Diagnosys of oxidative stress and antioxidant capacity

Transcription

1 Diagnosys of oxidative stress and antioxidant capacity Prof U Cornelli Loyola University School of Medicine Arezzo November 29th

2 Oxidative stress (OS) OS is caused by a poorly controlled oxidative aggression by Reactive Species (RS) These RS are uncorrectly defined as free radicals the main characteristics being the oxydant capacity The most common RS are: ROS, RNS, RSS, RCS, RClS* the most represented in the human environment are: ROS and RNS [*ROS = reactive oxygen species and respectively N= nitrogen, S = sulfur, Cl = chloride species] Arezzo November 29th

3 ROS and RNS Among RS there are those extremely aggressive characterized by a radical nature: O 2 - HO NO they react immediately, close to the site of their generation Other RS are less aggressive, such as: H 2 O 2, ROOH, ONOOˉ [respectively hydrogen peroxide, hydroperoxide, peroxynitrite ] that can react also far from the generation site. Arezzo November 29th

4 ROS and RNS H 2 O 2, ROOH, ONOOˉ are the real determinants of the transduction mechanisms impairment that govern the OS. ROOH are the most free to circulate and transfer the damage to the transcription factors. [Flohé R et al. Antiox Redox Sig 2011;15: ] ROOH do not derive from lipids only, but also from proteins, and DNA. Arezzo November 29th

5 OS mainmarkers PL Lipids Proteins Sugars DNA ROOH ROOH ROOH ROOH ROOH IsoPs React Ald Carb prot Ox Sugars Ox DNA Isotbx (MDA; 4-HNE) Isofur Isolevuglandins EpoxyIsoPs NeuroPs AdrenoPs Arezzo November 29th

6 Oxidation Paradigm [OX] death growth arrest mytogenesis incomplete growth 0 no growth Arezzo November 29th

7 The OB Let us have a look on the methods to determine the OB: Oxidative stress Antioxidant capacity Arezzo November 29th

8 Methods to determine oxidative stress (oxidized markers 1) DNA Deoxyribonucleic acid 1 SPC Serum protein carbonyls 1 ROOH Hydroperoxides d-roms test 1 TBARS Thiobarbituric acid reacting substances 1 LNO 2 Nitrolinoleate 1 MDA Malondialdehyde 1 4-HNE 4-hydroxynonenal 1 IsoPs F 2 /D 2 /E 2 isoprostanes 1 NeuroPs F 3 /F 4 isoprostanes 1 H 2 O 2 Hydrogen peroxide 1 BH Breath hydrocarbons 1 Arezzo November 29th

9 d-roms test d-roms test detects the ROOH and it is considered one of the most advanced measures of OS. [The number of pubblications exceeds 700; in the last four years, every months an average of > 5 publications appear on international journals] Arezzo November 29th

10 d-roms test execution Blood collection, a finger tip in an heparinized disposable microvette Centrifugation of the microvette for 90 sec using the portable device (Fras 4 Evolvo) 10 μl of the sample into the tube containing the buffer solution (1 ml given into a disposable tube), and then into the cuvette containing the chromogen. After 5 minutes the results in printed Arezzo November 29th

11 d-roms test: the distribution curve of CARR. U. in 4547 apparently healthy subjects Arezzo November 29th

12 d-roms test :Normal values = Carr.U. linearity in the range from 50 to 500 Carr.U. analytical imprecision between 2.2 and 4,5% can be done with plasma/serum stored at: +4 C until -20 C for 3 months -30/80 C up to 35 months Less than 5 % lower values than the immediate measure will be obtained Arezzo November 29th

13 d-roms values [Carr.U.] OS severity Normal value Borderline value Slight OS OS High OS >500 Very high OS* Arezzo November 29th

14 Hs Plasma Levels [d-roms test Carr.U.] During the Menstrual Cycle

15 Hs Plasma Levels [d-roms test Carr.U.] Normal Cycle and During OC (same 10 subjects)

16 Hs Plasma Levels [d-roms test Carr.U.] During OC + MF Templar

17 d-roms test Vs Other tests [Cornelli U et al. JCDSA 2011;1:64-70] The aim of the trial was to measure the tests sensitivity, the coefficients of variation (CV) of different OS markers in the following conditions: After a normal diet After an acute administration of an antioxidants pool [as a condition of antioxidant abundance ] Following a week of diet poor of antioxidants [as a condition of OS] Arezzo November 29th

