Evaluation of antioxidants: Scope, limitations and relevance of assays

Peroxidation of lipids, particularly polyunsaturated fatty acid residues (PUFA) of phospholipids and cholesterol esters, is a process of marked implications: it shortens the shelf-life of food and drugs, it causes fragmentation of DNA, it damages cellular membranes and it promotes the genesis of many human diseases. Much effort is therefore devoted to a search for "potent antioxidants", both synthetic and from natural sources, mostly plants. This, in turn, requires a reliable, simple, preferably high throughput assay of the activity of alleged antioxidants. The most commonly used assays are based on measurements of the total antioxidant capacity (TAC) of a solution, as evaluated either by determining the rate of oxidation of the antioxidant or by measuring the protection of an easily determined indicator against oxidation by the antioxidants. The commonly used assays utilized for ranking antioxidants share three common problems: (i) They usually evaluate the effects of those antioxidants that quench free radicals, which constitute only a part of the body's antioxidative network, in which enzymes play the central role. (ii) Both the capacity and potency of antioxidants, as obtained by various methods, do not necessarily correlate with each other. (iii) Most estimates are based on methods conducted in solution and are therefore not necessarily relevant to processes that occur at the lipid-water interfaces in both membranes and micro emulsions (e.g. lipoproteins). Given this "state of art", many researchers, including us, try to develop a method based on the formation of hydroperoxides (LOOH) upon peroxidation of PUFA in lipoproteins or in model membranes, such as liposomes. In these systems, as well as in lipoproteins, the most apparent effect of antioxidants is prolongation of the lag time preceding the propagation of a free radical chain reaction. In fact, under certain conditions both water soluble antioxidants (e.g. vitamin C and urate) and the lipid soluble antioxidant tocopherol (vitamin E), promote or even induce peroxidation. Based on the published data, including our results, we conclude that terms such as 'antioxidative capacity' or 'antioxidative potency' are context-dependent. Furthermore, criteria of the efficacy of antioxidants based on oxidation in solution are not necessarily relevant to the effects of antioxidants on peroxidation in biological systems or model lipid assemblies, because the latter processes occur at water/lipid interfaces. We think that evaluation of antioxidants requires kinetic studies of the biomarker used and that the most relevant characteristic of 'oxidative stress' in the biological context is the kinetics of ex vivo peroxidation of lipids. We therefore propose studying the kinetics of lipid-peroxidation in the absence of the studied antioxidant and in its presence at different antioxidant concentrations. These protocols mean that antioxidants are assayed by methods commonly used to evaluate oxidative stress. The advantage of such evaluation is that it enables quantization of the antioxidants' efficacy in a model of relevance to biological systems. In view of the sensitivity of the lag time preceding peroxidation, we propose studying how much antioxidant is required to double the lag observed prior to rapid peroxidation. The latter quantity (C(2lag)) can be used to express the strength of antioxidants in the relevant system (e.g. LDL, serum or liposomes).     

Seasonal Variation in Low Density Lipoprotein Oxidation and Antioxidant Status

Accumulating evidence indicates that oxidative modification of low-density lipoproteins is atherogenic and that antioxidants may play a role in protection of LDL against oxidation. Several studies have reported a seasonal fluctuation in antioxidant levels, but to date nothing is known about seasonal fluctuations in parameters of oxidizability. We collected blood from 10 volunteers at four different periods over one year (February, May, September and December), and measured the amount of plasma lip ids, plasma antioxidants, lipid and fatty acid composition of the LDL particle, LDL antioxidant content, LDL particle size and oxidation parameters (lag time and propagation rate). No seasonal fluctuation for lag time and propagation rate of copper ion-induced LDL oxidation was found. Small seasonal fluctuations were observed for some determinants of LDL oxidation, e.g. plasma and LDL vitamin E and LDL particle size, and for plasma lipids, plasma and LDL lutein and LDL p-carotene. Fatty acid composition of LDL did not change during the year. The main determinant of oxidation susceptibility was the fatty acid composition of LDL. We conclude that LDL oxidation parameters do not change over the year.     

