Effects of PGI2 and analogues (taprostene, iloprost) on oxidation of native and glycated LDL.

Oxidation and glycation of low-density lipoprotein (LDL) has been claimed to play a central role in the pathogenesis of atherosclerosis. Therefore, the inhibition of this processes is of major therapeutic importance. In the present paper the influence of prostaglandin (PG)I2, and its stable analogues taprostene and iloprost on copper-induced oxidation of native, glycated and glycoxidated LDL was investigated. The results show, that the most pronounced effect on inhibition of native LDL-oxidation was obtained by taprostene in the whole concentration range tested (0.2 microg-10 microg/ml) reaching a maximal inhibition of 95% at 10 microg/ml. Examining glycoxidated LDL the inhibitory effect on oxidation was less pronounced reaching only about 10%. In case of glycated LDL, however, no significant inhibitory effect on oxidation was seen. Iloprost was effective as inhibitory agent against oxidation of native LDL at concentrations of 10 and 20 microg/ml, showing a maximal inhibition of 86% at a concentration of 20 microg/ml. Iloprost was ineffective on oxidation of glycated and glycoxidated LDL. Examining the extremely short-lived PGI2 itself, no significant inhibitory effect on oxidation of native, glycated or glycoxidated LDL, however, was seen. This finding might be of relevance for patients with diabetes mellitus, showing a decreased endogenous PGI2-production in particular those with bad metabolic control and high concentrations of circulating advanced glycosylation end products (AGEs).     

Troglitazone-binding to LDL and its glycated modifications: its role in cell-catalysed and Cu-mediated LDL-oxidation

Troglitazone (T), an anti-diabetic drug improving insulin resistance, was studied as to its inhibition of copper ion-catalysed oxidation of native, glycated and glycoxidated low-density lipoprotein (LDL). A dose-dependent inhibition was noted in the concentration range 40-160 microg/ml. An almost complete inhibition of oxidation (2-8 h), as monitored by the formation of thiobarbituric acid-reactive substances, was observed for both native and glycated LDL at a concentration of 160 microg/ml T, while the maximal inhibition for glycoxidated LDL amounted only to 60% at this concentration of the drug. This is reflected by differences in the affinity of the drug for the different types of LDL modification: While the binding of T both to native or glycated LDL increased linearly with increasing T concentration and was not saturable in the concentration range tested (0-160 microg/ml), binding of the drug to glycoxidated LDL was already nearly saturated at 10 microg/ml. The nearly complete inhibitory action of T towards oxidation of native and glycated LDL was lost, however, upon increasing the total oxidation time to 24 h. In human umbilical vein endothelial cell-mediated oxidation of LDL, T at a concentration of 20 microg/ml significantly reduced formation of oxidation-dependent fluorescent chromophores and liberation of 8-epi-PGF2alpha. In contrast, generation of thiobarbituric acid-reactive substances was not significantly inhibited. As opposed to copper-mediated LDL-oxidation, different binding of T to LDL-modifications does not govern inhibition of human umbilical vein endothelial cell-mediated LDL-oxidation.     

Why is glycated LDL more sensitive to oxidation than native LDL? A comparative study

