Comparative study of antioxidants as quenchers or scavengers of reactive oxygen species based on quenching of MCLA-dependent chemiluminescence.

The quenching or scavenging effect of non-enzymatic antioxidants against reactive oxygen species (ROS) was studied by comparing the degree of suppression of chemiluminescence (CL) caused by the oxidation of MCLA (methoxylated Cypridina luciferin analogue) by ROS. MCLA-dependent CL caused by O2- was effectively quenched by ascorbic acid, beta-carotene, lycopene and astaxanthin, while it was enhanced by alpha-tocopherol. The CL by 1O2 was quenched effectively by beta-carotene, lycopene and astaxanthin, moderately by ascorbic acid, and slightly by alpha-tocopherol. beta-Carotene and alpha-tocopherol remarkably suppressed the CL when ROS was HO*. The present study revealed that MCLA-dependent CL assay provides a simple and rapid method for the evaluation of antioxidants as a quencher or scavenger against any kind of ROS.     

Hydroxyl radical scavenging action of capsaicin

We recently reported that capsaicin (CAP) is capable of scavenging peroxyl radicals derived from 2,2'-azobis(2,4-dimethylvaleronitrile) as measured by electron spin resonance (ESR) spectroscopy. The present study describes the hydroxyl radical (HO*) scavenging ability of CAP as measured by DNA strand scission assay and by an ESR spin trapping technique with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The Fenton reaction [Fe(II)+ H(2)O(2) --> Fe(III) + HO* + HO(-)] was used as a source of HO*. The incubation of DNA with a mixture of FeSO(4) and H(2)O(2) caused DNA strand scission. The addition of CAP to the incubation mixture decreased the strand scission in a concentration-dependent manner. To understand the antioxidative mechanism of CAP, we used an ESR spin trapping technique. Kinetic competition studies using different concentrations of DMPO indicated that the decrease of the oxidative DNA damage was mainly due to the scavenging of HO* by CAP, not to the inhibition of the HO* generation system itself. We estimated the second order rate constants in the reaction of CAP and common HO* scavengers with HO* by kinetic competition studies. By comparison with the common HO* scavengers, CAP was found to scavenge HO* more effectively than mannitol, deoxyribose and ethanol, and to be equivalent to DMSO and benzoic acid, demonstrating that CAP is a potent HO* scavenger. The results suggest that CAP may act as an effective HO* scavenger as well as a peroxyl radical scavenger in biological systems.     

Induction of DNA strand breakage and base oxidation by nitroxyl anion through hydroxyl radical production.

Nitroxyl anion (NO-), the one-electron reduction product of nitric oxide (NO*), has been reported to be formed under various physiological conditions and to be cytotoxic, although the mechanism responsible for the toxic effects has not been identified. We have studied the effects of NO- generated from Angeli's salt (sodium trioxodinitrate) or Piloty's acid (N-hydoxybenzenesulfonamide) on DNA strand breakage and DNA base oxidation in vitro. Induction of strand breakage was dose- and time-dependent upon incubation of plasmid pBR322 with Angeli's salt or Piloty's acid. Similarly, 8-oxo-2'-deoxyguanosine and malondialdehyde were formed when calf-thymus DNA or 2'-deoxyribose, respectively, were incubated with Angeli's salt. Electron acceptors (ferricyanide, 4-hydroxy-TEMPO), that convert NO to NO*, inhibited the reactions, indicating that NO , but not NO*, is responsible for the reactions. Furthermore, the reactions were also inhibited by the presence of hydroxyl radical (HO*) scavengers, antioxidants, metal chelators and superoxide dismutase and catalase, implying involvement of free HO*. These results suggest that NO- is a possible endogenous source of HO*, that may be formed either directly from the reaction product of NO- with NO* (N2O2*-) or indirectly through H2O2 formation. Thus NO may play an important role as a cause of diverse pathophysiological conditions such as inflammation and neurodegenerative diseases.     

