Role of group IIa and group V secretory phospholipases A(2) in the metabolism of lipoproteins. Substrate specificities of the enzymes and the regulation of their activities by sphingomyelin

Although many isoforms of secretory phospholipases A(2) (sPLA(2)) are known to be secreted by various inflammatory cells, and are present in plasma, their role in lipoprotein metabolism is unknown. We studied the in vitro hydrolysis of lipoprotein phospholipids by group IIa and group V sPLA(2), two structurally related enzymes with differing phospholipid specificities. The group V sPLA(2) was about 30 times more efficient than the group IIa enzyme in the hydrolysis of lipoprotein phosphatidylcholine (PC), and both enzymes were more active on high density liporotein (HDL) than on low density lipoprotein (LDL). The lower activity on LDL appears to be due to the higher sphingomyelin (SPH) concentration in this lipoprotein. PC hydrolysis in lipoproteins was stimulated significantly by enzymatic depletion of their SPH. The hydrolysis of PC in liposomes was inhibited by the incorporation of SPH, and this inhibition was reversed by treatment with sphingomyelinase. The incorporation of ceramide, on the other hand, stimulated the sPLA(2) activity significantly. Unlike most sPLA(2), which show no fatty acid preference, group V sPLA(2) released disproportionately more linoleate, and less arachidonate from lipoproteins. These studies show that group V sPLA(2) is physiologically more important than group IIa enzyme in lipoprotein metabolism, that the sPLA(2) activities are regulated by sphingomyelin and ceramide, and that the pathological effects of sPLA(2) may not be mediated through stimulation of eicosanoid synthesis.     

C-reactive protein inhibits in vitro oxidation of low-density lipoprotein

C-reactive protein (CRP) is elevated in cardiovascular disease and binds to oxidized phosphatidylcholine (oxPtC) in the low-density lipoprotein (LDL) surface. In the present study, we tested if CRP influences the susceptibility of LDL to oxidation. At physiological concentrations of 1-7mug/ml, CRP strongly inhibited copper-mediated oxidation of LDL and phospholipid liposomes in a concentration-dependent manner. Similar concentrations of different monoclonal antibodies or albumin did not influence LDL oxidation. Antioxidant activity of CRP was inhibited by phosphocholine (PC), indicating that the observed activity involves binding of CRP to oxPtC. These results suggest that CRP may limit atherogenic oxidation of LDL in vivo.     

Antioxidant treatment of diabetic rats inhibits lipoprotein oxidation and cytotoxicity

Increased lipid peroxidation products were detected in a lipoprotein fraction containing very low density lipoprotein (VLDL) and low density lipoprotein (LDL) obtained from rats made diabetic by streptozotocin injection. The enhanced oxidation in the diabetic VLDL plus LDL fraction correlated with the in vitro toxicity of this lipoprotein fraction to proliferating fibroblasts. In contrast, high density lipoprotein (HDL) was not cytotoxic. That the increased oxidation and development of cytotoxic activity in the diabetic VLDL + LDL was related to the diabetes was shown by the fact that insulin treatment of diabetic animals inhibited both oxidation and cytotoxicity of VLDL + LDL. In contrast, treatment of diabetic rats with the antioxidants vitamin E or probucol after diabetes was established also inhibited both the in vivo oxidation and in vitro cytotoxicity of diabetic VLDL + LDL, but without altering hyperglycemia. Vitamin E or probucol treatment thus allowed separation of the oxidation process from the hyperglycemia occurring in experimental diabetes. The mechanisms by which diabetes in humans or experimental animals leads to the various manifestations of tissue damage are unknown; however, these studies demonstrate for the first time that a relationship exists between the in vivo oxidation of lipoproteins in diabetes and the potential for tissue damage as monitored by in vitro cytotoxicity. Furthermore, these results suggest that the mechanism for certain aspects of tissue damage accompanying experimental diabetes may be mediated by lipid peroxidation products.     