It is well established that oxidative modification of low-density lipoprotein (LDL) plays a causal role in human atherogenesis and the risk of atherosclerosis is increased in patients with diabetes mellitus. To examine the influence of different agents which may influence LDL-glycation and oxidation, experiments including glycation with glucose, glucose 6-phosphate, metal chelators (EDTA) and antioxidants (BHT) were performed. The influence of time dependence on the glycation process and the alteration of the electrophoretic mobility of LDL under diverse glycation and/or oxidation conditions was also investigated. The formation of conjugated dienes and levels of lipid peroxides in these different LDL-modifications were estimated. The copper-induced oxidation of LDL in vitro was determined by measurement of thiobarbituric acid reactive substances (TBARS) and expressed as nmol MDA/mg of LDL protein. We found that glycated LDL is more prone to oxidation than native LDL. Using native LDL, the maximal oxidation effect was found to reach a value of 49.72 nmol MDA/mg protein after 8 h. The maximum oxidation of the 31 days, glycated LDL with glucose was 71.76 nmol MDA/mg protein amounting to 144.33% of the value found for native LDL. In the case of glucose 6-phosphate glycation, the maximum oxidation under the same conditions amounted to 173.77% of the value found for native LDL. To measure the extent of glycation, fluorescence of advanced glycation end products (AGEs) was determined (370 nm excitation and 440 nm emission). The most potent glycation agent was glucose 6-phosphate leading to the formation of very high amounts of AGEs. This process was promoted in the absence of EDTA, which prevents the oxidative cleavage of modified Amadori products (ketoamines) to AGEs. We therefore conclude that both processes, glycation and oxidation, result in the modification of LDL. The lower the glycation-rate (+/- EDTA) as measured by relative fluorescence units RFU (generation of AGEs), the lower the additional oxidation rate after glycation as measured by TBARS (generation of MDA equivalents). Glycation and/or oxidation change the electrophoretic mobility of LDL.     

Glycated phosphatidylethanolamine promotes macrophage uptake of low density lipoprotein and accumulation of cholesteryl esters and triacylglycerols.

Non-enzymatic glycation of low density lipoprotein (LDL) has been suggested to be responsible for the increase in susceptibility to atherogenesis of diabetic individuals. Although the association of lipid glycation with this process has been investigated, the effect of specific lipid glycation products on LDL metabolism has not been addressed. This study reports that glucosylated phosphatidylethanolamine (Glc-PtdEtn), the major LDL lipid glycation product, promotes LDL uptake and cholesteryl ester (CE) and triacylglycerol (TG) accumulation by THP-1 macrophages. Incubation of THP-1 macrophages at a concentration of 100 micrograms/ml protein LDL specifically enriched (10 nmol/mg LDL protein) with synthetically prepared Glc-PtdEtn resulted in a significant increase in CE and TG accumulation when compared with LDL enriched in non-glucosylated PtdEtn. After a 24-h incubation with LDL containing Glc-PtdEtn, the macrophages contained 2-fold higher CE (10.11 +/- 1.54 micrograms/mg cell protein) and TG (285.32 +/- 4.38 micrograms/mg cell protein) compared with LDL specifically enriched in non-glucosylated PtdEtn (CE, 3.97 +/- 0.95, p < 0.01 and TG, 185.57 +/- 3.58 micrograms/mg cell protein, p < 0.01). The corresponding values obtained with LDL containing glycated protein and lipid were similar to those of LDL containing Glc-PtdEtn (CE, 11.9 +/- 1.35 and TG, 280.78 +/- 3.98 micrograms/mg cell protein). The accumulation of both neutral lipids was further significantly increased by incubating the macrophages with Glc-PtdEtn LDL exposed to copper oxidation. By utilizing the fluorescent probe, 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI), a 1.6-fold increase was seen in Glc-PtdEtn + LDL uptake when compared with control LDL. Competition studies revealed that acetylated LDL is not a good competitor for DiI Glc-PtdEtn LDL (5-6% inhibition), whereas glycated LDL gave an 80% inhibition, and LDL + Glc-PtdEtn gave 93% inhibition of uptake by macrophages. These results indicate that glucosylation of PtdEtn in LDL accounts for the entire effect of LDL glycation on macrophage uptake and CE and TG accumulation and, therefore, the increased atherogenic potential of LDL in hyperglycemia.     

Evidence that lipid hydroperoxides inhibit plasma lecithin:cholesterol acyltransferase activity

