μM Trolox Research Articles

Plasminogen-induced IL-1β and TNF-α production in microglia is regulated by reactive oxygen species

Microglia, major immune effector cells in the central nervous system, become activated during brain injury. In this study we showed that the blood component plasminogen/plasmin activates microglia. Plasminogen-induced IL-1β, TNF-α, and iNOS mRNA expression in primary cultured rat microglia and BV2 murine microglial cells. Plasmin caused a similar response. Serine protease inhibitors suppressed both plasminogen- and plasmin-induced IL-1β and TNF-α expression, indicating the importance of serine protease activity in plasminogen/plasmin activation of microglia. Reactive oxygen species (ROS) appeared to play an important role in plasminogen-induced microglial activation, with ROS being generated within 15 min of plasminogen treatment, and antioxidants (100 μM trolox and 10 mM NAC) reducing IL-1β and TNF-α expression in plasminogen-treated cells. Furthermore, plasminogen stimulated CREB and NF-κB DNA binding activity, and this activation was also reduced by trolox and NAC. These results suggest that plasminogen activates microglia via stimulation of ROS production.

Addition of β-mercaptoethanol or Trolox ® at the morula/blastocyst stage improves the quality of bovine blastocysts and prevents induction of apoptosis and degeneration by prooxidant agents

This study was conducted to evaluate the effect of β-mercaptoethanol (a stimulator of glutathione synthesis) and Trolox ® (an hydrosoluble analogue of Vitamin E) on bovine embryos cultured from the morula stage (Day 5 post-insemination; pi) under oxidative stress conditions. Culture of embryos with increased doses of Trolox ® showed a dose-dependent embryotoxicity on Day 8 pi. The use of 400 μM Trolox ® as well as β-mercaptoethanol at 100 μM prevented at least partly ( P<0.05) the prooxidant-induced blastocyst degeneration on Day 8. Hatching rates of surviving blastocysts were significantly increased by both antioxidants and β-mercaptoethanol alone improved their mean cell numbers, which was significant in the ICM ( P<0.05). Analysis of their effect on Day 7 pi showed that both the antioxidants significantly reduced the prooxidant-induced apoptosis and β-mercaptoethanol diminished the physiological level of apoptosis as well as it stimulated the glutathione synthesis ( P<0.05). In addition, a comparison between in vitro- and in vivo-produced embryos showed that the levels of apoptosis were similar at the same age post-insemination (morulae and blastocysts) but increased steadily with the embryonic age in in vitro ones. In conclusion, β-mercaptoethanol and Trolox ® added separately from the morula stage protected embryos against oxidative stress and improved the quality of the resulting blastocysts.

Effect of hemodialysis on the antioxidative properties of serum

In patients with chronic renal failure undergoing regular hemodialysis (HD), oxidative stress is involved in the development of dialysis-related pathologies. The aim of the study was to measure the effect of HD treatment on the general antioxidative status of serum with special consideration of the specific oxidizability of lipids and proteins. Indicators for the oxidative/antioxidative status of plasma were monitored at the beginning and at the end of a dialysis session on the arterial and venous side of the dialyzer. A decrease in the antioxidant status was accompanied by an increased oxidizability of proteins as well as lipids during HD treatment. During the first passage of the dialyzer, the lag time of lipid oxidation decreased from 114.0±19.8 to 81.5±18.9 min, the lag time of protein oxidation decreased from 105.0±24.6 to 72.9±21.3 min and the total antioxidative status decreased from 518±24 to 252±124 μM trolox equivalents. The carbonyl content of serum proteins was high in patients with end stage renal disease (ESRD) (3.9±1.1 vs. 0.9±0.1 nmol/mg in controls) but did not change significantly during dialysis procedure. Our data demonstrate that the susceptibility of serum lipids and proteins to oxidative modification is severely increased by HD treatment.

