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

Eugenio Luigi IorioA short reviiewRellease 4.. 1 – 2010The BAP testand the globalassessmentof oxidative stressin clinical practice

2www.fras4evolvo.it

Table of ContentsPreface and acknowledgments P. 41 Introduction p. 52 Principle p. 63 Validation and comparative studies p. 84 Procedure p. 95 Analytical performances p. 106 Interpretation of results p. 127 Clinical studies on Humans p. 138 Clinical studies on Animals p. 169 Experimental studies in vitro p. 1710 Indications and clinical usefulness p. 1711 Concluding remarks p. 18References P. 19Info P. 243www.fras4evolvo.it

Antioxidants play a relevant role in the biochemical homeostasis of our body by defendingour cells and tissues against potentially toxic chemical oxidants species, including free radicals andreactive oxygen species (ROS).Therefore, in the light to prevent/to treat the possible unwanted effects of oxidative stress,many peoples currently take antioxidant supplements with the aim to restore and/or to maintainan optimal level of antioxidant defense effectiveness.However recent studies and clinical experience suggest that any antioxidant supplementshould be taken only after a biochemical evidence of its deficiency. Indeed high doses of someantioxidants may result pro-oxidant.On these basis, in order to establish the clinical indications for antioxidant treatment and toavoid concomitantly the unwanted side effects of unneeded supplements, some years ago theItalian researcher Mauro Carratelli developed a new test, i. e. the BAP test, where the initials meanBiological Antioxidant Potential. This test is based on the ability of a plasma blood sample toreduce ferric ions to ferrous ions as a FRAP assay (ferric reducing ability of plasma). Howevercompared to the classical FRAP assay firstly described by Benzie, BAP test appears quicker andsimpler to perform. Moreover according to the novel concept of global oxidative stress assessment,the BAP test showed able to integrate the results of the d-ROMs test, which allows the assessmentof serum/plasma total oxidant capacity.The aim of the present book was to describe the principle, the validation and the clinicalapplication of BAP test in Human and Veterinary Medicine.We acknowledge the help of all Authors of clinical and experimental studies reported in thisbook and, particularly, Mauro Carratelli, the “inventor” of BAP test.Dr. Eugenio Luigi Iorio, MD, PhDThe President of InternationalObservatory of Oxidative Stress4www.fras4evolvo.it

1. IntroductionThe blood of Vertebrates contains severalcompounds/activities which are able, takentogether, to oppose the oxidant potential ofoxidant chemical species, like the reactiveoxygen species (ROS) (1).Virtually, any “endogenous” compound (i.e. albumin, transferrin, ceruloplasmin, bilirubin,uric acid, reduced glutathione, etc.) or“exogenous” biomolecule (i. e. tocopherols,carotens, ubiquinol, ascorbate, methionine,flavonoids, polyphenols, etc.) which is able togive equivalent reducing units (e. g. electrons)is able also to block the potential damage ofROS. Indeed, the reactivity of a free radical isjust the result of a lack of electrons (2).Of course, any injury to such “blood barrierto oxidation” – e. g. exposure either toradiations, or xenobiotics or infectious agents –can result in oxidative tissue damage and,therefore, in early aging and in the so-calledoxidative stress related diseases (e. g.atherosclerosis, arterial hypertension, stroke,myocardial infarction, diabetes, arthritis,dementia, colitis, pancreatitis, respiratorydiseases, cancer, infections, etc.) (3).The velocity of these processes can beslowed down not only by specificpharmacotherapies (e. g. antihypertensive orlipid-lowering drugs), but also by modifyingsome lifestyles (e. g. diet, exercise, etc.) and,when indicated, by the intake of compoundswith antioxidant activity (4).However, before any medical intervention,the antioxidant effectiveness of plasma barrierto oxidation should be assessed, in order tolimit the eventual intake of antioxidants only topatients showing a reduced antioxidant plasmaactivity (5). In fact, an uncontrolled intake ofantioxidants has been shown to induce aparadoxical unwanted pro-oxidant action (6,7).Therefore, the assessment of antioxidantactivity of plasma/serum blood is the first stepof any medical intervention aimed to redressthe oxidative balance in oxidative stress relatedconditions.Over the last few years, a number of testsbased on different chemical principles has beenproposed in order to assess the wholeantioxidant activity of plasma or serum blood(5, 8-18).In this subject, one of the most knowexamples of oxidation in Nature is the changeof iron from ferrous to ferric form, as it happenin the generation of the rust. Therefore onecan consider as antioxidant a solution, such theserum/blood plasma, which is able to bringback the iron from its ferric form to the ferrousform.In other words from the merely analyticalpoint of view, the effectiveness of antioxidantplasma barrier can be evaluated by testing itsability to reduce a specific substrate, i. e. byassessing its ability to give oxidant agents (e.g. free radicals) to one or more electrons.For this purpose, several chemicalreducing-oxidizing couples are available. Forinstance, transition metals (i e. iron) exhibitthe property to receive one electron thusshifting from the oxidized state (Fe 3+ ) toreduced state (Fe 2+ ). Such agents can work as“meters” in tests aimed to assess antioxidantpower of biological systems. Indeed, the socalled“plasma antioxidant power” is ultimatelya measure of the reducing or “electron-giving”activity of blood plasma.On the other hand, some compounds sharethe ability to change their absorbance justwhen bound to compounds able to switch fromthe oxidized to reduced state. For instance,some thiocyanates are able to reversibly shiftfrom colored to uncolored derivatives, in thepresence of ferric or ferrous salts, respectively(19). Such “chromogens” can work as excellent“detectors” when coupled with adequate“oxidizing/reducing meters” in a test designedto assess antioxidant activity of biologicalsamples.Indeed, when a ferric salt is dissolved in auncolored solution containing a particularthiocyanate derivative, the resulting solutionbecomes red, as a function of the ferric ionsconcentration. This phenomenon is ascribableto the formation of a complex between ferricsalt and thiocyanate. Further adding of a smallamount of blood plasma will reduce ferric ions5www.fras4evolvo.it