Chemiluminescence evaluation of oxidative damage to biomolecules induced by singlet oxygen and the protective effects of antioxidants

A chemiluminescence (CL) method was developed for the evaluation of oxidative damage to biomolecules induced by singlet oxygen ((1)O(2)) and for the evaluation of the protective effects of antioxidants. The (1)O(2) was generated from the reaction of H(2)O(2)+OCl(-). Results showed that the CL signal from the reaction of H(2)O(2)+OCl(-) was weak, however, it was enhanced dose-dependently with the addition of DNA and unsaturated fatty acid, respectively. Spectra analysis indicated that the enhanced CL could be ascribed to the decay of triplet-excited carbonyl compounds, which were generated from the reaction of (1)O(2) plus the biomolecules. On the other hand, the enhanced CL produced in the above systems could be effectively inhibited by lycopene, beta-carotene, VC, and VE, but could not be inhibited by mannitol, SOD, and NaN(3). The mechanism therein was discussed.     

Activated oxygen generation by a primaquine metabolite: inhibition by antioxidants derived from Chinese herbal remedies.

Primaquine is an important antimalarial drug which causes hemolytic anemia in patients with glucose-6-phosphate dehydrogenase (G6PDH) deficiency, probably due to oxidant generation by its metabolites. One of primaquine's metabolites, 5,6-dihydroxy-8-aminoquinoline (AQD), was found to cause chemiluminescence (CL) in vitro when incubated in the presence of luminol. This CL is inhibited by catalase and deferoxamine, unaffected by mannitol, and stimulated by superoxide dismutase (SOD), suggesting that it is mediated by H2O2. Three antioxidants (daphnetin, ferulate, and maltol), derived from Chinese herbal remedies, inhibited AQD- and H2O2-mediated CL, whereas a fourth, anisodamine, had no effect. Daphnetin also potently inhibited H2O2-mediated lipid peroxidation as measured by the production of thibarbituric acid reacting substances (TBARS). Thus, the possibility is raised that an antioxidant might be able to mitigate the oxidant hemolytic effects of primaquine.     

Manganese complexes of curcumin analogues: evaluation of hydroxyl radical scavenging ability, superoxide dismutase activity and stability towards hydrolysis

In order to improve the antioxidant property of curcumin and its analogue, diacetylcurcumin, manganese was incorporated into the structures in order to enhance superoxide dismutase (SOD) activity. Manganese (Mn) complexes of curcumin (CpCpx) and diacetylcurcumin (AcylCpCpx) were synthesized and firstly investigated for SOD activity and hydroxyl radical (HO*) scavenging ability. SOD activity was evaluated by both the nitroblue tetrazolium (NBT) reduction assay and electron paramagnetic resonance (EPR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trapping agent. CpCpx and AcylCpCpx inhibited the NBT reduction and decreased the DMPO/OOH adduct much greater than corresponding antioxidants or ligands, with IC50 values of 29.9 and 24.7 microM (NBT), and 1.09 and 2.40 mM (EPR), respectively. For EPR, potassium superoxide (KO2) was used as a source of O2- where qualitative results suggested that CpCpx and AcylCpCpx were SOD mimics, which catalyze the conversion of O2- to dioxygen and hydrogen peroxide (H2O2). Additionally, CpCpx and AcylCpCpx exhibited the great inhibition of DMPO/OH adduct formation with an IC50 of 0.57 and 0.37mM, respectively, which were comparable to that of curcumin (IC50 of 0.64 mM), indicating that both Mn complexes are also an effective HO* scavenger. The stability against hydrolysis in water, various buffers and human blood/serum was carried out in vitro. It was found that both Mn complexes were pH and salt concentration dependent, being more stable in basic pH. In the human blood/serum test, CpCpx was more stable against hydrolysis than AcylCpCpx with about 10 and 20% of free Mn2+ releasing, respectively.     