The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. Recently, we found that polar lipids isolated from minimally oxidized LDL produced a dramatic inhibition of lecithin: cholesterol acyltransferase (LCAT) activity, suggesting that HDL-cholesterol transport may be impaired during early atherogenesis. In this study, we have identified molecular species of oxidized lipids that are potent inhibitors of LCAT activity. Treatment of LDL with soybean lipoxygenase generated small quantities of lipid hydroperoxides (20 +/- 4 nmol/mg LDL protein, n = 3); but when lipoxygenase-treated LDL (1 mg protein/ml) was recombined with the d > 1.063 g/ml fraction of human plasma, LCAT activity was rapidly inhibited (25 +/- 4 and 65 +/- 16% reductions by 1 and 3 h, respectively). As phospholipid hydroperoxides (PL-OOH) are the principal oxidation products associated with lipoxygenase-treated LDL, we directly tested whether PL-OOH inhibited plasma LCAT activity. Detailed dose-response curves revealed that as little as 0.2 and 1.0 mole % enrichment of plasma with PL-OOH produced 20 and 50% reductions in LCAT activity by 2 h, respectively. To gain insight into the mechanism of LCAT impairment, the enzyme's free cysteines (Cys31 and Cys184) and active site residues were "capped" with the reversible sulfhydryl compound, DTNB, during exposure to either minimally oxidized LDL or PL-OOH. Reversal of the DTNB "cap" after such exposures revealed that the enzyme was completely protected from both sources of peroxidized phospholipids. We, therefore, conclude that PL-OOH inhibited plasma LCAT activity by modifying the enzyme's free cysteine and/or catalytic residues. These studies are the first to suggest that PL-OOH may accelerate the atherogenic process by impairing LCAT activity.     

Low density lipoprotein (LDL) inhibits histamine release from human mast cells

We examined the effect of low density lipoprotein (LDL) on histamine release from purified human lung mast cells. LDL inhibited anti-IgE- induced histamine release in a dose-dependent manner, with 100 micrograms/ml LDL-protein inhibiting histamine release by 53 +/- 8% (mean +/- SEM); half-maximal inhibition occurred at 40-80 micrograms/ml. LDL also inhibited calcium ionophore A23187-induced histamine release in a dose-dependent manner, with 1 mg/ml of LDL inhibiting histamine release by 83 +/- 9%; half maximal inhibition occurred at 220-280 micrograms/ml. Inhibition by LDL was time-dependent: half-maximal inhibition of anti-IgE- induced histamine release by LDL occurred at 30-50 minutes of incubation. The inhibitory effect of LDL was independent of buffer calcium concentrations (0-5 mM) or temperature (0-37 degrees C). These data are consistent with a newly defined immunoregulatory role for LDL.     

Plasma thiols inhibit hemin-dependent oxidation of human low-density lipoprotein

Oxidative modification of human low-density lipoprotein (LDL) renders it atherogenic. Previous studies demonstrated that plasma thiols promote oxidation of LDL by free ferric iron (Fe3+). The current study investigated effects of plasma thiols on oxidation of LDL by hemin, a physiological Fe3+-protoporphyrin IX complex thought to be capable of initiating LDL oxidation in vivo. In contrast to free Fe3+ which is incapable of oxidizing LDL in the absence of an exogenous reductant, hemin readily promoted LDL oxidation. During incubation of LDL (0.2 mg of protein/ml) with hemin (10 microM) at 37 degrees C for 6 h, thiobarbituric acid-reactive substances (TBARS), a marker of lipid oxidation, increased from 0.3 (+/-0.1) nmol/mg of LDL protein to a maximal concentration of 45.8 (+/-5.2) nmol/mg of LDL protein. Under the same experimental conditions, lipid-conjugated dienes, another marker of lipid oxidation, increased from non-detectable to near-maximal levels of 78-187 nmol/mg of LDL protein, and lipoprotein polyunsaturated fatty acyl-containing cholesteryl ester content decreased to 15-36% of that present in native (i.e. unoxidized) LDL. Continued incubation of LDL with hemin for up to 24 h resulted in no further significant alterations in lipoprotein levels of TBARS, lipid-conjugated dienes, and cholesteryl esters. In addition to these chemical modifications indicative of lipoprotein oxidation, agarose gel electrophoretic analysis indicated that exposure of LDL to hemin resulted in conversion of the lipoprotein to an atherogenic form as evidenced by its increased anodic electrophoretic mobility. Addition of physiological concentrations of plasma thiols (either cysteine, homocysteine or reduced glutathione; 1-100 microM, each) inhibited hemin-mediated oxidation of LDL. Thus, whereas the maximal TBARS concentration was achieved following 6 h of incubation of LDL with hemin alone, addition of thiol extended the time required to attain maximal TBARS concentration to > or = 12 h. Similar antioxidant effects of thiols on formation of lipid-conjugated dienes, loss of cholesteryl esters, and lipoprotein anodic electrophoretic mobility were also observed. However, all thiols were not equally effective at inhibiting hemin-dependent LDL oxidation. Thus, whereas reduced glutathione was most effective at inhibiting hemin-dependent LDL oxidation, an intermediate effect was observed for homocysteine, and cysteine was least effective. The inhibition of hemin-mediated LDL oxidation by plasma thiols reported here confirms a previous observation that, under certain conditions, thiols can function as antioxidants, but contrasts with the previously documented pro-oxidant effect of the same thiols on oxidation of LDL by free Fe3+. These contrasting effects of plasma thiols on hemin- and free Fe3+-mediated LDL oxidation indicate that, in vivo, the ability of thiols to function as either anti- or pro-oxidants during LDL oxidation may, at least in part, be determined by the type of oxidant stress to which the lipoprotein is exposed.     

