Measurement Of Total Antioxidant Capacity Research Articles

Antioxidant inhibition of oxygen radicals for measurement of total antioxidant capacity in biological samples

Few methods for assessing total antioxidant capacity (TAC) include both the percentage of inhibition and the length of inhibition in the measurement. Available methods require above ambient constant temperature incubation, reaction preheating, and/or separate assays for testing hydrophilic and hydrophobic samples. We describe a high-throughput method, antioxidant inhibition of oxygen radicals (AIOR), that overcomes these difficulties. AIOR uses peroxyl radicals to trigger a decrease in fluorescence of the indicator molecule, uroporphyrin I, which is delayed by the presence of antioxidants. The area under the curve is measured by a fluorescence spectrophotometer in a 96-well microplate format, and TAC results are expressed as millimole/liter Trolox equivalents. AIOR is performed at ambient temperature and is applicable to samples in either aqueous or common organic solvents. The reaction between uroporphyrin I and the peroxyl radicals generated from 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) was found to be of first-order kinetics with a mean rate constant ( k) of 0.0254. Applications to measure antioxidant capacity are demonstrated on individual chemicals and biological samples. The method has good linearity, within- and between-assay precision, and recovery.

The use of Pholasin® as a probe for the determination of plasma total antioxidant capacity

Background and objectives Total antioxidant capacity (TAC) reflects the capacity of plasma, or other body fluid, to resist oxidation. The aim of the present study was to assess the prognostic value of Pholasin® as a probe for the determination of plasma TAC. Design and methods Plasma samples either oxidised using the free radical generator 2′-azobis-(2-amidinopropane) hydrochloride (AAPH) at 60°C for 180 min or obtained from diabetic (type 1 and type 2) patients ( n = 61 and 124, respectively) with or without polyneuropathy (PN) and/or cardiovascular autonomic neuropathy (CAN) and control subjects ( n = 70) were analysed for TAC status. TAC was assessed using two versions of Analysis By Emitted Light (ABEL®) tests including quenching of Pholasin® chemiluminescence (QPC) with peroxynitrite (ONOO − -QPC) and with superoxide anion (O 2 − -QPC). Results The utilisation of AAPH to induce peroxyl radical mediated plasma oxidation produced a significant decrease in TAC as assessed by ONOO − -QPC and O 2 − -QPC assays in a time-dependent manner. Type 1 and type 2 diabetic patients without PN and/or CAN had lower TAC (ONOO − -QPC and O 2 − -QPC) than in control subjects. Further alterations in TAC were noted in the presence of PN and/or CAN. Correlations were found between TAC (ONOO − -QPC and O 2 − -QPC) values on duration of diabetes and neurological impairment score-lower limb (NIS-LL) in type 1 diabetic patients. Conclusions This study has established that the ONOO − -QPC and O 2 − -QPC versions of the ABEL® test fulfil the criteria in terms of simplicity, sensitivity and reliability for the measurement of plasma TAC. These biomarkers may prove useful in studies evaluating the impact of therapeutic drugs or antioxidant interventions aimed at delaying the onset of complications in clinical conditions associated with oxidative stress.

Resistance to Paclitaxel Is Proportional to Cellular Total Antioxidant Capacity

Paclitaxel, one of the most commonly prescribed chemotherapeutic agents, is active against a wide spectrum of human cancer. The mechanism of its cytotoxicity, however, remains controversial. Our results indicate that paclitaxel treatment increases levels of superoxide, hydrogen peroxide, nitric oxide (NO), oxidative DNA adducts, G2-M arrest, and cells with fragmented nuclei. Antioxidants pyruvate and selenium, the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester, and the NO scavenger manganese (III) 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide all decreased paclitaxel-mediated DNA damage and sub-G1 cells. In contrast, the glutamylcysteine synthase inhibitor buthionine sulfoximine (BSO) and the superoxide dismutase (SOD) inhibitor 2-methoxyestradiol (2-ME) increased the sub-G1 fraction in paclitaxel-treated cells. These results suggest that reactive oxygen and nitrogen species are involved in paclitaxel cytotoxicity. This notion is further supported with the observation that concentrations of paclitaxel required to inhibit cell growth by 50% correlate with total antioxidant capacity. Moreover, agents such as arsenic trioxide (As2O3), BSO, 2-ME, PD98059, U0126 [mitogen-activated protein/extracellular signal-regulated kinase inhibitors], and LY294002 (phosphatidylinositol 3-kinase/Akt inhibitor), all of which decrease clonogenic survival, also decrease the total antioxidant capacity of paclitaxel-treated cells, regardless whether they are paclitaxel sensitive or paclitaxel resistant. These results suggest that paclitaxel chemosensitivity may be predicted by taking total antioxidant capacity measurements from clinical tumor samples. This, in turn, may then improve treatment outcomes by selecting out potentially responsive patients.

