Electrophoretic Mobility Of Low-density Lipoprotein Research Articles

숙성 마늘 유래 S-allylmercaptocysteine의 human low density lipoprotein (LDL)에 대한 항산화 효과

Oxidation of low density lipoprotein (LDL) plays an important role in the development and progression of atherosclerotic disease. In this study, human LDL was isolated and oxidized using CuSO4 in the presence or absence of S-allylmercaptocysteine. Oxidative modification of the LDL fraction was monitored by both the appearance of thiobarbituric acid substances (TBARS), an increase in electrophoretic mobility, and conjugated diene formation. The addition of S-allylmercaptocysteine reduced lipid peroxide formation, indicating it to be an effective antioxidant. The inhibition of LDL oxidation by 5~20 μg/ml S-allylmercaptocysteine occurred in a dose-dependent manner, as assessed by the TBARS assay. S-allylmercaptocysteine at 20 μg/ml almost completely inhibited the Cu2+ induced increases in electrophoretic mobility of LDL and almost completely inhibited conjugated diene ormation. A more potent antioxidative activity was observed for S-allylmercaptocysteine than for either Vitamin C or ㎗-α-tocopherol. Thus, S-allylmercaptocysteine aid in preventing the development and progression of atherosclerotic disease.

Photochemistry of Bacteriochlorophylls in Human Blood Plasma: 1. Pigment Stability and Light‐induced Modifications of Lipoproteins

Transmetalated derivatives of bacteriochlorophyll are promising sensitizers in photodynamic therapy. Protocols using short delay times between injection and irradiation cause interest in the photochemistry of these pigments in the blood. Using near-infrared irradiation where these pigments absorb strongly, we have studied the photochemistry of Zn- and Pd-bacteriopheophorbide (WST09), and of the highly polar taurinated Pd-derivative, WST11, in isolated fractions of human blood plasma. The stability of all pigments is increased in blood plasma, compared with monomeric solutions. Pd-bacteriopheophorbide is much more stable than the other two derivatives. It also has a higher capacity for inducing reactive oxygen species, yet the consumption of oxygen is comparable. There is furthermore evidence for photobleaching under anoxic conditions. The generation of hydroperoxides (ROOH) is faster with Pd- than with Zn-complexes; the formation of endoperoxides (ROOR'), measured as thiobarbituric acid reactive substances, is comparable with the two central metals. Formation of both ROOH and ROOR' is increased in low-density lipoproteins (LDL) compared with high-density lipoproteins (HDL), which is probably related to the higher concentration of target molecules in the former. In HDL, extensive cross-linking is induced among the apolipoproteins; judged from the electrophoretic mobility of LDL and HDL particles, there is also a gross structural change. Photosensitized cross-linking is much less pronounced with high-density proteins.

Tiliroside and gnaphaliin inhibit human low density lipoprotein oxidation

Two flavonoids, gnaphaliin and tiliroside, isolated from Helichrysum italicum, were studied in vitro for their capacity to inhibit Cu 2+-induced human low density lipoprotein (LDL) and diluted plasma oxidation. LDL oxidation was monitored by conjugated diene, thiobarbituric acid-reactive substances (TBARS) formation and electrophoretic mobility on agarose gel. Gnaphaliin and tiliroside increased the lag-phase for diene conjugate production in a dose-dependent manner. The reduction of TBARS production confirmed the antioxidant activity of gnaphaliin and tiliroside with 50% inhibitory concentration (IC 50) values of 8.0 ± 3.9 μM and 7.0 ± 2.6 μM respectively. Furthermore, the flavonoids negated the Cu 2+-induced increase in electrophoretic mobility of LDL. Antioxidant activity of gnaphaliin and tiliroside was significantly different when diluted plasma was oxidised by adding 1 mM CuSO 4. Although both flavonoids again reduced the TBARS production, tiliroside showed higher activity than gnaphaliin (IC 50 = 10.6 ± 2.5 μM vs. IC 50 > 50 μM). In conclusion, tiliroside and gnaphaliin are antioxidants against in vitro Cu 2+-induced LDL oxidation in the same order of magnitude compared to that of the reference drug, probucol.