The Src/PLC/PKC/MEK/ERK signaling pathway is involved in aortic smooth muscle cell proliferation induced by glycated LDL

Low density lipoproteins (LDL) play important roles in the pathogenesis of atherosclerosis. Diabetes is associated with accelerated atherosclerosis leading to cardiovascular disease in diabetic patients. Although LDL stimulates the proliferation of arterial smooth muscle cells (SMC), the mechanisms are not fully understood. We examined the effects of native LDL and glycated LDL on the extracellular signal-regulated kinase (ERK) pathway. Addition of native and glycated LDL to rat aorta SMCs (RASMCs) stimulated ERK phosphorylation. ERK phosphorylation was not affected by exposure to the Ca2+ chelator BAPTA-AM but inhibition of protein kinase C (PKC) with GF109203X, inhibition of Src kinase with PP1 (5 microM) and inhibition of phospholipase C (PLC) with U73122/U73343 (5 microM) all reduced ERK phosphorylation in response to glycated LDL. In addition, pretreatment of the RASMCs with a cell-permeable mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059, 5 microM) markedly decreased ERK phosphorylation in response to native and glycated LDL. These findings indicate that ERK phosphorylation in response to glycated LDL involves the activation of PKC, PLC, and MEK, but is independent of intracellular Ca2+.     

The role of antioxidants in the long-term glycation of low density lipoprotein and its Cu2+-catalyzed oxidation

In the present study we investigated the influence of antioxidants such as EDTA, α-tocopherol, troglitazone and acetylsalicylic acid on the long-term-glycation of LDL and its copper ion-catalyzed oxidation. We observed that (a) all antioxidants inhibited AGE-formation, while Amadori product formation was only diminished by extreme concentrations of acetylsalicylic acid, (b) glycated LDL was more susceptible to coppercatalyzed oxidation than unglycated LDL, and (c) the oxidation of native LDL was more dramatically inhibited by the antioxidants than that of glycated LDL. The observed differences may be a consequence of the significantly higher endogenous content in hydroperoxides of glycated LDL as compared to native LDL. Therapeutic implications of these findings regarding vitamin E, which is supposed to slow atherogenesis and the development of microvascular complications in diabetes, are obvious: Vitamin E-monotherapy, while blocking oxidative and AGE-modification of LDL, is unable to inhibit its AP-formation. As a consequence, tocopherol is susceptible to increased consumption by AP-associated radical production in hyperglycemic patients, which could be checked in part by the tocopherol-protecting agent troglitazone and/or by acetylsalicylic acid.     