Reactive oxygen species generation in gingival fibroblasts of Down syndrome patients detected by electron spin resonance spectroscopy

Oral manifestations of Down syndrome include high susceptibility to gingival inflammation with early onset and rapidly progressive periodontitis. The influence of reactive oxygen species (ROS) on periodontitis of Down syndrome is unclear. The aim of this study was to characterize ROS formation in Down syndrome-gingival fibroblasts (DS-GF) using electron spin resonance (ESR) spin trapping with 5,5-dimetyl-1-pyrolline-N-oxide (DMPO), and to determine whether ROS generation plays a role in the pathogenesis of periodontitis in Down syndrome patients. We observed formation of the DMPO-OH spin adduct, indicating HO* generation from cultured DS-GF and non-DS-GF. The increased HO* generation in cultured DS-GF was strongly decreased in the presence of the H2O2 scavenger, catalase, or the iron chelator, desferal. This may due to the enzymatic ability of over-expressed CuZn-superoxide dismutase in Down syndrome to catalyze the formation of H2O2 from O2*-, thereby increasing the availability of substrate H2O2 for the iron-dependent generation of HO* via the Fenton reaction, suggesting that HO* generated from DS-GF may be involved in progressive periodontitis of Down syndrome.     

A new technique for highly sensitive detection of superoxide dismutase activity by chemiluminescence

A stable enzymatic free radical generation system has been developed which allows a precise production of 02-. and its detection by chemiluminescence between 2 pmol and 8 nmol. This test has been used for assaying superoxide dismutase (SOD) by inhibition of the chemiluminescence (CL) signal. No inhibition was observed with catalase, which excludes the participation of H2O2 in lucigenin CL. N,N-Diethyldithiocarbamate gives 100% inhibition of SOD activity either from a purified enzymatic preparation or from biological samples, which confirms the specificity of the CL assay. SOD assay can be performed either on a purified enzymatic preparation or on biological materials such as cultured cells.     

A comparison of chemical systems for luminometric determination of antioxidant capacity towards individual reactive oxygen species.

The aim of the study was to investigate the reactive oxygen species (ROS) production in the hypoxanthine-xanthinoxidase (HX-XO), hydrogen peroxide-ferrous sulphate (H2O2-FeSO4) and hydrogen peroxide (H2O2) systems by using various concentrations of ROS scavengers, such as superoxide dismutase (SOD), dimethylthiourea (DMTU) or catalase (CAT). Luminol (0.8 mmol/L) was dissolved in a borate buffer, pH 9.0, and was used as a luminophor in the chemiluminescence (CL) measurements. In the HX-XO system SOD, CAT and DMTU deepened the CL signal, whereas in the H2O2-FeSO4 system, only CAT and DMTU deepened the CL signal, and in the H2O2 system SOD and CAT increased and DMTU deepened the CL signal. Electron spin resonance (ESR) measurements were performed only in the H2O2-FeSO4 system. 5,5-dimethyl-pyrroline-N-oxide (DMPO) was used as a spin trap. According to typical ESR spectra, .OH was produced in this chemical system. It can be concluded that the chemical systems do not produce single reactive oxygen species but a mixture of them.     

Nitric oxide decreases the stability of DMPO spin adducts.

The effect nitric oxide (NO*) on the stability of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) adducts has been investigated using EPR spectroscopy. We report that the DMPO/HO* adduct, generated by porcine pulmonary artery endothelial cells in the presence of H2O2 and DMPO, or by a Fenton system (Fe(II)+H2O2) is degraded in the presence of the NO*-donor, 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) or by bolus addition of an aqueous solution of NO*. A similar effect of DEANO was observed on other DMPO adducts, such as DMPO/*CH3 and DMPO/*CH(CH3)OH, generated in cell-free systems. Measurements of the loss of DMPO/HO* in the presence of DEANO in aerated and oxygen-free buffers showed that in both of these settings the process obeys first-order kinetics and proceeds with similar efficacy. This indicates that direct interaction of the nitroxide with NO*, rather than with NO2* (formed from NO* and O2 in aerated media), is responsible for destruction of the spin adduct. These results suggest that the presence of NO* may substantially affect the quantitative determination of DMPO adducts. We also show that NO2* radicals, generated by a myeloperoxidase/H2O2/nitrite system, also degrade DMPO/HO*. Because DMPO is frequently used to study generation of superoxide and hydroxyl radicals in biological systems, these observations indicate that extra caution is required when studying generation of these species in the presence of NO* or NO2* radicals.     