Glucose-Induced Enhancement of Hemin-Catalyzed LDL OxidationIn VitroandIn Vivo

Growing evidence indicates that oxidative modification of low-density lipoprotein (LDL) is increased in diabetes mellitus; however, the mechanism(s) of this phenomenon is still unclear. gamma-Glutamyl semialdehyde (gammaGSA) is a product of hemin (Fe(3+)-protoporphyrin IX)-catalyzed oxidation of apolipoprotein B-100 (apoB- 100) proline and arginine residues. On reduction, gammaGSA forms 5-hydroxy-2-aminovaleric acid (HAVA). This report describes the application of sensitive HAVA assay, to characterize gammaGSA formation in LDL under normo- and hyperglycemic conditions, both in vitro and in vivo. In vitro studies revealed that apoB-100 proline and arginine residues are not oxidized to HAVA by HOCl or the myeloperoxidase/hydrogen peroxide (H(2)O(2)) oxidation system. Cu(2+), Cu(2+)/H(2)O(2), and Fe(2+) induced only minor HAVA formation. In contrast, the hemin oxidation system appeared reactive toward LDL apoB-100 proline and arginine residues. The resulting significant HAVA formation was specifically inhibited by a redox-inert ferric iron chelator. Glucose further enhanced hemin-induced increase in relative electrophoretic mobility of LDL and apoB-100 HAVAformation. In vivo we observed elevated concentrations of HAVA in LDL apoB-100 in patients with impaired glucose tolerance and with manifest diabetes mellitus. In conclusion, glucose promotes iron-mediated oxidation of apoB- 100 proline and arginine residues via a superoxide-dependent mechanism, thus rendering the LDL particles more atherogenic. The findings (a) identify a potential mechanism of enhanced atherogenesis in subjects with diabetes mellitus and (b) support the value of HAVA as a specific marker of LDL apoB-100 oxidation. Antioxid. Redox Signal. 7, 1507-1512.

Antioxidant activity of piperlactam S: prevention of copper-induced LDL peroxidation and amelioration of free radical-induced oxidative stress of endothelial cells.

The protective effects of piperlactam S, an alkaloid isolated from Piper kadsura (Choisy) Ohwi, on lipid peroxidation and free radical-mediated cell injuries were investigated. Piperlactam S (1 to 20 microM) concentration-dependently prevented the copper-catalyzed oxidative modification of human low-density lipoproteins (LDL) measured through (i) the lag period, (ii) the slope of the propagation phase, (iii) the total amount of conjugated dienes formed, and (iv) the electrophoretic mobility of LDL. Fe2+-induced oxidative modification of cell membrane was also significantly attenuated by piperlactam S as measured by thiobarbituric acid-reactive substances (TBARS). Furthermore, piperlactam S effectively minimized the loss of cell viability induced by Fenton's reagent (H2O2/FeSO4) in cultured endothelial cells and significantly reversed H2O2/FeSO4-induced impairment of endothelium-dependent relaxation to acetylcholine in rat aorta. Since the oxidative modification of LDL plays an important role in the genesis of atherosclerosis, piperlactam S may help to reduce the risk of atherosclerosis, not only by protecting LDL and membrane lipids from oxidative modification but also by reducing free radical-induced endothelial injury and/or dysfunction.

Extracts of Ginkgo biloba L. leaves and Vaccinium myrtillus L. fruits prevent photo induced oxidation of low density lipoprotein cholesterol

Oxidation of low density lipoproteins (LDL) favours cholesterol loading in macrophages and formation of "foam cells", typical of the early atheroma lesions. LDL cholesterol oxidation generates oxysterols, extremely cytotoxic molecular species with diverse biological activities. Vegetable polyphenols are dietary components of pharmacological interest for their anti-oxidant properties. Ginkgo biloba L. (Gingkoaceae) leaves and Vaccinium myrtillus L. (Ericaceae) fruits are known for their beneficial effects in the treatment of various diseases involving free radicals and oxidative damage to biological lipids. In this study we investigated the effect of Ginkgo biloba L. and Vaccinium myrtillus L. extracts on the formation of cholesterol oxides during the photo induced oxidation of human LDL. The results demonstrate a concentration dependent inhibition of oxysterol formation in the presence of both extracts. Protection against oxidation was confirmed by the partial restoration of the normal electrophoretic mobility of LDL, which has been influenced by the UV irradiation. These effects extend knowledge of the therapeutic action of Ginkgo biloba L. and Vaccinium myrtillus L. as agents in anti-atherosclerotic regimens.