to ferrous ions thus making uncolored theinitial red solution. Such a chromatic change –may be due to the release of ferrous ions bythe colored thiocyanate complex – can be readby means of a photometer, previously set atthe wavelength of chromogen.Therefore, the entity of absorbance changewill directly correlate with the antioxidant“potential” of blood plasma against the specificsubstrate which has been used asoxidant/detector (ferric ions). In other words,the ability of the tested plasma to reduce ferricto ferrous ions may provide a direct measureof the ability of a sample of such plasma togive reducing equivalents and then neutralizechemical species lacking of electrons, like ROS– obviously in the reduction-oxidation potentialrange of chosen oxidant-reducing couple(Fe 3+ /Fe 2+ ).On the basis of this background, a noveltest, aimed to assess the antioxidant plasmaactivity, has been recently developed by theItalian Chemist Mauro Carratelli and firstdescribed by Dohi et al. (20).This test has been called “BAP” test whichinitials underline that the assay is designed toassess the Biological Antioxidant Potential ofblood plasma.The aim of this short review is to presentthe principle, the validation, the procedure, theindications and clinical usefulness of the BAPtest in the light of the available literature.2. PrincipleBAP test is based on the ability of a coloredsolution, containing a source of ferric (Fe 3+ )ions adequately bound to a specialchromogenic substrate, to decolour when Fe 3+ions are reduced to ferrous ions (Fe 2+ ), as itoccurs by adding a reducing/anti-oxidantsystem, i. e. a blood plasma sample (20).Therefore, in the BAP test, a small amountof blood plasma (10 µL) to be tested isdissolved in a colored solution, which has beenpreviously obtained by mixing a source offerric ions (i. e. ferric chloride, FeCl 3 , the R 2reagent) with a special chromogenic substrate(i. e. a thiocyanate derivative, the R 1 reagent)(19).After a short incubation (5 min), at 37 °C,such solution will decolor and the intensity ofthis chromatic change will be directlyproportional to the ability of plasma to reduce,during the incubation, ferric ions (initiallyresponsible for the color of solution) to ferrousions, according to these reactions:1. FeCl 3 + AT (uncolored) → FeCl 3 – AT (colored)2. FeCl 3 –AT (colored) + BP (e-) → FeCl 2 + AT (uncolored) + BPwhere:• FeCl 3 is ferric chloride;• AT (uncolored) is a thiocyanate derivative (uncolored);• FeCl 3 – AT (colored) is the colored complex of ferricchloride with the thiocyanate derivative;• BP (e-) is a molecule of blood plasma barrier withreducing/electron giving/antioxidant activityagainst ferric ions;• BP is the oxidized form of BP (e-) ;• FeCl 2 is the ferrous chloride obtained by thereducing activity of BP (e-) .By assessing photometrically the intensityof decoloration, the amount of reduced ferricions can be adequately evaluated thus allowingan effective measurement of reducing ability orantioxidant potential of tested blood plasma(20).Such a “potential” is obviously not absolutebut relative to the tested substrate, i. e. ferricions. Considering that such ions are naturallyoccurring components of the body, BAP test,as its initials suggest (Biological AntioxidantPotential), provides a reliable measure ofantioxidant power of the fraction of plasmabarrier to oxidation which is directly involved –due to the implicated reducing-oxidantpotentials – against the attack of reactivechemical species in “physiological” or“biological” conditions.However, since the main aim of BAP test isto provide a global assessment of antioxidantdefenses of our body, no studies has beenperformed in order to establish the exactcontribution to the assay of eachserum/plasma blood antioxidants, whichamount should be detected by means of6www.fras4evolvo.it

different analytical approach (e. g. massspectrometry, chromatography and so on).In any case by considering that BAP testexploits the same principle of FRAP assay (21)i. e. the reduction of ferric to ferrous ions, it islikely that BAP test measures the sameantioxidants of any assay based on ironreduction (Table 1).µEq/L1600120080040000 10 50 100µg/mLTable 1. Relative activity of individual plasmaantioxidants and their estimated contributions tototal ferric reducing activity of plasma (modified byBenzie et al, 1996) (21).PlasmaantioxidantsRelativeActivity 1Expectedplasma level 2Estimatedcontribution 3Uric acid 2.0 (2.0-2.4) 150 – 450 60Ascorbic acid 2.0 (1.9-2.1) 30 – 100 15Proteins 0.1(0.1-0.15) 800 – 1100 10α-Tocopherol 2.0 (1.7-2.1) 15 – 40 5Bilirubin 4.0 (4.2-4.6) < 20 5Others – – 51 Measured range. 2 Fasting concrentrations (µmol/L). 3 Tototal iron-reducing plasma activity.Indirect evidence that BAP test measuresat least some of these antioxidants has beenprovided by some experiments performedeither on standard solutions or blood plasmasamples.For instance BAP test was able to detect invitro the antioxidant potency of uric acid (UA)as ferric reducing capacity in solutionscontaining increasing amounts of UA (22)(Figure 1).µEq/L1600120080040000 10 20 30mg/dLFigure 1. Effects of aqueous solutions of uric acid(UA) on BAP test in vitro. For each concentrationUA in aqueous solution, BAP was measured threetimes, and values are presented as means ± SD. Alldifferences among solutions were statisticallysignificant (P

