Fresh Low-density Lipoprotein Research Articles

Formation mechanism of low-density lipoprotein gel induced by NaCl

Salted eggs, which are a traditional Chinese egg product, are favored by Chinese consumers and have become very popular in other Asian countries due to their unique features such as "fresh, fine, tender, loose, gritty and oily texture." In order to illuminate the forming process of salted egg, the gelation behavior and mechanism of low-density lipoprotein (LDL) induced by NaCl were investigated using marinated model outside the eggshell. Results showed that as the salting proceeded, the moisture content exhibited a decreasing trend. The NaCl content, oil exudation, hardness, and surface hydrophobicity showed constantly increasing trends. In the early stages of salting, the size of the LDL particles, soluble protein content, and T21 increased, whereas T21 (with D2O), T22, and the free sulfhydryl content declined. In the later stages of salting, LDL formed a multiple composite aggregate gel structure with filamentous apoproteins and non-spherical lipid particles intertwined with each other. The soluble protein content and T23 (without D2O) decreased, whereas T21 (with D2O), T22 and the free sulfhydryl content increased. Fourier transform infrared spectroscopy revealed that the fresh LDL mainly consisted of α-helix and β-sheet structures. After the gel becomes hardened, the LDL secondary structure was changed remarkably, characterized by the decrease of α-helix elements and increase of β-sheet elements. The results suggested that the oil exudation of salted LDL gels was mainly due to LDL destruction and the release of components (apoproteins, phospholipids, and neutral lipids) facilitated by increased interactions between apoproteins and lipids.

Low density lipoproteins added to an extender frozen or lyophilized are evenly efficient in cryoprotecting ovine sperm cells than when 16% whole egg yolk was added

The aim of this study was to test different concentrations of low density lipoprotein (LDL) in replacement of whole egg yolk in extenders preserved in the aqueous or lyophilized form, for ram sperm cryopreservation using two cooling curves (-40°C/min from 5 to –140°C and nitrogen vapor). One ejaculate from six Santa Ines rams was collected. Each ejaculate was divided into nine different diluents as follows: Tris-16% yolk (control), and Tris with 2, 4, 6 and 8% fresh LDL, and criopreserved in the aqueous or lyophilized form. The samples were diluted to a final concentration of 100 x 106 sperm/mL and filled into 0.25 ml straws. After thaw, sperm cells were evaluated for motility and sperm kinetics (CASA), and submitted to the hypoosmotic swelling test and the evaluation of the structural integrity of sperm membranes using fluorescent dyes (CFDA: PI), as well as sperm morphology and longevity. The experimental design was randomized blocks, and results were submitted to ANOVA and the averages were compared using the Scott-Knott test. There were no differences in progressive motility and functional and structural integrity of the membrane evaluated when different concentrations of aqueous or lyophilized low density lipoproteins or egg yolk were added to the extender (P>0.05). As for the velocity of sperm movement, the control medium had some kinetic scores similar to the extender containing LDL, both aqueous and lyophilized (P> 0.05). Results were similar between cooling curves. Therefore, we conclude that the media containing all concentrations of LDL, aqueous or lyophilized, were able to protect the ram sperm cells during the cryopreservation process, as whole egg yolk did.

Trans fatty acids influence the oxidation of LDL in ECV304 cells

The objectives of this study are to explore whether separate sources of trans fatty acids (TFA) have different effects on ECV304 cell line and to further elucidate the oxidation mechanism induced by TFA. ECV304 cells are used in the study because they display many endothelial features. Cell apoptosis rates increased in a dose-dependent manner following 24-h treatment with TFA from separate sources. Additionally, TFA stimulated human alpha-defensin 1 (HNP-1) expression and resulted in a significant increase in both malondialdehyde (MDA) and ROS levels. MDA levels reach their peak at 18 h. HNP-1 expression levels increase at 2 h and then reach their peak at 10 h. At the same time, the protein carbonyl (PCO) value declines slightly. After 10 h of TFA co-culture, the cells were washed and fresh low-density lipoprotein (LDL) was added. MDA generation significantly increased after 6 h and it could be inhibited by 4-aminobenzoic acid hydrazide (ABAH) or sodium ferulate. However, after the TFA co-culture for 2 h, adding LDL for 6 h just caused slight MDA generation change and the MDA generation could be inhibited by verapamil or sodium ferulate. TFA from different sources did not have different effects on ECV304. HNP-1 mediates the oxidation induced by TFA by activating ROS. Furthermore, TFA can stimulate the oxidation of LDL in ECV304 cells through both passive and active pathway. In the oxidation induced by linoelaidic acid, ABAH can decrease the MDA generation in active oxidation pathway and verapamil can decrease the MDA generation in passive oxidation pathway.