Antioxidant activity and total phenolic content of Iranian Ocimum accessions

Basil ( Ocimum basilicum L.) is used in traditional medicine, as a culinary herb and a well-known source of flavouring principles. Total antioxidant activity in 23 Iranian basil accessions was determined as Trolox equivalent antioxidant capacity (TEAC). Total phenolic contents were determined using a spectrophotometric technique, based on the Folin-Ciocalteau reagent, according to the method of Spanos and Wrolstad [Journal of Agricultural & Food Chemistry, 38 (1990) 1565] and calculated as gallic acid equivalents GAE/g dw. Total antioxidant activity varied from 10.8 to 35.7 μM Trolox, and total phenolic content ranged from 22.9 to 65.5 mg gallic acid/g dw in “Dezful I” and “Babol” accessions, respectively. A linear positive relationship existed between the antioxidant activity and total phenolic acids content of the tested basil accessions ( R 2=0.71). Iranian basils possess valuable antioxidant properties for culinary and possible medicinal use.

OXIDATIVE STRESS AFTER THREE DIFFERENT INTENSITIES OF RUNNING

The exercise induces oxidative stress (OS). However, it is not clear at which intensity it occurs, and if different fitness can influence OS levels. Many researchers studied the influence of different percent of oxygen uptake (VO2) on the mechanisms that underlie OS. However, ventilatory threshold is a better determinant of relative exercise intensity. PURPOSE To study the influence of three different exercise intensities on OS parameters in trained (T) and untrained (UT) subjects. METHODS Seventeen healthy, volunteered male subjects were assigned into a 2X3X2 factorial design according to training level [T (n=8)], exercise intensity (low, moderate and high intensity) and blood collect (before and after exercise). The subjects were evaluated through a maximal aerobic power test in a treadmill. The maximal oxygen uptake (VO2max) and ventilatory thresholds (VT1 and VT2) were obtained. Three exercise tests were performed in random order at a VO2 of 10% bellow VT1 (low intensity), 10% bellow VT2 (moderate intensity) and between VT2 and VO2max (high intensity). Chemiluminescence (CL), total antioxidant capacity (TRAP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were measured in the blood. RESULTS Exercise effect was observed on TRAP, which increased after exercise (p < 0.05); and fitness effect on GPx, which was greater in T group (p < 0.05). In plasma, CL decreased (3589±193 to 3274±223 cps.mg Hb−1, P < 0.05), and TRAP was increased (304±45 to 384±57 μM trolox, P < 0.05) after high intensity exercise in T group. SOD increased after low (8.35±0.85 to 9.23±1.03 U SOD.mg protein−1) and moderate (8.89±0.98 to 10.44±86 U SOD.mg protein−1) exercise intensity in UT (p < 0.05). CAT did not changed. CONCLUSIONS Our results indicate that exercise in this model did not increase LPO, despite alterations in some antioxidants. Probably the reactive oxygen species involved were scavengered by GPx and TRAP. Supported by CNPq, CAPES and SNE-MET-Brazil

Oxidative Stress and Cardiac Microvascular Structure in Ischemia and Reperfusion: The Protective Effect of Antioxidant Vitamins

Reperfusion of the ischemic myocardium results in structural changes in the capillary bed, which may contribute to decreased microcirculatory flow (“no reflow”). This study was designed to correlate the endothelial cell shape changes with both oxidative stress and lipid peroxidation and to evaluate the beneficial potential of Trolox (a hydrophilic analogue of α-tocopherol) and ascorbic acid. Isolated buffer-perfused rat hearts were made ischemic for 45 min and then reperfused with 100 μM Trolox and/or 100 μM ascorbic acid. Morphological changes were quantified by measuring capillary cross-sectional areas. Increased myocardial content of oxidized glutathione and its release into the coronary effluent were used as indices of oxidative stress. Myocardial MDA, an end product of lipid peroxidation, was also measured. Luminal membrane blebs and capillary “constriction” in the ischemic groups occurred when there was no change in either glutathione status or MDA concentrations. Reperfusion altered the redox state of the heart sufficiently to induce lipid peroxidation. It also induced endothelial cell swelling and a reduction in luminal area. Ascorbic acid was a more effective antioxidant than Trolox as it significantly reduced both oxidative stress and ultrastructural injury. The combined antioxidant treatment returned both the stress ratio and the capillary measurements to control values. We conclude that endothelial cell swelling correlates with the degree of oxidative stress and that antioxidant vitamins reduce membrane damage by preventing lipid peroxidation.

