Conjugated Diene Production Research Articles

A one-step method of 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid synthesis by soybean lipoxygenase

A product of lipoxygenase (LOX) oxidation of docosahexaenoic acid (DHA), 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid [10,17(S)-diH(P)DHA] was obtained through various reaction pathways that involved DHA, 17(S)-hydro(pero)xydocosahexa-4Z,7Z,11Z,13Z,15E,19Z-enoic acid [17(S)-H(P)DHA], soybean lipoxygenase (sLOX), and potato tuber lipoxygenase (ptLOX) in various combinations. The structure of the product was confirmed by HPLC, ultraviolet (UV) light spectrometry, GC-MS, tandem MS, and NMR spectroscopy. It has been found that 10,17(S)-diH(P)DHA formed by sLOX through direct oxidation of either DHA or 17(S)-H(P)DHA was apparently identical to the product of ptLOX oxidation of the latter. The sLOX- and ptLOX-derived samples of 10,17-diHDHAs coeluted under the conditions of normal, reverse, and chiral phase HPLC analyses, displayed identical UV absorption spectra with maxima at 260, 270, and 280 nm, and had similar one-dimensional and two-dimensional proton NMR spectra. Analysis of their NMR spectra led to the conclusion that 10,17-diHDHA formed by sLOX had solely 11E,13Z,15E configuration of the conjugated triene fragment, which was identical to the previously published structure of its ptLOX-derived counterpart. Based on the cis,trans geometry of the reaction products, the conclusion is made that in the tested conditions sLOX catalyzed direct double dioxygenation of DHA. Compared with the previously described two-enzyme method that involved sLOX and ptLOX, the current simplified one-enzyme procedure uses only sLOX as the catalyst of both dioxygenation steps.

Antrodia Camphoratain Submerged Culture Protects Low Density Lipoproteins Against Oxidative Modification

Antrodia camphorata is well known in Taiwan as a traditional Chinese medicine. In this study, we have investigated the antioxidant properties of a fermented culture broth of Antrodia camphorata (FCBA) and the aqueous extracts of mycelia from Antrodia camphorata (AEMA) on the oxidative modification of human low-density lipoproteins (LDL), as induced by either copper sulfate (CuSO(4)) or 2,2'-azo-bis(2-amidinopropane) hydrochloride (AAPH). Under such oxidant stress, FCBA and AEMA appear to possess antioxidant properties with respect to oxidation of LDL in a time-and concentration-dependent manner, as assessed by inhibition of thiobarbituric acid-reactive substances (TBARS) formation, conjugated diene production, and cholesterol degradation of oxidized LDL. In addition, both FCBA and AEMA exhibited a remarkable ability to rescue the relative electrophoretic mobility and fragmentation of the Apo B moiety of the oxidized LDL. Furthermore, FCBA and AEMA effectively protected the endothelial cells from the damaging effects of the CuSO(4)-oxidized LDL. Our findings suggest that the antioxidant properties of Antrodia camphorata may also provide effective protection from atherosclerosis.

Antioxidant Activities of a New Lignan and a Neolignan from Saururus chinensis.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Effect of homocysteine, folates, and cobalamin on endothelial cell‐ and copper‐induced LDL oxidation

Oxidation of LDL contributes to endothelial dysfunction and atherosclerosis. This process could be associated with hyperhomocysteinemia, a condition that can be reduced after folic acid treatment. Because a reduction in LDL oxidation may improve endothelial function, we studied the effect of some vitamins (folic acid, 5-methyltetrahydrofolic acid, and vitamin B-12) on LDL oxidation, either in the presence or absence of homocysteine. For this purpose, two in vitro systems were used: an endothelial cell-catalyzed LDL oxidation system and a cell-free copper-initiated LDL oxidation system. The kinetics of copper-catalyzed LDL oxidation was determined by continuous monitoring of the production of conjugated dienes in the reaction medium. TBARS production, a parameter of lipid peroxidation, was also evaluated. In both in vitro systems, only 5-methyltetrahydrofolic acid was able to decrease TBARS production in a concentration-dependent manner, independently of the presence or absence of homocysteine. In the copper-induced LDL oxidation system, vitamin B-12 and 5-methyltetrahydrofolic acid increased the lag time of conjugated diene production by 25 and 47%, respectively, suggesting that both vitamins in this system had antioxidant properties. Folic acid was unable to show antioxidant properties when included in either in vitro system. The results demonstrate that 5-methyltetrahydrofolic acid and vitamin B-12 are important protective agents against LDL oxidative modifications.