Russian Article pp. 055–069

The authors studied the action of inhibitor oxidation (antioxidant) reaction in the processes of cell proliferation. The data represented here concerned with the stimulating influence of antioxidants on the growth of microorganisms. Special consideration is given to the increase of biomass yield, productivity and yeast amount per substrate. This work established that synthetic antioxidants are able to carry out functions of natural antioxidants in growing cells.     

Kinetic analysis of copper-induced peroxidation of HDL, autoaccelerated and tocopherol-mediated peroxidation

Comparison of the kinetic profiles of copper-induced peroxidation of HDL and LDL at different copper concentrations reveals that under all the studied experimental conditions HDL is more susceptible to oxidation than LDL. The mechanism responsible for HDL oxidation is a complex function of the copper/HDL ratio and of the tocopherol content of the HDL. At high copper concentrations, the kinetic profiles were similar to those observed for LDL oxidation, namely, relatively rapid accumulation of oxidation products, via an autoaccelerated, noninhibited mechanism, was preceded by an initial "lag phase." Under these conditions, the maximal peroxidation rate (V(max)) of HDL and LDL depended similarly on the molar ratio of bound copper/lipoprotein. Analysis of this dependency in terms of the binding characteristics of copper to lipoprotein, yielded similar dissociation constant (K = 10(-6) M) but different maximal binding capacities for the two lipoproteins (8 Cu(+2)/HDL as compared to 17 Cu(+2)/LDL). Given the size difference between HDL and LDL, these results imply that the maximal surface density of bound copper is at least 2-fold higher for HDL than for LDL. This difference may be responsible for the higher susceptibility of HDL to copper-induced oxidation in the presence of high copper concentrations. At relatively low copper concentrations, the kinetic profile of HDL oxidation was biphasic, similar to but more pronounced than the biphasic kinetics observed for the oxidation of LDL lipids at the same concentration of copper. Our results are consistent with the hypothesis that the first phase of rapid oxidation occurs via a tocopherol-mediated-peroxidation (TMP) mechanism. Accordingly, enrichment of HDL with tocopherol resulted in enhanced accumulation of hydroperoxides during the first phase of copper-induced oxidation. Notably, the maximal accumulation during the first phase decreased upon increasing the ratio of bound copper/HDL. This behavior can be predicted theoretically for peroxidation via a TMP mechanism, in opposition to autoaccelerated peroxidation. The possible pathophysiological significance of these findings is discussed.     

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.     

Ranking antioxidants based on their effect on human serum lipids peroxidation

Evaluation of the activity of antioxidants is commonly based on measurements of the effect of a specific antioxidant on redox reactions conducted in a solution. Given the difference between reactions that occur in homogeneous solutions and those that occur at lipid–water interfaces, as in biological membranes and lipoproteins, the relevance of the commonly-used assays (such as TEAC and ORAC) to the antioxidative activity in biological systems is questionable. The aim of the present investigation is to develop a more relevant assay. Based on our results, we propose an assay based on prolongation of the lag preceding fast peroxidation of serum lipids. The assay employs our previously developed procedure for determination of susceptibility of serum lipids to peroxidation. The effect of antioxidants is expressed in terms of the relative prolongation of the lag preceding peroxidation. It can be considered reliable because it is only marginally dependent on the specific sera used for the assay. The resultant ranking of antioxidants may be expressed either as the relative prolongation of the lag per 1 μM of antioxidant or as the concentration of antioxidant required to double the lag. As expected, the observed ranking order is very different from that reported for TEAC or ORAC assays, undermining the relevance of these assays for oxidation that occurs at interfaces.     

Antioxidant activity: what do we measure?