Role of lipid peroxidation in the inhibition of mononuclear cell proliferation by normal lipoproteins

Stimulated peripheral blood mononuclear cells (PBMC) can oxidize normal lipoproteins, and sufficiently oxidized lipoproteins are cytotoxic. However, the role of lipid peroxidation in the inhibition of mitogen-stimulated PBMC proliferation by physiologic concentrations of normal lipoproteins is unclear. In the present investigation, normal low density lipoprotein (LDL) and very low density lipoprotein (VLDL) suppressed [3H]thymidine incorporation and gamma interferon production in concanavalin A-stimulated PBMC without causing cell death. This suppression was accompanied by parallel increases in lipid peroxidation products measured as thiobarbituric acid reactive substances (TBARS). In contrast, high density lipoprotein (HDL) failed to inhibit PBMC and TBARS remains low. Differences between the PBMC suppression from LDL, VLDL, and HDL were best accounted for by normalizing the lipoprotein concentrations by their total lipid content. Moreover, the antioxidants superoxide dismutase and butylated hydroxytoluene each substantially ameliorated the inhibition of PBMC caused by LDL, and reduced the levels of lipid peroxidation products that were generated. Altogether, these results suggest that reactive oxygen species generated by stimulated PMBC may cause oxidative alterations of normal lipoproteins that may, in turn, account for much of the previously reported inhibition of PBMC by normal lipoproteins.     

Concentration of neutral lipids in the phospholipid surface of substrate particles determines lipid transfer protein activity

To better understand the mechanism of lipid transfer protein (LTP) action and the effects of altered lipoprotein composition on its activity, we evaluated the dependence of LTP activity on the concentrations of cholesteryl ester (CE) and/or triglyceride (TG) in the phospholipid bilayer of substrate particles. Phosphatidylcholine (PC)-cholesterol liposomes containing up to 2 mole% TG and/or CE were prepared by cholate dialysis and used as either the donor of lipids to, or the acceptor of lipids from, low density lipoproteins (LDL). CE or TG transfer from liposomes of varying neutral lipid content to LDL showed saturation kinetics with an apparent Km of less than or equal to 0.2 mole%. Throughout this concentration-dependent response. PC transfer, which depended on the same LTP-donor particle binding interactions as those required for neutral lipid transfer, was essentially unchanged. Lipid transfer in the reverse direction (from LDL to liposomes of varying neutral lipid content) followed the same kinetics showing that transfer between the two particles is tightly coupled and bidirectional. When liposomes contained both TG and CE, these lipids competed for transfer in a manner analogous to that previously noted with lipoprotein substrates. In conclusion, CE and TG transfer activities are determined by the concentration of these lipids in the phospholipid surface of donor and acceptor particles. At low TG and CE concentrations, LTP bound to the liposome surface as indicated by PC transfer, but only a portion of these interactions actually facilitated a neutral lipid transfer event. Thus, the overall rate of neutral lipid transfer, and the competition between TG and CE for transfer, depend on the concentrations of these lipids in the phospholipid layer.     

Lipid fluidity at different regions in LDL and HDL of beta-thalassemia/Hb E patients

Atherosclerosis-related vascular complications in beta-thalassemia/hemoglobin E (beta-thal/Hb E) patients may result from iron induced oxidation of lipoproteins. To identify the specific site of oxidative damage, changes in lipid fluidity at different regions in LDL and HDL particle were investigated using two fluorescence probes and two ESR spin probes. The magnitude of increased lipid fluidity in thalassemic lipoproteins was dependent on the location of the probes. In hydrophobic region, the rotational correlation times for 16-doxyl stearic acid and DPH anisotropy were markedly changed in LDL and HDL of the patients. In the surface region, there was only a slight change in the order parameter (S) for 5-doxyl stearic acid and TMA-DPH anisotropy. Lipid fluidity at the core of LDL and HDL showed good correlation with oxidative stress markers, the ratio of CL/CO, and the level of alpha-tocopherol, suggesting that hydrophobic region of thalassemic lipoprotein was a target site for oxidative damage.     