Susceptibility to Hypoxia/Reoxygenation of Aged Rat Cardiomyocytes and Its Modulation by Selenium Supplementation

Since in the aged heart an increased basal production of reactive oxygen species (ROS) has been demonstrated, and the resistance to ROS attack could be ameliorated by antioxidant supplementation, we verified the protective effect of selenium, as sodium selenite (SS) or seleno methionine (SM), in cultured rat cardiomyocytes aged in vitro. In normoxia, glutathione peroxidase (GPx) activity and total antioxidant activity were higher in old than in young cardiomyocytes, suggesting the existence of a compensatory increase of antioxidant defenses. When aged cells were submitted to hypoxia/reoxygenation, GPx activity was not modified; while total antioxidant activity decreased, conjugated diene level increased. Selenium supplementation, particularly as SM, was able to increase GPx, and consequently total antioxidant activity, and to decrease conjugated diene production. The observed ability of selenium supplementation to protect aged cardiomyocytes from hypoxia/reoxygenation damage underlines the importance of an optimal selenium dietary intake, particularly in the elderly.

Antioxidant activities of a new lignan and a neolignan from Saururus chinensis

A new diarylbutane lignan, 2′-hydroxy dihydroguaiaretic acid ( 4), and a known 8- O-4′-type neolignan, machilin D ( 5), were isolated from the ethyl acetate extracts of the underground parts of Saururus chinensis. Compounds 4 and 5 exhibited low-density lipoprotein (LDL)-antioxidant activity in the thiobarbituric acid-reactive substances (TBARS) assay ( 4: IC 50 = 3.3 μM and 5: IC 50 = 3.8 μM), the lag time of conjugated diene production, the relative electrophoretic mobility (REM) of ox-LDL, the apoB-100 fragmentation on copper-mediated LDL oxidation and the macrophage-mediated LDL oxidation, and radical DPPH scavenging activity.

Antioxidant activity of dulse ( Palmaria palmata) extract evaluated in vitro

Palmaria palmata (dulse) is traditionally consumed as a snack food and garnish; but, little is known about its potential as a source of antioxidants. A 1-butanol soluble fraction extracted from dulse exhibited OH scavenging activity ± EDTA (non-site and site specific activity) in a deoxyribose assay. EC 50 concentrations of dulse extract to quench DPPH and ABTS + free radicals were 12.5 and 29.5 mg/ml. Dulse extract inhibited ( p < 0.05) conjugated diene production in a linoleic acid emulsion at 24, 48 and 52 h, 38 °C; and inhibited ( p = 0.044) thiobarbituric acid reactive substances (TBARS) production at 52 h. One milligram dulse extract exhibited reducing activity = 9.68 μg l-ascorbic acid and total polyphenol content = 10.3 μg gallic acid; the dulse extract did not chelate transition metal ions. The antioxidant activity of the dulse extract was associated with aqueous/alcohol-soluble compounds characterized by phenolic functional groups with reducing activity.

Novel 3,5‐Diaryl Pyrazolines and Pyrazole as Low‐Density Lipoprotein (LDL) Oxidation Inhibitor.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Thyroid hormone (T 3) and its acetic derivative (TA 3) protect low-density lipoproteins from oxidation by different mechanisms

Triiodothyronine (T 3) and triiodothyroacetic acid (TA 3) 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, TA 3 is more antioxidant than T 3 on LDL oxidation induced by copper ions (Cu 2+), 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 Cu 2+ (0.5–4 μM) or AAPH (0.25–2 mM) concentrations in the presence of T 3, TA 3, butylated hydroxytoluene (BHT) (a free radical scavenger) or ethylenediaminetetracetic acid (EDTA) (a metal chelator). From the kinetics were estimated: length of the lag phase ( T lag), maximum velocity of conjugated diene production ( V max), and maximum amount of generated dienes ( D max). 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 Cu 2+ 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. T 3 increased LDL-copper whereas TA 3 slightly decreased it. The whole data suggest that T 3 and TA 3 are free radical scavengers that also differently disturb LDL-copper binding, an essential step for LDL lipid peroxidation. The most likely mechanisms are that T 3 induces new copper binding sites inside the LDL particle, increasing the LDL-copper amount but in a redox-inactive form, whereas TA 3 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.

