Comparison of the effect of alpha-lipoic acid and alpha-tocopherol supplementation on measures of oxidative stress

In vitro studies have shown that alpha-lipoic acid (LA) is an antioxidant. There is a paucity of studies on LA supplementation in humans. Therefore, the aim of this study was to assess the effect of oral supplementation with LA alone and in combination with alpha-tocopherol (AT) on measures of oxidative stress. A total of 31 healthy adults were supplemented for 2 months either with LA (600 mg/d, n = 16), or with AT (400 IU/d, n = 15) alone, and then with the combination of both for 2 additional months. At baseline, after 2 and 4 months of supplementation, urine for F2-isoprostanes, plasma for protein carbonyl measurement and low-density lipoprotein (LDL) oxidative susceptibility was collected. Plasma oxidizability was assessed after incubation with 100 mM 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) for 4 h at 37 degrees C. LDL was subjected to copper- and AAPH-catalyzed oxidation at 37 degrees C over 5 h and the lag time was computed. LA significantly increased the lag time of LDL lipid peroxide formation for both copper-catalyzed and AAPH-induced LDL oxidalion (p < .05), decreased urinary F2-isoprostanes levels (p < .05), and plasma carbonyl levels after AAPH oxidation (p < .001). AT prolonged LDL lag time of lipid peroxide formation (p < .01 ) and conjugated dienes (p < .01) after copper-catalyzed LDL oxidation, decreased urinary F2-isoprostanes (p < .001), but had no effect on plasma carbonyls. The addition of LA to AT did not produce an additional significant improvement in the measures of oxidative stress. In conclusion, LA supplementation functions as an antioxidant, because it decreases plasma- and LDL-oxidation and urinary isoprostanes.     

Antioxidative activity on human low density lipoprotein (LDL) oxidation by pentagalloic acid

The aim of this study was to investigate the efficiency of the pentagalloic acid compound in inhibiting the metal ions and cell lines that mediate in low density lipoprotein (LDL) oxidation. Pentagalloic acid prolonged the lag time preceeding the onset of conjugated diene formation. In chemically induced LDL oxidation by Cu²⁺ plus hydrogen peroxide or peroxyl radical generated by 2, 2′-azo-bis (2-amidino propane) hydrochloride (AAPH), pentagalloic acid inhibited LDL oxidation as monitored by measuring the thiobarbituric acid reactive substances (TBARS), malondialdehyde (MDA), and gel electrophoretic mobility. The physiological relevance of the antioxidative activity was validated at the cellular level where pentagalloic acid inhibited mouse macrophage J774 and endothelial cell-mediated LDL oxidation. When compared with several other antioxidants, pentagalloic acid showed a much higher ability than naturally occuring antioxidants, α-tocopherol and ascorbic acid, and the synthetic antioxidant, probucol.     

Selenoprotein P protects low-density lipoprotein against oxidation

Selenoprotein P (SeP) is an extracellular glycoprotein with 8-10 selenocysteines per molecule, containing approximately 50% of total selenium in human serum. An antioxidant function of SeP has been postulated. In the present study, we show that SeP protects low-density lipoproteins (LDL) against oxidation in a cell-free in-vitro system. LDL were isolated from human blood plasma and oxidized with CuCl₂, 2,2'-azobis(2-amidinopropane) (AAPH) or peroxynitrite in the presence or absence of SeP, using the formation of conjugated dienes as parameter for lipid peroxidation. SeP delayed the CuCl₂- and AAPH-induced LDL oxidation significantly and more efficiently than bovine serum albumin used as control. In contrast, SeP was not capable of inhibiting peroxynitrite-induced LDL oxidation. The protection of LDL against CuCl₂- and AAPH-induced oxidation provides evidence for the antioxidant capacity of SeP. Because SeP associates with endothelial membranes, it may act in vivo as a protective factor inhibiting the oxidation of LDL by reactive oxygen species.     

