Chemoattractant efficacy: oxidation of stimulus by responding cells

Although all human neutrophils have receptors for formyl-methionyl-leucyl-phenylalanine, only 20-30% migrate in vitro at optimal attractant concentrations. Since this attractant can be oxidized by myeloperoxidase from stimulated neutrophils, we determined if its efficacy was increased by protection from oxidation. Efficacy was increased by reducing agents and by molecules with oxidizable sulfur, 10(-5) M methionine or 1.5 X 10(-6) M albumin. The antioxidant effect was on the attractant, not the cells: albumin in the cell compartment did not increase responses. Furthermore, antioxidants had no effect on efficacy of fNle-Leu-Phe-Nle-Tyr-Lys, an attractant that also stimulated superoxide release but had no oxidizable sulfur.     

Assignment of endogenous substrates to enzymes by global metabolite profiling

Enzymes regulate biological processes through the conversion of specific substrates to products. Therefore, of fundamental interest for every enzyme is the elucidation of its natural substrates. Here, we describe a general strategy for identifying endogenous substrates of enzymes by untargeted liquid chromatography-mass spectrometry (LC-MS) analysis of tissue metabolomes from wild-type and enzyme-inactivated organisms. We use this method to discover several brain lipids regulated by the mammalian enzyme fatty acid amide hydrolase (FAAH) in vivo, including known signaling molecules (e.g., the endogenous cannabinoid anandamide) and a novel family of nervous system-enriched natural products, the taurine-conjugated fatty acids. Remarkably, the relative hydrolytic activity that FAAH exhibited for lipid metabolites in vitro was not predictive of the identity of specific FAAH substrates in vivo. Thus, global metabolite profiling establishes unanticipated connections between the proteome and metabolome that enable assignment of an enzyme's unique biochemical functions in vivo.     

Postprandial oxidative stress

Consumption of a meal containing oxidized and oxidizable lipids gives rise to an increased plasma concentration of lipid hydroperoxides, detectable by a sensitive chemiluminescence procedure. This is associated with increased susceptibility of LDL to oxidation, apparently due a structural perturbation at the particle surface brought about by lipid oxidation products. The postprandial modification of LDL is at least partially accounted for by an increase of LDL-, a subfraction containing lipid oxidation products where apoprotein-B-100 (apoB-100) is denatured. Consuming the meal with a suitable source of antioxidants, such as those found in red wine, minimizes this postprandial oxidative stress. The inhibition of peroxidation of lipids present in the meal during digestion is a possible mechanism for the observed protection of LDL. The in vivo oxidatively modified LDL- has numerous features that correspond to the atherogenic minimally modified LDL produced in vitro. These modified particles could account for a relevant link between nutrition and early biological processes that foster the development of atherosclerosis.     

Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays

Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specific detection of compounds with alkyne or azido groups. Here we describe the use of alkyne lipids as substrates for in vitro enzymatic assays of lipid modifying enzymes. The small alkyne moiety is introduced synthetically at the terminus of the hydrocarbon chain of various substrate lipids. After the assay, the label is click-reacted with the azide-bearing fluorogenic dye 3-azido-7-hydroxycoumarin, followed by the separation of the lipid mix by thin-layer chromatography and fluorescence detection, resulting in high sensitivity and wide-range linearity. Kinetic analyses using alkyne-labeled substrates for lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases, and ceramide synthases resulted in Michaelis-Menten constants similar to those for radiolabeled or natural substrates. We tested additional alkyne substrates for several hydrolases and acyltransferases in lipid metabolism. In this pilot study we establish alkyne lipids as a new class of convenient substrates for in vitro enzymatic assays.     