Novel 3,5-diaryl pyrazolines and pyrazole as low-density lipoprotein (LDL) oxidation inhibitor

Compounds 4a– j and 5 were synthesized by cyclocondensation of 3a– j and hydrazine and showed significant LDL-antioxidant activities in the TBARS assay, the lag time of conjugated diene production, the relative electrophoretic mobility (REM) of ox-LDL, the apoB-100 fragmentation, and the macrophage-mediated LDL oxidation. Among compounds 4a– j and 5, 4a was found to be the most active compound as an inhibitor of LDL oxidation and 4a (IC 50 = 0.1 μM) was 6-fold more potent than probucol (IC 50 = 0.6 μM) in the TBARS assay.

HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages

Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy ( r) by DPH probe where lower ( r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid (( r): 0.263±0.001, 0.279±0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) (( r): 0.206±0.001) ( P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes (( r): 0.231±0.001, 0.243±0.002, respectively) than those incubated with LP-HDL and nat-HDL (( r): 0.223±0.001) ( P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy ( r) and free cholesterol efflux from these cells (−0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL.

Selenium supplementation can protect cultured rat cardiomyocytes from hypoxia/reoxygenation damage.

The possibility of enhancing glutathione peroxidase (GPx) activity and cytosolic total antioxidant activity (TAA) in normoxia and hypoxia/reoxygenation (H/R) by the supplementation of different concentrations of sodium selenite (SS) or selenomethionine (SM) was investigated in cultured rat cardiomyocytes. To assess the entity of oxidative stress due to H/R, levels of conjugated dienes containing lipids were determined. In normoxia, GPx activity and TAA increased in parallel with the increase in SS and SM supplementation. H/R did not influence GPx activity but lowered TAA; both SS and SM supplementations were effective in increasing GPx activity, the most effective concentration being 1 microM. At this SS and SM concentration, TAA returned to a normoxic value. Conjugated diene production, increased by H/R, was reduced by SS and SM supplementation, the 1 microM concentration appearing to be the most effective one. According to these data Se supplementation represents another possibility to counteract oxidative damage in the myocardium.

Doxorubicin induces early lipid peroxidation associated with changes in glucose transport in cultured cardiomyocytes

Doxorubicin (DOX) has not only chronic, but also acute toxic effects in the heart, ascribed to the generation of reactive oxygen species (ROS). Focusing on the DOX-induced early biochemical changes in rat cardiomyocytes, we demonstrated that lipid peroxidation is an early event, in fact conjugated diene production increased after 1-h DOX exposure, while cell damage, evaluated as lactate dehydrogenase (LDH) release, was observed only later, when at least one third of the cell antioxidant defences were consumed. Cell pre-treatment with α-tocopherol (TC) inhibited both conjugated diene production and LDH release. In cardiomyocytes, DOX treatment caused a maximal increase in glucose uptake at 1 h, demonstrating that glucose transport may represent an early target for DOX. At longer times, as the cell damage become significant, the glucose uptake stimulation diminished. Immunoblotting of glucose transporter isoform GLUT1 in membranes after 1-h DOX exposure revealed an increase in GLUT1 amount similar to the increase in transport activity; both effects were inhibited by αTC. Early lipid peroxidation evokes an adaptive response resulting in an increased glucose uptake, presumably to restore cellular energy. The regulation of nutrient transport mechanisms in cardiomyocytes may be considered an early event in the development of the cardiotoxic effects of the anthracycline.

Green tea extracts can counteract the modification of fatty acid composition induced by doxorubicin in cultured cardiomyocytes

Doxorubicin cardiotoxicity is associated with the generation of free radicals, and involves not only lipid peroxidation but also a decreased biosynthesis of highly unsaturated fatty acids, leading to significant modification in cardiomyocyte fatty acid composition. We have evaluated whether naturally occurring antioxidants could counteract this side-effect. Green tea is an excellent source of catechins; we supplemented cultured rat cardiomyocytes with different green tea extracts to relate their catechin content and composition to their ability in protecting cells against doxorubicin-induced damage. The determination of total lipid fatty acid composition, of conjugated diene production (indicator of lipid peroxidation), and of lactate dehydrogenase release revealed that supplementation with tea extracts could counteract significant modifications in the fatty acyl pattern due to doxorubicin exposure, although to different extents. These differences could be ascribed to the different total catechin content and to qualitative differences among the tea extracts, determined by HPLC analysis.