18 d-roms test Vs Other tests 12 apparently healthy volunteers (6 M and 6F) age between years No food supplements were allowed Under dietary control for food Intake assessment Arezzo November 29th

19 d-roms Vs other test Day Run-in ND AO LD ND= normal diet AO=antioxidants pool LD= diet poor in Aos sampling (blood,urine, saliva: a.m) Arezzo November 29th

20 d-roms Vs other tests d-roms [Cornelli U et al J Nutr 2001;131: ] 8-Iso-PGF 2α(plasma) [Dillon SA et al. J Nutr 2002;132: ] 8-OHdG (urine) [MeiS etal. J ChromatB AnalytTechnolLife Sci 2005;827:83-87] Carbonylated proteins(plasma) [Buss H et al. Free Rad Biol Med 1997;23: ] hs-crp(plasma) [Rifai N et al. Clin Chem 2001;47: ] Arezzo November 29th

21 d-roms test Vs Other Test Normal diet AO pool Low AO diet 8 am 12 am 8 am 12 am 8 am 12 am d-roms CARR. U. 8-iso PGF2α nmol/l 8-OHdG nmol/l Carbonyl P pmol/mg pr hs-crp mg/l 268 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.75 Arezzo November 29th

22 d-romstest VsOtherCV % Test Normal diet AO pool Low AO diet d-roms CARR. U. 8 am 12 am 8 am 12 am 8 am 12 am iso PGF2α nmol/l 8-OHdG nmol/l Carbonyl P pmol/mg pr hs-crp mg/l Arezzo November 29th

23 Conclusions All the markers can pick up the OS. The CV % of d-roms is at least 5 times lower than comparative tests to measure OS. Execution time (6 min) is 10 times lower than the average of all the other tests. Cost is 10 times lower than the average cost of all the other tests. Can be carried out with a simple portable system without any reactive manipulation, minimizes the inter-laboratory variations. Arezzo November 29th

24 d-roms test applications Some of the most common applications: -sport medicine; - cardiovascular disease (CV) and dyslipidemia; -diabetes; - neurological diseases. However, the test has been used for many other diseases in which OS represents one of the clinical outcomes. Arezzo November 29th

25 Oxidative stress as a predictor for CVE < 480 Carr.U. 0.6 > 480 Carr.U months Vassalle C et al. Clin Lab Med 2012;50:1463-8

26 Methods to measure antioxidant capacity (PAT/SAT test) Prof Umberto Cornelli Loyola University School of Medicine-Chicago Arezzo November 29th

27 Methods to determine the oxidative balance (through spin traps 2, fluorescence 3, antioxidant capacity 4) ONOO - Peroxynitrite 2 PTN Alpha-phenyl- N-tert- butylnitrone 2 AHS Aromatic hydroxylation of salicilate 2 TRAP Total peroxyl radical scavenging antioxidant capacity 3 TOSCA Total oxyradical scavenging capacity assay 4 SAT Soluble antioxidant test in saliva 4 UAM Uric acid metabolite allantoin 4 TEAC Trolox equivalent antioxidant capacity 4 FRAP Ferric reducing ability 4 ORAC Oxygen radical absorbance capacity 4 PAT Plasma antioxidant test 4 DPPH 1,1-diphenyl-2-picrylhydrazyl 4 TRX Thioredoxine and glutaredoxine 4 Arezzo November 29th

28 PAT/SAT PAT allows PLASMA Aox determination SAT allows SALIVA Aox determination Arezzo November 29th

29 The tests for antioxidant capacity: PAT and SAT PAT and SAT allow respectively the plasma and saliva AO capacity. BotharebasedonthedifferentcolorofFe on the different of 3+ -thiocyanate complexes(from light red to brown-reddish) The AOs contained in the sample determine the decrease ofthecolorasameasureoftheantioxidantcapacity Arezzo November 29th

30 The innovations: PAT and SAT Time of execution: 1 minute Precision Linearity : umol/l Vit C Robustness and. High specificity: the tests avoid phosphates interference Arezzo November 29th

