.916.98D (II)0.060.030.160.113.60E0.060.102.650.823.28F0.260.050.290.183.65G0.020.030.290.592.88H0.140.095.850.8210.4I0.080.040.580.105.35J0.040.030.560.036.90K0.080.030.510.044.52Avg.0.08 ± 0.060.08 ± 0.051.61 ± 2.390.37 ± 0.345.62 ± 2.87 Open table in a new tab In agreement with previous findings (33Morel D. DiCorleto P. Chisolm G. Endothelial and smooth muscle cells alter low density lipoprotein in vitro by free radical oxidation.Arteriosclerosis. 1984; 4: 357-364Google Scholar, 34Steinbrecher U. Parthasarathy S. Leake D. Witztum J. Steinberg D. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids.Proc. Natl. Acad. Sci. USA. 1984; 83: 3883-3887Google Scholar), EaHy-1 human endothelial cells, when incubated in Ham's F-10 medium, initiated significant lipid oxidation in the LDL (Fig. 1). Oxidation products measured using the FOX assay showed that a small amount of oxidation also took place in LDL incubated without cells, however, the amount of oxidation resulting from its incubation with the cells was considerably more pronounced. In addition, cell protein remained relatively constant over the incubation period (data not shown). Fig. 2 shows the results of oxidation of LDL samples from three donors (I, J, and K), containing very similar carotenoid and α-tocopherol profiles (Table 1). The kinetics of oxidation as measured by the TBARS and FOX assays were markedly different. Using the FOX assay, lipid hydroperoxides, a direct product of lipid peroxidation, began to increase after a 4- to 6-h lag phase, reached a maximum at 24 h, and then decreased sharply. In the TBARS assay, short-chain aldehydes like malondialdehyde, breakdown products of lipid hydroperoxides, began to increase after a 10- to 12-hour lag phase, at least 2 h later than lipid hydroperoxides. They increased sharply for the first 20 h, then began leveling off through 36 h. Both the endogenous carotenoids and α-tocopherol in the LDL were destroyed before significant oxidation took place (Fig. 2). In comparison to lipid hydroperoxides as measured by the FOX assay, about 50% of the carotenoids and α-tocopherol were destroyed before lipid oxidation began to increase by a measurable amount. Also note that the rates of destruction of α-tocopherol and the combined carotenoids (Fig. 2) were virtually indistinguishable. In contrast to the relatively rapid destruction of LDL carotenoids in the presence of cells, LDL carotenoids were quite stable in culture medium when incubated in the absence of cells. Under these conditions, 90% of the total initial carotenoid was present at 18 h and thereafter declined to about 40% at 36 h (data not shown). The averaged rates of destruction of the individual carotenoids and α-tocopherol from the same three LDL samples (from donors I–K) were also compared (Fig. 3). Results show that the rates of destruction of α-tocopherol, as compared to the individual carotenoids, were quite similar (Fig. 3). Likewise, there was little difference among the rates of destruction of individual carotenoids. While the results in Fig. 3 show that β-carotene was destroyed at a slightly lower rate than others, in additional experiments β-carotene was destroyed at the same rate as the other carotenoids. LDL collected from a given individual was divided into two samples. One sample served as the unenriched control and received no treatment beyond storage at 4°C. The other sample was enriched in vitro with a given carotenoid by the method described above in Experimental Procedures. Both samples were incubated with the same endothelial cells (subcultures of the same flask) at the same time. Five LDL samples from four different donors (donors B, C, D, and H) were enriched in vitro with lutein. LDL from donor B was sampled twice, about 6 months apart. Treatment with lutein resulted in a 5- to 18-fold increase in LDL lutein. Representative results from two donors, donors B and C, are shown in Fig. 4. Fold enrichments in LDL lutein in these two samples were 13 and 11, respectively. In four out of five experiments, there was a dramatic enhancement of lipid oxidation in the sample of enriched LDL as compared to its unenriched control. In the LDL sample with the highest enrichment (18-fold, donor H), there was a very slight decrease in oxidation as compared to the unenriched LDL (data not shown). Three samples of LDL from three different donors (donors A, F, and H) were enriched in vitro with lycopene and were incubated with EaHy-1 cells. Enrichment resulted in a 2- to 8-fold increase in lycopene. Results from two representative samples (donors A, H) are shown i