Ibuprofen protects low density lipoproteins against oxidative modification

Oxidative modification of LDL by vascular cells has been proposed as the mechanism by which LDL become atherogenic. The effect of ibuprofen on LDL modification by copper ions, monocytes and endothelial cells was studied by measuring lipid peroxidation products. Ibuprofen inhibited LDL oxidation in a dose-dependent manner over a concentration range of 0.1 to 2.0 mM. Ibuprofen (2 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides formed during 2 and 6 h incubation in the presence of copper ions by 52 and 28%, respectively. Weak free radical scavenging activity of ibuprofen was observed in the DPPH test. The protective effect of ibuprofen was more marked when oxidation was induced by monocytes or endothelial cells. Ibuprofen (1 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides generated in LDL during monocyte-mediated oxidation by 40%. HUVEC-mediated oxidation of LDL in the absence and presence of Cu2+ was reduced by 32 and 39%, respectively. More lipid peroxides appeared when endothelial cells were stimulated by IL-1beta or TNFalpha and the inhibitory effect of ibuprofen in this case was more pronounced. Ibuprofen (1 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides formed during incubation of LDL with IL-1beta-stimulated HUVEC by 43%. The figures in the absence and presence of Cu2+ for HUVEC stimulated with TNFalpha were 56 and 59%, respectively. To assess the possibility that ibuprofen acts by lowering the production rate of reactive oxygen species, the intracellular concentration of H2O2 was measured. Ibuprofen (1 mM) reduced intracellular production of hydrogen peroxide in PMA-stimulated mononuclear cells by 69%. When HUVEC were stimulated by IL-1beta or TNFalpha the reduction was 62% and 66%, respectively.     

The role of antioxidants in the long-term glycation of low density lipoprotein and its Cu2+-catalyzed oxidation

In the present study we investigated the influence of antioxidants such as EDTA, alpha-tocopherol, troglitazone and acetylsalicylic acid on the long-term-glycation of LDL and its copper ion-catalyzed oxidation. We observed that (a) all antioxidants inhibited AGE-formation, while Amadori product formation was only diminished by extreme concentrations of acetylsalicylic acid, (b) glycated LDL was more susceptible to copper-catalyzed oxidation than unglycated LDL, and (c) the oxidation of native LDL was more dramatically inhibited by the antioxidants than that of glycated LDL. The observed differences may be a consequence of the significantly higher endogenous content in hydroperoxides of glycated LDL as compared to native LDL. Therapeutic implications of these findings regarding vitamin E, which is supposed to slow atherogenesis and the development of microvascular complications in diabetes, are obvious: Vitamin E-monotherapy, while blocking oxidative and AGE-modification of LDL, is unable to inhibit its AP-formation. As a consequence, tocopherol is susceptible to increased consumption by AP-associated radical production in hyperglycemic patients, which could be checked in part by the tocopherol-protecting agent troglitazone and/or by acetylsalicylic acid.     

Lysophosphatidylcholine: essential role in the inhibition of endothelium-dependent vasorelaxation by oxidized low density lipoprotein

Endothelial cells are known to play an important role in the regulation of vascular tone. Here we demonstrate that modified low density lipoprotein (LDL) with copper oxidation or phospholipase A2 treatment elicits a potent inhibitory action on endothelium-dependent relaxations evoked by acetylcholine, although native LDL does not affect endothelium-dependent relaxations. Phosphatidylcholine of native LDL is converted to lysophosphatidylcholine during these modifications. Furthermore, lysophosphatidylcholine fraction separated from oxidized LDL (0.5mg.protein/ml) by thin layer chromatography abolished endothelium-dependent relaxations, although the remaining lipid fraction had little effects on endothelium-dependent relaxations. These results indicate that lysophosphatidylcholine is the principal substance for the impairment of endothelium-dependent relaxations by oxidized LDL and phospholipase A2 treated LDL.     