Thyroid hormone (T3) and its acetic derivative (TA3) protect low-density lipoproteins from oxidation by different mechanisms

Triiodothyronine (T3) and triiodothyroacetic acid (TA3) are thyroid compounds that similarly protect low-density lipoprotein (LDL) against oxidation induced by the free radical generator 2,2'-azobis-[2-amidinopropane] dihydrochloride (AAPH). However, TA3 is more antioxidant than T3 on LDL oxidation induced by copper ions (Cu2+), suggesting that these compounds act by different mechanisms. Here we measured conjugated diene production kinetics during in vitro human LDL (50 mg LDL-protein per l) oxidation induced by various Cu2+ (0.5-4 microM) or AAPH (0.25-2 mM) concentrations in the presence of T3, TA3, butylated hydroxytoluene (BHT) (a free radical scavenger) or ethylenediaminetetracetic acid (EDTA) (a metal chelator). From the kinetics were estimated: length of the lag phase (Tlag), maximum velocity of conjugated diene production (Vmax), and maximum amount of generated dienes (Dmax). Thyroid compound effects on these oxidation parameters were compared to those of the controls BHT and EDTA. In addition we measured by atomic absorption spectrometry copper remaining in LDL after a 30 min incubation of LDL with Cu2+ and the compounds followed by extensive dialysis, i.e. copper bound to LDL. As expected, LDL-copper was decreased by EDTA in a concentration-dependent manner, whereas it was not affected by BHT. T3 increased LDL-copper whereas TA3 slightly decreased it. The whole data suggest that T3 and TA3 are free radical scavengers that also differently disturb LDL-copper binding, an essential step for LDL lipid peroxidation. The most likely mechanisms are that T3 induces new copper binding sites inside the LDL particle, increasing the LDL-copper amount but in a redox-inactive form, whereas TA3 blocks some redox-active copper binding sites highly implicated in the initiation and the propagation of lipid peroxidation. Alternatively, we also found that a little amount of copper is tightly bound in LDL, which may be essential for the propagation of lipid peroxidation induced by free radical generators.     

Effects of some flavonoids on the susceptibility of low-density lipoprotein to oxidative modification

The effects of six flavonoids viz., apigenin, genistein, morin, naringin, pelargonidin and quercetin on the susceptibility of low-density lipoprotein (LDL) to oxidative modification were investigated. Flavonoids were added to plasma and incubated for 3 hr at 37 degrees C, and the LDL fraction was separated by ultracentrifugation. Oxidizability of LDL was estimated by measuring conjugated diene (CD), lipid peroxides and thiobarbituric acid-reactive substances (TBARS), after cupric sulfate solution was added. Quercetin and morin significantly (P<0.01 by ANOVA) prolonged the lag time before initiation of oxidation reaction in dose-dependent manner. They also suppressed the formation of lipid peroxides and TBARS more markedly than other flavonoids. The ability to prolong lag time and suppression of lipid peroxides and TBARS formation was in the following order: quercetin >morin >pelargonidin >genistein >naringin >apigenin. LDL exposed to flavonoids reduced oxidizability. These findings suggest that flavonoids may have a role in ameliorating atherosclerosis.     

花背蟾蜍角膜早期发育中氨基多糖的电镜细胞化学研究

Glycosaminoglycans (GAGs) and their changes in early corneal development of Bufo raddei Strauch (from stage 16, neural tube, to stage 25, operculum completely closed) were studied with electron microscopic cytochemical method. Results show that synthesis of GAGs changes from non-sulfated to sulfated, and its content increased gradually with the development of cornea. Hyaluronic acid (HA) in each part of cornea begins to increase gradually from stage 16 to 21 (mouth open stage), with its peak at stage 20 (gill circulation stage) to 21, then decreases. In the mean time, contents of dermatam sulfate (DS), chondroitin sulfate (CS), heparan sulfate (HS) and heparin (Hep) increase gradually. It is considered that HA, HS and collagen may be related to the migration of mesenchymal cells, and HA promotes the expansion and hydration of corneal stroma; sulfated GAGs are correlated with dehydration of cornea, cell density and corneal transparency; DS, CS, HS and Hep deposited among collagen fibrils could adjust their arrangement. All these changes would enhance transparency of cornea.     

