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

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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
On the other hand, in experimental trials, ithas been shown that the BAP resultssatisfyingly correlate with those of FRAP, whichis considered the comparison method the mostsimilar to BAP test, in the actual panorama ofall the assays proposed to evaluate the plasmaantioxidant capacity.Indeed, the FRAP assay is based on thereduction of ferric tripyridyltriazine (Fe III -TPTZ)to ferrous tripyridyltriazine (Fe II -TPTZ), whichdevelops an intense blue color, phometricallymeasurable by setting the wavelength to 593nm (21). The calibration may be performed bymeans of solutions with known concentrationsof Fe II (FeSO 4 . 7H 2 O). Analogously, the BAP testis based on the use of a colored solution whichis obtained by adding a source of ferric ions toa chromogenic mixture.The entity of decoloration is then assessedby measuring the absorbance at 505 nm,which is proportional to the reducing activity ofthe sample.According to these observations, Vassalle(unpublished data) recently assessedcomparatively analytical performance of BAPand FRAP test on 18 samples of human bloodplasma.According to the result of this study, meanvalue (± SD) of BAP test was 3163±770 µEq/L,while the mean value (± SD) of FRAP test was521±132 µmol/L.The correlation between test was shown tobe acceptable in 16/18 tested sample(88.89%).The remaining two samples were shown tonot correlate, maybe to the higher level ofplasma lipids, which can interfere with thephotometric readings of every photometric test(Figure 7).FRAP test (µmol/L)FRAP (µmol/l)80070060050040030020010001500 2000 2500 3000 3500 4000 4500BAP test (µEq/L)Figure 7. Comparative evaluation of BAP test andFRAP assay (modified by Vassalle et al, 2006)(Unpublished data).4. ProcedureBAP test can be performed either onwhole blood or heparinised blood plasma orserum. Such samples should be fresh, in orderto avoid an eventual underestimation of theresults due to possible auto-oxidationphenomena.Therefore, while waiting further data bystudies in progress, it is suggested tocentrifuge as soon as possible (within 1 hour)the blood and to recover plasma or serum. Ifthe sample (plasma/serum) cannot beimmediately processed it should be stored at+4 °C and tested within 1-to-3 days oraccording to the guidelines of laboratory goodpractice.However very recent evidence suggeststhat BAP test maintains its analytical reliabilityeven on previous frozen samples (at -80°C)The BAP test can be performed with acommon photometer, according to the exactstandardized work conditions (Table 2).Table 2. Work conditions of BAP test.Wavelenght Optical path Temperature Mode505 nm 1 cm 37 °C DifferentialHowever, due to some practical reasons,the test can be performed also with dedicatedanalytical devices, like FREE system (DIACRONInternational s. r. l., Grosseto, Italy) (24), forblood plasma or serum, or FRAS 4 system(H&D s. r. l., Parma, Italy) (20) by startingform whole (capillary) blood. Both systems,which require dedicated kits, substantially limitmanual intervention to a few steps comparedto common photometers, thus reducing apossible source of analytical imprecision.Moreover, either FREE or FRAS systemallows a global assessment of oxidative stress,due to the possibility to perform not only theBAP test (antioxidant status assessment) butalso the d-ROMs test (oxidant statusassessment) (20, 24).9www.fras4evolvo.it
Page 1 and 2: Eugenio Luigi IorioA short reviiewR
Page 3 and 4: Table of ContentsPreface and acknow
Page 5 and 6: 1. IntroductionThe blood of Vertebr
Page 7: different analytical approach (e. g
Page 12 and 13: In particular, 140 Labrador Retriev
Page 14 and 15: µEq/L25002000150010005000Before di
Page 16 and 17: 8. Clinical studies on AnimalsGrowi
Page 18 and 19: absorption of nutrients, etc.). In
Page 20 and 21: 21. Benzie IFF, Strain JJ. The ferr
Page 22 and 23: 53. Carratelli M, Iorio EL, Bianchi
Page 24: F R A S 4 EVOLVO SYSTEMFREE RADICAL

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