Formation of cholesterol ozonolysis products through an ozone-free mechanism mediated by the myeloperoxidase-H2O2-chloride system

The presence of the cholesterol ozonolysis products, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (atheronal-A) and its aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (atheronal-B) in human atherosclerotic tissues was recently reported as evidence for the generation of ozone by activated human neutrophils. However, the mechanism for the formation of atheronals in atherosclerotic tissues is unknown. In this study, we found that atheronals were formed by the reaction of cholesterol with human myeloperoxidase (MPO) in the presence of its substrates H₂O₂ and Cl⁻. The omission of either H₂O₂ or Cl⁻ from the MPO-H₂O₂-Cl⁻ system resulted in a significant reduction in yields. The formation of atheronals by the MPO-H₂O₂-Cl⁻ system was inhibited by an inhibitor of MPO and scavengers of reactive oxygen species such as sodium azide, methionine, β-carotene, and vinylbenzoic acid. Our results suggest that MPO produces atheronals at least partly through an ozone-free mechanism, via the reaction of cholesterol with singlet oxygen generated from HOCl and H₂O₂.     

Formation of myeloperoxidase compound II during aerobic stimulation of rat neutrophils

We have made simultaneous spectrophotometric and O₂ measurements on suspensions of rat neutrophils during activation of the respiratory burst. Under aerobic conditions an absorption increase attributable to myeloperoxidase compound II was observed in parallel with the rapid phase of O₂ uptake. Identification of this compound was confirmed by analysis of a spectrum obtained with purified myeloperoxidase and H₂O₂. Whereas a second addition of stimulus did not increase O₂ uptake any further, a second phase of myeloperoxidase release and compound II formation was observed. These results suggest thatin vivo myeloperoxidase reacts with H₂O₂ generatedvia the respiratory burst to form compound II under conditions in which the chlorination reaction would be the expected major pathway.Abbreviations FMLP N-formylmethionylleucyl phenylalanine - MPO²⁺.H₂O₂ Myeloperoxidase compound II - MPO³⁺.H₂O₂ Myeloperoxidase compound I - {ei275-1} superoxide     

Proatherogenic effects of the cholesterol ozonolysis products, atheronal-A and atheronal-B

The proatherogenic properties of the cholesterol 5,6-secosterols (atheronal-A and atheronal-B), recently discovered in atherosclerotic arteries, have been investigated in terms of their effects on monocyte/macrophage function. A fluorescent analogue of atheronal-B (1) (50 microM), when cultured in either aqueous buffer (PBS) or in media containing fetal calf serum (10%), is rapidly taken-up into cultured macrophage (J774.1 or RAW 264.7) cells and accumulates at perinuclear sites (within 1 h). Co-incubation of macrophage cells (J774.1) with atheronal-A (25 microM) and atheronal-B (25 microM) when complexed with low-density lipoprotein (LDL) (100 microg/mL) leads to a significant upregulation of scavenger receptor class A (approximately 3-fold increase relative to LDL alone, p < 0.05) but not CD36, showing that cultured macrophages respond to LDL-complexed atheronals in a manner highly analogous to acetylated LDL rather than oxidized LDL. Both atheronal-A and atheronal-B in solution exhibit a dose-dependent (0-25 microM) induction of chemotaxis of cultured macrophages (p < 0.001). When complexed with LDL (100 microg/mL), atheronal-A (but not atheronal-B) induces a dose-dependent (0-25 microM, p < 0.05) upregulation of the cell-surface adhesion molecule endothelial (E)-selectin on vascular endothelial cells (HUVECs). LDL (100 microg/mL) complexed atheronal-B (25 microM) but not atheronal-A induces cultured human monocytes (THP-1) to differentiate into macrophage cell lineage. When these in vitro data are taken together with the already known effects of cholesterol 5,6-secosterols on foam cell formation and macrophage cytotoxicity, the atheronals possess biological effects that if translated to an in vivo setting could lead to the recruitment, entrapment, dysfunction, and ultimate destruction of macrophages, with the major leukocyte player in inflammatory artery disease. As such, the atheronal molecules may be a new association, in the already complex inter-relationship, between inflammation, cholesterol oxidation, the tissue macrophage, and atherosclerosis.     