Inhibition of oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by free radicals generated by decomposition of 2,2'-azobis(2-amidopropane) (ABAP) by antioxidants and biological material was studied. A correlation was found between the ability of various substances to delay the onset of ABTS oxidation and their rapid reduction of the ABTS+* cation radical, and between the ability to reduce the maximal rate of ABTS oxidation and slow reduction of ABTS+*. The length of the lag period of ABTS oxidation was found to be independent of ABTS concentration. Similar decrease of peroxynitrite-induced ABTS+* formation by antioxidants was observed when the antioxidants were added before and after peroxynitrite. All these findings indicate that the main effect of antioxidants in this system is reduction of ABTS+* and not prevention of its formation. Reduction of oxidation products rather than inhibition of their formation may be the predominant mode of action of antioxidants in various assays of antioxidant activity.     

High-throughput fluorescence screening of antioxidative capacity in human serum.

Diphenylhexatriene-labeled phosphatidylcholine and propionic acid have been established as selective fluorescence markers for the continuous determination of oxidation processes in the lipid and aqueous phases of unfractionated human serum. Oxidation of the respective fluorophores leads to a decrease in fluorescence intensity from which the time-dependent degradation of the marker molecule can be determined. The lag times preceding the propagation of oxidation are representative for the antioxidative capacity of the system, which may be influenced by exogenous factors, e.g., the antioxidants from the diet. Supplementation of human serum by quercetin, rutin, vitamin E, vitamin C, or total apple phenolics in vitro led to a decrease in oxidizability depending on the oxidation marker and the hydrophobicity of the antioxidant. Quercetin and vitamin E showed a higher in vitro capacity of protecting lipoproteins against oxidation. In contrast, rutin and vitamin C were more efficient as inhibitors in the aqueous phase. The same effect on serum was found after dietary consumption of apples. This result is in line with the known observation that intake of plant polyphenols leads to an increase in serum levels of hydrophilic antioxidants.     

Kinetics and mechanisms of oxidation of some antioxidants with photochemically generated tert-butoxyl radicals

Dietary antioxidants play an important role in the prevention of several chronic diseases including cardiovascular diseases, cancer, ageing and diabetes. In order to understand the mechanism of oxidation of antioxidants viz., gallic acid (GA), caffeic acid (CA), rosmarinic acid (RA) and chlorogenic acid (CGA), a systematic kinetic study of these antioxidants with photochemically generated tertiary butoxyl (t-BuO) radicals was carried out. The oxidation of antioxidants by t-BuO radicals was followed spectrophotometrically by measuring the absorbance of GA (266 nm), CA (310 nm), RA (324 nm) and CGA (328 nm) at their respective lambda(max). The initial rates of oxidation of antioxidants were calculated from the plot of absorbance vs time and were found to increase with increase in [antioxidant], [t-BuOOH] and light intensity in all the cases. The quantum yields (phi) were calculated from the initial rates of oxidation of antioxidant and the measured light intensity at 254 nm, the wavelength at which the tert-butyl hydroperoxide (t-BuOOH) was activated to radicals. The quantum yields were found to depend on [antioxidant] and [t-BuOOH], and were independent of light intensity. The order with respect to [antioxidant], [t-BuOOH] were found to be fractional whereas order with respect to intensity was one. The order of reactivity was found to be: CA > CGA > RA > GA. The products were identified by mass spectral data. On the basis of kinetic results and product analysis, probable mechanisms were suggested.     

Comparison of the antiradical activity of ionol, components of fresh ginger, and its extracts

The antiradical properties of three samples of ginger (Zingiber officinale R.)—juice from fresh rhizome, essential oil, and extracts (oleoresin)—were studied and compared with the properties of synthetic antioxidant ionol (butylatedhydroxy-toluene, BHT). Reaction antioxidants with stable free 2,2-diphenyl-1-picrylhydrozyl radicals were used as model systems. DPPH equivalents per gram of ginger sample, EC₅₀, and antiradical efficiency (AE) were determined. The EC₅₀ and AE values for ginger oleoresin and BHT were similar. They were the same as those of highly active natural antioxidants, and the values for essential oil and ginger juice were lower by two orders of magnitude. On the base of kinetic parameters, the ginger samples may belong to antiradical compounds with prolonged action.     