Involvement of HNP‐1 in different oxidation mechanisms in human endothelial cells

AbstractThe objective of this study was to elucidate the different mechanisms of low‐density lipoprotein (LDL) oxidation by human endothelial cells. High level of LDL stimulated human alpha‐defensin 1 (HNP‐1) expression strongly, activated myeloperoxidase (MPO), and accumulated malondialdehyde (MDA). In addition, lipopolysaccharides (LPS) had similar effect, however the cell response occurred even earlier. After 12 h of LDL co‐culture, the cells were washed and fresh LDL was added again. MPO activity and MDA generation were increased. Adding fresh LDL after the LPS co‐culture for 3 h, a similar trend, however weaker effect was observed. Culturing cells with LDL and adding various kinds of calcium antagonists at the same time did not make obvious changes to the expression of HNP‐1. In contrast, culturing cells with LPS, calcium antagonists increased HNP‐1 expression. Adding the anti‐radical drug sodium ferulate had no significant effect on oxidative function activated by LDL, but the oxidation activated by LPS was suppressed significantly and the HNP‐1 expression was not changed significantly. The endogenous irritant LDL and the exogenous irritant LPS activated human endothelial cells in different manners and HNP‐1 expression changes were involved.

Flavonoid-Induced Ability of Minimally Modified Low-Density Lipoproteins to Support Lymphocyte Proliferation

Low-density lipoprotein (LDL) peroxidation appears to be involved in atherogenesis. We studied the ability of minimally modified LDL (MM-LDL) to be used by proliferating lymphocytes and the effects of antioxidant flavonoids on this lipoprotein. MM-LDL were obtained by storing LDL at 4° for 1 month, which resulted in a decrease in lipophilic antioxidants and an increased susceptibility to oxidation when incubated with cells. MM-LDL were not cytotoxic; however, in cells treated with lovastatin that require cholesterol for cell growth, they were much less efficient than fresh LDL in sustaining proliferation as determined by [ 3H]thymidine incorporation into DNA. Pure quercetin and grape-derived beverages restored proliferation in the presence of MM-LDL and prevented the apoptosis otherwise induced by lovastatin. These effects of flavonoids correlated with their activity in inhibiting LDL peroxidation. The results demonstrate that potent antioxidants, such as flavonoids, protect MM-LDL from lipoperoxidation and preserve their ability to efficiently deliver cholesterol to cells.

Endothelial cells prevent accumulation of lipid hydroperoxides in low-density lipoprotein.

A variety of cell types, including endothelial cells, oxidize low-density lipoprotein (LDL). To investigate the mechanisms by which endothelial cells modulate LDL oxidation states, endothelial cell cultures were incubated with LDL (240 mg cholesterol/dL) for 24 hours in M199 supplemented with fetal bovine serum (FBS, 16.7%). These conditions were not toxic to endothelial cells over the time frame of the study. Changes in LDL oxidation were monitored by measuring thiobarbituric acid-reactive substances (TBARS), lipid hydroperoxide (LOOH), and conjugated dienes (A234nm). LDL medium incubated in the absence of endothelial cells contained higher TBARS than did LDL medium incubated with endothelial cells (0.35 +/- 0.08 versus 0.23 +/- 0.08 nmol MDA/mg, respectively). LOOHs were higher in LDL medium incubated without endothelial cells than in LDL medium incubated with endothelial cells (6.8 +/- 4.4 versus 0.49 +/- 0.89 nmol/mg, respectively). Conjugated diene formation, based on changes in absorbance at 234 nm, increased to a greater extent in LDL medium incubated in the absence of endothelial cells than when endothelial cells were present. To increase oxidative stress on the endothelial cell cultures, increasing concentrations of Cu2+ (0 to 4 mumol/L) were added to LDL medium. Endothelial cells prevented LOOH accumulation until the concentration of Cu2+ exceeded 0.75 mumol/L. At 1.5 and 4 mumol/L Cu2+, endothelial cells enhanced LOOH formation nearly 3 and 2.5 times the LOOH values in the corresponding medium incubated in the absence of endothelial cells. This loss of protective function however, was not permanent. Endothelial cells, preincubated for 24 hours with Cu(2+)-containing LDL medium, were still able to prevent LOOH accumulation in fresh LDL medium. Endothelial cells prevented LOOH accumulation even when exposed to LDL medium that contained low concentrations of LOOHs (< 22 nmol/mg). However, endothelial cells accelerated the accumulation of LOOHs in LDL when exposed to LDL medium that contained slightly higher concentrations of preexisting LOOHs (approximately equal to 33 nmol/mg). These data indicate that endothelial cells have a limited capacity for preventing LOOH formation and that small increases in LOOHs may play a critical role in enhancing the potential of endothelial cells for oxidative modification of LDL.

Cellular cysteine generation does not contribute to the initiation of LDL oxidation.