How to Characterize a Biological Antioxidant

An antioxidant is a substance that, when present at low concentrations compared to those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate. Many substances have been suggested to act as antioxidants in vivo, but few have been proved to do so. The present review addresses the criteria necessary to evaluate a proposed antioxidant activity. Simple methods for assessing the possibility of physiologically-feasible scavenging of important biological oxidants (superoxide, hydrogen peroxide, hydroxyl radical, hypochlorous acid, haem-associated ferryl species, radicals derived from activated phagocytes, and peroxyl radicals, both lipid-soluble and water-soluble) are presented, and the appropriate control experiments are described. Methods that may be used to gain evidence that a compound actually does function as an antioxidant in vivo are discussed. A review of the pro-oxidant and anti-oxidant properties of ascorbic acid that have been reported in the literature leads to the conclusion that this compound acts as an antioxidant in vivo under most circumstances.     

Relevance of apple polyphenols as antioxidants in human plasma: contrasting in vitro and in vivo effects

Apples are a major source of flavonoids in the Western diet, and flavonoid-rich foods may help protect against chronic diseases by antioxidant mechanisms. In the present study we investigated: (1) the antioxidant capacity of representative apple polyphenols and their contribution to the total antioxidant capacity of apple extracts; (2) the effects of adding apple extract to human plasma in vitro on oxidation of endogenous antioxidants and lipids; and (3) the effects of apple consumption by humans on ex vivo oxidation of plasma antioxidants and lipids. We found that the apple-contained flavonols and flavanols, quercetin, rutin, (-)-epicatechin, and (+)-catechin, had a higher antioxidant capacity than the dihydrochalcones, phloridzin and phloretin, and the hydroxycinnamate, chlorogenic acid. However, together these apple polyphenols contributed less than 20% to the total antioxidant capacity of aqueous apple extracts. When human plasma was exposed to a constant flux of aqueous peroxyl radicals, endogenous ascorbate (70.0 +/- 10.3 microM) was oxidized within 45 min of incubation, while endogenous urate (375 +/- 40 microM) and alpha-tocopherol (24.7 +/- 1.2 microM) were oxidized after ascorbate. Addition of 7.1 or 14.3 micrograms/ml total phenols of apple extract did not protect ascorbate from oxidation, but increased the half-life (t1/2) of urate from 136 +/- 15 to 192 +/- 16 and 208 +/- 23 min, respectively (p < 0.05 each), and t1/2 of alpha-tocopherol from 141 +/- 18 to 164 +/- 8 min (p = ns) and 188 +/- 8 min (p < 0.05). Lipid peroxidation started after ascorbate depletion, and addition of apple extract increased the lag time preceding detectable lipid peroxidation from 36.3 +/- 3.7 to 50.9 +/- 2.7 min (p < 0.05) and 70.4 +/- 4.2 min (p < 0.001). However, when six healthy volunteers ate five apples and plasma was obtained up to 4 h after apple consumption, no significant increases in the resistance to oxidation of endogenous urate, alpha-tocopherol, and lipids were found. Thus, despite the high antioxidant capacity of individual apple polyphenols and apple extracts and the significant antioxidant effects of apple extract added to human plasma in vitro, ingestion of large amounts of apples by humans does not appear to result in equivalent in vivo antioxidant effects of apple polyphenols.     

Characterization of the oxidation products of BO-653 formed during peroxyl radical-mediated oxidation of human plasma

4,6-Di-tert-butyl-2,3-dihydro-2,2-dipentyl-5-benzofuranol (BO-653) is a novel antioxidant synthesized by theoretical findings and considerations. Here we report on the aqueous peroxyl radical-induced oxidation of human plasma in the presence of BO-653. When BO-653 was given to healthy human subjects at 400 mg twice daily for 28 days, lipids in the resulting plasma were protected from oxidation compared with lipids present in plasma from subjects receiving placebo. Similarly, BO-653 added in vitro at 50 muM inhibited the peroxyl radical-induced accumulation of cholesteryl ester hydroperoxides that occurred in the presence of alpha-tocopherol, although BO-653 did not decrease the rate of consumption of ascorbate, albumin-bound bilirubin, and uric acid. The antioxidant action of in vivo and in vitro added BO-653 was associated with the formation of two major reaction products of BO-653, the structures of which were elucidated by mass spectrometry and nuclear magnetic resonance analyses. The products were identified as stereoisomers of dioxybis(4,6-di-tert.-butyl-2,3,5,7a-tetrahydro-2,2-dipentylbenzofuran-5-one). These dialkylperoxides of BO-653 might be useful markers to assess the antioxidant function of BO-653 in biological systems in vivo.     