1063 Automated measurement of total antioxidant capacity of seminal plasma

1063 Automated measurement of total antioxidant capacity of seminal plasma

Impact of marathon running on cardiac structure and function in recreational runners.

The present study examined the relationship between LV (left ventricular) function, markers of cardiac-specific damage and markers of oxidative stress in recreational runners following a marathon. Runners (n=52; 43 male and nine female; age, 35+/-10 years; height, 1.74+/-0.08 m; body mass, 75.9+/-8.9 kg) were assessed pre- and immediately post-marathon. LV function was assessed using standard M-mode two-dimensional Doppler echocardiography and TDI (tissue-Doppler imaging) echocardiography. Serum was analysed for cTnT (cardiac troponin-T), TEAC (Trolox equivalent antioxidant capacity; a measure of total antioxidant capacity), MDA (malondealdehyde) and 4-HNE (4-hydroxynonenal). A strong relationship was observed between standard and TDI echocardiography for all functional measures. Diastolic function was altered post-marathon characterized by a reduction in E (peak early diastolic filling: 0.79+/-0.11 compared with 0.64+/-0.16 cm/s; P<0.001), an increase in A (peak late diastolic filling: 0.48+/-0.11 compared with 0.60+/-0.12 cm/s; P<0.001) and a resultant decrease in E/A (ratio of E to A; 1.71+/-0.48 compared with 1.10+/-0.31; P<0.001). Ejection fraction remained unchanged post-marathon. Thirty-two runners presented with cTnT values above the lower limit of detection for the assay (0.01 microg/l), and 20 runners presented post-marathon with cTnT values above the acute myocardial infarction cut-off value (0.05 microg/l). No significant correlations were observed between cTnT and any functional measurements. MDA (2.90+/-1.58 compared with 3.59+/-1.47 micromol/l) and TEAC (1.80+/-0.12 compared with 1.89+/-0.21 mmol/l) were significantly increased post-marathon, but were unrelated to changes in function or cTnT. In conclusion, the present study demonstrated a reduction in diastolic function and widespread evidence of minimal cardiac damage following a marathon in recreational runners. The mechanism(s) underpinning the altered function and appearance of cTnT appear unrelated to reactive oxygen species.

Measurement of total antioxidant capacity in gingival crevicular fluid and serum in dogs with periodontal disease

To determine whether gingival crevicular fluid (GCF) and serum total antioxidant capacities (TACs) correlate with the degree of severity of periodontal disease in dogs. 41 Toy and Miniature Poodles. After assessment of the degree of severity of naturally occurring periodontitis, GCF samples from both maxillary fourth premolars and a blood sample were collected from each dog. The condition of the periodontium of the entire dentition and at each site of GCF collection was recorded. Clinical parameters assessed included plaque index, gingival index, and probing depth. Radiographic analysis of alveolar bone level was also performed. Total antioxidant capacity was measured in GCF and serum samples by use of a commercial kit. Dogs with gingivitis and minimal periodontitis had significantly higher TAC in GCF than dogs with advanced periodontitis. Bivariate regression analysis revealed significant negative correlations between TAC in GCF and clinical parameters and age. The TAC in serum was significantly negatively correlated with the degree of gingival inflammation but was not significantly correlated with age. TAC in GCF is related to the degree of severity of periodontal disease in dogs. This is likely the result of release of reactive oxygen species by activated phagocytes and fibroblasts in the inflamed periodontal tissues. The results of our study suggest that the local delivery of antioxidants may be a useful adjunctive treatment for periodontitis in dogs.