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

The FREE system is an “open” systemconsisting of a photometer and a drythermostating block. Both the tools aremanaged by a computerized system able toreceive, to elaborate and to export the data.The initials FREE means Free Radical ElectiveEvaluator, i. e. system for the elective study offree radicals.The dedicated kit of the BAP test for FREEcontains cuvettes ready-to-use with a solutionof a thiocyanate derivative (R 1 reagent) and avial of ferric chloride (R 2 reagent).According to the procedure for the FREEsystem, at least 10 min before the analyticalsession, it is preferable to seat the cuvettes,ready to use for analysis, in the thermostatedhousing of instrument. It is also very importantto bring the reagents to working temperaturebefore the use.When ready, the operator must select,through the keyboard of instrument, the presetchannel.For each analytical series, the procedureinvolves, before testing plasma or serum, thezeroing and the subsequent automaticsubtraction of reagent blank absorbance.Practically, after having adjusted the zeroagainst distilled water in the photometer, 50 µLof R 2 reagent (ferric chloride) is transferredinto the cuvette containing the R 1 reagent (athiocyanate derivative). The resulting coloredsolution, after gently mixing by inversion,undergoes photometric reading. Then, 10 µL ofsample is added in the same cuvette and theobtained solution is gently mixed andincubated into the thermostatic block for 5 minat 37 °C. After incubation, the sample can beread for absorbance.FRAS 4 is a new integrated analyticalsystem consisting of a dedicated photometerwith incorporate centrifuge designed to allowfor the global assessment of oxidative stresson whole blood, by means of two tests, i. e. d-ROMs test and BAP test. The initials FRASmeans Free Radical Analytical System, i. e.system for the analytical study of free radicals.The most innovative technological featureof FRAS 4 is the integration of centrifuge withthe analytical module. This system enables theoperator to perform not only thecentrifugation but also the photometricanalysis.The dedicated kit of BAP test for FRAS 4 issimilar to that one of FREE system; moreover,it contains also the tools for taking the wholeblood from a finger.Practically, a small amount of plasma (10µL), which has been obtained after centrifugingthe whole blood sample (100-150 µL), isdissolved in the colored solution, previouslyprepared by mixing the R 2 reagent (FeCl 3 ) withR 1 reagent (a thiocyanate derivative). After 5min of incubation at 37 °C, the solutionundergoes photometric reading.To periodically calibrate FREE and FRAS 4systems, a lyophilized human control serum isavailable with a known antioxidant titleexpressed as µmol/L.Both devices directly provide the results ofthe BAP test – that is the “biologicalantioxidant potential” of plasma – asµEquivalents/L (µEq/L) (by using ascorbate asiron reducing agent).Data of Producer from healthy individualsindicated initially that the optimal value of BAPtest should be higher than 2.200 µEq/L. Thestudies performed in the last 3 yearssubstantially confirmed this statement (seebelow), although every laboratory shoulddetermine its own reference value.5. Analytical performancesAnalytical performances were initiallyevaluated with a first set of experiments onstandard solutions of ascorbic acid (AsA) withknown values, by using a FREE equipment(Diacron International, Grosseto, Italy), inorder to establish the lowest limits of detectionand quantification, respectively, and linearity.The lowest detection limit, i. e. the lowestconcentration of a signal that can bedistinguished by the blank (as calculated bymultiplying 3 times the standard deviation, asobtained by the average of 10 replicatedanalyses of the blank) was 118 (mean value).The lowest quantification limit, i. e. the lowest10www.fras4evolvo.it

In particular, 140 Labrador Retriever dogs,75 females and 55 males, between 1 and 5years old, fed with the same super-premiumdry food and with the same lifestyle, wereincluded in a study (25). All the subjects werein good health conditions at the physical andlaboratory examination (haemogram,biochemical profile, urine analysis and serumprotein electrophoresis) (25).The BAP test showed linear in the testedinterval (Figure 9), with a 0,95 “r” coefficient.µEq/L2500.002000.001500.001000.00500.00R 2 =0.950.000 20 40 60 80 100 120%Figure 9. Linearity of BAP test (data from canineplasma) (modified by Pasquini et al, 2008) (25).The recovery values of accuracy turned out106,41%. The within-run CV WAS 6,4 %, whilethe between-run CV was 8,1% (25). Thesedifferences, compared to the lower data shownin Humans, can be at least partially ascribableto pre-analytical errors. In fact, preliminaryreports on rats indicate that the intake of foodand/or antioxidants significantly increases thebasal value of the BAP test. Therefore, it ismandatory to perform the BAP test in the samepre-analytical conditions of other biochemicaltests, like blood glucose (the morning, at rest).This should significantly decrease anyvariability of the BAP test even in the sameindividual.Taken together, these findings indicate thatthe BAP test shows good to excellent analyticalperformances as a reliable tool to assessantioxidant status in blood samples either fromHumans or Animals.These performances were shown improvedrecently by automating the test on a multipleanalyzer (OLYMPUS 450) (data submitted forpublication).Interestingly, BAP test showed excellentrepeatability on other biological matrices, likewater-soluble antioxidant formulas, with anintra-assay coefficient of variation 1.9% (seebelow, Studies in Vitro).At the moment, the only describedinterfering factor as well as for all photometricassays is a very high blood level of lipids (seebelow).6. Interpretation of resultsAccording to the data of the Producer(Diacron International, Grosseto, Italy), the“optimal value” of BAP test in healthy peoplesshould be at least 2.200 µEq/L.Benedetti et al. confirmed this finding, byshowing values 2169 ± 105 µEq/L on plasmablood samples (n=12) from apparently healthypeoples, selected among a more largepopulation of healthy blood donors(unpublished data).Noticeably these values are very similar tothese very recently reported by Nagata et al. in50 healthy volunteers (2137 ± 228 µEq/L)(26).Therefore, the normal range proposed bythe producer is substantially confirmed bythese preliminary data (see also below, ClinicalStudies on Humans).In other words, values lower than 2.200µEq/L indicate a reduced “biological potential”and hence a decreased effectiveness of theantioxidant plasma barrier, according to anarbitrary scale of severity (Table 5).Table 5. BAP test results and antioxidant barrierimpairment.BAP test results Impairment degree of(µEq/L) plasma antioxidant barrier2.200 – 2.000 Border line condition2.000 – 1.800 Slight reduction1.800 – 1.600 Moderate reduction1.600 – 1.400 Strong reduction< 1.400 Very strong reductionOptimal value > 2.200 µEq/L12www.fras4evolvo.it