Inhibition of lipid synthesis in isolated rat hepatocytes by serum lipoproteins

The incorporation of labeled acetate into lipids was studied in rat hepatocytes isolated after treatment of liver with collagenase and hyaluronidase. About 60% of the lipid radioactivity was in free cholesterol and 13% was in triglycerides. Acetate incorporation was markedly inhibited when human serum lipoproteins were present in the incubation medium. Very low, high, and low density lipoproteins, at concentrations of 1.0 mg/ml, inhibited acetate incorporation by 70, 55, and 35%, respectively. Chylomicrons, at similar concentrations, did not inhibit acetate incorporation. The distribution of radioactivity into lipid classes was unchanged by the addition of lipoproteins. Lipoproteins did not produce a nonspecific toxic effect on hepatocytes, since their addition did not alter the rate of leucine incorporation into protein. The addition of the delipidated protein from low density lipoprotein or of lecithin in amounts comparable to those present in inhibitory concentrations of lipoproteins failed to diminish acetate incorporation. Artificial cholesterol-lecithin emulsions containing small amounts of free cholesterol did not inhibit lipid synthesis. Although the mechanism for the inhibition of acetate incorporation by lipoproteins is unclear, such effects may play some physiological role in the control of lipid biosynthesis in the liver.     

Lipolytic degradation of human very low density lipoproteins by human milk lipoprotein lipase: the identification of lipoprotein B as the main lipoprotein degradation product

Although the direct conversion of very low density lipoproteins (VLDL) into low density (LDL) and high density (HDL) lipoproteins only requires lipoprotein lipase (LPL) as a catalyst and albumin as the fatty acid acceptor, the in vitro-formed LDL and HDL differ chemically from their native counterparts. To investigate the reason(s) for these differences, VLDL were treated with human milk LPL in the presence of albumin, and the LPL-generated LDL1-, LDL2-, and HDL-like particles were characterized by lipid and apolipoprotein composition. Results showed that the removal of apolipoproteins B, C, and E from VLDL was proportional to the degree of triglyceride hydrolysis with LDL2 particles as the major and LDL1 and HDL + VHDL particles as the minor products of a complete in vitro lipolysis of VLDL. In comparison with native counterparts, the in vitro-formed LDL2 and HDL + VHDL were characterized by lower levels of triglyceride and cholesterol ester and higher levels of free cholesterol and lipid phosphorus. The characterization of lipoprotein particles present in the in vitro-produced LDL2 showed that, as in plasma LDL2, lipoprotein B (LP-B) was the major apolipoprotein B-containing lipoprotein accounting for over 90% of the total apolipoprotein B. Other, minor species of apolipoprotein B-containing lipoproteins included LP-B:C-I:E and LP-B:C-I:C-II:C-III. The lipid composition of in vitro-formed LP-B closely resembled that of plasma LP-B. The major parts of apolipoproteins C and E present in VLDL were released to HDL + VHDL as simple, cholesterol/phospholipid-rich lipoproteins including LP-C-I, LP-C-II, LP-C-III, and LP-E. However, some of these same simple lipoprotein particles were present after ultracentrifugation in the LDL2 density segment because of their hydrated density and/or because they formed, in the absence of naturally occurring acceptors (LP-A-I:A-II), weak associations with LP-B. Thus, the presence of varying amounts of these cholesterol/phospholipid-rich lipoproteins in the in vitro-formed LDL2 appears to be the main reason for their compositional difference from native LDL2. These results demonstrate that the formation of LP-B as the major apolipoprotein B-containing product of VLDL lipolysis only requires LPL as a catalyst and albumin as the fatty acid acceptor. However, under physiological circumstances, other modulating agents are necessary to prevent the accumulation and interaction of phospholipid/cholesterol-rich apolipoprotein C- and E-containing particles.     

Trans unsaturated fatty acids are less oxidizable than cis unsaturated fatty acids and protect endogenous lipids from oxidation in lipoproteins and lipid bilayers

Epidemiological data suggest that dietary trans unsaturated fatty acids increase the risk of heart disease; however, the underlying mechanisms are unclear. In this study, we investigated one possible mechanism, namely, their effect on LDL oxidation. Supplementation of LDL with 10% 16:1 trans-cholesteryl ester (CE) inhibited the oxidation compared to that with 16:1 cis-CE. Total replacement of core lipids with 18:2 trans,trans-CE decreased the rate of LDL oxidation by 19% compared to replacement with 18:2 cis,cis-CE. When the surface phosphoglycerides were replaced with either 16:0-18:2 cis,cis-phosphatidylcholine (PC) or 16:0-18:2 trans,trans-PC, the latter was found to inhibit the rate and increase the lag time of oxidation to a greater extent than the former. To confirm these findings, we studied the oxidation of PC liposomes by assessing the formation of conjugated dienes or the degradation of a fluorescently labeled PC. By both methods, the 16:0-18:2 trans,trans-PC exhibited greater resistance to oxidation than the 16:0-18:2 cis,cis-PC. Eliminating the fluidity differences did not completely eliminate the differences in oxidation rates, suggesting that the trans double bond is inherently resistant to oxidation. The composition of the conjugated hydroperoxy products formed after oxidation differed markedly for the two 18:2 isomers. Supplementation of 16:0-18:2 cis,cis-PC liposomes with 20 mol % di16:1 trans-PC retarded oxidation rates to a greater extent than supplementation with di16:1 cis-PC. These studies show that dietary trans unsaturated fatty acids decrease the rate of lipid peroxidation, an effect that may mitigate the atherogenic effect of these fatty acids.     