Lipid Peroxidation in Rat Brain During Focal Cerebral Ischemia: Prevention of Malondialdehyde and Lipid Conjugated Diene Production by a Novel Antiepileptic, Lamotrigine

The effects of Lamotrigine (LTG) which blocks ischemia induced glutamate (Glu) release, on lipid peroxidation have been evaluated in cortical and cerebellar tissues of rat brain during focal cerebral ischemia. A total of 45 rats were randomly assigned into one of four groups; sham operated animals as controls, animals subjected to middle cerebral artery occlusion (MCAO) and treatment groups with LTG (20 mg/kg i.p.) either 30 min before or just after MCAO. Changes in lipid peroxides were expressed as nanomoles of malondialdehyde (MDA) and conjugated diene (CD) per milligram of protein. MDA values following 60 min of ischemia relative to contralateral cortex and CD levels in 0, 10 and 60 min of ischemia were found to be higher in the ipsilateral cortex than those in the contralateral cortex. On the other hand, contralateral cerebellar MDA levels after 0 and 60 min of ischemia and CD levels after 0, 10 and 60 min of ischemia were higher than those in the ipsilateral cerebellum. Pharmacological inhibition of Glu release significantly decreased the MDA and CD production in both cortex and cerebellum. Pre- or post-ischemic administration of LTG did not significantly change CD levels, but MDA levels in contralateral cortex were found to be significantly decreased than those in ischemic cortex in both pre- and post-treated group.

Green tea protection of hypoxia/reoxygenation injury in cultured cardiac cells

Antioxidant-rich diets exert a protective effect in diseases involving oxidative damage. Among dietary components, green tea is an excellent source of antioxidants. In this study, cultured neonatal rat cardiomyocytes were used to clarify the protective effect of a green tea extract on cell damage and lipid peroxidation induced by different periods of hypoxia followed by reoxigenation. Cultures of neonatal rat cardiomyocytes were exposed to 2–8 hr hypoxia, eventually followed by reoxygenation, in the absence or presence of α-tocopherol or green tea. LDH release and the production of conjugated diene lipids were measured, and appeared linearly related to the duration of hypoxia. During hypoxia, both LDH release and conjugated diene production were reduced by α-tocopherol and, in a dose dependent manner, by green tea, the 50 μg/ml being the most effective dose. Reoxygenation caused no further increase in LDH leakage, while it caused a significant increase in conjugate dienes, which absolute value was lower in antioxidant supplemented cells. Anyway, the ratio between conjugated diene production after hypoxia and after reoxygenation was similar in all groups, indicating that the severity of free radical-induced reoxygenation injury is proportional to the severity of previous hypoxic injury. Since hypoxic damage is reduced by α-tocopherol and green tea, our data suggest that any nutritional intervention to attenuate reoxygenation injury must be directed toward the attenuation of the hypoxic injury. Therefore, recommendations about a high dietary intake of antioxidants may be useful not only in the prevention, but also in the reduction of cardiac injury following ischemia.

Quantity versus quality of LDL cholesterol in patients with familial hypercholesterolemia—which is more important?

Background: The aim of this study was to determine the effects of low-density lipoprotein (LDL) particle size and composition on the susceptibility of LDL to oxidation in subjects with Familial Hypercholesterolemia (FH). Methods: LDL was isolated from 20 FH homozygotes, 20 FH heterozygotes and 20 normal controls. Susceptibility of LDL to ex vivo copper-mediated oxidation was assessed by measuring conjugated diene production at 234 nm. Other factors known to influence LDL oxidation, namely particle size, vitamin E levels, and fatty acid composition of the LDL particles were also measured. Results: The mean duration of the lag phase was 1.42-fold longer in the FH homozygotes, and 1.21-fold longer in the FH heterozygotes than in the normal controls. LDL particle size was significantly larger in the FH homozygotes (26.45±0.37 nm) and FH heterozygotes (26.01±0.40 nm) compared to the normal control group (25.17±0.39 nm). LDL vitamin E concentrations, when expressed relative to LDL cholesterol concentrations, were similar in all the groups. In addition, no significant differences were observed in the total saturated, monounsaturated or polyunsaturated fatty acid content of LDL in the three groups of subjects. Conclusion: These results suggest that it is the great excess in LDL quantity, rather than LDL ‘quality’, that is responsible for the severe and premature atherosclerosis in patients with FH.