Hemin: a possible cause of oxidative stress in blood circulation of beta-thalassemia/hemoglobin E disease

A correlation between endogenous hemin and pro-oxidant activity was revealed in serum of beta-thalassemia/hemoglobin E disease (beta-thal/Hb E), which is the most common prevalent type of thalassemia in Thailand. The technique of low temperature electron spin resonance spectroscopy was used for characterization and quantification of high spin ferric heme, which had been identified as hemin (iron (III)-protoporphyrin IX). Hemin was present at levels ranging from 50 to 280 microM in serum of beta-thal/Hb E but not detectable in serum of non-thalassemia. Pro-oxidant activity in serum of beta-thal/Hb E was demonstrated by luminol-mediated chemiluminescence, a sensitive method for screening of free radical generation in vitro. In the presence of H2O2, the chemiluminescence intensity (CL) was about 20 fold enhanced in serum of beta-thal/Hb E, indicating its extensive pro-oxidant activity. The CL showed a good correlation with serum heroin, r = 0.778 (p < 0.001), while the correlations with total serum iron and serum ferritin were 0.260 (p = 0.259) and 0.519 (p = 0.004), respectively. Our finding suggested that serum hemin readily catalyzed free radical reactions and it may contribute a major pro-oxidant in blood circulation of beta-thal/Hb E.     

Chemiluminescence assay for reactive oxygen species scavenging activities and inhibition on oxidative damage of DNA in Deinococcus radiodurans.

Free radical scavenging effects of the cellular protein extracts from two strains of Deinococcus radiodurans and Escherichia coli against O2-, H2O2 and *OH were investigated by chemiluminescence (CL) methods. The cellular protein extracts of D. radiodurans R1 and KD8301 showed higher scavenging effects on O2- than that of E. coli. D. radiodurans R1 and KD8301 also strongly scavenged H2O2 with an EC50 (50% effective concentration) of 0.12 and 0.2 mg/mL, respectively, compared to that of E. coli (EC50 = 3.56 mg/mL). The two strains of D. radiodurans were effective in scavenging *OH generated by the Fenton reaction, with EC50 of 0.059 and 0.1 mg/mL, respectively, compared to that of E. coli (EC50 > 1 mg/mL). Results from the chemiluminescence assay of *OH-induced DNA damage and the plasmid pUC18 DNA double-strand break (DSB) model in vitro showed that D. radiodurans had remarkably inhibitory effect on the *OH-induced oxidative damage of DNA. The scavenging effects of D. radiodurans on reactive oxygen species (ROS) played an important role in the response to oxidation stress and preventing against DNA oxidative damage, and may be attributed to intracellular scavenging proteins, including superoxide dismutase (SOD) and catalase.     

The antioxidant action of taurine, hypotaurine and their metabolic precursors.

It has been suggested that taurine, hypotaurine and their metabolic precursors (cysteic acid, cysteamine and cysteinesulphinic acid) might act as antioxidants in vivo. The rates of their reactions with the biologically important oxidants hydroxyl radical (.OH), superoxide radical (O2.-), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) were studied. Their ability to inhibit iron-ion-dependent formation of .OH from H2O2 by chelating iron ions was also tested. Taurine does not react rapidly with O2.-, H2O2 or .OH, and the product of its reaction with HOCl is still sufficiently oxidizing to inactivate alpha 1-antiproteinase. Thus it seems unlikely that taurine functions as an antioxidant in vivo. Cysteic acid is also poorly reactive to the above oxidizing species. By contrast, hypotaurine is an excellent scavenger of .OH and HOCl and can interfere with iron-ion-dependent formation of .OH, although no reaction with O2.- or H2O2 could be detected within the limits of our assay techniques. Cysteamine is an excellent scavenger of .OH and HOCl; it also reacts with H2O2, but no reaction with O2.- could be measured within the limits of our assay techniques. It is concluded that cysteamine and hypotaurine are far more likely to act as antioxidants in vivo than is taurine, provided that they are present in sufficient concentration at sites of oxidant generation.     