Protective effects of endomorphins, endogenous opioid peptides in the brain, on human low density lipoprotein oxidation

Neurodegenerative disorders are associated with oxidative stress. Low density lipoprotein (LDL) exists in the brain and is especially sensitive to oxidative damage. Oxidative modification of LDL has been implicated in the pathogenesis of neurodegenerative diseases. Therefore, protecting LDL from oxidation may be essential in the brain. The antioxidative effects of endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid peptides in the brain, on LDL oxidation has been investigated in vitro. The peroxidation was initiated by either copper ions or a water-soluble initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). Oxidation of the LDL lipid moiety was monitored by measuring conjugated dienes, thiobarbituric acid reactive substances, and the relative electrophoretic mobility. Low density lipoprotein oxidative modifications were assessed by evaluating apoB carbonylation and fragmentation. Endomorphins markedly and in a concentration-dependent manner inhibited Cu2+ and AAPH induced the oxidation of LDL, due to the free radical scavenging effects of endomorphins. In all assay systems, EM1 was more potent than EM2 and l-glutathione, a major intracellular water-soluble antioxidant. We propose that endomorphins provide protection against free radical-induced neurodegenerative disorders.     

Apolipoprotein B stimulates formation of monocyte-macrophage surface-connected compartments and mediates uptake of low density lipoprotein-derived liposomes into these compartments

Much of the cholesterol that accumulates in atherosclerotic plaques is found within monocyte-macrophages transforming these cells into "foam cells." Native low density lipoprotein (LDL) does not cause foam cell formation. Treatment of LDL with cholesterol esterase converts LDL into cholesterol-rich liposomes having >90% cholesterol in unesterified form. Similar cholesterol-rich liposomes are found in early developing atherosclerotic plaques surrounding foam cells. We now show that cholesterol-rich liposomes produced from cholesterol esterase-treated LDL can cause human monocyte-macrophage foam cell formation inducing a 3-5-fold increase in macrophage cholesterol content of which >60% is esterified. Although cytochalasin D inhibited LDL liposome-induced macrophage cholesteryl ester accumulation, LDL liposomes did not enter macrophages by phagocytosis. Rather, the LDL liposomes induced and entered surface-connected compartments within the macrophages, a unique endocytic pathway in these cells that we call patocytosis. LDL liposome apoB rather than LDL liposome lipid mediated LDL liposome uptake by macrophages. This was shown by the findings that: 1) protease treatment of the LDL liposomes prevented macrophage cholesterol accumulation; 2) liposomes prepared from LDL lipid extracts did not cause macrophage cholesterol accumulation; and 3) purified apoB induced and accumulated within macrophage surface-connected compartments. Although apoB mediated the macrophage uptake of LDL liposomes, this uptake did not occur through LDL, LDL receptor-related protein, or scavenger receptors. Also, LDL liposome uptake was not sensitive to treatment of macrophages with trypsin or heparinase. Cholesterol esterase-mediated transformation of LDL into cholesterol-rich liposomes is an LDL modification that: 1) stimulates uptake of LDL cholesterol by apoB-dependent endocytosis into surface-connected compartments, and 2) causes human monocyte-macrophage foam cell formation.     