Aminoglycosides as substrates and inhibitors of peroxidases: a possible role of these antibiotics against myeloperoxidase-dependent cytotoxicity

The kinetics of the catalytic cycle of myeloperoxidase and of horseradish peroxidase reacting with aminoglycosides have been studied by conventional and stopped-flow spectrophotometry. Aminoglycosides acted as one-electron reducing substrates converting compound I, formed when stoichiometric amounts of hydrogen peroxide were added to the enzyme, to compound II, and compound II to the resting, ferric enzyme. The latter gradually decayed into a further spectroscopic derivative (_max = 540 and 403 nm) tentatively identified as a complex of ferric heme with the antibiotic oxidation product(s), and the resulting enzyme was fully inactivated. Since myeloperoxidase is the only human enzyme known to convert chloride ions into the cytotoxic hypochlorous acid, the data presented in this paper bear relevance to the pharmacological effects of aminoglycoside antibiotics, which, while inhibiting bacterial growth, also prevent oxidative cellular damage caused by hypochlorous acid aging as substrates and inhibitors of myeloperoxidase.     

Formation of biologically active oxysterols during ozonolysis of cholesterol present in lung surfactant

Exposure of the lung to concentrations of ozone found in ambient air is known to cause toxicity to the epithelial cells of the lung. Because of the chemical reactivity of ozone, it likely reacts with target molecules in pulmonary surfactant, a lipid-rich material that lines the epithelial cells in the airways. Phospholipids containing unsaturated fatty acyl groups and cholesterol would be susceptible to attack by ozone, which may lead to the formation of cytotoxic products. Whereas free radicalderived oxidized cholesterol products have been frequently studied for their cytotoxic effects, ozonized cholesterol products have not been studied, although they could reasonably play a role in the toxicity of ozone. The reaction of ozone with cholesterol yielded a complex series of products including 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al, 5-hydroperoxy-B-homo-6-oxa-cholestan-3beta,7a-diol, and 5beta,6beta-epoxycholesterol. Mass spectrometry and radioactive monitoring were used to identify the major cholesterol-derived product during the reaction of 2 ppm ozone in surfactant as 5beta,6beta-epoxycholesterol, which is only a minor product during ozonolysis of cholesterol in solution. A dose-dependent formation of 5beta,6beta-epoxycholesterol was also seen during direct exposure of intact cultured human bronchial epithelial cells (16-HBE) to ozone. Studies of the metabolism of this epoxide in lung epithelial cells yielded small amounts of the expected metabolite, cholestan-3beta,5alpha,6beta-triol, and more abundant levels of an unexpected metabolite, cholestan-6-oxo-3beta,5alpha-diol. Both 5beta,6beta-epoxycholesterol and cholestan-6-oxo-3beta,5alpha-diol were shown to be cytotoxic to cultured 16-HBE cells. A possible mechanism for cytotoxicity is the ability of these oxysterols to inhibit isoprenoid-based cholesterol biosynthesis in these cells.     

Inactivation of alpha-1-proteinase inhibitor in serum by stimulated human polymorphonuclear leucocytes. Evidence for a myeloperoxidase-dependent mechanism

Triggered polymorphonuclear leucocytes (PMNL) can decrease the elastase inhibitory capacity of serum by inactivating the main inhibitor of elastase alpha-1-proteinase inhibitor (alpha-1-PI). Maximal inactivation occurs with stimuli that release myeloperoxidase from PMNL along with hydrogen peroxide. Specific protection of alpha-1-PI function is obtained with antioxidants that interfere with this system. PMNL that are activated with phorbol myristate acetate release hydrogen peroxide but not myeloperoxidase, and only inactivate alpha-1-PI in the presence of exogenously-added PMNL-derived supernatants which contain this enzyme. Cell-free inactivation requires both active enzyme and hydrogen peroxide, and is greatest at pH 6.2, the pH optimum for myeloperoxidase-catalysed inactivation of alpha-1-PI. This data supports the notion that leucocyte myeloperoxidase may act to suppress the antiprotease screen afforded by alpha-1-PI by generating hypochlorous acid in the presence of chloride and respiratory burst-derived hydrogen peroxide, and in the microenvironment of lowered pH associated with degranulation. Pulmonary emphysema seems to be associated with an imbalance between elastase and its inhibitors at the lung surface. PMNL are likely to play an important role in the pathogenesis of emphysema since they contain both elastase, which can solubilize connective tissue elastin, and the constituents of an oxidative system which can inactivate the most important antielastase, alpha-1-PI.     