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.     

Influence of oligomer chain length on the antioxidant activity of procyanidins

The antioxidant activity of catechin monomers and procyanidin (dimers to hexamers) fractions purified from cocoa was studied in two in vitro systems: liposomes and human LDL. Liposome oxidation (evaluated as formation of 2-thiobarbituric acid reactive substances) was initiated with 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN), iron/ascorbate, or UV-C; LDL oxidation (evaluated as formation of conjugated dienes) was initiated with Cu(2+) or AAPH. Catechin monomers and procyanidin fractions inhibited both liposome and LDL oxidation. Monomers, dimers, and trimers fractions were the most effective antioxidants when liposome oxidation was initiated in the aqueous phase. When oxidation was initiated in the lipid domains, higher molecular weight procyanidins were the most effective. All fractions significantly inhibited Cu-mediated LDL oxidation; no significant effect of procyanidin molecular weight was observed. The hexamer fraction was the least effective with respect to preventing AAPH initiated LDL oxidation. Results reported herein give further evidence on the influence of the oligomer chain length on the antioxidant protection by procyanidins.     

Glycosaminoglycans of Rat Cerebellum: II. A Developmental Study

Total and individual glycosaminoglycans (GAGs) were determined in rat cerebellum in tissue explants at various postnatal ages. The major constituents of GAGs were chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). Dermatan sulfate (DS) and keratan sulfate (KS) could not be detected and therefore each amounts to less than 5% of all GAGs at all ages studied. HA was the prominent GAG during postnatal development and only a minor constituent at adult ages, whereas CS was the predominant GAG in adulthood. HS remained relatively constant throughout development. The incorporation of [3H]glucosamine into individual GAGs was highest for HS at postnatal day 6, whereas HA showed intermediate and CS the lowest levels of incorporation during the first postnatal week. All major GAGs showed the lowest incorporation values at adult ages.     

Effects of flavonoids on the susceptibility of low-density lipoprotein to oxidative modification

Dietary flavonoid intake has been reported to be inversely associated with the incidence of coronary artery disease. To clarify the possible role of flavonoids in the prevention of atherosclerosis, we investigated the effects of some of these compounds on the susceptibility of low-density lipoprotein (LDL) to oxidative modification. In this study, six flavonoids, "apigenin, genistein, morin, naringin, pelargonidin and quercetin", were added to plasma and incubated for 3h at 37 degrees C. Then, the LDL fraction was separated by ultracentrifugation. The oxidizability of LDL was estimated by measuring conjugated diene (CD), lipid peroxides and thiobarbituric acid-reactive substances (TBARS) after cupric sulfate solution was added. We showed that among flavonoids used, quercetin and morin significantly (P<0.01 by ANOVA) and dose-dependently prolonged the lag time before initiation of oxidation reaction. Also, these two flavonoids suppressed the formation of lipid peroxides and TBARS more markedly than others. Their ability to prolong lag time and suppression of lipid peroxides and TBARS formation resulted to be in the following order: quercetin>morin>pelargonidin>genistein>naringin>apigenin. LDL exposed to flavonoids in vitro reduced oxidizability. These findings show that flavonoids may have a role in ameliorating atherosclerosis.     