Three different pathways for human LDL oxidation are inhibited in vitro by water extracts of the medicinal herb Achyrocline satureoides

In this study we investigated the antioxidant properties of one herbal preparation widely used in complementary and alternative medicine in large areas of the world: Achyrocline satureoides (AS), popularly known as "marcela". Although rich in flavonoids, the ethnopharmacological uses of this plant do not include atherosclerosis prevention. Furthermore, no study had been conducted so far exploring the antioxidant activity of Achyrocline satureoides vis-à-vis human LDL oxidation, which is the compelling issue in pinpointing potential cardioprotective new uses for a traditional remedy. We explored the effects of AS extracts on human LDL oxidation, employing 3 different systems which are thought to play a role in oxidation of LDL in the arterial wall: copper, peroxynitrite, and lipoxygenase. Oxidation was monitored by conjugate dienes, TBARS formation and aggregation of apoB using SDS-PAGE. In copper-initiated oxidation a dose dependent inhibition of the initiation and propagation of lipid oxidation is shown by an increase in the lag phase for conjugate diene production which was 60 +/- 15 min in the absence and 120 +/- 20 min in the presence of 4 microg/ml AS extracts (p < 0.001). TBARS production was reduced by 95% after 3 h incubation at 5 microg/ml. Aggregation of apoB was abolished at the same concentrations. SIN-1 (3-morpholinosydnonimine) produces peroxynitrite via generation of NO and O2-. When LDL was incubated in its presence, a milder oxidation was observed as compared with Cu2+, and AS produced over 70% inhibition. Finally, we show a striking dose-dependent inhibitory effect of lipoxygenase conjugate diene production, which is over 95% at AS concentrations of 5 microg/ml. When compared with other antioxidants, AS effect is greater but in the same order of magnitude than that of ascorbic acid and similar to the popular herbal tea Ilex paraguariensis. In all three systems employed an effect is already substantiated at a concentration of the AS extract of 4 microg/ml, which corresponds to a 1/100 dilution of the preparations usually drunk.     

Suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and of incorporation of acetate into cholesterol in homozygous hypercholesterolemic fibroblasts by ferritin-low density lipoprotein conjugates.

Conjugates of ferritin with low density lipoproteins (LDL) were prepared and separated by sucrose gradient centrifugation. These conjugates, at cholesterol concentration of 100--132 microgram/ml, caused a greater than 90% suppression of hydroxymethylglutaryl coenzyme A reductase activity and of acetate incorporation into cholesterol in cultured skin fibroblasts from a normal subject as well as from a subject with homozygous familial hypercholesterolemia. The half maximal inhibition concentration was approx. 10 microgram/ml cholesterol for LDL and ferritin . (LDL)2 and 5 microgram/ml for (ferritin)2 . LDL in both cell lines. In contrast, native low density lipoproteins have only a minimal inhibitory effect in homozygous cells. The ability of the conjugates to stimulate the incorporation of oleate into cholesteryl esters was also equal in the two cell lines, although the conjugates were only 10% as active as low density lipoproteins in the normal cells. LDL reduced the ferritin . (LDL)2-mediated suppression of hydroxymethylglutaryl-CoA reductase activity in homozygous cells while ferritin . (LDL)2 reduced the LDL-mediated stimulation of cholesteryl ester formation in normal cells.     

Inhibition of human low density lipoprotein oxidation by active principles from spices

Spice components and their active principles are potential antioxidants. In this study we examined the effect of phenolic and non-phenolic active principles of common spices on copper ion-induced lipid peroxidation of human low density lipoprotein (LDL) by measuring the formation of thiobarbituric acid reactive substance (TBARS) and relative electrophoretic mobility (REM) of LDL on agarose gel. Curcurriin, capsaicin, quercetin, piperine, eugenol and allyl sulfide inhibited the formation of TBARS effectively through out the incubation period of 12 h and decreased the REM of LDL. Spice phenolic active principles viz. curcumin, quercetin and capsaicin at 10 M produced 40–85% inhibition of LDL oxidation at different time intervals while non-phenolic antioxidant allyl sulfide was less potent in inhibiting oxidation of LDL. However, allyl sulfide, eugenol and ascorbic acid showed pro-oxidant activity at lower concentrations (10 M) and antioxidant activity at higher concentrations (50 M) only. Among the spice principles tested quercetin and curcumin showed the highest inhibitory activity while piperine showed least antioxidant activity at equimolar concentration during initiation phase of oxidation of LDL. The inhibitory effect of curcumin, quercetin and capsaicin was comparable to that of BHA, but relatively more potent than ascorbic acid. Further, the effect of curcurnin, quercetin, capsaicin and BHA on initiation and propagation phases of LDL oxidation showed that curcurnin significantly inhibited both initiation and propagation phases of LDL oxidation, while quercetin was found to be ineffective at propagation phase. These data suggest that the above spice active principles, which constitute about 1–4% of above spices, are effective antioxidants and offer protection against oxidation of human LDL.     