Susceptibility of plasma to ferrous iron/hydrogen peroxide-mediated oxidation: demonstration of a possible Fenton reaction

The aim of our study was to evaluate a model system by using iron in the peroxidation of plasma. Lipid peroxidation was monitored by fluorometric measurement of lipid peroxides (LPO). Plasma coincubated with Fe2+ and H2O2 had a 268% increase in plasma LPO after 1 h. The optimum concentrations were 0.42 mmol/L Fe2+ and 0.73 mol/L H2O2. Coincubation of plasma with these concentrations of Fe2+ and H2O2 separately resulted in no increase in plasma LPO. The increase in plasma LPO after oxidation with Fe2+/H2O2 was paralleled by a decrease in plasma polyunsaturated fatty acids, an increase in the relative electrophoretic mobility of low-density lipoprotein (LDL), and decreases in apolipoprotein (apo) B-100 and apo A-I immunoreactivity. In vitro oxidation of LDL and high-density lipoprotein separately with this system produced increases of LPO of 246% and 128%, respectively. LPO formation in plasma was inhibited by catalase, desferrioxamine, and mannitol, but not by superoxide dismutase. Hydroxyl radical generation with Fe2+/H2O2 was evidenced by fragmentation of deoxyribose. We conclude that the Fe2+/H2O2 system, possibly by a Fenton reaction mechanism, resulted in significant plasma oxidation. This model system may be useful for examining lipid peroxidation in clinical investigations.

Grape extract inhibits lipid peroxidation of human low density lipoprotein.

Some epidemiological data have linked dietary polyphenols with lower risk of coronary heart disease. Polyphenols might impair lipoprotein oxidation which is believed to be an important step in initiating atherogenesis. The purpose of this study was to determine if grape extract known to contain polyphenolic substances can block copper-induced oxidative modification of human low density lipoprotein (LDL).LDL oxidation was monitored spectrophotometrically by measurement of change in absorbance at 234 nm. Incubation of LDL (0.05 mg protein/ml) with 1.66 microM cupric chloride produced a lag phase of 130 min before onset of the propagation phase where polyunsaturated fatty acids undergo conversion to conjugated lipid hydroperoxides. However, in the presence of grape extract at a final concentration equal to an 8000-fold dilution, the lag phase was extended to 185 min. A 4000-fold and 2000-fold dilution of grape extract produced lag phases of 250 and 465 min, respectively. LDL oxidation was essentially blocked for at least 10 h with a 1000-fold dilution of grape extract. In other experiments, incubation of LDL (0.2 mg protein/ml) with 5 microM cupric chloride for 1-4 h increased both thiobarbituric acid-reactive substances and electrophoretic mobility of LDL on agarose gel. In addition, there was loss of immunoreactivity of LDL with a murine monoclonal antibody against human apolipoprotein B-100. However, these oxidative changes to LDL by copper were prevented when diluted grape extract was present during incubation. It is concluded that grape extract contains antioxidants in the form of polyphenols with the capacity to inhibit oxidative modification of LDL.

マクロファージによる酸化低密度リポ蛋白生成およびマクロファージ内コレステリルエステル蓄積におよぼす糖尿病状態の影響について

The influence of diabetic state on the generation of macrophage-mediated oxidized low-density lipoprotein (LDL) and the accumulation of cholesteryl ester (CE) in the macrophages were studied in peritoneal macrophages taken from rats with streptozocin-induced diabetes. Electrophoretic mobility of LDL after a 20-hrs incubation with peritoneal macrophages from diabetic and control rats (9.8±0.4mm, 9.8±0.4mm, mean±SD) was significantly higher than that in cell-free medium (8.6±0.6mm). The lipid hydroperoxide (LHPO) level of LDL after a 20-hrs incubation with peritoneal macrophages from diabetic and control rats (1.97±0.57nmol/ml, 2.21±0.44nmol/ml) was significantly higher than that in cell-free medium (0.34±0.14nmol/ml). There were no significant differences in the electrophoretic mobility or LHPO level between diabetic rat macrophage-mediated oxidized LDL and control rat macrophage-mediated oxidized LDL. The CE content of peritoneal macrophages of diabetic rats after the incubation (1.28±0.31μg/106 cells) was significantly higher than that of control rats (0.84±0.08μg/106 cells). These findings suggest that macrophages under the endothelium in diabetes play a role in the accumulation of CE and in the development of atherosclerosis.

Production of oxidized lipids during modification of low-density lipoprotein by macrophages or copper.