Comparison of two methods for radioiodination on the oxidizability properties of low density lipoprotein

Radiolabeling of low density lipoprotein (LDL) apoB100 with 125I, an oxidative process, is commonly used in lipoprotein investigation. Since 1) LDL is unstable and oxidation-prone, 2) the modification of apoB100 by oxidation increases the negative charge of particles and leads to the uptake of modified LDL through the scavenger receptor pathway, and 3) oxidized LDL is cytotoxic, it is relevant to investigate whether the oxidative stability of LDL is influenced by its labeling with 125I. The aim of this study was to investigate and compare lipid and protein oxidation markers in human LDL after labeling with 125I by two widely adopted methods that use ICl or the chloramide 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycoluril as the oxidizing agent. Native LDL served as a common control and sham-iodinated LDL as a handling control for each procedure. The resistance against copper-induced oxidation of 125I-LDL labeled with ICl was similar to that of controls with regard to the lag time and maximal amount of conjugated diene formed, as there were levels of initial conjugated diene, alpha-tocopherol, and tryptophan. However, radioiodination with the chloramide accelerated the onset of the rapid phase of LDL oxidation due to a drastic depletion of alpha-tocopherol and increased conjugated diene content. Measurements of copper-induced LDL oxidizability showed enhanced indices of lipid oxidation. The lag time and the time to maximal diene production were 65% and 30% shorter than controls. This was accompanied by a 50% reduced tryptophan fluorescence. The anionic surface charge of the LDL particle increased moderately with both labeling procedures. The results indicate that labeling of LDL with 125I may oxidize lipids and apoB100 to a variable extent, depending on the nature of the iodinating agent. This is why assessment of the oxidizability properties of 125I-labeled LDL is recommended for reliable biological studies.     

Evidence for inhibition of low density lipoprotein oxidation and cholesterol accumulation by apolipoprotein H (beta2-glycoprotein I)

Low density lipoprotein (LDL) oxidation and lipid accumulation are thought to enhance the progression of atherosclerosis. Apolipoprotein H (apoH) has been implicated in the development of human atherosclerosis. However, the roles of apoH in the oxidative modification of LDL and cellular accumulation of lipid constituents remained uncharacterized. In this study, the level of plasma apoH was found to be significantly associated with the oxidative susceptibility of LDL in human subjects. Plasma levels of apoH were positively correlated with the lag time but negatively correlated with LDL oxidation rate in conjugated diene formation. By using a J774 A.1 macrophage culture system, we found that apoH could not only inhibit the formation of conjugated diene and thiobarbituric acid-reactive substances, but also reduce the electrophoretic mobility of oxidized LDL. Furthermore, apoH decreased cellular accumulation of cholesterol via a reduction in cholesterol influx and an increase in cholesterol efflux. This is the first demonstration that apoH appears to have "antioxidant"-like effects on LDL oxidation. The results also suggest that apoH can inhibit the translocation of cholesterol from extracellular pools to macrophages, suggesting that apoH may play an important role in the prevention of atherosclerosis.     

Increased deposition of glycosaminoglycans and altered structure of heparan sulfate in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant deposition of extracellular matrix (ECM) constituents, including glycosaminoglycans (GAGs), that may play a role in remodelling processes by influencing critical mediators such as growth factors. We hypothesize that GAGs may be altered in IPF and that this contribute to create a pro-fibrotic environment. The aim of this study was therefore to examine the fine structure of heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA) in lung samples from IPF patients and from control subjects. GAGs in lung samples from severe IPF patients and donor lungs were analyzed with HPLC. HS was assessed by immunohistochemistry and collagen was quantified as hydroxyproline content. The total amount of HS, CS/DS and HA was increased in IPF lungs but there was no significant difference in the total collagen content. We found a relative increase in total sulfation of HS due to increment of 2-O, 6-O and N-sulfation and a higher proportion of sulfation in CS/DS. Highly sulfated HS was located in the border zone between denser areas and more normal looking alveolar parenchyma in basement membranes of blood vessels and airways, that were immuno-positive for perlecan, as well as on the cell surface of spindle-shaped cells in the alveolar interstitium. These findings show for the first time that both the amount and structure of glycosaminoglycans are altered in IPF. These changes may contribute to the tissue remodelling in IPF by altering growth factor retention and activity, creating a pro-fibrotic ECM landscape.     

