Methods For Evaluation Of Biological Reducing Power

Due to the difficulties in measuring each reducing antioxidant component separately and the interactions among different antioxidant components, several methods have been developed to assess the total reducing capacity of biological tissues and fluids. These methods consist of two general categories: direct and indirect approaches [2]. Direct methods for measuring reducing capacity in biological tissues and fluids are those that use an external probe as a measure of the reducing or oxidizing capacity of a system. An example of a direct method is an electrode at which the current is proportional to the concentrations of the species present. Indirect methods are those that measure consequential factors of redox capacity, such as oxidation products formed, or concentrations of major redox couples in the biological environment, such as glutathione/glutathione disulfide. Within this category, a few techniques are available whereby free radicals are produced at a known rate, and their removal by scavenger, which is monitored by an endpoint [2,25].

It must be understood that each method measures different factors using various technologies; therefore, different results for identical samples should be expected. The results are then given as a measure of the specific factor of a certain method and not as a definite intrinsic parameter.

A. Indirect Methods for Measuring Reducing Power in Biological Tissue and Fluids

Indirect methods include those that measure the concentration of a specific redox couple in the biological tissue/fluid using fluorescent or spectrophotometric techniques. In this approach it is assumed that a biological redox buffer exists in the form of a redox couple that is sensitive to changes in the redox environment and thus reflects changes in reducing power. The concentration of a redox couple (its reduced and oxidized forms) reflects the reducing capacity of the sample but is not, by definition, the reducing power, which is measured by other methods, such as voltammetry. Other methods for measuring reducing power are inhibition methods that consist of adding a radical species to the sample together with a scavenger that can be detected due to its optical properties.

1. NADP/NADPH

The redox state of the cell was defined by the concentration ratio of free NAD+/ NADH2 [3]. Due to the fact that classical enzymatic techniques fail to distinguish between free and protein-bound nucleotides, this ratio cannot be obtained by direct measurement of free NAD+ and NADH2 in tissue [26]. The NAD + /NADH2 ratio is calculated using the concentrations of other linked redox couples that interact with the NAD + /NADH2 at a known equilibrium constant, such a lactate/pyruvate [26].

2. Lactate/Pyruvate

The widest clinically used method for measuring tissue redox state is the lactate/ pyruvate redox sytem [1]. Analyses of lactate and pyruvate are carried out by extraction and spectrophotometry [27].

In human blood, concentrations of the lactate/pyruvate couple are 300-1300 and 30-70 mM, respectively [1]. Due to its ability to permeate cell walls, coronary venous lactate/pyruvate was postulated to reflect the NAD/NADH ratio in cells, but this is not always the case [28,29]. The lactate/pyruvate ratio remains extraordinarily constant, even when both lactate and pyruvate are raised above normal levels [29]. For this reason this redox couple was considered a redox buffer in blood and to some extent in tissue. Though unaffected by nutritional factors, the lactate/pyruvate ratio varies as a result of muscular work (excess lactate) [29].

3. Glutathione/Glutathione Disulfide

Glutathione (GSH) is the major free thiol in most living cells and participates in major biological processes. Oxidation of GSH results in glutathione disulfide (GSSG). Intracellular GSH is effectively maintained in the reduced state by GSSG reductase. GSH and GSSG have been considered as the redox buffer of the living cell and tissue, and their absolute and relative concentrations a measure of the system's redox state [1,19].

There are many methods for the measurement of GSH. The most common consists of complexation of a substance with GSH, which results in a product/complex that can be detected by spectrophotometry or fluorescence. Examples of complexating agents are methylglyoxal [30] and ortho-phthaldialdehyde [30]. Some methods involve redox reaction with a reactant so that it or its product can be detected by spectropho-tometry or fluorescence. Examples are NADPH in presence of GSSG reductase [30] or NADPH and DTNB [5,5'-dithiobis (2-nitrobenzoic acid)] [30,31]. For HPLC analysis of GSH and GSSG, derivatization of thiol compounds with fluorescent labeling agents such as monobromobimane [30] or 2,4-dinitrofluorobenzene [32] is necessary, although direct detection using an electrochemical detector is possible.

4. Ascorbic Acid/Ascorbate in Blood Plasma

Table 2 shows the major antioxidants found in blood plasma. Since the concentration of ascorbic acid in human blood plasma is relatively high compared to other anti-oxidants, it has been considered a potential blood buffer, and a variety of assays for blood ascorbic acid exist [33]. The most widely used method for measuring both uric acid and ascorbic acid is paired ion, reversed phase HPLC with an electrochemical detector [34]. Most other antioxidants in blood plasma are measured using HPLC as well, with either spectrophotometric, fluorescent, or electrochemical detectors. However, in contrast to GSH or NADH, ascorbic acid is an antioxidant supplied by a dietary source. Therefore, the BRP in blood, as measured by this method, will be influenced by dietary factors as well as by changes in redox active components.

5. Trolox Equivalent Antioxidant Capacity

Trolox equivalent antioxidant capacity (TEAC) [34,35] is a measure of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble vitamin E mimic) antioxidant equivalents. The sample is placed in a solution with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and H2O2, which generate ABTS

cation radicals, in the presence of metmyoglobin as a peroxidase. The decay in ABTS cation radicals absorbance as a result of quenching by antioxidants in the sample is monitored in the absence and the presence of Trolox. Only a lag phase or inhibition percentage at a fixed time point can be quantified as the result. The direct interaction of an added sample of antioxidants with H2O2 cannot be excluded as the molar ratios of H2O2:metmioglobin:ABTS:Trolox are 10.2:0.25:25:1. Direct interaction between H2O2 and ABTS cation radical should also be taken into account. The TEAC results for blood plasma are not linear with concentration of sample [36].

6. Total Radical Trapping Potential

The total radical trapping potential (TRAP) [2] has been widely used during the last decade and is based on O2 measurement with an O2 electrode. 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) that undergoes spontaneous decomposition and produces peroxyl radicals at a temperature-dependent rate [37], is added to the sample, and the O2 consumed is monitored both in the absence and the presence of Trolox. This method is based on the assumption that all damage resulting from the attack of the peroxyl radicals involves O2 consumption. As this is not always the case, the TRAP assay result cannot be considered as a measure of the BRP. Furthermore, the use of the O2 electrode, which is not sufficiently stable during prolonged measurement, sheds doubt on the value of this particular measurement of peroxyl radicals damage control [35].

7. Chemiluminescence

The chemiluminescence method [2,38] is based on the reaction of AAPH radicals with Luminol to produce a cation radical, which is monitored by chemiluminescence. Like previous methods discussed in this category, the sample capacity to inhibit the luminescence of luminol is measured relative to Trolox. The lag time is proportional to the total antioxidants present in the sample.

8. Oxygen Radical Absorbance Capacity

Oxygen radical absorbance capacity (ORAC) [2] is based on the fluorescence properties of phycoerythrin (PE). The fluorescence of PE is highly sensitive to the conforma-tional and chemical integrity of the protein. The loss of PE fluorescence under appropriate conditions is indicative of oxidative damage. The fluorescene decay of PE in the presence of radicals relates a lag phase or rate to antioxidant capacity of an added sample. The results are compared to those in the presence of Trolox.

Unlike TEAC and TRAP, this method takes the reactive species to complete reaction and uses the area under the curve technique for quantification, thus combining both the inhibition time and inhibition percentage of the reactive species action by antioxidants into a single quantity.

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