Vitamin C protects low-density lipoprotein from homocysteine-mediated oxidation

Homocysteine, an atherogenic amino acid, promotes iron-dependent oxidation of low-density lipoprotein (LDL). We investigated whether vitamin C, a physiological antioxidant, could protect LDL from homocysteine-mediated oxidation. LDL (0.2 mg of protein/ml) was incubated at 37 degrees C with homocysteine (1000 microM) and ferric iron (10-100 microM) in either the absence (control) or presence of vitamin C (5-250 microM). Under these conditions, vitamin C protected LDL from oxidation as evidenced by an increased lag time preceding lipid diene formation (> or = 5 vs. 2.5 h for control), decreased thiobarbituric acid-reactive substances accumulation (< or = 19 +/- 1 nmol/mg when vitamin C > or = 10 microM vs. 32 +/- 3 nmol/mg for control, p <.01), and decreased lipoprotein anodic electrophoretic mobility. Near-maximal protection was observed at vitamin C concentrations similar to those in human blood (50-100 microM); also, some protection was observed even at low concentrations (5-10 microM). This effect resulted neither from altered iron redox chemistry nor enhanced recycling of vitamin E in LDL. Instead, similar to previous reports for copper-dependent LDL oxidation, we found that vitamin C protected LDL from homocysteine-mediated oxidation through covalent lipoprotein modification involving dehydroascorbic acid. Protection of LDL from homocysteine-mediated oxidation by vitamin C may have implications for the prevention of cardiovascular disease.     

Stepwise degradation of serum low denisty lipoprotein by sodium dodecyl sulfate.

The structure of human serum low density lipoprotein (LDL) was investigated by perturbing the LDL structure with sodium dodecyl sulfate (SDS). The change in LDL structure induced by the addition of SDS was monitored by sedimentation velocity measurements, ultraviolet difference spectroscopy, fluorescence spectroscopy and proteolytic digestion of apo-LDL with subtilisin BPN' [EC 3.4.21.14]. As the concentration of SDS was increased from 0.1 mg/ml to 3 mg/ml with LDL concentrations between 2.0 mg/ml and 4.4 mg/ml, the sedimentation coefficient of LDL changed in three distinct steps. It was found by chemical analyses that not more than 30% of the total lipid was lost from LDL in the second step, whereas the final step in the change of sedimentation coefficient corresponded to the complete removal of apo-LDL from the constituent lipids of LDL. The ultraviolet difference spectrum between the native and SDS-treated LDL and the quenching of LDL fluorescence underwent about 80% of the total change while the SDS concentration was only sufficient to cause the second of the three step changes in sedimentation coefficient. SDS-polyacrylamide gel electrophoresis of apo-LDL treated with subtilisin BPN' also showed that more than 70% of apo-LDL became susceptible to proteolysis under the same conditions. These results were interpreted as indicating that the solubilization of 20 to 30% of the lipids on the surface of LDL exposed nearly 80% or more of apo-LDL to the solvent. A small portion of apo-LDL was, however, still firmly anchored to the remaining lipid micelle as long as the concentration of SDS was less than that required to cause the final step of the change in sedimentation coefficient.     

