The BAP test and the global assessment of oxidative stress ... - Medial

The result of a clinical study on hyperbarictherapy seems to confirm this hypothesis (23)(see below, clinical studies on Humans andFigure 11 ). In any case BAP test was able todetect in vitro increased amounts of bilirubin aminor iron-reducing agent (20) (Figure 4).10000µEq/L5000400030002000R=0.7µEq/L80006000400020000Control 0.1 0.5 1.0 2.0 5.0Bilirubin (mg/dL)Figure 4. Effects of solutions of bilirubin on BAPtest in vitro (modified by Dohi et Al, 2005) (20).Finally, experiments ex-vivo performed onhuman plasma showed a direct and significantcorrelation between BAP test values and α-tocopherol levels (24) (Figure 5).00 2 4 6 8 10 1214µMFigure 5. Correlation between BAP test values andα-tocopherol concentration in a sample of rat bloodplasma (modified by Dohi et al, 2007) (24).Taken together the evidence now availableindicates that BAP test provides a reliablemeasure of all the antioxidant plasmacompounds/activities able to reduce ferric ionsto ferrous ions including UA, AsA, bilirubin andα−tocopherol, as previously described for FRAPassay (21) (see also below, paragraph 3).3. Validation and comparative studiesThe validation of BAP test has beenachieved by means of experiments performedwith the Electron Spin/ParamagneticResonance Spectrometry (ESR/EPR) that isconsidered the “golden standard” technique tostudy free radicals ex vivo.According to its principle, BAP test providesan evaluation of the whole antioxidant capacityof blood plasma, measured as reducingpotential against ferric ions.Several compounds may contribute to this“biological antioxidant potential” of whomsome – like bilirubin (see Table 1) – exhibits a“scavenger” activity against free radicals,having the ability to neutralize directly freeradicals.On these basis, an ESR/EPR studyperformed showed that the bilirubin exhibits“in vitro” direct scavenging activity againsteither the hydroxyl radical (HO • ), the mostpotentially dangerous oxygen free radicals inliving organisms, or the 1,1-diphenylpicrylhydrazyl radical already atphysiological conditions (20).In experiments performed in parallel withthe BAP test it was shown that the results ofboth assays (as conducted either by ESR/EPRor photometry) overlapped (20) (Figure 6).Figure 6. Comparison between photometric BAPassay (histograms) and Electron Spin ResonanceSpectrometry (ESR/EPR) (box): increasingconcentrations of bilirubin are able to increase BAPtest values (because bilirubin acts anantioxidant/iron reducing compound) and to reduceconcomitantly ESR/EPR signal (because bilirubinacts as a scavenger against hydroxyl radical)(modified by Dohi et al, 2005) (20).By considering that ESR/EPR is thereference technique to study the free radicals,BAP test must be considered, thanks to thisauthoritative validation, a test really able todetect and quantify in a specific and suitablemanner scavenging/antioxidant activities, as aconfirmation of the above proposed principle ofits mechanism.8www.fras4evolvo.it