Comparison of spectrophotometric and enhanced chemiluminescent assays of serum antioxidant capacity

Background: Over recent years, interest in total antioxidant capacity measurement in biological fluids has increased. A number of assays are now available, and we wished to compare an enhanced chemiluminescence (ECL) method to a spectrophotometric method, the total antioxidant status (TAS) assay. Methods: Serum urate concentration, ECL and TAS were measured in 34 healthy subjects. Additionally, 10 subjects participated in a two-way, randomised crossover study, and received urate 1000 mg or vitamin C 1000 mg intravenously over 1 h. Serum ECL and TAS were measured at 0, 15, 30, 45, 60, 90 and 120 min after commencing infusion. Results: Baseline measurements were poorly correlated between ECL and TAS assays, and between serum urate concentration and each antioxidant assay. There was good correlation between the change in antioxidant capacity detected by both assays during urate infusion ( R=0.79, p<0.001, n=60), but not vitamin C infusion. Conclusions: ECL and TAS measures of serum antioxidant capacity correlate poorly in a healthy population, although both are sensitive to increases in circulating urate concentrations. Therefore, ECL and TAS appear sensitive to different factors. The comparative strengths and weaknesses of various antioxidant assays should be reviewed.

Wine, diet, antioxidant defenses, and oxidative damage.

Oxidative stress is a central mechanism for the pathogenesis of ischemic heart disease and atherogenesis, for cancer and other chronic diseases in general, and it also plays a major role in the aging process. Dietary antioxidants constitute a large group of compounds that differ in mechanism of action, bioavailability and side effects. A systematic analysis of the role of the various antioxidants in chronic diseases is hampered by the difficulty of employing death or clinical events as end points in intervention studies. Therefore, valid markers for oxidative stress, which show dose response and are sensitive to changes in dietary supply of antioxidants, are potentially of great value when trying to establish healthy dietary patterns, or when one component, like red wine, is evaluated specifically. To evaluate potential oxidative stress markers we have studied the effect of different diets plus wine supplementation on antioxidant defenses and oxidative damage. In three experimental series, four groups of young male university students, one of older men and other of older women, 20-24 volunteers each, received Mediterranean or occidental (high-fat) diets alone or supplemented with red wine, white wine, or fruits and vegetables. Measurements included, leukocyte DNA 8-OH-deoxyguanosine (8OHdG), plasma 7 beta-hydroxycholesterol, TBARS and well-characterized antioxidants, and plasma and urine polyphenol antioxidants. In all experimental groups that received red wine, consumption resulted in marked decrease in 8OHdG. The changes observed in 8OHdG correlate positively with the other markers of oxidative damage, and shows a clear inverse correlation with the plasma level of well established antioxidants and with measurements of total antioxidant capacity. Urinary total polyphenol content as well as the sum of some specific plasma species also correlate inversely with 8OHdG. In conclusion, the results identify 8OHdG as a very promising general marker of oxidative stress in nutrition intervention studies in humans, and red wine shows a remarkable protective effect.

Total Antioxidant Capacity (TAC) Values and Their Correlation with Individual Antioxidants in Healthy Beagles