Protective activity of plicatin B against human LDL oxidation induced in metal ion-dependent and -independent processes. Experimental and theoretical studies

Oxidation of low-density lipoproteins (LDL) is thought to be a major factor in the pathophysiology of atherosclerosis. Natural antioxidants have been shown to protect LDL from oxidation and to inhibit atherogenic developments in animals. Structurally related prenylated pterocarpans, erybraedin C and bitucarpin A, and the prenylchalcone plicatin B were examined for their ability to inhibit LDL oxidation in vitro. The kinetic profile of peroxidation is characterized by the lag time of oxidation (t_lag), the maximal rate of oxidation (V_max) and the maximal accumulation of oxidation products (OD_max). Specific variation of the set of kinetic parameters by antioxidants may provide important information about the mechanism of inhibitory action of a given compound. At equimolar concentrations (1 μM) the prenylated derivatives tested were found to inhibit 1 μM copper sulphate-induced oxidation of LDL (50 μg protein/ml) in accordance with the following order of activity: plicatin B>erybraedin Cbitucarpin A. Structural aspects, such as hydrogen-donating substituents, their number and arrangement in the aromatic ring moieties, and the prenyl and methoxy substituents, were investigated in order to explain the findings obtained. It is well known that the antioxidant activity of flavonoids is believed to be caused by a combination of transition metal chelation and free-radical-scavenging activities. To investigate these differences we comparatively studied the protective mechanism of plicatin B in copper-dependent or -independent LDL oxidation. The latter was mediated by 2,2’-azo-bis-(2-amidinopropane) dihydrochloride (ABAP). We measured the formation of conjugated dienes (OD_234 nm). Plicatin B (0.2-1.5 μM) delayed the Cu²⁺ (1 μM) promoted oxidation as conjugate diene formation (t_lag) of the LDL by 45.2-123.5 min and reduced V_max by 0.46-0.29 μM/min. In the ABAP (0.2 mM) promoted LDL oxidation t_lag increased by 67.2-110.2 min through plicatin B (0.5-2.5 μM). In experiments in which Cu²⁺ concentrations increased (0.5 - 3 μM) and the amount of plicatin B (1 μM) was maintained constant, a significant decrease in t_lag and an increase in V_max was observed. In this study plicatin B appeared to exhibit a mixed mechanism, interfering with the formation of the radicals by chelating copper involved in the initiation/propagation reaction, but also by scavenging free hydroperoxyl radicals resulting from ABAP thermolysis. In addition, theoretical analysis indicated that plicatin B preferentially established the chelating complex with Cu²⁺, because its affinity value is notably higher (by a factor of 5) than that for Cu⁺.     

Natural and synthetic antioxidants: An updated overview

Abstract

The current understanding of the complex role of ROS in the organism and pathological sequelae of oxidative stress points to the necessity of comprehensive studies of antioxidant reactivities and interactions with cellular constituents. Studies of antioxidants performed within the COST B-35 action has concerned the search for new natural antioxidants, synthesis of new antioxidant compounds and evaluation and elucidation of mechanisms of action of both natural and synthetic antioxidants. Representative studies presented in the review concern antioxidant properties of various kinds of tea, the search for new antioxidants of herbal origin, modification of tocopherols and their use in combination with selenium and properties of two promising groups of synthetic antioxidants: derivatives of stobadine and derivatives of 1,4-dihydropyridine.     