Comparison of LDL trap assay to other tests of antioxidant capacity; effect of vitamin E and lovastatin treatment

Oxidized low density lipoprotein (LDL) has a major impact in the development of atherosclerosis. Risk for oxidative modification of LDL is usually determined indirectly by measuring the capability of LDL to resist radical insult. We compared three different methods quantifying the antioxidative capacity of LDL ex vivo in dyslipidemic patients with coronary heart disease. Plasma samples were obtained from two double-blinded cross-over trials. The duration of all interventions (placebo, lovastatin 60 mg/day, RRR-alpha-tocopherol 300 mg/day and lovastatin + RRR-alpha-tocopherol combined) was 6 weeks. The total radical capturing capacity of LDL (TRAP) in plasma was determined using 2,2-azo-bis(2,4-dimethyl-valeronitrile) (AMVN) -induced oxidation, and measuring the extinction time of chemiluminescence. TRAP was compared to the variables characterizing formation of conjugated dienes in copper-induced oxidation. Also the initial concentrations and consumption times of reduced alpha-tocopherol (alpha-TOH) and ubiquinol in AMVN-induced oxidation were determined. Repeatability of TRAP was comparable to that of the lag time in conjugated diene formation. Coefficient of variation within TRAP assay was 4.4% and between TRAP assays 5.9%. Tocopherol supplementation produced statistically significant changes in all antioxidant variables except those related to LDL ubiquinol. TRAP increased by 57%, the lag time in conjugated diene formation by 34% and consumption time of alpha-TOH by 88%. When data of all interventions were included in the analyses, TRAP correlated with the lag time (r = 0.75, p < 10(-6)), with LDL alpha-TOH (r = 0.50, p < 0.001) and with the consumption time of alpha-TOH (r = 0.58, p < 0.0001). In the baseline data, the associations between different antioxidant variables were weaker. TRAP correlated with the lag time (r = 0.55, p < 0.001) and alpha-TOH consumption time (r = 0.48, p < 0.05), and inversely with apolipoprotein Al (r = -0.51, p < 0.05). Lag time at the baseline did not correlate with ubiquinol or tocopherol parameters, or with any plasma lipid or lipoprotein levels analyzed. Lovastatin treatment did not significantly affect the antioxidant capacity of LDL. In conclusion, TRAP reflects slightly different properties of LDL compared to the lag time. Thus, LDL TRAP assay may complement the other methods used to quantify the antioxidant capacity of LDL. However, TRAP and the lag time react similarly to vitamin E supplementation.     

花背蟾蜍角膜早期发育中氨基多糖的电镜细胞化学研究

本文用钌红显示氨基多糖的电镜细胞化学方法,较细致地研究了花背蟾蜍(Bufo raddeiStrauch)胚胎发育过程中(16—25期,即神经管至鳃盖完全封闭期)氨基多糖(GAG)在角膜早期发育中的变化。结果表明:GAG的合成由非硫酸化向硫酸化转变,且随着角膜的发育其含量逐渐增加。角膜各部位的HA在16—21期(胚胎开口期),其含量逐渐增加,且20(鳃血循环期)-21期达最高水平,此后含量下降,同时DS、CS、HS和Hep的含量上升。据分析,HA、HS和胶原与间充质细胞迁移有关,HA还可促使基质的膨胀和水化。硫酸化GAG在角膜的脱水、致密、细胞密度及角膜透明中起作用。胶原纤维间的硫酸化GAG能调节胶原纤维的规则化,故也有助于角膜的透明。     

Follicular fluid glycosaminoglycans inhibit degradation of low-density lipoproteins and progesterone production by porcine granulosa cells