Preliminary data also suggests that elderlypeoples have results of the BAP test lower thanhealthy adults (unpublished data).Interestingly, dogs showed a normal rangevery similar to that one of Humans (2446 ±585 µEq/L) with a normal-like distribution of allvalues, ranging from 1440 to 3260 µEql/L (25)(Table 6).Table 6. Referral range of BAP test in dogs(modified by Pasquini et al, 2008) (25).Unitsµmol/LKurtosis – 1.4Skewness – 0.2Media 2466Mediana 2610SD 585Reference range 1440 – 32607. Clinical studies on HumansThe clinical usefulness of the BAP test inHumans bas been demonstrated in severalstudies, either in physiological or pathologicalconditions. Most of these papers wereperformed by coupling d-ROMs test with BAPtest. However, because this short review isfocused on BAP test we will describeparticularly clinical studies on this test with areference to d-ROMs test when indicated.Due to the close relationships betweenoxygen pressure and oxidative stresspreliminary studies evaluated BAP test inconditions mimicking hyperoxia.It has been suggested that hyperbaricoxygen (HBO) causes oxidative stress throughthe production of reactive oxygen metabolites,as also detected by d-ROMs test (27). Howeversome patients with decompression illness (DCI)need repeated HBO treatments which can leadto concerns about an accumulation of oxidativestress (28). Therefore 9 DCI patients wereexposed to two session of U. S. NavyTreatment Table 6 at an interval of 3 days(23). BAP significantly increased after the 1 stand after the 2 nd HBO compared with before 1 stHBO (23) (Figure 10).µEq/L25002000150010005000P

µEq/L25002000150010005000Before divingP

specific advise on lifestyle the score of aspecific visual analogical scale (VAS) for fatiguesignificantly increase with a trend to BAPincreased values (although the change did notreach the statistical significance cut-off) (36).No significant changes were noted in theresults of the BAP test after acupuncture inspite of the decrease of d-ROMs test values(37) while Japanese females suffering fromhypotension showed increase values of BAPtest (38).Postmenopausal women also showedsignificantly higher BAP test values comparedto men in a clinical trial performed onmetabolic syndrome (2531±323 µEq/L vs.2345±245 µEq/L, respectively; P

8. Clinical studies on AnimalsGrowing evidence suggest the potentialusefulness of BAP test in the monitoring ofoxidative stress in different animal species,including rats, mice, piglets and dogs (44).In particular, liver regeneration afterhepatectomy was followed in rats by asignificant decrease of BAP values, may be dueto the concomitant increase of d-ROMs testvalues (45) (Figure 17).µEq/LFigure 17. Changes in BAP test values afterhepatectomy in rats. Values are reported as mean± SD. P values

9. Experimental studies in vitroA recent challenge for antioxidantsupplements is the determination ofantioxidant power in vitro. Indeed in the lightto develop new formulas, the producer whenpossible should report on the label at least anindication on the ability of the formula toneutralize effectively one or more oxidants.Among the available assays BAP testproven useful in the assessment of antioxidantcapacity of some formulas destined to patientsor healthy peoples (Table 7).Table 7. BAP test values of some antioxidant formulasFormulas BAP test values N CV (%)GIN PENT TM (1) 48500±2880 9 4.1DEUTROSULFAZYME TM (2) 64747± 3660 8 5.7DEUTROSULFAZYME TM (3) 235500± 9161 8 3.9DEUTROSULFAZYME TM (4) 274500± 6009 8 2.2ARAMEN TM (5) 22440 ± 908 7 4.0BAP test values are expressed as mean µEq/L±SD. N isthe number of determinations. CV is the within-runcoefficient of variation. (1) Gimnostemma pentaphyllumplus fermented papaya plus reduced glutathione (2)Deutrosulfazyme TM basic formula. (3) Deutrosulfazyme TMvitamin C-enriched formula. (4) Deutrosulfazyme TMmultivitamins-enriched formula. (5) Herbal product basedon Laminaria digitata, Condrus crispus, Sambucus nigra,Vaccinium myrtillus, Rosa canina and Feniculus vulgareMiller.Among these was also Deutrosulfazyme TM(CELLFOOD TM , NUSCIENCE CORPORATION,US) a non-addictive, non-invasive, andcompletely non-toxic proprietary colloidal-ionicformula containing finest all-natural, plantbasedorganic substances like ionic minerals,enzymes, amino acids and deuterium sulphateas traces (51, 52).More recently BAP test was performedcomparatively on samples of hand-made beersagainst to industrial beers(Table 8) (Bianchiand Iorio, unpublished data).Table 8. BAP test values and polyphenols levels inhand-made and industrial beers.Beer BAP test values* Polyphenols levels**Label 1 10035 583Label 2 9750 517Label 3 9655 631Label 4 9645 904Label 5 10375 700Label 6 7280 477Label 7 9515 451Label 8 11040 938Label 9 9860 629Label 10*** 6080 316*As µEq/L. **As mg catechin/kg. ***IndustrialInterestingly hand made beers showedhigher values of both BAP test and polyphenolscompared to industrial beers. Noticeably BAPtest values correlated with polyphenolscontent.10. Indications and clinical usefulnessThe BAP test, by exploiting the samechemical principle of the well-known FRAP test– i. e. the reduction of ferric to ferrous ions –provides a reliable measure of biologicalantioxidant potential of blood plasma (53).Therefore, the BAP test should beperformed with the aim of assessing theantioxidant status either in healthy peoples, inorder to prevent oxidative stress, or in patientswith oxidative stress-related diseases, in orderto monitor the efficacy of specific and/orantioxidant therapies (54, 55).All healthy peoples should undergo the BAPtest because all the individuals are potentiallyexposed to the risk of producingexaggerate amounts of free radicals, aphenomenon which requires a very effectiveantioxidant barrier. Indeed, the primary aim ofthis test is to identify and to prevent oxidativestress and its unwanted consequences (earlyaging, diseases) (56).All the more so, the BAP test should besystematically performed on all clinicallyasymptomatic subjects, who are exposed for anumber of reasons to factors able to reducephysiological antioxidant plasma defenses (e.g. inadequate exercise, unbalanced diet,chronic inflammatory diseases with reduced17www.fras4evolvo.it