Synthesis and Antioxidant Capacity of Some Derivatives of Sesamol at the C-6 Position

Several synthetic approaches (aminomethylation, alkylation, condensation, etc.) have been used to synthesize derivatives based on the sesamol ( 1 ), natural phenol. The set of methods, including the study of antioxidant activity (AOA) by the ability to inhibit the initiated oxidation of animal lipids, radical scavenging activity, Fe²⁺-chelation ability, as well as a comparative assessment of membrane-protective activity under the conditions of H₂O₂-induced hemolysis of mice red blood cells (RBCs), was used to analyze the antioxidant potential of the synthesized compounds. The synthesized derivatives have demonstrated different activity in the listed test systems, and we have identified compounds which appear to be most promising for a detailed study of their pharmacological properties.     

Adaptation of a manometric biosensor to measure glucose and lactose

A manometric sensor previously developed to measure urea was modified to measure glucose and lactose through enzymatic oxidation. Change in pressure in an enclosed cavity was correlated to the depletion of oxygen resulting from the enzymatic oxidation of glucose or lactose. The response of the sensor was linear and could be made adjustable over a large range by adjusting the amount of sample loaded into the fixed volume reactor. Because of the slow mutarotation of glucose, the oxidation of glucose was not allowed to proceed to completion. Therefore, the precision of the sensor (approximately 0.2 mM in a range from 0 to 5 mM) was limited by variations in the oxidation rate of glucose by glucose oxidase. Because the assay for lactose measured glucose subsequent to the hydrolysis of lactose by beta-galactosidase, the same degree of precision was observed in lactose. Milk lactose, typically at concentrations of about 150 mM, was estimated using the lactose assay after first diluting the samples. For many fluids such as milk, the use of manometric sensors for oxidizable substrates may be preferable to optical and electrochemical methods because they are robust and suffer a low degree of optical and chemical interferences. Glucose and lactose are representative of many important oxidizable substrates, which may be determined in this manner, many of which do not suffer from limitations caused by mutarotation. In theory, detection limits less than 1 microM may be achieved using these methods.     

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).     

Nitric oxide dissociates lipid oxidation from apoptosis and phosphatidylserine externalization during oxidative stress

Oxidative stress in biological membranes can regulate various aspects of apoptosis, including phosphatidylserine (PS) externalization. It is not known, however, if the targets for these effects are lipids or proteins. Nitric oxide (NO), a bifunctional modulator of apoptosis, has both antioxidant and prooxidant potential. We report here that the NO donor PAPANONOate completely protected all phospholipids, including PS, from oxidation in HL-60 cells treated with 2,2'-azobis(2,4-dimethylisovaleronitrile) (AMVN), presumably via the ability of NO to react with lipid-derived peroxyl radicals and terminate the propagation of lipid peroxidation. PAPANONOate, however, had no effect on PS externalization or other markers of apoptosis following AMVN. Therefore, PS oxidation is not required for PS externalization during AMVN-induced apoptosis. PS externalization was accompanied by inhibition of aminophospholipid translocase (APT). NO potentiated AMVN inhibition of APT. Treatment with PAPANONOate alone produced modest (20%) inhibition of APT without PS externalization. NO did not reverse AMVN-induced oxidation of glutathione and protein thiols. We speculate that APT was sensitive to AMVN and/or NO via modification of protein thiols critical for functional activity. Therefore, the lipoprotective effects of NO were insufficient to prevent PS externalization and apoptosis following oxidative stress. Other targets such as protein thiols may be important redox-sensitive regulators of apoptosis initiation and execution. Thus, in the absence of significant peroxynitrite formation, NO's antioxidant effects are restricted to protection of lipids, while modification of protein substrates continues to occur.     