Flavonoid metabolites and susceptibility of rat lipoproteins to oxidation

Flavonoids are ingested with vegetables and beverages and exert a beneficial effect on cardiovascular disease. Studies in animals in vitro and in humans ex vivo on the resistance of lipoproteins to oxidation are not consistent and the mechanisms by which flavonoids protect against atherosclerosis are a matter of debate. In the present study, we investigated the effects of administering diets containing 0.3% (wt/wt) quercetin, 0.3% (wt/wt) catechin, or 35% (vol/wt) dealcoholated red wine (DRW) for 10 days in healthy rats on markers of oxidative damage in lipoproteins and in plasma. The antioxidant levels in low-density lipoproteins (LDL) or the lag phase, oxidation rate, and maximum level of conjugated dienes during ex vivo LDL oxidation did not differ between control and treated rats. Plasma levels of alpha-tocopherol and retinol were similar in all groups. The total antioxidant status of the plasma from rats fed either quercetin or DRW diet was higher than in control rats. Only glucuronide and sulfate compounds of quercetin were detected in plasma from rats fed the quercetin-rich diet, and no flavonoids or their metabolites were detected in plasma or LDL from rats fed the catechin- or the DRW-rich diet. No significant differences in malondialdehyde or in conjugated dienes in plasma were observed. These results indicate that although metabolites from quercetin are present in plasma, they are not detected in lipoproteins and do not modify the level of other antioxidants. In conclusion, in the absence of any pathology or of oxidative stress the intake of quercetin, catechin, or DRW did not protect lipoproteins from oxidation ex vivo.     

Phenolics as potential antioxidant therapeutic agents: mechanism and actions

Accumulating chemical, biochemical, clinical and epidemiological evidence supports the chemoprotective effects of phenolic antioxidants against oxidative stress-mediated disorders. The pharmacological actions of phenolic antioxidants stem mainly from their free radical scavenging and metal chelating properties as well as their effects on cell signaling pathways and on gene expression. The antioxidant capacities of phenolic compounds that are widely distributed in plant-based diets were assessed by the Trolox equivalent antioxidant capacity (TEAC), the ferric reducing antioxidant power (FRAP), the hypochlorite scavenging capacity, the deoxyribose method and the copper-phenanthroline-dependent DNA oxidation assays. Based on the TEAC, FRAP and hypochlorite scavenging data, the observed activity order was: procyanidin dimer > flavanol > flavonol > hydroxycinnamic acids > simple phenolic acids. Among the flavonol aglycones, the antioxidant propensities decrease in the order quercetin, myricetin and kaempferol. Gallic acid and rosmarinic acid were the most potent antioxidants among the simple phenolic and hydroxycinnamic acids, respectively. Ferulic acid displayed the highest inhibitory activity against deoxyribose degradation but no structure–activity relationship could be established for the activities of the phenolic compounds in the deoxyribose assay. The efficacies of the phenolic compounds differ depending on the mechanism of antioxidant action in the respective assay used, with procyanidin dimers and flavan-3-ols showing very potent activities in most of the systems tested. Compared to the physiologically active (glutathione, -tocopherol, ergothioneine) and synthetic (Trolox, BHA, BHT) antioxidants, these compounds exhibited much higher efficacy. Plant-derived phenolics represents good sources of natural antioxidants, however, further investigation on the molecular mechanism of action of these phytochemicals is crucial to the evaluation of their potential as prophylactic agents.     

Effect of exercise intensity and training on antioxidants and cholesterol profile in cyclists