absorption of nutrients, etc.). In these casesalso, the aim of the BAP test is to identify andto prevent oxidative stress and its unwantedconsequences (early aging, diseases) (57).Moreover, the BAP test should be carriedout on all patients with oxidative stress-relateddiseases (more than one hundred), such asAlzheimer’s disease, Parkinson’s disease,stroke, infarction, Crohn’s diseases, rheumatoidarthritis, AIDS, some cancers, etc. In all thesecases, the aim of the BAP test is to monitoroxidative stress and to prevent itsconsequences, by monitoring the efficacy ofspecific therapy, either alone or with aconcomitant antioxidant treatment (54). Onthis subject, it must be underlined that in manyof above mentioned diseases, all almost havinga chronic course, oxidative stress tends toraise the role of an additional health riskfactor. Therefore, it must be controlled in orderto optimize results of any treatment. Forinstance, the evidence, by means of BAP test,of a reduced biological antioxidant potential inhypertensive patients indicates a persistentimpairment of physiological defenses againstROS, which can be responsible for anincomplete control of the current disease bythe current anti-hypertensive treatment. Sucha finding suggests to the clinician an integratedtherapeutic approach, in which should be takeplace not only conventional, medical and/orsurgical treatments, but also the correction oflife-style and, eventually, the intake ofantioxidants.Finally, ideal candidates for the BAP testare all the subjects that undergopharmacotherapy (e. g. with chemotherapeuticals,hormone replacement therapy,contraceptive pill, etc.), surgical interventions(e. g. organ transplantation, by-pass, etc.),including dialysis, all conditions being able tofavor oxidative stress by increasing the dailyrequirements of antioxidants (53, 54).In all these conditions, the aim is toidentify and to prevent oxidative stress and itsconsequences, and, particularly, to monitor theefficacy of eventual current measures with theultimate objective of preventing oxidativetissue damage (56).Furthermore, according to the guidelines ofthe International Observatory of OxidativeStress, Free Radicals and Antioxidant Systems,the BAP test is very useful, together d-ROMstest, in the so-called global assessment ofoxidative stress, now very easy to performgrace to a specific diagnostic algorithm (41,58).In this situation, when d-ROMs value isincreased, an optimal value of BAP test shouldrequire the dosing of a single antioxidant.Alternatively, if such a dosing is not available,we suggest a mix of antioxidants according tothe RDA tables. This should enable theantioxidant defenses to balance the increasedoxidative status, as detected by the increasedd-ROMs test values, especially when more thanone risk factor is present (e. g. exposition toradiations, cigarette smoke, pollutants, etc.).11. Concluding remarksScientific evidence shows that BAP test isvery useful in the assessment of antioxidantactivity of blood plasma. AAccording to the wide available scientificliterature BAP test was validated by ESR/EPRand provided results that largely parallel tothose of FRAP test, against which it appearsquicker and simpler to perform. Indeed, thepreparation and management of the FRAPreagents is a step somewhat complex,compared to that of BAP test, so that manualprocedure of FRAP test can increase analyticalvariability. Moreover, a further increase ofanalytical accuracy was recently reached bythe application of BAP test to automaticanalytical devices.Finally, according to the novel concept ofglobal oxidative stress assessment, the BAPtest is a reliable assay able to integrate theresults of d-ROMs test, which allows for theassessing of the total oxidant status of bloodplasma.18www.fras4evolvo.it

References1. Halliwell B, Gutteridge JMC. Free radicals inbiology and medicine. 2 nd Edn, ClarendonPress, Oxford. 1989.2. Cadenas E, Packer L. Handbook ofantioxidants. Marcel Dekker, Inc. 1996.3. Halliwell B, Gutteridge JMC, Cross CE. Freeradicals, antioxidants, and human disease:where are we now? J Clin Lab Med. 1992.119: 598–620.4. Valko M, Leibfritz D, Moncol J, Cronin MT,Mazur M, Telser J. Free radicals andantioxidants in normal physiological functionsand human disease. Int J Biochem Cell Biol.2007. 39 (1): 44–84.5. Cao G, Prior RL. Comparison of differentanalytical methods for assessing totalantioxidant capacity of human serum. ClinicalChemistry. 1998. 44 (6): 1309–1315.6. Olson J L. Benefits and liabilities of vitamin Aand carotenoids. J Nutr. 1996. 126: 1208S–1212S.7. Schwartz JL. The dual role of nutrients asantioxidants and prooxidants: their effect ontumor cell growth. J Nutr. 1996. 126: 1221S–1227S8. Wayner DDM, Burton GW, Ingold KU, LockeS. Quantitative measurement of the total,peroxyl radical-trapping antioxidant capacity ofhuman blood plasma by controlledperoxidation. FEBS Lett. 1985. 187: 33–7.9. Glazer AN. Phycoerythrin fluorescence-basedassay for reactive oxygen species. MethodsEnzymol. 1990. 186. 161–8.10. Cao G, Alessio HM, Cutler RG. Oxygenradicalabsorbance capacity assay forantioxidants. Free Rad Biol Med. 1993.14:303–11.11. Cao G, Verdon CP, Wu AHB, Wang H, PriorRL. Automated oxygen radical absorbance19capacity assay using the COBAS FARA II. ClinChem. 1995. 41: 1738–44.12. Rice-Evans C, Miller NJ. Total antioxidantstatus in plasma and body fluids. MethodsEnzymol. 1994. 234: 279–93.13. Miller NJ, Rice-Evans C, Davies MJ,Gopinathan V, Milner A. A novel method formeasuring antioxidant capacity and itsapplication to monitoring the antioxidant statusin premature neonates. Clin Sci. 1993. 84:407–12.14. Ghiselli A, Serafini M, Maiani G, Assini E,Ferro-Luzzi A. A fluorescence-based method formeasuring total plasma antioxidant capability.Free Rad Biol Med. 1994. 18: 29–36.15. Whitehead TP, Thorpe GHG, Maxwell SRJ.Enhanced chemiluminescent assay forantioxidant capacity in biological fluids. AnalChim Acta. 1992. 266: 265–77.16. Trotti R, Carratelli M, Barbieri M, Micieli G,Bosone D, Rondanelli P, Bo P. Oxidative stressand thrombophilic condition in alcoholicswithout severe liver disease. 2001.Haematologica. 86: 85-91.17. Carratelli M, Porcaro R, Ruscica M, DeSimone E, Bertelli AAE, Corsi MM. ReactiveOxygen Metabolites (ROMs) and prooxidantstatus in children with Down Syndrome.International Journal of Clinical PharmacologyResearch. 2001. 21 (2): 79-84.18. Brambilla G, Fiori M, Archetti LI. Evaluationof the oxidative stress in growing pigs bymicroplate assays. J Vet Med A. 2001. 48: 33-38.19. MERCK CHEMICAL Database. 2003.20. Dohi K, Satoh K, Ohtaki H, Shioda S,Miyake Y, Shindo M, Aruga T. Elevated plasmalevels of bilirubin in patients with neurotraumareflect its pathophysiological role in free radicalscavenging. In Vivo. 2005. 19 (5): 855–860.www.fras4evolvo.it