Protective properties of butanolic extract of the Calendula officinalis L.(marigold) against lipid peroxidation of rat liver microsomes and action as free radical scavenger

Calendula officinalis (marigold) has many pharmacological properties. It is used for the treatment of skin disorders, pain and also as a bactericide, antiseptic and anti-inflammatory. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are known to participate in the pathogenesis of various human diseases and may be involved in the conditions which C. officinalis is used to treat. The aim of this study was to investigate the relationship between the beneficial properties of this plant and its antioxidant action. The butanolic fraction (BF) was studied because it is non-cytotoxic and is rich in a variety of bioactive metabolites including flavonoids and terpenoids. Superoxide radicals (O2•-) and hydroxyl radicals (HO•) are observed in decreasing concentrations in the presence of increasing concentrations of BF with IC50 values of 1.0 ± 0.09 mg/ml and 0.5 ± 0.02 mg/ml, respectively, suggesting a possible free radical scavenging effect. Lipid peroxidation in liver microsomes induced by Fe2+/ascorbate was 100% inhibited by 0.5 mg/ml of BF (IC50 = 0.15 mg/ml). Its total reactive antioxidant potential (TRAP) (in μM Trolox equivalents) was 368.14 ± 23.03 and its total antioxidant reactivity (TAR) was calculated to be 249.19 ± 14.5 μM. The results obtained suggest that the butanolic fraction of C. officinalis possesses a significant free radical scavenging and antioxidant activity and that the proposed therapeutic efficacy of this plant could be due, in part, to these properties.

NT-4/5 Exacerbates Free Radical-Induced Neuronal Necrosis in Vitro and in Vivo

Neurotrophins render neurons highly vulnerable to certain injuries. We examined the possibility that NT-4/5 would enhance free radical neurotoxicity in vivo as well as in vitro. Striatal neurons exposed to 10 μM Fe2+ or 1 mM l-buthionine-[S, R]-sulfoximine (BSO) underwent mild degeneration within 24 h. With concurrent addition of 10–100 ng/ml NT-4/5, neuronal death following exposure to Fe2+ or BSO was significantly increased and suppressed by addition of 100 μM trolox, an antioxidant. In the adult brain, the intrastriatal injections of 20 nmol Fe2+ revealed features of neuronal necrosis such as swelling cell body and mitochondria, fenestration of plasma membrane prior to nuclear membrane, and scattering condensation of nuclear chromatin. Cotreatment with 1.8 μg NT-4/5 augmented the striatal damage 24 h following the injections of Fe2+. This study implies that free radicals produce necrotic degeneration in vivo as well as in vitro that becomes more sensitive in the presence of neurotrophins.

Melatonin but not vitamins C and E maintains glutathione homeostasis in t-butyl hydroperoxide-induced mitochondrial oxidative stress.