The aim of this work was to evaluate the effect of different intensity of exercise and different training status on antioxidants and cholesterol profile in cyclists. 33 male cyclists (17 amateur and 16 professional cyclists) participated in this study. The amateurs all trained 14 +/- 1 h each week, and their VO(2) max was 62.5 +/- 1.8 ml/Kg x min; the professionals all trained 24 +/- 1 h each week, and their VO(2) max was 80.2 +/- 1.6 ml/Kg x min. Amateurs were submitted to the maximal and submaximal prolonged exercise tests. Professionals were submitted to a mountain stage (170 km) of cycling competition. Serum lipid and cholesterol profile (triglycerides, total cholesterol, HDL-cholesterol, LDL-cholesterol and VLDL-cholesterol) and plasma antioxidant capacity (ascorbic acid, alpha-tocopherol, retinol, beta-carotene and others) were measured before and after exercise tests. Hematological determinations (number of erythrocytes, hematocrit and hemoglobin concentration) and dietary intake were also measured. No significant differences were observed in basal values (before exercise tests) of amateur and professional cyclists. Negligible differences were found between dietary intake of amateur and professional cyclists, and also the results of hematological values showed there was no effect of degree of hydration or dietary intake on blood levels of studied antioxidant and lipid parameters. An increase in plasma levels of vitamin C, vitamin E, triglycerides and VLDL-cholesterol levels, and also a decrease of beta-carotene and LDL-cholesterol. were observed in well-trained professional cyclists after the cycling stage - an endurance exercise--but not in amateur cyclists. Amateur cyclists showed only mild increases in total cholesterol after maximal and submaximal exercise, while a rise in HDL-cholesterol was only observed after maximal exercise; none of these changes were observed in professional cyclists. Plasma levels of antioxidant vitamins and carotenes, and also serum lipids, total cholesterol and lipoprotein-cholesterol showed an overall response to exercise, and their increase and/or decrease must be explained as a consequence of the different training status of sportsmen and intensity and duration of exercise tests.     

Lipoprotein lipase-catalyzed hydrolysis of phosphatidylcholine of guinea pig very low density lipoproteins and discoidal complexes of phospholipid and apolipoprotein: effect of apolipoprotein C-II on the catalytic mechanism

To elucidate the mechanism by which apolipoprotein C-II (apoC-II) enhances the activity of lipoprotein lipase (LpL), discoidal phospholipid complexes were prepared with apoC-III and di[(14)C]palmitoyl phosphatidylcholine (DPPC) and containing various amounts of apoC-II. The rate of DPPC hydrolysis catalyzed by purified bovine milk LpL was determined on the isolated complexes. The rate of hydrolysis was optimal at pH 8.0. Analysis of enzyme kinetic data over a range of phospholipid concentrations revealed that the major effect of apoC-II was to increase the maximal velocity (V(max)) some 50-fold with a limited effect on the Michaelis constant (K(m)). V(max) of the apoC-III complex containing no apoC-II was 9.2 nmol/min per mg LpL vs. 482 nmol/min per mg LpL for the complex containing only apoC-II. The effect of apoC-II on enzyme kinetic parameters for LpL-catalyzed hydrolysis of DPPC complexes was compared to that on the parameters for hydrolysis of DPPC and trioleoylglycerol incorporated into guinea pig very low density lipoproteins (VLDL(p)) which lack the equivalent of human apoC-II. Tri[(3)H]oleoylglycerol-labeled VLDL(p) were obtained by perfusion of guinea pig liver with [(3)H]oleic acid. Di[(14)C]palmitoyl phosphatidylcholine was incorporated into the VLDL(p) by incubation of VLDL(p) with sonicated vesicles of di[(14)C]palmitoyl phosphatidylcholine and purified bovine liver phosphatidylcholine exchange protein. The rates of LpL-catalyzed hydrolysis of trioleoylglycerol and DPPC were determined at pH 7.4 and 8.5 in the presence and absence of apoC-II. In the presence of apoC-II, the V(max) for DPPC hydrolysis in guinea pig VLDL(p) increased at both pH 7.4 and pH 8.5 (2.4- and 3.2-fold, respectively); the value of K(m) did not change at either pH (0.23 mm). On the other hand, the kinetic value of K(m) for triacylglycerol hydrolysis in the presence of apoC-II decreased at both pH 7.4 (3.05 vs. 0.54 mm) and pH 8.5 (2.73 vs. 0.62 mm). These kinetic studies suggest that apoC-II enhances phospholipid hydrolysis by LpL in apoC-III-DPPC discoidal complexes and VLDL(p) mainly by increasing the V(max) of the enzyme for the substrates, whereas the activator protein primarily causes a decrease in the apparent K(m) for triacylglycerol hydrolysis.-Shirai, K., T. J. Fitzharris, M. Shinomiya, H. G. Muntz, J. A. K. Harmony, R. L. Jackson and D. M. Quinn. Lipoprotein lipase-catalyzed hydrolysis of phosphatidylcholine of guinea pig very low density lipoproteins and discoidal complexes of phospholipid and apolipoprotein: effect of apolipoprotein C-II on the catalytic mechanism.     