Porcine granulosa cells were incubated with commercially available glycosaminoglycans (GAGs) or GAGs purified from porcine follicular fluid to evaluate the effects of GAGs on degradation of low-density lipoproteins (LDL) and progesterone production. Commercially available heparin and chondroitin sulfates (CS) as well as follicular CS and heparan sulfate (HS) inhibited degradation of LDL in dose-dependent manners. Doses of follicular CS and HS required to inhibit 50% of the LDL degradation corresponded to concentrations found in follicular fluid (less than 1 mg/ml). Progesterone production was also inhibited in a dose-dependent fashion by follicular GAGs at concentrations found in follicles. The ability of the follicular GAGs to inhibit degradation of LDL could represent a mechanism by which the utilization of LDL-derived sterol is temporarily restricted following permeabilization of the ovulatory follicle. Follicular GAGs may also modulate utilization of apoprotein E-containing high-density lipoproteins in unruptured follicles.     

Impaired resistance to oxidation of low density lipoprotein in cystic fibrosis: Improvement during vitamin E supplementation

Antioxidants such as vitamin E protect unsaturated fatty acids of LDL against oxidation. In the ex vivo model used, LDL was exposed to Cu²⁺ ions, a potent prooxidant capable of initiating the oxidation of LDL. The lag time, indicating the delay of conjugated diene formation in LDL due to antioxidant protection, was measured in 54 cystic fibrosis (CF) patients with plasma -tocopherol levels below (Group A, n = 30) or above (Group B, n = 24) 15.9 μmol/L (mean - 2 SD of Swiss population). Patients were reevaluated after 2 months on 400 IU/d of oral RRR--tocopherol. In group A, -tocopherol concentrations in LDL increased significantly from 3.2 ± 1.6 mol/mol LDL to 8.2 ± 2.8 mol/mol (P < 0.001) and lag times increased from 79 ± 33, min to 126 ± 48 min (P < 0.001), whereas in the vitamin E sufficient group B no further increase neither in LDL -tocopherol concentrations or in lag times was observed. LDL oleic acid concentrations were higher, and linoleic acid concentrations were lower in patients than in controls. After efficient vitamin E supplementation, lag times were positively related to LDL -tocopherol (P < 0.01) and negatively to LDL linoleic and arachidonic acid content (P < 0.001). The maximum rate of oxidation correlated positively with linoleic and arachidonic acid concentrations, as did the maximum conjugated diene absorbance. These results indicate that LDL resistance to oxidation is impaired in vitamin E deficient CF patients but can be normalized within 2 months when -tocopherol is given in sufficient amounts. Linoleic and arachidonic acid content exhibit a major influence on the LDL resistance to oxidation.     

Antioxidant effects of phyto-and synthetic-estrogens on cupric ion-induced oxidation of human low-density lipoproteins in vitro

Oxidation of low-density lipoproteins (LDL) promotes the formation of atherosclerotic plaques. Estrogenic compounds (EC) from foods and other natural products, and synthetic estrogenic compounds (SECs) may prevent heart disease by inhibiting LDL oxidation. In the present study, we tested the antioxidant capacities of two phytoestrogens, daidzein (DAI) and genistein (GEN), and four SECs, (+)- and (-)-Z-bisdehydrodoisynolic acid (ZBDDA), and (+)- and (-)-hydroxy-allenoic acid (HAA), on isolated human LDL subjected to oxidation by cupric sulfate. The effects of these estrogenic compounds on the kinetics of conjugated diene formation in LDL undergoing oxidation were evaluated with a lag-time assay with continuous monitoring of absorbance at 234 nm. Lag-time data revealed that (+)-HAA, (-)-HAA, (+)-ZBDDA, and (-)-ZBDDA had similarly stronger antioxidant activities than either GEN or DAI. We also found that (+)-HAA, (-)-HAA, (+)-ZBDDA, and (-)-ZBDDA strongly inhibited the formation of Cu+-induced thiobarbituric acid reactive substances (TBARS) in LDL, and that GEN and DAI were less effective for inhibiting LDL lipid peroxidation. Finally, electrophoretic evaluation suggested that (+)-HAA, (-)-HAA, (+)-ZBDDA, and (-)-ZBDDA protected the apolipoprotein B-100 of LDL against oxidation better than did GEN or DAI. In summary, the four SECs, (+)-HAA, (-)-HAA, (+)-ZBDDA, and (-)-ZBDDA, were more potent antioxidants than the phytoestrogens, DAI and GEN.     