Effect of feed deprivation on hepatic membrane and lipoprotein fluidity and binding of lipoproteins to hepatic membranes in the chick (Gallus domesticus)

1. Male chicks were deprived of feed for 48 hr to study the effect of metabolic stress on hepatic membrane and lipoprotein fluidity and binding of radioiodinated lipoproteins to hepatic membranes. 2. Plasma levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) were markedly and slightly elevated, respectively. 3. There was a reduction in lipoprotein and hepatic membrane fluidity. 4. Binding of [125I]LDL, but not [125I]HDL, to hepatic membranes was decreased. 5. It is suggested that a reduction in the fluidity of LDL and/or hepatic membranes impedes LDL catabolism in vivo.     

The specificity of lipoxygenase-catalyzed lipid peroxidation and the effects of radical-scavenging antioxidants

The oxidation of low density lipoprotein (LDL) by lipoxygenase has been implicated in the pathogenesis of atherosclerosis. It has been known that lipoxygenase-mediated lipid peroxidation proceeds in general via regio-, stereo- and enantio-specific mechanisms, but that it is sometimes accompanied by a share of random hydroperoxides as side reaction products. In this study we investigated the oxidation of various substrates (linoleic acid, methyl linoleate, phosphatidylcholine, isolated LDL, and human plasma) by the arachidonate 15-lipoxygenases from rabbit reticulocytes and soybeans aiming at elucidating the effects of substrate, lipoxygenase and reaction milieu on the contribution and mechanism of random oxidation and also the effect of antioxidant. The specific character of the rabbit 15-lipoxygenase reaction was confirmed under all conditions employed here. However, the specificity by soybean lipoxygenase was markedly dependent on the conditions. When phosphatidylcholine liposomes and LDL were oxygenated by soybean lipoxygenase, the product pattern was found to be exclusively regio-, stereo-, and enantio-random. When free linoleic acid was incorporated into PC liposomes and oxidized by soybean lipoxygenase, the free acid was specifically oxygenated, whereas esterified linoleate gave random oxidation products exclusively. Radical-scavenging antioxidants such as alpha-tocopherol, ascorbic acid and 2-carboxy-2,5,7,8-tetramethyl-6-chromanol selectively inhibited the random oxidation but did not influence specific product formation. It is assumed that the random reaction products originate from free radical intermediates, which have escaped the active site of the enzyme and thus may be accessible to radical scavengers. These data indicate that the specificity of lipoxygenase-catalyzed lipid oxidation and the inhibitory effects of antioxidants depend on the physico-chemical state of the substrate and type of lipoxygenase and that they may change completely depending on the conditions.     

Effects of oxidation on the structure and stability of human low-density lipoprotein

Oxidation of low-density lipoprotein (LDL), the major cholesterol carrier in plasma, is thought to promote atherogenesis via several mechanisms. One proposed mechanism involves fusion of oxidized LDL in the arterial wall; another involves oxidation-induced amyloid formation by LDL apolipoprotein B. To test these mechanisms and to determine the effects of oxidation on the protein secondary structure and lipoprotein fusion in vitro, we analyzed LDL oxidized by nonenzymatic (Cu2+, H2O2, and HOCl) or enzymatic methods (myeloperoxidase/H2O2/Cl- and myeloperoxidase/H2O2/NO2-). Far-UV circular dichroism spectra showed that LDL oxidation induces partial unfolding of the secondary structure rather than folding into cross-beta amyloid conformation. This unfolding correlates with increased negative charge of oxidized LDL and with a moderate increase in thioflavin T fluorescence that may result from electrostatic attraction between the cationic dye and electronegative LDL rather than from dye binding to amyloid. These and other spectroscopic studies of low- and high-density lipoproteins, which encompass amyloid-promoting conditions (high protein concentrations, high temperatures, acidic pH), demonstrate that in vitro lipoprotein oxidation does not induce amyloid formation. Surprisingly, turbidity, near-UV circular dichroism, and electron microscopic data demonstrate that advanced oxidation inhibits heat-induced LDL fusion that is characteristic of native lipoproteins. Such fusion inhibition may result from the accumulation of anionic lipids and lysophospholipids on the particle surface and/or from protein cross-linking upon advanced lipoprotein oxidation. Consequently, oxidation alone may prevent rather than promote LDL fusion, suggesting that additional factors, such as albumin-mediated removal of lipid peroxidation products and/or LDL binding to arterial proteoglycans, facilitate fusion of oxidized LDL in vivo.     