21. Benzie IFF, Strain JJ. The ferric reducingability of plasma (FRAP) as a measure of“antioxidant power”: the FRAP assay. AnalBiochem. 1996. 239: 70-76.22. Nakayama K, Terawaki H, Nakayama M,Iwabuchi M, Sato T, Ito S. Reduction of serumantioxidative capacity during haemodialysis.Clin Exp Nephrol. 2007. 11: 218–224.23. Kongoji J, Yamami N, Togawa S, Suzuki N,Yamamoto K, Mano Y. Indexes of oxidativestress during HBO treatment. ProceedingsUndersea and Hyperbaric Medical Society.Scientific Meeting & Associates/BNA. AnnualScientific Meeting. 22-24 June 2007. Orlando,Florida (USA). 2007. P 112.24. Dohi K, Satoh K, Nakamachi T, Yofu S,Hiratsuka K, Nakamura S, Ohtaki H, YoshikawaT, Shioda S, Aruga T. Does Edaravone (MCI-186) act as an antioxidant and aneuroprotector in experimental traumatic braininjury? Antioxid. Redox Signal. 2007. 8: 281–287.25. Pasquini A, Luchetti E, Marchetti V, CardiniG, Iorio EL. Analytical performances of d-ROMstest and BAP test in canine plasma. Definitionof the normal range in healthy Labrador dogs.Vet Res Commun. 2008. 32 (2): 137–143.26. Nagata K, Hasegawa T, Hirokado Y,Kiyama K, Otsuki C. Lifestyle-related diseasesand the oxidative stress regulation system. JpnJ Psychosom Med. 2008. 4(3): 176-183.27. Benedetti S, Lamorgese A, Piersantelli M,Pagliarani S, Benvenuti F, Canestrari F.Oxidative stress and antioxidant status inpatients undergoing prolonged exposure tohyperbaric oxygen. Clinical Biochemistry.2004. 37: 312–317.28. Klingmann C, Gonnermann A, Dreyhaupt J,Vent J, Praetorius M, Plinkert PK.Decompression illness reported in a survey of429 recreational divers. Aviat Space EnvironMed. 2008. 79(2):123–128.29. Buonocore G, Perrone S, Longini M, VezzosiP, Marzocchi B, Paffetti P, Bracci R. Oxidativestress in preterm neonates at birth and on theseventh day of life. Pediatric Research. 2002.52 (1): 46–49.30. Ezaki S, Kurishima C, Suzuki K, Kondo T,Tamura M. Resuscitation of preterm infantswith reduced concentration of inspired oxygen.Proceedings 11 th Annual Congress PerinatalSociety of Australia and New Zealand (PSANZ2007). 2007. April, 1–4. Melbourne ConventionCentre. Melbourne, Victoria (Australia). 2007.31. Yamami N, Nakayama H, Kongoji J,Togawa S, Nozawa T, Yagishita K, ShibayamaM, Nakayama T, Suzuki N, Yamamoto K,Kawashima M, Mano Y. Free radicals andantioxidant potential in scuba divers.Proceedings Undersea and Hyperbaric MedicalSociety. Scientific Meeting & Associates/BNA.Annual Scientific Meeting. 22-24 June 2007.Orlando, Florida (USA). 2007. P 98–99.32. Giganti MG, Iorio EL, Verna R, Zenobi R.Oxidative balance in scuba divers. Journal ofMolecular and Clinical Pathology. 2007. 3:101.33. Zazryn TR, Finch CF, McCrory P. A 16 yearstudy of injuries to professional kickboxers inthe state of Victoria, Australia. Br J Sports Med.2003. 37:448–51.34. Cavallini M, Iorio EL. Effect of training andantioxidant supplementation on oxidativebalance in martial arts. Journal of Molecularand Clinical Pathology. 2007. 2: 14-15.35. Karlsson J. Antioxidants and exercise.Human Kinetics. 1997.36. Nagata K, Kasuya N, Hayashi H. Oxidativestress and anti-oxidant potential of thestudents complaining chronic fatigue. CampusHealth. 2006. 43 (2): 83-88.37. Hirokado Y, Nagata K, Aoyama Y, KiyamaK, Okano K, Hasegawa. Prohomeostatic effectsof acupuncture. 2006. International CongressSeries. 2006. 1287: 107–110.38. Hasegawa T. Oxidative stress andantioxidant potential in Japanese females withhypotension. Proceedings 19 th World Congress20www.fras4evolvo.it