Formation of reactive cyclopentenone compounds in vivo as products of the isoprostane pathway

Cyclopentenone prostaglandins A2 and J2 are reactive compounds that possess unique biological activities. However, the extent to which they are formed in vivo remains unclear. In this study, we explored whether D2/E2-isoprostanes undergo dehydration in vivo to form A2/J2-isoprostanes. Oxidation of arachidonic acid in vitro generated a series of compounds that were confirmed to be A2/J2-isoprostanes by mass spectrometric analyses. A2/J2-isoprostanes were detected in vivo esterified to lipids in livers from normal rats at a level of 5. 1 +/- 2.3 ng/g, and levels increased dramatically by a mean of 24-fold following administration of CCl4. An A2-isoprostane, 15-A2t-isoprostane, was obtained and found to readily undergo Michael addition with glutathione and to adduct covalently to protein. A2/J2-isoprostanes could not be detected in the circulation, even following CCl4 administration, which we hypothesized might be explained by rapid formation of adducts. This was supported by finding that essentially all the radioactivity excreted into the urine following infusion of radiolabeled 15-A2t-isoprostane into a human volunteer was in the form of a polar conjugate(s). These data identify a new class of reactive compounds that are produced in vivo as products of the isoprostane pathway that can exert biological effects relevant to the pathobiology of oxidant injury.     

Leukocyte Mobilization, Chemiluminescence Response, and Antioxidative Capacity of the Blood in Intestinal Ischemia and Reperfusion

Intestinal ischemia and reperfusion elicits changes in leukocyte counts and increased production of reactive oxygen species (ROS). The purpose of this study was to investigate whether these changes were followed by and/or connected with changes in the extracellular antioxidative capacity in a rat superior mesenteric artery (SMA) occlusion/reperfusion model. The SMA was occluded for 45 min and then allowed to be reper-fused. Changes of leukocyte, polymorphonuclear (PMN), and lymphocyte counts, chemiluminescence (CL) of whole blood samples as a marker of ROS production, and the total antioxidative capacity of the serum were quantified at the end of ischemia and in 1 h intervals during the postischemic period up to 4 h. The myeloperoxidase (MPO) activity in the serum and intestinal tissue samples was also determined. The MPO activity in the intestinal tissue samples was significantly elevated at the end of ischemia, and this elevation lasted for the whole postischemic period. The oxidative challenge to the body induced a fast mobilization of extracellular antioxidative mechanisms already at the end of ischemia, which was followed by a significant increase in PMN counts and whole blood CL starting at the 2nd hour after reperfusion. The increased CL activity of whole blood was attributed to the increase of the circulating PMNs. No significant changes were observed in leukocyte and lymphocyte counts. It is concluded that compensatory mechanisms of the oxidative-antioxidative balance of the body react very quickly if challenged.     

Metabolic products of soluble epoxide hydrolase are essential for monocyte chemotaxis to MCP-1 in vitro and in vivo

Monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis is a major event in inflammatory disease. Our prior studies have demonstrated that MCP-1-dependent chemotaxis requires release of arachidonic acid (AA) by activated cytosolic phospholipase A₂ (cPLA₂). Here we investigated the involvement of AA metabolites in chemotaxis. Neither cyclooxygenase nor lipoxygenase pathways were required, whereas pharmacologic inhibitors of both the cytochrome-P450 (CYP) and the soluble epoxide hydrolase (sEH) pathways blocked monocyte chemotaxis to MCP-1. To verify specificity, we demonstrated that the CYP and sEH products epoxyeiscosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs), respectively, restored chemotaxis in the presence of the inhibitors, indicating that sEH-derived products are essential for MCP-1-driven chemotaxis. Importantly, DHETs also rescued chemotaxis in cPLA₂-deficient monocytes and monocytes with blocked Erk1/2 activity, because Erk controls cPLA₂ activation. The in vitro findings regarding the involvement of CYP/sEH pathways were further validated in vivo using two complementary approaches measuring MCP-1-dependent chemotaxis in mice. These observations reveal the importance of sEH in MCP-1-regulated monocyte chemotaxis and may explain the observed therapeutic value of sEH inhibitors in treatment of inflammatory diseases, cardiovascular diseases, pain, and even carcinogenesis. Their effectiveness, often attributed to increasing EET levels, is probably influenced by the impairment of DHET formation and inhibition of chemotaxis.     