31 The innovations: PAT and SAT Plasma and saliva contain phosphates that bind Fe and influence the reaction. Phosphates content: -Plasma: 2.6 and 4.5 mg/dl -Saliva: mg/dl Zirconium salt avoids phosphates interference. Arezzo November 29th

32 PAT Procedure [FRAS 4 Evolvo] Small amount of blood (about 200 µl) is taken by finger tip in heparinized microvette. Plasma is obtained by centrifugation (6000 rpm for 90s). 40 µl of Fe 3+ solution (R2) is added to a 1 ml of SCN- chromogenic solution (R1) in a photometric cuvette (disposable). 10 µl of plasma is added to the cuvette. The reaction proceeds for 1 minutes and the plasma AO amount is given as Cor U (1 Cor U= 1.4 µmol/l Vit C) Arezzo November 29th

33 PAT (Cor U) [251 cases:m/f] Apparently Healthy Subjects Arezzo November 29th

34 PAT normal values Range(Cor U) Indication <1800 Deficiency status Slight deficiency status Borderline values Normal values >2800 Very high values Arezzo November 29th

35 PAT Vs TAS Normal values of TAS [total antioxidant status] are around 1700 μmol/l (reducent eq) Normal values of PAT are around 2400 Cor.U 1.4 Cor.U corresponds to 1 μmol/l (reducent eq) now 2400/1.4 = 1714it means that TAS and SAT are equivalent Arezzo November 29th

36 O 3 and OB The OB balance following O 3 infusion was tested at different ex vivo concentrations in healthy volunteers d-roms test and PAT test were used for the OB Arezzo November 29th

37 O 3 addition ex vitro tests 6 volunteers: overnight fasting 50 ml of blood between 8-9 am 4 sample 10 ml each with heparin for O 3 addition 1:1 blood /gas (O 2 -O 3 ) [90-95 %;1-5 %] 5 min monodirectional oscillator (60 cycles/min) Arezzo November 29th

38 General characteristics of volunteers: blood variables Variables age RC Hb Ht WC Pl PP ALB/Glob years 10 6 mm 3 g/dl % 10 3 mm mm 3 g/dl ±6.3 ±0.08 ±0.55 ±1.33 ±0.49 ±9.5 ±0.18 ±0.08 Arezzo November 29th

39 Variables OB variables following O 3 addition O 3 µg/ml d-roms[carr.u.] 282 ± ± ± ±11.1 PAT [CorU] 2239 ± ± ± ±40.3 PAT/d-ROMs % decrease PAT % decrease ratio Arezzo November 29th

40 Conclusions O 3 addiction increases proportionally the d-roms and decreases proportionally PAT The ratio PAT/d-ROMs seems to be a more sensible index of OB Arezzo November 29th

41 The antioxidant capacity in saliva: SAT Saliva Acts as a barrier for the external environment and includes a very complex antioxidant system (AO). AO system consists of different molecules (e.c. uric acid, Vit E, Vit C) and enzymes (e.c. POXs, catalase). The three bias of the AO determination in saliva are: FLOW rate, URIC ACID and PHOSPHATES content. Arezzo November 29th

42 SAT test Procedure through MiniSAT Saliva is taken by chewing for 1 minute a 250 mg cotton square (60 ±2 bites/min). This procedure allows to obtain in 90 % of the cases volumes between 1 to 1.4 ml Saliva sample is collected by squeezing the cotton. 40 µl of Fe 3+ solution (R2) is added to a 1 ml of SCNchromogenic solution (R1) in a photometric cuvette (disposable) and 10 µl of saliva is added to the cuvette. The reaction procedes for 1 minutes and the saliva AO amount is given as µmol/l of Vitamin C. Arezzo November 29th

43 SAT normal values Range(Cor U) Indication <1000 Severe shortage Optimal normal values Normal values Borderline values >2500 Possibleinflammatoryprocesson place Arezzo November 29th

44 SAT (145 cases M/F) Arezzo November 29th

45 Conclusions In chemical terms death is nothing more than an advanced state of oxidation. and life is nothing more than the tentative to counteract an excessive oxidation Arezzo November 29th