The oxidation of low-density lipoprotein (LDL) is implicated in atherosclerosis. Lipids and oxidized lipids were analysed by gas chromatography and gas chromatography-mass spectrometry in human LDL incubated with mouse peritoneal macrophages (MPM) or copper (II) sulphate in Ham's F-10 medium or medium alone (control). MPM-modification and copper-catalysed oxidation of LDL resulted in the formation of oxysterols, mainly cholest-5-en-3 beta,7 beta-diol (7 beta-OH-CHOL); 7%-19% of the initial cholesterol was converted to 7 beta-OH-CHOL in 24 h. 7 beta-OH-CHOL levels in control LDL were very low. The increase in 7 beta-OH-CHOL in MPM and copper-oxidized LDL was accompanied by decreases in linoleate and arachidonate and increases in the electrophoretic mobility and degradation of LDL protein by 'target' macrophages. The concerted occurrence of these processes and their similarity in both MPM-modification and copper-catalysed oxidation of LDL were suggested by the highly significant cross-correlations. The fall in polyunsaturated fatty acid (PUFA) was accompanied by a directly proportional increase in electrophoretic mobility of the LDL. Production of 7 beta-OH-CHOL and protein degradation by macrophages showed modest elevations during the initial steep fall in PUFA, and showed their greatest increases as the levels of PUFA slowly approached zero. The levels of 7 beta-OH-CHOL and the degradation of LDL by macrophages were directly proportional. The degradation of LDL by macrophages increased rapidly as the electrophoretic mobility of LDL was slowly approaching its maximum level.

Protective actions of tamoxifen and 4-hydroxytamoxifen against oxidative damage to human low-density lipoproteins: a mechanism accounting for the cardioprotective action of tamoxifen?

Tamoxifen and 4-hydroxytamoxifen protect isolated human low-density lipoproteins (LDLs) against copper-ion-dependent lipid peroxidation: 4-hydroxytamoxifen is more protective than tamoxifen or 17 beta-oestradiol. 4-Hydroxytamoxifen and 17 beta-oestradiol also prevent the increase in the electrophoretic mobility of LDL caused by exposure to copper ions, presumably by protection of the apoprotein B of LDL against oxidative modification. Our observations may help to account for the cardioprotective benefits reported to be associated with tamoxifen therapy and prophylaxis in breast cancer.

Carvedilol, a new antihypertensive, prevents oxidation of human low density lipoprotein by macrophages and copper.

Growing evidence indicates that oxidized low-density lipoprotein (LDL) may promote atherogenesis. Therefore, inhibition of LDL oxidation may impede this process. Carvedilol is a vasodilating, beta-adrenoceptor blocking agent. As a new antihypertensive drug, carvedilol is unique by virtue of its potent antioxidant activity. Therefore, we tested the ability of carvedilol to inhibit the oxidation of LDL by either macrophages or Cu2+. Carvedilol inhibited LDL oxidation by macrophages in a dose-dependent manner, with an IC50 value of 3.8 microM, as assessed by a thiobarbituric acid reactive substance (TBARS) assay. Under the same conditions, propranolol showed only a mild inhibitory effect (IC50 > 100 microM), while pindolol, atenolol and labetalol had almost no effect. Carvedilol, at 10 microM, almost completely inhibited the macrophage-induced increase in electrophoretic mobility of LDL, while other beta-blockers at 50-300 microM had no significant effect. Carvedilol inhibited superoxide release from mouse macrophages, which correlated well with its inhibition of LDL oxidation. Carvedilol also inhibited Cu(2+)-induced LDL oxidation with an IC50 value of 17 microM, while all other beta-blockers were inactive up to 300 microM. These observations suggest that carvedilol might not only be an effective antihypertensive drug, but might also be effective in prevention of atherosclerosis.

Characterization of low-density lipoproteins from patients with recessive X-linked ichthyosis