PTEN improve renal fibrosis in vitro and in vivo through inhibiting FAK/AKT signaling pathway

Renal fibrosis, the ultimate common pathway of progressive nephropathy, is characterized by excess accumulation and deposition of extracellular matrix (ECM) within the renal interstitium and glomeruli, finally resulting in end-stage kidney failure. TGFβ1 is not only abnormally increased during fibrosis but also involved in ECM induction and accumulation. Based on the bioinformative analyses, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and focal adhesion kinase (FAK) signaling pathway might be involved in TGFβ1 functions on renal fibrosis development. In the present study, fibrosis was induced in HK-2 cells using TGFβ1 and PTEN expression was significantly suppressed by 24 or 48 hours TGFβ1 treatment. PTEN overexpression in HK-2 cells improved TGFβ1-induced fibrosis within α-SMA and E-cadherin. According to the KEGG signaling pathway annotation analyses on microarray profiles (GSE23338 and GSE20247) and immunoblotting validation, FAK signaling might be involved in PTEN functions in TGFβ1-induced fibrosis. PTEN overexpression significantly inhibited TGFβ1- or unilateral ureteral obstruction (UUO)-induced FAK signaling pathway activation both in vitro and in vivo; more importantly, PTEN silence enhanced TGFβ1- or UUO-induced fibrosis, while FAK inhibitor PF567721 significantly reversed the effects of PTEN silence, indicating that PTEN exerted its effects on TGFβ1- and UUO-induced fibrotic development in vitro and in vivo via inhibiting FAK signaling pathway. In summary, these findings indicate that PTEN could improve cellular fibrotic changes and renal fibrosis via inhibiting FAK/AKT signaling pathway. Restoring PTEN expression to target FAK/AKT signaling pathway might be a potent strategy for renal fibrosis treatment.     

Wnt pathway targeting reduces triple-negative breast cancer aggressiveness through miRNA regulation in vitro and in vivo

Triple-negative breast cancer, devoid of estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER-2) expression, is deprived of commonly used targeted therapies. MicroRNAs (miRNAs) are undergoing a revolution in terms of potentially diagnostic or therapeutic elements. Combining computational approaches, we enriched miRNA binding motifs of Wnt pathway-associated upregulated genes. Our in-depth bioinformatics, in vitro and in vivo analyses indicated that miR-381 targets main genes of the Wnt signaling pathway including CTNNB1, RhoA, ROCK1, and c-MYC genes. The expression level of miR-381 and target genes was assessed by quantitative real-time polymerase chain reaction (RT-qPCR) in MCF-7, MDA-MB-231, and MCF-10A as well as 20 breast cancer samples and normal tissues. Luciferase reporter assay was performed. Lentiviral particles containing miR-381 were used to evaluate the effect of miR-381 restoration on cell proliferation, migration, and invasion of the invasive triple-negative MDA-MB-231 cell line and also in a mouse model of breast cancer. The expression of miR-381 was lower than that of normal cells, especially in TNBC cell line and breast tissues. Luciferase assay results confirmed that miR-381 targets all the predicted 3′-untranslated regions (3′-UTRs). Upon miR-381 overexpression, the expression of target genes declined, and the migration and invasion potential of miR-381-receiving MDA-MB-231 cells decreased. In a mouse model of triple-negative breast cancer, miR-381 re-expression inhibited the invasion of cancer cells to lung and liver and prolonged the survival time of cancer cell-bearing mice. Therefore, miR-381 is a regulator of Wnt signaling and its re-expression provides a potentially effective strategy for inhibition of TNBC.     

Effects of hydrogen peroxide on neutrophil ability to generate reactive oxygen and chlorine species and to secrete myeloperoxidase in vitro

In this work, the effects of H₂O₂ at concentrations of 10^?8–10^?2 mol/l on the neutrophil ability to generate reactive oxygen and chlorine species (ROCS) and to secrete myeloperoxidase (MPO) were studied, as well as the H₂O₂ damaging action on neutrophils. It was found that H₂O₂ at concentrations of 2 × 10^?3–10^?2 mol/l led to disturbances of neutrophil membrane barrier properties and to a lactate dehydrogenase release. Incubation of neutrophils with an addition of 10^?4–10^?7 mol/l H₂O₂ was accompanied by an increase of the cell ability to generate ROCS during phagocytosis and a decrease of neutrophil ability to secrete MPO and ROCS into the extracellular medium during adhesion. Mechanisms of the H₂O₂ action are coupled with arachidonic acid metabolism. Inhibition of the 5-lipoxygenase or cyclooxygenase metabolism pathways produced an enhancement of the H₂O₂ destructive effect. Block of 5-lipoxygenase pathway led to elimination of the H₂O₂ action on MPO and ROCS secretion and to an enhancement of the H₂O₂ effect on the neutrophil ability to generate ROCS during phagocytosis. The obtained data indicate a high blood neutrophil resistance to the H₂O₂ destructive action and confirm the H₂O₂ regulatory role with respect to the neutrophil functions.     