Regulation of Fas (CD95)-induced apoptotic and necrotic cell death by reactive oxygen species in macrophages

Although reactive oxygen species (ROS) have long been suspected to play a key role in Fas (CD95)-induced cell death, the identity of specific ROS involved in this process and the relationship between apoptotic and necrotic cell death induced by Fas are largely unknown. Using electron spin resonance (ESR) spectroscopy, we showed that activation of Fas receptor by its ligand (FasL) in macrophages resulted in a rapid and transient production of hydrogen peroxide (H2O2) and hydroxyl radicals (*OH). The response was visible as early as 5 min and peaked at approximately 45 min post-treatment. Morphological analysis of total death response (apoptosis vs. necrosis) showed dose and time dependency with apoptosis significantly increased at 6 h after the treatment, while necrosis remained at a baseline level. Only at a 35-fold increase in apoptosis did necrosis become significant. Inhibition of apoptosis by a pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (zVAD-fmk), significantly inhibited cell necrosis, indicating the linkage between the two events. Catalase (H2O2 scavenger) and deferoxamine (*OH scavenger) effectively inhibited the total death response as well as the ESR signals, while superoxide dismutase (SOD) (O2*- scavenger) had minimal effects. These results established the role for H2O2 and *OH as key participants in Fas-induced cell death and indicated apoptosis as a primary mode of cell death preceding necrosis. Because the Fas death pathway is implicated in various inflammatory and immunologic disorders, utilization of antioxidants and apoptosis inhibitors as potential therapeutic agents may be advantageous.     

Chemiluminescence study of active oxygen species produced by TiO2 photocatalytic reaction.

Two chemiluminescence approaches have been used for study of active oxygen species produced by the TiO2 photocatalytic reaction. One is based on flow injection analysis (FIA)-luminol chemiluminescence (CL); another is a time-resolved CL method. In the FIA-CL experiment, an UV-illuminated TiO2 suspension and water were passed into a mixing cell by two separate flow lines. Luminol solution was injected into the water flow line at different times. The injected luminol reacted with active oxygen species generated by the TiO2 photocatalytic reaction in a mixing coil and produced CL. It was found that the maximum CL was detected at the first injection of luminol. CL intensity decreased with time of injection. When the luminol was injected after 5 min, the CL intensity was almost unchanged. Addition of scavengers of active oxygen species indicated that the CL produced early in the 5 min was caused by O2- and H2O2, while CL after 5 min was only from H2O2. In the time-resolved CL, the third harmonic wavelength of Nd:YAG laser (355 nm) was used as a UV light source, and CL was detected by a PMT and recorded in a millisecond time scale using a digital oscilloscope. It was found that CL induced by the photocatalytic reaction increased with concentration of the TiO2 suspension. Scavengers of active oxygen species of *OH, O2- and H2O2 were added to study the involvement of the active oxygen species.     

活性氧诱发人类11号染色体基因突变

对体外产生的和内源性刺激产生的活性氧 (ROS)诱发人类 11号染色体 (Hchr 11)基因突变规律及其突变谱进行研究 .体外羟自由基 (·OH)用过氧化氢 (H2 O2 )与Fe2 + 反应产生 ,并用化学发光(CL)进行相对定量分析 ;内源性ROS用佛波醇酯 (PMA)刺激人外周血白细胞产生 ,并用CL和特异性抗氧化物检测和鉴定 ;用包含单条Hchr 11的人 中国仓鼠卵巢细胞 (AL)为靶 ,经CD59表面抗原抗体筛选突变细胞克隆 ,研究ROS诱发的Hchr 11基因突变 ;突变克隆细胞DNA用Hchr 11上 5种标志基因引物进行多重PCR分析 ,结合琼脂糖凝胶电泳绘制基因突变谱 .结果表明 ,体外ROS可诱发Hchr 11基因突变 ,且·OH诱发基因突变的能力明显强于H2 O2 ,两者的突变谱也存在明显差异 ;PMA可刺激人外周血白细胞产生大量的多种ROS ,并诱发Hchr 11基因突变 ,突变谱综合了H2 O2 和·OH的所有特征 ;一些抗氧化物对内源性产生的ROS诱发Hchr 11基因突变有明显抑制作用 .提示体外和内源性ROS可诱发Hchr 11基因突变 ,不同的活性氧分子诱发的基因突变可能具有特异性     