组合生物催化

自然界最有效的分子是由酶催化的反应所产生,并对这些产物进行自然选择,使其具有优化的生理活性,组合生物催化（Combinatorial Biocatalysis)利用酶反应的多样性,完成有机库（Organic Library)的反复合成,这些反复的反应,可以用分离的酶或全细胞,在天然或非天然的环境中、在溶液或固相中与底物进行反应。组合生物催化是组合方法的在药物发现和发展中产生和优化先导化合物（LeadCompound)的一个有力补充。     

Flavonoids and their oxidation products protect efficiently albumin-bound linoleic acid in a model of plasma oxidation

Although LDL esterified polyunsaturated fatty acids (PUFA) contribute largely to the pool of oxidizable lipids in plasma, they coexist with a non-negligible content of free PUFA. In some pathological conditions, the free PUFA/albumin ratio becomes abnormally elevated. Modeling was performed in a system constituted of linoleic acid bound to human serum albumin (HSA) in which oxidation was initiated by hydrophilic AAPH. Inhibition of lipid peroxidation was evaluated for various flavonoids. The accumulations of hydroperoxyoctadecadienoic acids (HPODE), hydroxyoctadecadienoic acids (HODE) and ketooctadecadienoic acids (KODE) were similarly inhibited: isoquercitrin>quercetin>catechin=isorhamnetin>kaempferol>quercetin-4'-beta-D-glucoside=quercetin-3,4'-di-beta-D-glucoside. Surprisingly, quercetin and isorhamnetin afforded a protection to linoleic acid long after their consumption. Elucidation by mass spectrometry and NMR of the quercetin oxidation products and assessment of their antioxidant capacity pointed out that 3,4-dihydroxybenzoic acid and 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one are major contributors to the apparent quercetin antioxidant capacity.     

Triuret: a novel product of peroxynitrite-mediated oxidation of urate

Urate is an efficient antioxidant and has recently emerged as a competitive inhibitor of tyrosine nitration by peroxynitrite. In vivo and in vitro studies demonstrate the large extent to which urate prevents nitration and establish the biological importance of the reaction between urate and peroxynitrite. The existing lack of characterization of this reaction has led us to focus our studies upon the mechanism of urate oxidation and the products formed. An oxidation product has been previously isolated and mass spectrometry revealed a mass of 146, which spontaneously fragmented into several other ion peaks without use of MS/MS mode. Here, we propose the novel oxidation product to be triuret (H(2)NCONHCONHCONH(2)). Triuret accurately reproduced the peculiar mass spectrum. Identification of the oxidation product helps to develop the mechanism of peroxynitrite-mediated oxidation of urate and can help explain urate's potential as both an antioxidant for tyrosine nitration while paradoxically acting as a pro-oxidant for lipids and sulfhydryls.     

Biological activities and potential cosmeceutical applications of bioactive components from brown seaweeds: a review

Seaweeds are the primary producers of all aquatic ecosystems. Chemical constituents isolated from diverse classes of seaweeds exert a wide range of nutritional, functional and biological activities. Unique metabolites of seaweeds possess specific biological properties that make them potential ingredients of many industrial applications such as functional foods, pharmaceuticals and cosmeceuticals. Cosmeceuticals of natural origin are becoming more popular than synthetic cosmetics. Hence, the investigation of new seaweeds derived functional components, a different source of natural products, has proven to be a promising area of cosmeceutical studies. Brown seaweeds also produce a range of active components including unique secondary metabolites such as phlorotannins and many of which have specific biological activities that give possibilities for their economic utilization. Brown seaweeds derived active compounds have been shown various functional properties including, antioxidant, antiwrinkling, whitening, antiinflammatory and antiallergy. It is well-known that these kind of biological effects are closely associated with cosmeceutical preparations. This communication reviews the current knowledge on brown seaweeds derived metabolites with various biological activities and the potential use as cosmeceutical ingredients. It is hoped that the reviewed literature on multifunctional properties of brown seaweeds will improve access to the seaweed based natural products specially the ability to incorporate these functional properties in cosmeceutical applications.     