Interactions betweenpanax quinquefoliumsaponis and vitamin C are observed in vitro

Inasmuch as the oxidation of low-density lipoprotein (Ox-LDL) may play a key role in the initiation and progression of atherosclerosis, it has become increasingly important to identify potential antioxidants. Panax quinquefolium saponins (PQS) are extracted from the stems and leaves of the North American form of ginseng,Panax quinquefolium. Our previous studies have indicated that PQS (0.25-1 mg/ml) can protect against oxidation of LDLin vitro. The purpose of the current work was to investigate the potential interaction of lower concentrations of PQS (1-100 g/ml) with vitamin C on the reduction of LDL oxidation. LDL was isolated from the plasma of healthy human donors by sequential ultracentrifugation. Native LDL (0.05 or 0.2 mg/ml) was incubated with PQS and/or vitamin C for 30 min at 20°C. Oxidative modification was initiated with 2 M or 5 M CuSO₄ at 37°C for 0-24 h. Pretreatment with PQS (100 g/ml) reduced alterations in phospholipids, lipid peroxide levels and relative electrophoretic mobility of Ox-LDL. The presence of vitamin C (1-10 M) significantly enhanced the protective effects of PQS. Pretreatment with PQS (1-100 g/ml) resulted in concentration-dependent inhibition of LDL oxidation and prolongation of lag time as determined from measurements of conjugated lipid hydroperoxide content in Ox-LDL samples. Interestingly, the inhibitory actions of lower amounts of PQS (1 and 10 g/ml) on the formation of conjugated dienes were significantly increased when vitamin C (0.1 or 1 M) was present. In conclusion, our results suggest that PQS not only have direct antioxidant property but at low concentrations, their actions can be enhanced by vitamin C.     

Factors Affecting Events During Oxidation of Low Density Lipoprotein: Correlation of Multiple Parameters of Oxidation

The present study shows that copper oxidation of LDL is a tightly-ordered process which can be finely controlled by appropriate selection of duration of oxidation and of concentrations of LDL and copper. Oxidation of LDL (0.1-2.0 mg LDL protein/ml) was carried out by copper catalysis (in the ratio of 2.5 μM Cu²⁺ to 0.1 mg LDL protein/ml) in phosphate-buffered saline, and was monitored by agarose gel electro-phoresis, gas chromatography (GC), anion exchange fast protein liquid chromatography (FPLC), fluorescence spectroscopy and dynamic light scattering. Analysis of the data showed strong cross correlations between many of the parameters of oxidation. Oxidation was more rapid for lower concentrations than for higher concentrations of LDL, despite the same ratio of copper to LDL being employed. Chemical kinetics analysis of the GC data suggested that 7β-hydroxycholesterol formation occurred as a first order (or pseudo first order) consecutive reaction to the oxidation of linoleate. The first order rate constants for decomposition of lioleate and production of 7β-hydroxycholesterol correlated closely with the theoretically-calculated times between collision of LDL particles. LDL particle diameter, measured by dynamic light scattering, increased by ca. 50% over 24 h oxidation, suggesting unfolding of apo B-100.

Prolonged oxidation of LDL at low concentration suggested that the radical chain reaction was able to propagate, albeit slowly, on cholesterol after all the polyunsaturated fatty acid was consumed. For higher concentrations of LDL, prolonged oxidation resulted in partial aggregation. These findings are applicable to preparing oxidised LDL with different degrees of oxidation, under controlled conditions, for studying its biological properties.     

Native low-density lipoproteins stimulate leukotriene B4 production by human monocyte-derived macrophages.

We have evaluated the effect of native low-density lipoproteins (LDL) on the production of leukotriene B4 (LTB4), a potent inflammatory and chemotactic factor, by human monocyte-derived macrophages. The capacity of LDL (d, 1.024-1.050 g/ml) to increase LTB4 secretion was dose-dependent with an optimal response at 100 micrograms LDL protein/ml, representing an approx. 7.5-fold stimulation over basal levels at 10 days of culture; the half-maximal response occurred at 20 micrograms/ml. The effect of LDL on LTB4 production was rapid (within 15 min) and was maintained for at least 21 h. The generation of LTB4 in response to LDL was partially inhibited (approx. 70% inhibition) by EDTA (5 mM) and by a monoclonal antibody (IgG-C7; 160 micrograms/ml) directed against the binding site of the cellular LDL receptor. In addition, the effects of native LDL and acetylated LDL were additive. These findings suggest that the specific interaction of LDL with its high affinity receptor represents a major component in the stimulation of the production of LTB4 by human monocyte-derived macrophages.