In vivo total antioxidant capacity: comparison of two different analytical methods

Abstract

Several methods to assess the total antioxidant capacity (TAC) are available. However, the final value of measured TAC in the sample depends on the procedure used in every specific assay. This makes crucial the comparison of different analytical methods. The aim of our study was to evaluate analytical characteristics and laboratory reliability of two different assays: the ferric-reducing ability (FRAP) assay and a new spectrophotometric test (OXY-adsorbent test, Diacron, Italy).

Unselected outpatients referred to the Institute of Clinical Physiology were studied (n=187, 58 females, 129 males, mean age: 65±13 years). All blood samples were maintained on ice, centrifuged within 15 minutes after blood collection and then stored at −80°C until performance of assay procedures.

OXY assay: The lower limit of sensitivity was 6 μmol HClO/ml. The assay was found to be linear up to 440 μmol HClO/ml (r=−0.99, p<0.001). Absorbance was linear over a wide concentration range with solutions containing uric acid in purified form (0–1000 μmol/l, r=−0.996, p<0.001), serum (r=−0.99, p<0.01) or plasma serially diluted (r=−0.99, p<0.01). Mean value in plasma samples accounted for 366.2±7.2 μmol HClO/ml. Mean OXY value in females (353.4±13.2 mmol HClO/ml) was not different from that detected in males (372±8.6 mmol HClO/ml). A significant difference was observed between subjects without and with hypertension in serum OXY levels (344.8±9.9 and 383.2±10 μmol HClO/ml, p<0.01, respectively).

FRAP assay: The lower limit of sensitivity was 15 μmol/l. Linearity was observed up to 1000 μmol/l (r=−0.998, p<0.001). Absorbance was linear over a wide concentration range with solutions containing uric acid in purified form (0–1000 μmol/l, r=0.997, p<0.001), serum (r=0.99, p<0.01) or plasma serially diluted (r=0.99, p<0.01). FRAP mean value in plasma samples, evaluated in 102 patients, accounted for 514.1±19.1 μmol/l. Mean FRAP in females (469±22.5 μmol/l) was not different from that detected in males (535±25.6 mmol/l).

FRAP vs. OXY: A significant direct relationship was observed when comparing FRAP with OXY levels in the whole population (r=0.22, p<0.05).

Neither of the methods are expensive and they are speedy and simple to perform. Values are reproducible and linearly correlated to the concentration of antioxidants present in the samples. For this reason, these methods may be considered practicable indicators of total antioxidant capacity, for routinely potential use in every laboratory and useful in all the studies concerning the evaluation of oxidative stress.