Metabolism of oxidized phosphatidylcholines formed in oxidized low density lipoprotein by lecithin-cholesterol acyltransferase.

The possible involvement of lecithin-cholesterol acyltransferase (LCAT) in the metabolism of oxidized phosphatidylcholine (PC) in plasma was investigated. A variety of oxidized products are formed from PC following oxidation of low density lipoproteins (LDL). A significant increase in LDL oxidation levels in patients with familial LCAT deficiency (FLD) has been previously demonstrated by a sensitive sandwich ELISA for oxidized LDL using the monoclonal antibody DLH3 which recognizes oxidized products of PC. In the present study, we found that LCAT produces various metabolites from oxidized PC and that oxidized PC molecules in LDL particles serve as substrates. When the neutral lipid fraction was separated by TLC after the incubation of oxidized 1-palmitoyl-2-[1-14C]linoleoyl PC with human plasma, a number of radioactive bands were formed in addition to cholesteryl ester. These products were not formed from native 1-palmitoyl-2-[1-14C]linoleoyl PC. Plasma from FLD patients also failed to form the additional products from oxidized PC. The addition of dithio-bis(nitrobenzoate) (DTNB), an LCAT inhibitor, or the inactivation of LCAT activity by treating the plasma at 56 degrees C for 30 min abolished the generation of these products from oxidized PC. The activity was recovered in the high density lipoprotein (HDL) fraction but not in the LDL fraction separated from normal plasma. When 1-palmitoyl-2-[1-14C](9-oxononanoyl) PC and 1-stearoyl-2-[1-14C](5-oxovaleroyl)PC, PC oxidation products that contain short chain aldehydes, were incubated with human plasma, radioactive products in the neutral lipid fraction were observed on TLC. LDL containing oxidized PC was measured by sandwich ELISA using an anti-apolipoprotein B antibody and DLH3. The reconstituted oxidized PC-LDL particles were found to have lost their ability to bind DLH3 upon incubation with HDL, while the reactivity of the reconstituted oxidized PC-LDL remained unchanged in the presence of DTNB. These results suggest that LCAT is capable of metabolizing a variety of oxidized products of PC and preventing the accumulation of oxidized PC in circulating LDL particles.     

Sphingomyelinase activity associated with human plasma low density lipoprotein

Isolated human plasma low density lipoprotein (LDL) was observed to possess sphingomyelinase activity. Accordingly, the formation of ceramide was catalyzed by LDL at 37 degrees C using tertiary liposomes composed of sphingomyelin (mole fraction (x) = 0.2), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (x = 0.7), 1, 2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (x = 0.1), and either the fluorescent sphingomyelin analog Bodipy-sphingomyelin or [(14)C]sphingomyelin as substrates. However, this activity was not present in either very low density lipoprotein or the high density lipoprotein subfractions HDL(2) and HDL(3). Oxidation of LDL abrogated its sphingomyelinase activity. Aggregation of the liposomes upon incubation with LDL was evident from the light scattering measurements. Microinjection of LDL to the surface of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-d-sphingomyelin (C16:0-sphingomyelin), and Bodipy-sphingomyelin as a fluorescent tracer (0.75:- 0.20:0.05, respectively) revealed the induction of vectorial budding of vesicles, resembling endocytosis.     

Molecular action of vitamin E in lipoprotein oxidation: implications for atherosclerosis

The oxidation theory of atherosclerosis proposes that the oxidative modification of low-density lipoproteins (LDL) plays a central role in the disease. Although a direct causative role of LDL oxidation for atherogenesis has not been established, oxidized lipoproteins are detected in atherosclerotic lesions, and in vitro oxidized LDL exhibits putative pro-atherogenic activities. alpha-Tocopherol (alpha-TOH; vitamin E), the major lipid-soluble antioxidant present in lipoproteins, is thought to be antiatherogenic. However, results of vitamin E interventions on atherosclerosis in experimental animals and cardiovascular disease in humans have been inconclusive. Also, recent mechanistic studies demonstrate that the role of alpha-TOH during the early stages of lipoprotein lipid peroxidation is complex and that the vitamin does not act as a chain-breaking antioxidant. In the absence of co-antioxidants, compounds capable of reducing the alpha-TOH radical and exporting the radical from the lipoprotein particle, alpha-TOH exhibits anti- or pro-oxidant activity for lipoprotein lipids depending on the degree of radical flux and reactivity of the oxidant. The model of tocopherol-mediated peroxidation (TMP) explains the complex molecular action of alpha-TOH during lipoprotein lipid peroxidation and antioxidation. This article outlines the salient features of TMP, comments on whether TMP is relevant for in vivo lipoprotein lipid oxidation, and discusses how co-antioxidants may be required to attenuate lipoprotein lipid oxidation in vivo and perhaps atherosclerosis.     