Mechanism of electron transfer processes photoinduced by lumazine

UV-A (320-400 nm) and UV-B (280-320 nm) radiation causes damage to DNA and other biomolecules through reactions induced by different endogenous or exogenous photosensitizers. Lumazines are heterocyclic compounds present in biological systems as biosynthetic precursors and/or products of metabolic degradation. The parent and unsubstituted compound called lumazine (pteridine-2,4(1,3H)-dione; Lum) is able to act as photosensitizer through electron transfer-initiated oxidations. To get further insight into the mechanisms involved, we have studied in detail the oxidation of 2'-deoxyadenosine 5'-monophosphate (dAMP) photosensitized by Lum in aqueous solution. After UV-A or UV-B excitation of Lum and formation of its triplet excited state ((3)Lum*), three reaction pathways compete for the deactivation of the latter: intersystem crossing to singlet ground state, energy transfer to O(2), and electron transfer between dAMP and (3)Lum* yielding the corresponding pair of radical ions (Lum˙(-) and dAMP˙(+)). In the following step, the electron transfer from Lum˙(-) to O(2) regenerates Lum and forms the superoxide anion (O(2)˙(-)), which undergoes disproportionation into H(2)O(2) and O(2). Finally dAMP˙(+) participates in subsequent reactions to yield products.     

NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-alpha

We tested the hypothesis that the NAD(P)H oxidase-dependent generation of superoxide anion (O2-*) mediates tumor necrosis factor-alpha (TNF)-induced alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The NAD(P)H oxidase subcomponents p47phox and p22phox were assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species O2-* was measured by the fluorescence of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetatedi(acetoxymethyl ester), 5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester, and dihydroethidium. TNF treatment (50 ng/ml for 4.0 h) induced 1) p47phox translocation, 2) an increase in p22phox protein, 3) increased localization of p47phox with p22phox, 4) O2-* generation, and 5) increased permeability to albumin. p22phox antisense oligonucleotide prevented the TNF-induced effect on p22phox, p47phox, O2-*, and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in O2-* and permeability to albumin was also prevented by the O2-* scavenger Cu-Zn superoxide dismutase (100 U/ml). The results indicate that the activation of NAD(P)H oxidase, via the generation of O2-*, mediates TNF-induced barrier dysfunction in PMEM.     

Scavenging of reactive oxygen species by novel indolin-2-one and indoline-2-thione derivatives

The antioxidant behavior of a series of new synthesized substituted indoline-2-ones and indolin-2-thiones was investigated in this study using an oxygen radical absorbance capacity assay (ORAC(ROO*-) and 2,2'-azobis(2-amidino-propane) dihydrochloride (AAPH) as the radical generator; system generating superoxide anion radical, O2*- (18-crown-6/KO(2)/DMSO), and the Fenton-like reaction [Co(II) + H(2)O(2) --> Co(III) + HO(*) + HO(-)]. Measurements were done using fluorescence, chemiluminescence methods, and a deoxyribose assay based on the spectrophotometry method, respectively. The results obtained indicated that the examined indoline derivatives had effective activities as radical scavengers and may be considered as an effective source for combating oxidative damage.     

Photochemiluminescent detection of antiradical activity. VII. Comparison with a modified method of thermo-initiated free radical generation with chemiluminescent detection.

The method of photosensitized chemiluminescence (PCL) allows the quantification of water- and lipid-soluble antioxidants and activity of superoxide dismutase (SOD) in the same measuring system. However, it needs a special device, which we have described in a previous paper in this series. Another method suitable for the assay of water- and lipid-soluble antioxidants is the thermo-initiated decay of azo-compounds combined with the measurement of O2 consumption (Niki, 1985; Wayner et al., 1985). Its long duration and the complicated measuring procedure is not acceptable for routine medical applications. We show that a modification using CL detection of free radicals with luminol, has results comparable with PCL for the determination of non-enzymic water- and lipid-soluble antioxidants, SOD activity and oxidative modification of proteins. In contrast to PCL, it is possible to use any luminometer with a heatable measuring cell and to investigate coloured samples. While the new method has an overall higher sensitivity and is scalable to microtitre plates, PCL measurements can be made at different pH. The advantages and analytical information content of certain components of the integral antioxidative capacity of blood plasma are discussed in comparison with other methods.