of Psychosomatic Medicine. Québec City(Québec) Canada. 2007. August 26–31. 2007.P 30.39. Tsuchiya M, Tosaka M. There is a genderdifference in the relationship betweenmetabolic syndrome and oxidative stressamong Japanese. Clinical Report. 2005.Masako Medical Clinic, Tokuyama, Japan.40. Nagata K, Hasegawa T, Kiyama K, AoyamaY, Okano K, Hirokado Y. A therapy forfibromyalgia syndrome using Yi-Gan San.Journal of Japanese Association of OrientalPsychosomatic Medicine. 2006. 21 (1-2): 46–47.41. Iorio EL. A diagnostic algorithm to manageoxidative stress (original title “Un algoritmodiagnostico per la gestione dello stressossidativo”). Proceedings InternationalCongress of Antiaging Medicine. 2006,Starhotels Business Palace, 20 th May. Milan.2006.42. Pober BR, Morris CA. Diagnosis andmanagement of medical problems in adultswith Williams-Beuren syndrome. Am J MedGenet C Semin Med Genet. 2007. 145(3):280-290.43. Komatsu F, Kudoh H, Kagawa Y. Evaluationof oxidative stress and effectiveness of lowdoseglucocorticoid therapy on exacerbation ofchronic obstructive pulmonary disease. Journalof Gerontology. 2007. 62A (4): 459–464.44. Iorio EL. Oxidative stress assessment inAnimals. Actuality and perspectives [originaltitle “La valutazione dello stress ossidativo neglianimali. Attualità e prospettive”]. Proceedings“67° Convegno Nazionale Unione ZoologicaItaliana”. Napoli (Italy). 2006, September12 nd -15 th . Pp. 28.45. Han SY, Chang EJ, Choi HJ, Nam SI, LeeNH, Kwak CS, Park SB, Kim HC, Mun KC. Totalantioxidant status and oxygen free radicalsduring hepatic regeneration. TransplantationProceedings. 2006. 38: 2214–2215.46. Fujiwara T, Nakabayashi M, Goto M,Koyama S, Fujimoto A, Kugu K, Yano T,Tsutsumi O, Taketani Y, Okamoto T.Preliminary study on action of coenzyme Q 10 infemale reproductive system. Proceedings 5 thConference of the International Coenzyme Q 10Association. 2007. November 9 to 12. KobeGakuin University at Port Island. Kobe, Japan.2007. JP-039 (1–2).47. Kakita H, Hussein Mh, Daoud Ga, Kato T,Murai H, Sugiura T, Mizuno K, Yamada Y, ItoT, Fukuda S, Kato I, Suzuki S, Togar H. Totalhydroperoxide and biological antioxidantpotentials in a neonatal sepsis model. PediatricResearch. 2006. 60 (6): 675–679.48. Cardini G, Pasquini A, Luchetti E, MarchettiV, Voltini B. Free radicals oxygen (d-ROMs test)and Biological Antioxidant Potential (BAP test)assessment in serum: dog’s reference ranges[original title “La determinazione dei radicaliliberi dell’ossigeno (d-ROMs test) e delpotenziale antiossidante biologico (BAP test)nel siero: intervalli di riferimento nella speciecanina”]. Proceedings LIX Convegno Nazionale– Società Italiana delle Scienze VeterinarieViareggio (Italy). 2005, 21 – 24 September.2005. 331–332.49. Pasquini A, Luchetti E, Cardini G. Oxidativestress and recovery of hunting dogs [originaltitle: “Stress ossidativo e tempi di recupero nelcane da caccia”]. Annali Fac Med Vet. 2006.LIX: 229–235.50. Pasquini A, Simonetti P, Luchetti E,Bardana C, Cardini G. The Ginkgo biloba in thediet of ageing dog. [original title “Il Ginkgobiloba nella dieta del cane anziano”]. PraxisVeterinaria. 2005. 26 (2): 1-10.51. EL. Deutrosulfazyme TM (CELLFOOD TM ). Aclinical pharmacological overview [original titleDeutrosulfazyme ® (CELLFOOD ® ). Overviewclinico-farmacologica].ProceedingsInternational Conference Safety Evaluation ofComplementary and Alternative Medicine.2003. Empoli (Italy). 2003, October 24 – 25.52. Iorio EL, Bianchi L, Storti A.Deutrosulfazyme ® : a powerful antioxidant[original title “Deutrosulfazyme ® : un potenteantiossidante”]. La Medicina Estetica. 2006. 30(1): 115 – 116.21www.fras4evolvo.it