SPECIFIC AIMTo assess whether the antioxidant properties of melatonin are suitable to maintain GSH homeostasis counteracting mitochondrial oxidative stress.PRINCIPAL FINDINGS1. Effect of melatonin, NAC, ascorbate, and Trolox on mitochondrial GSH and GSSG levelsIn basal conditions, i.e., mitochondria incubated in the absence of t-BHP, melatonin (100 nM) significantly (P<0.001) increased the content of GSH and decreased that of GSSG in brain (GSH: 6.82±0.78 to 10.13±0.42 nmol/mg prot) and liver (GSH: 11.54±0.89 to 19.43±0.80 nmol/mg prot; GSSG: 1.44±0.22 to 0.06±0.007 nmol/mg prot) mitochondria. The other antioxidants tested were unable to exert a similar effect of melatonin, and only the dose of 1000 μM Trolox increased GSH levels (11.50 ± 0.43 nmol/mg prot) in brain mitochondria.After incubation with 100 μM t-BHP, practically all GSH was oxidized to GSSG (Fig. 1)⤻ . Melatonin (100 nM) counteracted these toxic effects, recovering the basal levels of GSH and GSSG. In this situation, i.e., t-BHP-induced oxid...

Is the endogenous peroxyl-radical scavenging capacity of plasma protective in systemic inflammatory disorders in humans?

Systemic inflammatory response syndrome (SIRS) in humans is associated with heightened intravascular oxidative stress. The clinical significance of plasma endogenous antioxidative capability in SIRS remains undetermined. Time-sequence changes of plasma total radical-trapping antioxidant parameter (TRAP) and its components were measured in 135 patients with various clinical conditions leading to SIRS. The results were correlated with clinical parameters. Plasma TRAP significantly depressed upon diagnosis of SIRS (SIRS vs. healthy subjects (n = 50), 605.7 ± 20.4 vs. 803.4 ± 30.8 μM Trolox equivalent, p < .001). In survivors (n = 86), TRAP declined further during the course of SIRS, followed by a mild recovery at the end of follow-up. General linear mixed model analysis revealed that uric acid, vitamin C, vitamin E and unidentified antioxidants contributed to most of the changes in TRAP (each factor p < .001). In nonsurvivors (n = 49), TRAP increased steadily until death, and the increase was predominantly the result of the increased contribution of bilirubin ( p < .01). Higher TRAP levels were not correlated with diminished blood oxidants formation ( r = −0.13, p > .05), lower intensity of lipid peroxidation ( r = 0.261, p < .05) or lesser disease severity of SIRS. The results do not support the hypothesis that the endogenous peroxyl radical scavenging ability of plasma plays a protective role in the course of SIRS.

H 2O 2 mediates O 2 toxicity in cultured fetal rat distal lung epithelial cells

It is unknown which of the reactive oxygen species is primarily responsible for the cytotoxicity of 95% O 2 for rat distal fetal lung epithelial cells in vitro. Incubation of cells with 25 U/ml polyethylene glycol (PEG)-conjugated SOD and 50 U/ml PEG-catalase, but not PEG-SOD or SOD mimics alone, significantly reduced 95% O 2-mediated cytotoxicity. Liposome-entrapped catalase, without SOD, also significantly reduced 95% O 2-mediated cytotoxicity. Increased formation of lipid hydroperoxides, as assessed by the formation of 8-isoprostane and aldehydes, was attenuated by both 100 μM Trolox, a vitamin E analogue, and by 5 μM U74389G, an amino steroid. Trolox, but not U74389G, prevented an increase in cell-derived H 2O 2, hydroxyl radical and 95% O 2-mediated cytotoxicity. An increase in hydroxyl radical formation, but not cell death, observed in 95% O 2, was prevented by 0.1 μM phenanthrolene, a cell permeant iron chelator. DNA extracts of rat distal fetal lung epithelial cells maintained under serum-free conditions had an electrophoretic pattern consistent with some degree of apoptosis. However, no increase in laddering was seen with exposure to 95% O 2. These data are consistent with hydrogen peroxide, but not lipid hydroperoxides or hydroxyl radical, being a critical effector of O 2-mediated necrotic cell death in distal lung epithelial cells.