Extent of copper LDL oxidation depends on oxidation time and copper/LDL ratio: chemical characterization

The aim of our study was to determine, as a function of [Cu(2+)]/[LDL] ratios (0.5 and 0.05) and of oxidation phases, the extent of LDL oxidation by assessing the lipid and apo B oxidation products. The main results showed that: (i) kinetics of conjugated diene formation presented four phases for Cu(2+)/LDL ratio of 0.5 and two phases for [Cu(2+)]/[LDL] ratio of 0.05; (ii) oxidation product formation (cholesteryl ester and phosphatidylcholine hydroperoxides, apo B carbonyl groups) occurred early in the presence of endogenous antioxidants, under both copper oxidation conditions; (iii) apo B carbonylated fragments appeared when antioxidants were totally consumed at [Cu(2+)]/[LDL] ratio of 0.5; and (iv) antioxidant concentrations were stable, oxysterol formation was negligible, and no carbonylated fragment was detected at [Cu(2+)]/[LDL] ratio of 0.05. Depending on the copper/LDL ratio, oxidized LDL differ greatly in the nature of lipid peroxidation product and the degree of apo B fragmentation.     

Comparison of LDL TRAP assay to other tests of antioxidant capacity; Effect of vitamin E and lovastatin treatment

Oxidized low density lipoprotein (LDL) has a major impact in the development of atherosclerosis. Risk for oxidative modification of LDL is usually determined indirectly by measuring the capability of LDL to resist radical insult. We compared three different methods quantifying the antioxidative capacity of LDL ex vivo in dyslipidemic patients with coronary heart disease. Plasma samples were obtained from two double-blinded cross-over trials. The duration of all interventions (placebo, lovastatin 60 mg/day, RRR-α-tocopherol 300 mg/day and lovastatin + RRR-α-tocopherol combined) was 6 weeks. The total radical capturing capacity of LDL (TRAP) in plasma was determined using 2,2-azobis(2,4-dimethyl-valeronitrile) (AMVN)-induced oxidation, and measuring the extinction time of chemiluminescence. TRAP was compared to the variables characterizing formation of conjugated dienes in copper-induced oxidation. Also the initial concentrations and consumption times of reduced α-tocopherol (α-TOH) and ubiquinol in AMVN-induced oxidation were determined.

Repeatability of TRAP was comparable to that of the lag time in conjugated diene formation. Coefficient of variation within TRAP assay was 4.4% and between TRAP assays 5.9%. Tocopherol supplementation produced statistically significant changes in all antioxidant variables except those related to LDL ubiquinol. TRAP increased by 57%, the lag time in conjugated diene formation by 34% and consumption time of α-TOH by 88%. When data of all interventions were included in the analyses, TRAP correlated with the lag time (r = 0.75, p < 10^-6), with LDL α -TOH (r = 0.50, p < 0.001) and with the consumption time of α-TOH (r = 0.58, p < 0.0001). In the baseline data, the associations between different antioxidant variables were weaker. TRAP correlated with the lag time (r = 0.55, p < 0.001) and α-TOH consumption time (r = 0.48, p < 0.05), and inversely with apolipoprotein Al (r = -0.51, p < 0.05). Lag time at the baseline did not correlate with ubiquinol or tocopherol parameters, or with any plasma lipid or lipoprotein levels analyzed. Lovastatin treatment did not significantly affect the antioxidant capacity of LDL. In conclusion, TRAP reflects slightly different properties of LDL compared to the lag time. Thus, LDL TRAP assay may complement the other methods used to quantify the antioxidant capacity of LDL. However, TRAP and the lag time react similarly to vitamin E supplementation.