Nemec A. , M. Drobnia-Ko‰orok, M. Ski tek, Z. Pavl ica , S. Galac, J . But inar : Total Antioxidant Capacity (TAC) Values and Their Correlation with Individual Antioxidants in Serum of Healthy Beagles. Acta Vet. Brno, 2000, 69: 297–303. The paper aims to establish a range for serum Total Antioxidant Capacity (TAC) and to determine the correlation between TAC and some individual antioxidants (vitamin A and E, lipid standardised vitamin E [Vit E/LS/], β-carotene, total bilirubin and albumin) in a uniform population of beagle dogs. Emphasis was directed on their general health status to establish a basis for future investigation of the role of TAC in diseases of dogs as species most frequently studied in veterinary medicine. The animal body possesses a variety of protective antioxidant substances that act as a harmoniously and finely tuned mechanism to neutralise harmful oxidants. TAC measurements provide a tool for establishing links between antioxidant capacity and the risk of disease as well as for monitoring of antioxidant therapy. Serum samples of 19 healthy beagles were assayed for TAC on LP 700 photometer (Dr. Lange, Germany) with a commercially available TAS kit (“Total Antioxidant Status” – TAS; Randox, Crumlin, UK). TAS kit measures the capacity of all of the antioxidants present in serum or plasma sample. The assay is based on the suppression of the absorbance of the radical cation of 2,2“-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS•+) by antioxidants. Assay results are expressed as mmol/l of Trolox (6-hydroxy-2, 5, 7, tetramethylchroman-2-carboxylic acid – a water-soluble analogue of α-tocopherol) equivalents. The range for TAC expressed as mean ± SD resulted in 1.08 ± 0.08 mmol/l. TAC correlated positively with albumin (r = 0.18), vitamin E (r=0.14) and Vit E (LS) (r = 0.20), and negatively with total bilirubin (r = -0.30), vitamin A (r = -0.15) and β-carotene (r = -0.13) although no significant correaltion has been found. Antioxidant capacity, antioxidants, free radicals, dog A wide range of substances, known as Reactive Oxygen Species (ROS), consisting of free radicals such as O2•, OH•, and other non-radical oxygen derivatives such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and singlet Oxygen ( O2) are constantly generated in vivo as an integral part of metabolism, as part of a controlled inflammatory reaction and by exposure to environmental factors. A free radical can be defined as any species capable of independent existence that contains one or more unpaired electrons (Hal l iwel l 1996). Non-radical oxygen derivatives are substances capable of radical formation in intraand extra-cellular environments (Cao and Prior 1998; Chapple 1997; Hall iwel l 1997). The pathological increase of ROS generation has already been recognised in over one hundred human and animal diseases including cancer, cardiovascular disease, diabetes mellitus, male infertility, renal disease and dialysis, cataracts, neurological, liver, periodontal, lung and inflammatory diseases (Hal l iwel l 1996; Lantos et al. 1997; Moore et al. 1994; Pinzani et al. 1998). ROS circulate freely in the body with access to all organs and tissues. They cause tissue damage by a variety of different mechanisms, which ACTA VET. BRNO 2000, 69: 297–303 Address for correspondence: Alenka Nemec Clinic for Small Animal Medicine and Surgery Veterinary Faculty Gerbiaeva 60, 1115 Ljubljana, Slovenia Phone: + 386 01 4779 301 Fax: + 386 01 2833 708 E-mail: nemecal@mail.vf.uni-lj.si http://www.vfu.cz/acta-vet/actavet.htm

Plasma chain-breaking antioxidants in Alzheimer's disease, vascular dementia and Parkinson's disease.

We studied the plasma chain-breaking antioxidants alpha carotene, beta carotene, lycopene, Vitamin A, Vitamin C, Vitamin E and a measure of total antioxidant capacity, TAC, in 79 patients with Alzheimer's disease (AD), 37 patients with vascular dementia (VaD), 18 patients with Parkinson's disease and dementia (PDem), and 58 matching controls, together with 41 patients with Parkinson's disease (PD) and 41 matching controls. Significant reductions in individual antioxidants were observed in all dementia groups. When compared to controls, the following were reduced: Vitamin A in AD (p < 0.01) and VaD (p < 0.001); Vitamin C in AD (p < 0.001), VaD (p < 0.001) and PDem (p < 0.01); Vitamin E in AD (p < 0.01) and VaD (p < 0.001); beta carotene in VaD (p = 0.01); lycopene in PDem (p < 0.001). Lycopene was also reduced in PDem compared to AD (p < 0.001) and VaD (p < 0.001). Antioxidant levels in PD were not depleted. No significant change in TAC was seen in any group. The reduction in plasma chain-breaking antioxidants in patients with dementia may reflect an increased free-radical activity, and a common role in cognitive impairment in these conditions. Increased free-radical activity in VaD and PDem could be associated with concomitant AD pathology. Individual antioxidant changes are not reflected in TAC.