Effect of in vitro culture and maternal insulin-like growth factor-i on development of bovine conceptuses

This study was conducted to evaluate the effect of β-mercaptoethanol (a stimulator of glutathione synthesis) and Trolox® (an hydrosoluble analogue of Vitamin E) on bovine embryos cultured from the morula stage (Day 5 post-insemination; pi) under oxidative stress conditions. Culture of embryos with increased doses of Trolox® showed a dose-dependent embryotoxicity on Day 8 pi. The use of 400 μM Trolox® as well as β-mercaptoethanol at 100 μM prevented at least partly (P<0.05) the prooxidant-induced blastocyst degeneration on Day 8. Hatching rates of surviving blastocysts were significantly increased by both antioxidants and β-mercaptoethanol alone improved their mean cell numbers, which was significant in the ICM (P<0.05). Analysis of their effect on Day 7 pi showed that both the antioxidants significantly reduced the prooxidant-induced apoptosis and β-mercaptoethanol diminished the physiological level of apoptosis as well as it stimulated the glutathione synthesis (P<0.05). In addition, a comparison between in vitro- and in vivo-produced embryos showed that the levels of apoptosis were similar at the same age post-insemination (morulae and blastocysts) but increased steadily with the embryonic age in in vitro ones.In conclusion, β-mercaptoethanol and Trolox® added separately from the morula stage protected embryos against oxidative stress and improved the quality of the resulting blastocysts.

Effect of Hydroxytyrosol Found in Extra Virgin Olive Oil on Oxidative DNA Damage and on Low-Density Lipoprotein Oxidation

Hydroxytyrosol found in extra virgin olive oil strongly inhibited low-density lipoprotein oxidation stimulated by 2,2‘-azobis(2-amidinopropane) hydrochloride (AAPH), suggesting the ability to scavenge the AAPH-derived peroxyl radicals. Hydroxytyrosol inhibited iron-dependent phospholipid liposome peroxidation at low concentrations (IC50 = 50 ± 1.3 μM). In similar experiments, the calculated, IC50 values for other antioxidants compared are 1.5 ± 0.05 μM (carnosol), 2.25 ± 0.08 μM (carnosic acid), 65 ± 2.6 μM (Trolox C), and 250 ± 10 μM (vitamin E). Hydroxytyrosol and ascorbate reduced copper(II) ions to their copper(I) prooxidant form, but this was not reflected by their abilities to induce oxidative DNA damage in the complex copper−phenanthroline. Only high, nonphysiological, millimolar concentrations of pure hydroxytyrosol weakly stimulated copper-dependent chemical modification to DNA bases. The prooxidant (redox actions on metal ions) concentrations in vitro may never be achieved in vivo (following con...

Protective effects of the thiophosphate amifostine (WR 2721) and a lazaroid (U83836E) on lipid peroxidation in endothelial cells during hypoxia/reoxygenation

Little is known about pharmacological interventions with thiophosphates or lazaroids in endothelial cells injured by hypoxia/reoxygenation with respect to membrane lipid peroxidation (LPO) caused by reactive oxygen species. Therefore, a cell line of bovine aortic endothelial cells was studied after 120-min hypoxia followed by 30-min reoxygenation, resulting in moderate and predominantly reversible injury (energy depression/cytosolic Ca 2+-accumulation during hypoxia, which almost normalized during reoxygenation; membrane blebs, an increasing amount of lysosomes, vacuolization, lipofuscin formation, alterations in mitochondria size, some lyzed cells). 18.9 ± 4.3% of the cells died. Radical-induced LPO measured as malondialdehyde continuously increased to 2.18 ± 0.17 nmol/mg of protein after reoxygenation vs control (0.41 ± 0.13, P < 0.05). Simultaneously, the content of 4-hydroxynonenal, a novel indicator of LPO, increased from 0.02 ± 0.01 to 0.11 ± 0.02 nmol/mg of protein ( P < 0.01). The results support the assumption that reoxygenation injury is accompanied by an increase in membrane LPO, causing structural and functional disturbances in the monolayer. The thiophosphate WR 2721 [S-2-(3-aminopropylamino) ethylphosphorothioic acid] and the lazaroid U83836E {(-)-2-[[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl] methyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol (dihydrochloride)} were effective scavengers of ·OH, being more efficient than trolox C (6-hydroxy-2,5,7,8-tetramethylchroman-2-carbon acid) used as standard ( ec 50: 12, 5 and 15 μM, respectively, measured by electron spin resonance spectroscopy). One mM WR 2721, 10 μM U83836E, and 5 μM trolox C reduced formation of malondialdehyde during hypoxia/reoxygenation to 53 ± 7, 51 ± 10 and 48 ± 6%, respectively ( P < 0.05 each, versus control). In general, WR 2721 and U83836E prevent radical-induced membrane LPO in a model of endothelial cells injured by hypoxia/reoxygenation. The use of these two agents is a new approach to protect the endothelium against oxidative stress.