We investigated lipoprotein metabolism in 14 patients with recessive X-linked ichthyosis (RXLI), a metabolic disease characterized by scaly skin, corneal opacity and steroid sulfatase deficiency. Plasma total cholesterol (TC) levels ranged from normal to slightly low (mean ± SD: 156 ± 28 mg/dl). Four patients showed a mild or moderate elevation of plasma triglyceride (TG) levels ranging from 150 to 365 mg/dl. The apoprotein B (apo B) to TC ratio was higher than in normal controls (0.63 ± 0.11 vs. 0.52 ± 0.07, P < 0.01), while plasma apoB levels were within the normal range (99 ± 17 mg/dl). Polyacrylamide gel electrophoretic mobility of low-density lipoprotein (LDL) was markedly increased in all patients, and further analyses showed that this finding was not due to a change in the particle size of the LDL but to an increased content of cholesterol sulfate (1.0–2.3% of the LDL-cholesterol content). In addition to the alteration of electrophoretic mobility, marked changes in the lipid and apoprotein compositions of the LDL fraction were observed; cholesterol ester content in LDL (LDL-CE) was significantly lower than that of control subjects (37 ± 4% vs. 41 ± 2% of total lipids, P < 0.01), while the triglyceride content (LDL-TG) and apo B to cholesterol ratios in LDL were significantly higher than those of controls (18 ± 7 vs. 10 ± 2, P < 0.001; 1.21 ± 0.19 vs. 0.73 ± 0.05, P < 0.001, respectively). This anionized LDL, in which cholesterol sulfate was increased, was shown to bind to the LDL receptor of fibroblasts to much the same extent as normal LDL. In conclusion, the increase in cholesterol sulfate in LDL fraction not only alters the electrophoretic moiety but also the relative contents of apoB, cholesterol, and triglyceride in the lipoprotein. It does not change the affinity of LDL for the LDL receptor.

In vitro incubation of low-density lipoproteins with inflammatory cells causes enhanced degradation by macrophages in culture

Utilizing the polyvinyl sponge-implant model, we have reported in vivo modification of low-density lipoproteins (LDL) isolated from interstitial inflammatory fluid (IF) of the rabbit. Further studies on the metabolism of IF-LDL by resident mouse peritoneal macrophages (MPM), demonstrated enhanced uptake and degradation of these modified lipoproteins by scavenger mechanisms. Based upon these studies, we attempted to examine the mechanisms of these observed in vivo modifications in IF-LDL by in vitro incubation of plasma LDL with inflammatory fluid subfractions. Incubation of LDL with inflammatory cells at 37 degrees C resulted in an increased anodal electrophoretic mobility and alteration in apolipoprotein (APO) composition. Subsequent incubation of this modified plasma LDL with MPM resulted in a significant increase in cell surface binding and an increase in the appearance of degradation products in the medium. The formation of lipid peroxides, measured as thiobarbituric acid-reacting substances (T Bars), increased with the time of LDL incubation with inflammatory cells. Conversely, incubation of LDL with cell-free, lipoprotein-deficient IF (LPDIF, d greater than 1.210 g/ml) significantly inhibited LDL degradation by MPM. LPDIF did not alter the electrophoretic mobility of LDL or result in the appearance of T Bars in the medium. These results implicate peroxidative reactions associated with an inflammatory response as mediators of the in vivo modifications in IF-LDL which facilitates enhanced uptake via the scavenger receptor in MPM.

Low-density lipoprotein concentration in interstitial fluid from human atherosclerotic lesions: Relation to theories of endothelial damage and lipoprotein binding

Increased endothelial permeability to low-density lipoprotein (LDL) is believed to be an initiating factor for atherosclerotic lesions. Concentrations of LDL, α 2-macroglobulin and albumin were measured by immunoassay in interstitial fluid collected from normal intima and atherosclerotic lesions of human aortas. The concentration of LDL in interstitial fluid from normal intima was twice the concentration in the patient's serum. In early proliferative (gelatinous) lesions the amount of interstitial fluid was consistently increased but its LDL concentration varied between 80 and 200% of adjacent normal intima. Highest concentrations of LDL were found in interstitial fluid from more advanced proliferative lesions, but the amount was reduced, suggesting a shift in tissue water. LDL was consistently low in interstitial fluid from fatty streaks comprised of lipid-filled cells, and in four of 12 lesions it was absent although α 2-macroglobulin and albumin concentrations were normal. Electrophoretic mobility of LDL, reflecting surface charge, was unchanged or increased in interstitial fluid from normal intima and fatty streaks, but decreased in gelatinous lesions. The ratio of LDL to α 2-macroglobulin and albumin in interstitial fluid was higher than in adjacent intact tissue. The results do not support the idea that increased endothelial permeability to LDL initiates atherogenesis.

X-linked ichthyosis: increased blood cholesterol sulfate and electrophoretic mobility of low-density lipoprotein.

Plasma cholesterol sulfate concentration is increased in patients with recessive X-linked ichthyosis, a disease in which steroid sulfatase activity is absent. In these patients, cholesterol sulfate is found primarily in the low-density lipoprotein fraction of plasma, and the electrophoretic mobility of these lipoproteins is greatly increased.