Formation and metabolism in vitro of 5,6-epoxides of cholesterol and beta-sitosterol

The formation of 5alpha,6alpha- and 5beta,6beta-epoxides of cholesterol and beta-sitosterol in rat liver subcellular fractions has been studied. The results show that the epoxidation seems to occur only in connection with the nonspecific tissue oxidation of the sterols. The beta-epoxides were formed in three- to fourfold excess over the alpha-epoxides. Both cholesterol epoxides were efficiently converted by a microsomal hydrolase into the 3beta,5alpha,6beta-triol. The conversion was less extensive with beta-sitosterol epoxides, especially the beta-epoxide. The possible biological significance in the formation of the sterol epoxides and the triols was evaluated by their ability to inhibit the microsomal cholesterol 7alpha-hydroxylase. Only the cholesterol epoxides and especially the beta-epoxide were active in this respect.     

Sema4C participates in myogenic differentiation in vivo and in vitro through the p38 MAPK pathway

Sema4C is a member of transmembrane semaphorin proteins which regulate axonal guidance in the developing nervous system. The expression of Sema4C was dramatically induced not only during differentiation of C2C12 mouse myoblasts, but also during injury-induced skeletal muscle regeneration. C2C12 cells stably or transiently expressing Sema4C both showed increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. Overexpression of Sema4C elicited p38 phosphorylation directly, and the effects of Sema4C during myogenic differentiation could be abolished by the p38alpha-specific inhibitor SB203580. Knockdown of Sema4C by siRNA transfection during C2C12 myoblasts differentiation could suppress the phosphorylation of p38 followed by dramatically diminished myotube formation. Sema4C could activate the myogenin promoter during myogenic differentiation. This activation could be abolished by p38 inhibitor SB203580. Taken together, these observations reveal novel functional potentialities of Sema4C which suggest that Sema4C promotes terminal myogenic differentiation in a p38 MAPK-dependent manner.     

Inhibition of Pyk2 blocks lung inflammation and injury in a mouse model of acute lung injury

Background

Proline-rich tyrosine kinase 2 (Pyk2) is essential in neutrophil degranulation and chemotaxis in vitro. However, its effect on the process of lung inflammation and edema formation during LPS induced acute lung injury (ALI) remains unknown. The goal of the present study was to determine the effect of inhibiting Pyk2 on LPS-induced acute lung inflammation and injury in vivo.

Methods

C57BL6 mice were given either 10 mg/kg LPS or saline intratracheally. Inhibition of Pyk2 was effected by intraperitoneal administration TAT-Pyk2-CT 1 h before challenge. Bronchoalveolar lavage analysis of cell counts, lung histology and protein concentration in BAL were analyzed at 18 h after LPS treatment. KC and MIP-2 concentrations in BAL were measured by a mouse cytokine multiplex kit. The static lung compliance was determined by pressure-volume curve using a computer-controlled small animal ventilator. The extravasated Evans blue concentration in lung homogenate was determined spectrophotometrically.

Results

Intratracheal instillation of LPS induced significant neutrophil infiltration into the lung interstitium and alveolar space, which was attenuated by pre-treatment with TAT-Pyk2-CT. TAT-Pyk2-CT pretreatment also attenuated 1) myeloperoxidase content in lung tissues, 2) vascular leakage as measured by Evans blue dye extravasation in the lungs and the increase in protein concentration in bronchoalveolar lavage, and 3) the decrease in lung compliance. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. By contrast, production of neutrophil chemokines MIP-2 and keratinocyte-derived chemokine in the bronchoalveolar lavage was not reduced by TAT-Pyk2-CT. Western blot analysis confirmed that tyrosine phosphorylation of Pyk2 in LPS-challenged lungs was reduced to control levels by TAT-Pyk2-CT pretreatment.

Conclusions

These results suggest that Pyk2 plays an important role in the development of acute lung injury in mice and that pharmacological inhibition of Pyk2 might provide a potential therapeutic strategy in the pretreatment for patients at imminent risk of developing acute lung injury.     