53. Carratelli M, Iorio EL, Bianchi L. Methods tomeasure the oxidative stress [original title“Metodi di misurazione dello stressossidativo”]. ADI Magazine. 2006. 4 (10): 405– 414.54. Iorio EL. Carratelli M, D’Amicantonio T.Oxidative stress and diseases [original title“Stress ossidativo e malattia”]. ADI Magazine.2006. 4 (10): 399 – 404.55. Pece A, Marchetti M, Pallotti R. The freeradicals [original title: “I radicali liberi”]. SOOFTItalia Ed. 2006.56. Iorio EL. Oxidative stress and ageing[original title “Stress ossidativo e senescenza”].In “Il manifesto della lunga vita. La rivoluzionedella medicina predittiva. Marandola P, MarottaF. Sperling and Kupfer. 2007. 318–322.57. Iorio EL. Oxidative stress and sport[original title: “Stress ossidativo e sport”].European Journal of Health, Sport andNutrition. 2007. 1: 102–103.58. Iorio EL, Cinquanta L, Pisano R. Adiagnostic algorithm to manage oxidativestress. Australasian J Cosmet Surg. 2006. 2(1) : 26-30.59. Bertone M, Rizzo M, Assorgi R, MuscatelloA, Crisostomi S, De Blasis E, Di Giacinto B,Spataro A, Iorio EL. Plasma oxidant andantioxidant capacity at rest, after exerciseinduceddehydration, and after rehydrationwith an antioxidant drink in elite rowers. MedSport. 2008. 61: 487–495.60. Celi P, Sullivan M, Evans D. The stability ofthe reactive oxygen metabolites (d-ROMs) andbiological antioxidant potential (BAP) tests onstored horse blood. The Veterinary Journal.2008. Doi:10.1016/j.tvjl.2008.09.018.61. Cesarone MR, Di Renzo A, Erpichi S,Schönlau F, Wilmer JL, Blumenfeld J.Improvement in circulation and incardiovascular risk factors with a proprietaryisotonic bioflavonoid formula OPC-3TM.Angiology. 2008. 59(4): 408–414.2262. Costantino M, Filippelli A. Efficacy ofthermal therapy in osteoarthritis [original title“Efficacia della terapia termalenell’osteoartrosi.“]. Eur Med Phys. 2008.44(Suppl. 1 to No. 3): 1–3.63. Di Marco P, Priori A, Finoia MG, Petochi T,Marino G, Lemarié G, Alexis M, Alberti A,Macciantelli D. Plasma totaloxidant/antioxidant status in Dicentrarchuslabrax after exposure to experimental hypoxia,hyperoxia and hypercapnia. ComparativeBiochemistry and Physiology - Part A:Molecular & Integrative Physiology. 2008.151(1, Suppl 1): S15.64. Du Plessis LH, van der Westhuizen FH,Kotzé HF. The protective effect of plasmaantioxidants during ozone autohemotherapy.African Journal of Biotechnology. 2008. 7(14):2472–2477.65. Ezaki S, Ito T, Suzuki K, Tamura M.Association between total antioxidant capacityin breast milk and postnatal age in days inpremature infants. J Clin Biochem Nutr. 2008.42: 133–137.66. Fujimoto H, Katayama K, Hayashi T,Kimura K, Yano T. Effect of electroacupunctureon exercise-induced oxidative stress. ZenNippon Shinkyu Gakkai Zasshi (Journal of theJapan Society of Acupuncture and Moxibustion:JJSAM). 2008. 58(2): 203–212.67. Barassi A, Colpi GM, Piediferro G, DogliottiG, Melzi D'Eril GV, Corsi MM. Oxidative stressand antioxidant status in patients with erectiledysfunction. J Sex Med. 2009. DOI:10.1111/j.1743-6109.2009.01279.x68. Buico A, Cassino C, Ravera M, Betta PG,Osella D. Oxidative stress and total antioxidantcapacity in human plasma. Redox Re. 2009.14(3): 125–131.69. Costantini D, Verhulst S. Does highantioxidant capacity indicate low oxidativestress? Functional Ecology. 2009. 23(3): 506–509.70. Iwabayashi M, Fujioka N, Nomot K,Miyazaki R, Takahashi H, Hibino S, TakahashiY, Nishikawa K, Nishida M, Yonei Y. Efficacywww.fras4evolvo.it

and safety of eight-week treatment withastaxanthin in individuals screened forincreased oxidative stress burden. Anti-AgingMedicine. 2009. 6(4): 15–21.71. Kaneko K, Taniguchi N, Tanabe Y, NakanoT, Hasui M, Nozu K. Oxidative imbalance inidiopathic renal hypouricemia. Pediatr Nephrol.2009. 24(4): 869–871.72. Iwata N, Okazaki M, Kamiuchi S, Hibino Y.Protective effects of oral administrated ascorbicacid against oxidative stress and neuronaldamage after cerebral ischemia/reperfusion indiabetic rats. J Health Sci. 2009. 56(1). INPRESS.73. Landoni G, Mariani E, Oriani G, Donarini C,Guerrerio T, Iorio EL. Improvement ofantioxidant status in women conventionallytreated for breast cancer after 12 months of acow milk whey-based supplementation. Apreliminary study. Mediterranean Journal ofNutrition and Metabolism. 2009. 2(2): 127-131.athletes. J Sports Med Phys Fitness. 2009.49(1): 122–127.76. Mochizuki T, Amenomori Y, Miyazaki R,Hasegawa T, Watanabe M, Fukuike K, Yonei Y.Evaluation of exercise programs at a fitnessclub in female exercise beginners using antiagingmedical indicators. Anti-Aging Medicine.2009. 6(8): 66–78.77. Sakamoto R, Matsubayashi K, Kimura Y,Ishine M, Kosaka Y, Wada T, Wada C,Nakatsuka M, Ishimoto Y, Hirosaki M, KasaharaY, Konno A, Chen W, Fujisawa M, Otsuka K,Nakashima M, Wang H, Dai Q, Yang A, Qiao H,Gao J, Li Z, Zhang Y, Ge R-L, Okumiya K.Comprehensive geriatric assessment of elderlyhighlanders in Qinghai, China, III: oxidativestress and aging in Tibetan and Han elderlyhighlanders. Geriatr Gerontol Int. 2009. doi:10.1111/j.1447-0594.2009.00544.x74. Mandas A, Iorio EL, Congiu MG, BalestrieriC, Mereu A, Cau D, Dessì S, Correli N.Oxidative imbalance in HIV-1 infected patientstreated with antiretroviral therapy. Journal ofBiomedicine and Biotechnology. 2009. ArticleID 749575, 7 pages.doi:10.1155/2009/749575.75. Martarelli D, Pompei P. Oxidative stressand antioxidant changes during a 24-hoursmuntain bike endurance exercise in master23www.fras4evolvo.it

F R A S 4 EVOLVO SYSTEMFREE RADICAL ANALYTICAL SYSTEMThe ideal tool for global oxidative stress assessmentin routinary clinical practice“Dedicated” integrated analytical system• Photometer (wavelenght 505 nm)• Peltier dry thermostating system• Incorporated mini-centrifuge• Liquid crystal display• Mini-printer with output3 dedicated kits ready to use for analyses on whole blood• d-ROMs test: determination of reactive oxygen metabolites (ROMs)• BAP test: determination of biological antioxidant plasma potential• SAT test: determination of biological antioxidant saliva potentialH & D s. r. l.Strada Langhirano 264/1A – 43124 Parma, ItalyPhone +39 0521 – 462607FAX +39 0521 – 467083e–mail: info@hedsrl.itweb site: www.hedsrl.itwww.fras4evolvo.comDepending on the permission of the Author(s), most abstractsor full texts of above reported references are available.Both the Editor and the Author do not answer for eventual errors contained in this paper and for the improper use of its content.24www.fras4evolvo.it