Enhanced Susceptibility to Oxidation and Diminished Vitamin E Content of LDL from Patients with Stable Coronary Artery Disease

Convincing evidence points to oxidative modification of LDL as an important trigger in a complex chain of events leading to atherosclerosis. We investigated the occurrence of enhanced susceptibility of LDL to oxidation and decreased vitamin E concentration in LDL as additional risk factors promoting atherosclerosis among patients with established coronary artery disease (CAD). We examined 132 patients with angiographically confirmed CAD and compared them with 111 healthy control individuals. We measured conjugated diene production to assess susceptibility of LDL to copper-mediated oxidation. Vitamin E content of LDL was measured by HPLC. The mean lag time of LDL oxidation and LDL alpha-tocopherol/LDL-cholesterol ratio were lower in the patients with CAD (55 +/- 14 min and 2.4 +/- 1.0 mmol/mmol) than in the controls (63 +/- 13 min and 2.9 +/- 1.1 mmol/mmol; P <0.0001 and <0.001, respectively). Multiple stepwise regression analysis demonstrated the lag time (odds ratio, 1.96; 95% confidence interval, 1.34-2.87; P <0.0001) and concentration of vitamin E in LDL (odds ratio, 1.65; 95% confidence interval, 1.16-2.33; P <0.005) as independent determinants of CAD. Significant inverse Spearman rank correlations were found between lag time (r = -0.285; P <0.001) or concentration of vitamin E in LDL (r = -0.197; P <0.002) and severity of CAD. Lag times were not significantly correlated with serum C-reactive protein or ferritin. Our data suggest that a short LDL oxidation lag time and a low concentration of vitamin E in LDL might be independent coronary risk factors for stable CAD in Iranian people.

Specific antioxidant activity of caffeoyl derivatives and other natural phenolic compounds: LDL protection against oxidation and decrease in the proinflammatory lysophosphatidylcholine production.

Specific antioxidant activity (SAA) (i.e., activity related to the molar or gallic acid equivalent amount of antioxidant) of natural polyphenolic mixtures or pure phenolic compounds was studied using their capacity to delay the conjugated diene production brought about by in vitro LDL copper-mediated or AAPH-mediated oxidation. The cinnamic acid series (caffeic, sinapic, ferulic acids) displayed a constant SAA over a large range of concentrations, whereas the benzoic acid series (gallic and protocatechuic acids) showed much higher SAA at low concentrations. The natural phenolic mixtures had a constant SAA. The highest SAA was obtained with caffeoyl esters (caffeoylquinic, rosmarinic, and caffeoyltartaric acids) and catechin for the copper-oxidation and the AAPH-oxidation system, respectively. Phenolic mixtures and acids delayed vitamin E depletion and decreased proinflammatory lysophosphatidylcholine production. As with polyphenols, probucol delayed lysophosphatidylcholine and conjugated dienes production, at higher concentrations, but was not effective at preventing vitamin E depletion. Polyphenols prevent the oxidation of LDL and its constituents (vitamin E, phosphatidylcholine), which is compatible with an antiinflammatory and antiatherosclerotic role in pathophysiological conditions.

Daily cocoa intake reduces the susceptibility of low-density lipoprotein to oxidation as demonstrated in healthy human volunteers

Nine male volunteers were given 36 g of cocoa powder (containing 2610 mg of polyphenols) per day with sugar and 6 volunteers received an equivalent amount of sugar for 2 weeks. Conjugated diene production in LDL induced by 2-2′ azobis(4-methoxy-2,4-dimethyl-valeronitrile) (V-70) and copper ion were evaluated. The lag time was significantly prolonged at 1 and 2 weeks in V-70 and at 2 weeks in copper ion after cocoa powder consumption. The level of excretion of epicatechin in urine was significantly higher in the cocoa group than that in the control group. In conclusion, the antioxidants in cocoa powder might be absorbed and increase the resistance of human LDL to oxidation.