Antioxidant and free radical scavenging effects in extracts of the medicinal herb Achyrocline satureioides (Lam.) DC. ("marcela")

Achyrocline satureioides (Lam.) DC. (Compositae) is a medicinal herb used in Argentina, Uruguay, Brazil and Paraguay for its choleretic, antispasmodic and hepatoprotective properties. The presence of the flavonoid quercetin and its derivatives, and of different phenolic acids such as caffeic, chlorogenic and isochlorogenic acids in the aerial parts of this plant has led us to study the antioxidant activity of its extracts using different bioassays. The inhibition of luminol-enhanced chemiluminescence by the aqueous and methanolic extracts was used to show that their total reactive antioxidant potential index (TRAP; in microM Trolox equivalents) was 91.0 +/- 15.4 and 128.1 +/- 20.1 microM, respectively, while the total antioxidant reactivity index (TAR) was calculated to be 1537 +/- 148 and 1910 +/- 171 microM. Only the methanolic extract was capable of reducing iron (II)-dependent DNA damage. Lipid peroxidation was assessed by two different methods. The aqueous extract reduced hydroperoxide-initiated chemiluminescence in rat liver homogenates at all concentrations in a dose-dependent manner, with a calculated IC50 = 225 micrograms/ml, while the methanolic extract was only effective at higher concentrations (100 and 1000 micrograms/ml). Both aqueous and methanolic extracts were capable of reducing the production of thiobarbituric acid reactive substances (TBARS) in rat liver homogenates, with an IC50 > 1000 micrograms/ml. The results obtained suggest that the extracts of A. satureioides possess significant free radical scavenging and antioxidant activity in vitro, a fact that should encourage future in vivo studies.

Glutamate neurotoxicity in mouse cortical neurons: atypical necrosis with DNA ladders and chromatin condensation

The possibility that glutamate may induce neuronal apoptosis was examined in cultured cortical neurons. Neurons underwent widespread death 24 h following exposure to 50 μM glutamate. The glutamate neurotoxicity was blocked by inclusion of the glutamate antagonists, 10 μM MK-801 and 50 μM CNQX. The death was characterized by swelling cell body and bursting cytoplasmic membrane in the early phase of degeneration, suggesting that glutamate produces receptor-mediated excitotoxic necrosis. With blockade of excitotoxicity by addition of 10 μM MK-801 and 50 μM CNQX, cortical neurons exposed to 2 mM glutamate underwent necrosis morphologically identical to excitotoxicity but sensitive to 100 μM trolox, an antioxidant, suggesting that high doses of glutamate produce oxidative neuronal necrosis via non-receptor-mediated mechanisms. Interestingly, internucleosomal DNA fragmentation and chromatin condensation were observed in the course of glutamate-induced neuronal necrosis.