Surface-bound myeloperoxidase is a ligand for recognition of late apoptotic neutrophils by human lung surfactant proteins A and D

Surfactant proteins A (SP-A) and D (SP-D), both members of the collectin family, play a well established role in apoptotic cell recognition and clearance. Recent in vitro data show that SP-A and SP-D interact with apoptotic neutrophils in a distinct manner. SP-A and SP-D bind in a Ca²⁺-dependent manner to viable and early apoptotic neutrophils whereas the much greater interaction with late apoptotic neutrophils is Ca²⁺-independent. Cell surface molecules on the apoptotic target cells responsible for these interactions had not been identified and this study was done to find candidate target molecules. Myeloperoxidase (MPO), a specific intracellular defense molecule of neutrophils that becomes exposed on the outside of the cell upon apoptosis, was identified by affinity purification, mass-spectrometry and western blotting as a novel binding molecule for SP-A and SP-D. To confirm its role in recognition, it was shown that purified immobilised MPO binds SP-A and SP-D, and that MPO is surface-exposed on late apoptotic neutrophils. SP-A and SP-D inhibit binding of an anti-MPO monoclonal Ab to late apoptotic cells. Fluorescence microscopy confirmed that anti-MPO mAb and SP-A/SP-D colocalise on late apoptotic neutrophils. Desmoplakin was identified as a further potential ligand for SP-A, and neutrophil defensin as a target for both proteins.     

A fluorescent cholesterol analog traces cholesterol absorption in hamsters and is esterified in vivo and in vitro.

The fluorescent cholesterol analog 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (fluoresterol) was characterized as a tool for exploring the biochemistry and cell biology of intestinal cholesterol absorption. Hamsters absorbed fluoresterol in a concentration- and time-dependent manner, with an efficiency of about 15-30% that of cholesterol. Fluoresterol absorption was blocked by compounds known to inhibit cholesterol absorption, implying that fluoresterol interacts with those elements of the normal pathway for cholesterol absorption on which the inhibitors act. Confocal microscopy of small intestinal tissue demonstrated that fluoresterol was taken up by absorptive epithelial cells and packaged into lipoprotein particles, suggesting a normal route of intracellular trafficking. Uptake of fluoresterol was confirmed by biochemical analysis of intestinal tissue, and a comparison of [(3)H] cholesterol and fluoresterol content in the mucosa suggested that fluoresterol moved through the enterocytes more rapidly than did cholesterol. This interpretation was supported by measurements of fluoresterol esterification in the mucosa. Four hours after hamsters were given fluoresterol and [(3)H]cholesterol orally, 44% of the fluoresterol in the intestinal mucosa was esterified, compared to 8% of the [(3)H]cholesterol. Caco-2 cells took up 2- to 5-fold more [(3)H]cholesterol than fluoresterol from bile acid micelles, and esterified 21-24% of the fluoresterol but only 1-4% of the [(3)H]cholesterol. Thus fluoresterol apparently interacts with the proteins required for cholesterol uptake, trafficking, and processing in the small intestine.     

Effect of dietary cholesterol on erythrocyte peroxidant stress in vitro and in vivo

The effect of dietary variation of plasma cholesterol concentrations on the susceptibility of erythrocytes to in vitro and in vivo peroxidant stress was studied in rats. Malonyldialdehyde, produced in vivo (endogenous malonyldialdehyde) or following in vitro exposure of cells to 10 mM H2O2 (H2O2 malonyldialdehyde), was used as a measure of peroxidant stress. After 5 weeks, the plasma cholesterol concentrations in rats receiving 1.2% cholesterol + 0.6% cholic acid in their diet rose to 6-times that of control rats receiving a diet without added cholesterol; at the same time, erythrocyte H2O2 malonyldialdehyde in the cholesterol-fed rats decreased significantly relative to the control rats. During subsequent exposure of both groups to in vivo peroxidant stress with phenylhydrazine in two separate dose trials, erythrocyte peroxidant stress remained significantly lower in the cholesterol-fed rats: at a dose of 100 mumol/100 g body weight, H2O2 malonyldialdehyde was lower; at a dose of 25 mumol/100 g body weight, both endogenous and H2O2 malonyldialdehyde were lower. Erythrocyte membrane cholesterol concentrations were 12% higher in the cholesterol-fed rats than in controls. The effects of in vivo peroxidant stress on plasma cholesterol were also studied. In vivo peroxidant stress at the higher dose of phenylhydrazine produced a decrease in plasma cholesterol concentrations of control rats. The lower dose had no effect on this group and the plasma cholesterol concentrations were unchanged in the cholesterol-fed rats during both treatments. The data suggest that elevated plasma cholesterol concentrations are protective against erythrocyte peroxidant stress. The mechanism of cholesterol's protective effect is probably mediated through elevated membrane cholesterol concentrations.