Oxidation of cholesterol in low density and high density lipoproteins by cholesterol oxidase

The cholesterol oxidase-catalyzed oxidation of cholesterol in native low density (LDL) and high density lipoproteins (HDL3) as well as in monolayers prepared from surface lipids of these particles, has been examined. The objective of the study was to compare the oxidizability of cholesterol, and to examine the effects of lipid packing on oxidation rates. When [3H]cholesterol-labeled lipoproteins were exposed to cholesterol oxidase (Streptomyces sp.), it was observed that LDL [3H]cholesterol was oxidized much faster than HDL3 [3H]cholesterol. This was true both at equal cholesterol concentration per enzyme unit, and at equal amounts of lipoprotein particles per enzyme unit. About 95% of lipoprotein [3H]cholesterol was available for oxidation. The complete degradation of lipoprotein sphingomyelin by sphingomyelinase (Staphylococcus aureus) resulted in a 10-fold increase in the rate of LDL [3H]cholesterol oxidation, whereas the effects on rates of HDL3 [3H]cholesterol oxidation were less dramatic. A monolayer study with LDL surface lipids indicated that degradation of sphingomyelin loosened the lipid packing, because the ceramide formed occupied a smaller surface area than the parent sphingomyelin, and since the condensing effect of cholesterol on sphingomyelin packing was lost. The effects of sphingomyelin degradation on lipid packing in monolayers of HDL3-derived surface lipids were difficult to determine from monolayer experiments. Based on the finding that cholesterol oxidases are surface pressure-sensitive with regard to their catalytic activity, these were used to estimate the surface pressure of intact LDL and HDL3. The cut-off surface pressure of a Brevibacterium enzyme was 25 mN/m and 20 mN/m in monolayers of LDL and HDL3-derived surface lipids, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Implications of Lag Time Concept in the Oxidation of LDL

Oxidation of low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. The most common technique for measuring the oxidation of lipoproteins is the continuos measurement of the formation of conjugated diene at OD 234 nm. The concept of “lag time”, derived from such measurements, has been used to test the efficacy of various antioxidants for their ability to inhibit the oxidation of LDL. This review will elaborate on some of the factors that might affect the lag time.     

Reconstitution of sodium channels in large liposomes formed by the addition of acidic phospholipids and freeze-thaw sonication

Phosphatidylcholine (PC) alone or with phosphatidylethanolamine (PE) are sufficient for the reconstitution of Na+ channels in planar lipid bilayers. However, when Na+ channels were first reconstituted into liposomes using the freeze-thaw-sonication method, addition of acidic phospholipids, such as phosphatidylserine (PS), to the neutral phospholipids was necessary to obtain a significant toxin-modulated 22Na uptake. To further investigate the acidic phospholipid effect on reconstitution into liposomes, Na+ channels purified from Electrophorus electricus electrocytes were reconstituted into liposomes of different composition by freeze-thaw sonication and the effect of batrachotoxin and tetrodotoxin on the 22Na flux was measured. The results revealed that, under our experimental conditions, the presence of an acidic phospholipid was also necessary to obtain a significant neurotoxin-modulated 22Na influx. Though neurotoxin-modulated 22Na fluxes have been reported in proteoliposomes made with purified Na+ channels and PC alone, the 22Na fluxes were smaller than those found using lipid mixtures containing acidic phospholipids. Electron microscopy of negatively stained proteoliposomes prepared with PC, PC/PS (1:1 molar ratio), and PS revealed that the acidic phospholipid increases the size of the reconstituted proteoliposomes. The increment in size caused by the acidic phospholipid, due to the associated increase in internal volume for 22Na uptake and in area for Na+ channel incorporation, appears to be responsible for the large neurotoxin-modulated 22Na fluxes observed.