Melatonin: A peroxyl radical scavenger more effective than vitamin E

We have compared the peroxyl radical scavenger ability of melatonin with that of vitamin E, vitamin C and reduced glutathione (GSH). In the assay system, β-phycoerythrin (β-PE) was used as fluorescent indicator protein, 2-2′-azo-bis(2-amidinopropane)dihydrochloride as a peroxyl radical generator and the water soluble vitamin E analogue, Trolox, as reference standard. Results are expressed as oxygen radical absorbing capacity (ORAC perox) units, where 1 ORAC unit equals the net protection produced by 1 μM Trolox. A linear correlation of ORAC values with concentration (0.5–4 μM) of all the substances tested has been observed. However, on molar basis, the relative ORAC perox of Trolox, vitamin C, GSH and melatonin was 1 : 1.12 : 0.68 : 2.04, respectively. Thus, melatonin, which is a lipid-soluble compound, was twice more active than vitamin E, believed to be the most effective lipophilic antioxidant.

Unconjugated bilirubin inhibits the oxidation of human low density lipoprotein better than trolox

Oxidative modification of human low density lipoprotein (LDL) has been implicated in plaque formation in blood vessels leading to atherogenesis. Conversely, there is increasing evidence that prevention of LDL oxidation reduces the incidence of coronary artery disease. Here, we have compared the effect of unconjugated bilirubin (Bu) and Trolox (a vitamin E analogue) on the oxidation of LDL after treatment with Cu 2+ under defined conditions. We observed that Bu, at or near the normal serum level (i.e. 17 μM) effectively inhibits oxidation of LDL, while it takes at least 500 μM Trolox to achieve a similar effect. This means that, on a per mole basis, Bu is >20 times more effective than Trolox in preventing LDL oxidation. The oxidation of LDL was assessed by agarose gel electrophoresis. This was further corroborated by assaying the malondialdehyde formed upon reacting the presumptive peroxidation product (s) of LDL with thiobarbituric acid. Thus, we have directly verified that Bu and, less so, Trolox, can each prevent the oxidative damage of LDL in vitro . Our result supports the contention that Bu as an endogenous antioxidant can prevent LDL oxidation and hence reduce the risk of atherogenesis.

Oxygen-radical absorbance capacity assay for antioxidants

A relatively simple but sensitive and reliable method of quantitating the oxygen-radical absorbing capacity (ORAC) of antioxidants in serum using a few μl is described. In this assay system, β-phycoerythrin (β-PE) is used as an indicator protein, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble vitamin E analogue) as a control standard. Results are expressed as ORAC units, where 1 ORAC unit equals the net protection produced by 1 μM Trolox. The uniqueness of this assay is that total antioxidant capacity of a sample is estimated by taking the oxidation reaction to completion. At this point all of the nonprotein antioxidants (which include α-tocopherol, vitamin C, β-carotene, uric acid, and bilirubin) and most of the albumin in the sample are oxidized by the peroxyl radical. Results are quantified by measuring the protection produced by antioxidants. This solves many problems associated with kinetics or lag-time measurements. A linear correlation of ORAC value with concentration of serum, Trolox, vitamin C, uric acid, and bovine albumin is demonstrated. The coefficient of variation within a run is found to be about 2% and from run to run about 5%. Trolox, α-tocopherol, vitamin C, β-carotene, uric acid, and bilirubin completely protect β-PE from oxidation, while bovine albumin protects β-PE only partially. On a molar basis, the relative peroxyl radical absorbance capacity of Trolox, α-tocopherol acid succinate, uric acid, bilirubin, and vitamin C is 1:1:0.92:0.84:0.52. Bovine albumin per unit weight has a lower peroxyl absorbing capacity than these antioxidants. However, the serum protein fraction, containing some lipid-soluble antioxidants, represents the major contributor to the ORAC value found in whole serum. The minimum amount of vitamin C and uric acid which could still be detectable when added to a serum supernatant fractiion is 1.5 μg and 0.59 μg, respectively, which account for about 1% of the total ORAC value of the serum supernatant fraction.