Effects of an antioxidant-rich juice (sea buckthorn) on risk factors for coronary heart disease in humans

There is increasing evidence to support the hypothesis that free radical-mediated oxidative processes contribute to atherogenesis. More recently the ability of antioxidant nutrients to affect cell response and gene expression has been reported in vitro, providing a novel mechanistic perspective for the biological activity of antioxidants. Sea buckthorn (Hippopha? rhamnoides L.) is a rich source of antioxidants both aqueous and lipophilic, as well as polyunsaturated fatty acids. The objective of the study was to characterize the antioxidant profile of Sea buckthorn juice (SBJ) and to evaluate its effect on plasma lipids, LDL oxidation, platelet aggregation and plasma soluble cell adhesion protein concentration. Twenty healthy male volunteers were given either a placebo or SBJ for 8 weeks. Additional daily intakes of vitamin C, alpha-tocopherol, beta-carotene and flavonoids through SBJ supplementation were 462, 3.2, 1.0 and 355 mg respectively. There were no significant changes in plasma total cholesterol, LDL-C, platelet aggregation or plasma intercellular cell adhesion molecule 1 (ICAM-1) levels between treatment groups. Although not significant, a 20% and 17% increase in plasma HDL-C and triacylglycerol (TAG) concentrations were observed. SBJ supplementation also resulted in a moderate decrease in the susceptibility of LDL to oxidation.     

Kinetic behaviour of free lipase and mica-based immobilized lipase catalyzing the synthesis of sugar esters

The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K(m) and V(max), were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K(m) values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V(max,app)>V(max)). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values.     

Features of lipid peroxidation and antioxidant defense of hens in ontogenesis and as a result of antioxidant action

The peculiarities of lipid peroxidation (LPO) and antioxidant defence of Leghorn hens in ontogenesis were studied. The action of stibyl biogeneous preparation in complex with dymethylsulfoxide and distinol synthetic preparation on hen organism antioxidant defence parameters were estimated. The results show that the antioxidant preparations don't render any effect on CAT and GPX activities, meanwhile SOD activity declines under antioxidant influence in the first period of feeding (7-35 days) but increases in the repeated administration admixtures (98-126 days) in comparison with the control group. Administration of antioxidants with fodder is accompanied by increasing the carotinoids, vitamins A and E content and mM levels decrease in hepatic tissue.     

In vitro inhibition by estrogens of the oxidative modifications of human lipoproteins

Estrogens exert protective actions against atherosclerosis, part of these effects having been ascribed to their antioxidant properties. The aim of this work was to assess the ability of estrogens to prevent the oxidative modifications of low density lipoproteins (LDL) and other plasma lipoprotein fractions whose relationship with atherosclerosis has been less studied. For this purpose, different estrogen compounds were used: natural and synthetic estrogens, and catecholestrogens. The molecules were added in vitro to human LDL and very low density lipoproteins (VLDL) in the presence of Cu2+. The lipoprotein oxidative modifications were determined by measuring the formation of thiobarbituric acid reactive substances, the appearance of conjugated dienes and the degradation of tryptophan groups from the apoproteins. In VLDL, 2-hydroxyestradiol and diethylstilbestrol exerted potent antioxidant effects similar to those found for alpha-tocopherol and probucol. 17beta-Estradiol and 4-hydroxyestradiol also prevented VLDL oxidation, but to a lesser extent. When LDL were used, estrogens similarly exerted antioxidant actions, 2-hydroxyestradiol being the most potent inhibitor. These results show that estrogens, whose antioxidant actions have been demonstrated in other experimental models, also possess the ability to prevent in vitro the oxidative modifications of human plasma LDL and VLDL.