It has been suggested that the generation of cysteine (Cys-SH) by cells may play a role in the initiation of oxidation of low density lipoprotein (LDL). Cysteine has long been considered as an antioxidant. We studied the effect of Cys-SH on the oxidation of LDL by copper. The presence of Cys-SH had a profound inhibitory effect on the formation of conjugated dienes when fresh LDL was used. However, when we used LDL samples that were subjected to pre-incubation with copper, a progressive decrease in the inhibition and an actual enhancement of oxidation by Cys-SH could be demonstrated. The oxidation of freshly prepared LDL by RAW macrophages as compared to older LDL was considerably less. The addition of Cys-SH inhibited the oxidation of LDL by cells. In contrast, the addition of cystine (Cys-S-S) enhanced the oxidation of older LDL preparations while such additions had no effect on the oxidation of freshly prepared LDL. When pre-incubated LDL was subjected to oxidation by cells an enhancement of oxidation by Cys-S-S could be noted. These results demonstrate that the role of Cys-SH generated as a result of cellular recycling of Cys-S-S in the oxidation of LDL may not relate to the initiation of oxidation reactions. However, Cys-SH may enhance the rate of oxidation of LDL that may contain peroxides.

Involvement of Preexisting Lipid Hydroperoxides in Cu 2+-Stimulated Oxidation of Low-Density Lipoprotein

Oxidative modification of human low-density lipoprotein (LDL) is thought to play an important role in the development of atherosclerosis. LDL oxidizability is believed to be strongly influenced by factors such as (a) content of preexisting lipid hydroperoxides (LOOHs) and (b) content of endogenous antioxidants such as α-tocopherol and β-carotene. The purpose of this study was to examine the prooxidant role of preexisting LDL-LOOHs, using a recently developed method for ultrasensitive and selective LOOH analysis: high-performance liquid chromatography with mercury drop electrochemical detection (HPLC-EC). Exceedingly low detection limits for LDL-LOOHs have been achieved by HPLC-EC, e.g., ∼100 fmol for cholesteryl ester hydroperoxide (CEOOH). This sensitivity has allowed us to monitor LDL-LOOHs at levels that are undetectable by most other methods. Fresh LDL prepared with the utmost care to prevent autoxidation was found to contain small, yet significant amounts of CEOOH, 6-12 pmol/mg protein. Our data suggest that these peroxides could not have arisen during LDL isolation or sample work-up for HPLC-EC. Incubation with GSH and phospholipid hydroperoxide glutathione peroxidase resulted in nearly complete reduction of the CEOOH. This LDL was found to be much more resistant to Cu 2+-induced peroxidation than starting material, exhibiting a lag period that was at least six times greater. We have also determined that LDL becomes progressively more susceptible to Cu 2+-induced lipid peroxidation (as evidenced by a shortened lag) when it is preloaded with increasing amounts of photochemically generated LOOHs. Taken together, these results provide strong support for the idea that preexisting LOOHs in LDL are important determinants of its overall oxidizability.

Tocopherol-mediated peroxidation. The prooxidant effect of vitamin E on the radical-initiated oxidation of human low-density lipoprotein

Oxidation of human low-density lipoprotein (LDL) is implicated as an initiator of atherosclerosis. α-Tocopherol (α-TocH) may thus inhibit atherosclerosis beause it is the major and most active chain-breaking antioxidant in extracted LDL lipid. Our studies show, however, that α-TocH can be a strong prooxidant for the LDL itself, i.e., an aqueous dispersion of lipid-bearing particles. Thus, a steady flux (R g ) of alkylperoxyl radicals (ROO . ) generated from a water-soluble azo initiator induced lipid peroxidation in LDL which was faster in the presence of α-TocH than in its absence (for R g <2 NM S -1 ), insensitive to R g and [O 2 ], and inhibited by vitamin C, ubiquinol-10 (normally present in fresh LDL), and small phenolic antioxidants but not inhibited by the aqueous radical scavenger uric acid

Noninvasive assays for oxidative RNA damage

Oxidative modification of human low-density lipoprotein (LDL) is thought to play an important role in the development of atherosclerosis. LDL oxidizability is believed to be strongly influenced by factors such as (a) content of preexisting lipid hydroperoxides (LOOHs) and (b) content of endogenous antioxidants such as α-tocopherol and β-carotene. The purpose of this study was to examine the prooxidant role of preexisting LDL-LOOHs, using a recently developed method for ultrasensitive and selective LOOH analysis: high-performance liquid chromatography with mercury drop electrochemical detection (HPLC-EC). Exceedingly low detection limits for LDL-LOOHs have been achieved by HPLC-EC, e.g., ∼100 fmol for cholesteryl ester hydroperoxide (CEOOH). This sensitivity has allowed us to monitor LDL-LOOHs at levels that are undetectable by most other methods. Fresh LDL prepared with the utmost care to prevent autoxidation was found to contain small, yet significant amounts of CEOOH, 6-12 pmol/mg protein. Our data suggest that these peroxides could not have arisen during LDL isolation or sample work-up for HPLC-EC. Incubation with GSH and phospholipid hydroperoxide glutathione peroxidase resulted in nearly complete reduction of the CEOOH. This LDL was found to be much more resistant to Cu2+-induced peroxidation than starting material, exhibiting a lag period that was at least six times greater. We have also determined that LDL becomes progressively more susceptible to Cu2+-induced lipid peroxidation (as evidenced by a shortened lag) when it is preloaded with increasing amounts of photochemically generated LOOHs. Taken together, these results provide strong support for the idea that preexisting LOOHs in LDL are important determinants of its overall oxidizability.