Changes in oxidant-antioxidant status in young diabetic patients from clinical onset onwards

Oxidative stress has been implicated as a mechanism underlying hyperglycaemia-associated cellular damage and could play a role in the development of diabetes-related complications. This study aimed to evaluate the significance of changes in oxidant-antioxidant status in 176 child and adolescent diabetic patients at clinical onset, during disease progression and when early microvascular complications appeared. Indicative lipid and protein oxidation markers and antioxidant defence activity were measured in plasma and correlated with clinical data, diabetes duration, long-term glycometabolic control and serum lipids. Compared with their respective age-matched controls, diabetic patients had greater oxidative damage to lipids and proteins, demonstrated through the analysis of hydroperoxides, lipoperoxides and oxidation protein products, all of which were significantly raised at onset, decreased during the first 1.5 years of evolution and rose progressively thereafter. Plasma levels of oxidizable lipids were significantly associated with lipid and protein oxidation products. Overall, plasma antioxidant capacity was significantly and consistently lower from clinical onset onwards. These results suggest that insulin therapy in the first year improved metabolic and oxidant homeostasis and consequently hyperglycaemia-derived biomolecular oxidative damage. Diabetes-associated hyperlipidaemia is related to lipid and protein oxidation processes, which supports the concept of glucose toxicity and lipotoxicity being interrelated. The greatest increase in lipid and protein oxidative damage biomarkers in young diabetic patients with premature microangiopathy points to oxidative stress as a possible contributing mechanism of microvascular dysfunction. Consequently, tight lipid and glycometabolic control may have therapeutic potential by diminishing oxidative tissue-damaging effects of hyperglycaemia.     

Production of manganic chelates by laccase from the lignin-degrading fungus Trametes (Coriolus) versicolor.

Many ligninolytic basidiomycete fungi have been shown to secrete a group of peroxidase isozymes whose sole function appears to be the peroxide-dependent oxidation of manganous [Mn(II)] to manganic [Mn(III)] ions. Manganic chelates and these Mn peroxidases have been implicated as central to the degradation of various natural and synthetic lignins and lignin-containing effluents by white rot (ligninolytic) fungi. Another group of enzymes, the laccases, are commonly secreted by wood-rotting fungi, but are generally regarded as being able to oxidize (and usually polymerize) only phenolic substrates. In this report it is shown that in the presence of appropriate oxidizable phenolic accessory substances or primary substrates, a variety of laccases and peroxidases catalyzing one-electron oxidations can also produce Mn(III) chelates from Mn(II).     

Production of manganic chelates by laccase from the lignin-degrading fungus Trametes (Coriolus) versicolor.

Many ligninolytic basidiomycete fungi have been shown to secrete a group of peroxidase isozymes whose sole function appears to be the peroxide-dependent oxidation of manganous [Mn(II)] to manganic [Mn(III)] ions. Manganic chelates and these Mn peroxidases have been implicated as central to the degradation of various natural and synthetic lignins and lignin-containing effluents by white rot (ligninolytic) fungi. Another group of enzymes, the laccases, are commonly secreted by wood-rotting fungi, but are generally regarded as being able to oxidize (and usually polymerize) only phenolic substrates. In this report it is shown that in the presence of appropriate oxidizable phenolic accessory substances or primary substrates, a variety of laccases and peroxidases catalyzing one-electron oxidations can also produce Mn(III) chelates from Mn(II).