Article pubs.acs.org/JAFC

Oxidation of Carbon Monoxide by Perferrylmyoglobin Silvia H. Libardi,† Leif H. Skibsted,*,§ and Daniel R. Cardoso*,† †

Instituto de Quı ́mica de São Carlos, Universidade de São Paulo, Av. Trabalhador São Carlense 400, CP 780, CEP 13560-970 São Carlos, SP, Brazil § Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark S Supporting Information *

ABSTRACT: Perferrylmyoglobin is found to oxidize CO in aerobic aqueous solution to CO2. Tryptophan hydroperoxide in the presence of tetra(4-sulfonatophenyl)-porphyrinate-iron(III) or simple iron(II)/(III) salts shows similar reactivity against CO. The oxidation of CO is for tryptophan hydroperoxide concluded to depend on the formation of alkoxyl radicals by reductive cleavage by iron(II) or on the formation of peroxyl radicals by oxidative cleavage by iron(III). During oxidation of CO, the tryptophan peroxyl radical was depleted with a rate constant of 0.26 ± 0.01 s−1 for CO-saturated aqueous solution of pH 7.4 at 25 °C without concomitant reduction of the iron(IV) center. Carbon monoxide is as a natural metabolite accordingly capable of scavenging tryptophan radicals in myoglobin activated by peroxides with a second-order rate constant of (3.3 ± 0.6) × 102 L mol−1 s−1, a reaction that might be of importance in cellular membranes of the intestine for protection of tissue against radical damage during meat digestion. KEYWORDS: carbon monoxide, carbon dioxide, perferrylmyoglobin, tryptophan radical, meat digestion

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INTRODUCTION Carbon monoxide (CO) is formed in heme metabolism and seems to protect the gastrointestinal tract against noxious injury and inflammation.1−3 Prior to the more recent interest in CO as a signaling molecule in the gastrointestinal tract and in a potential use of CO as a therapeutic agent, CO interaction with oxidized heme proteins has been studied in relation to autoreduction of cytochrome c oxidase.4,5 Autoreduction processes seem important for the function of heme-based enzymes activated by H2O2 and also for regeneration of myoglobin and hemoglobin exposed to H2O2 during metabolic processes under ischemia-induced injury.6,7 Negative health effects of a high intake of red meat, recognized as an increased frequency of gastrointestinal cancers in certain geographical regions and population segments, have been linked to the prooxidative effects of the meat pigments.8,9 Cooking temperature and species variation for myoglobin affect the digestibility of meat and prooxidative effects.10,11 Myoglobin as the most prominent meat pigment forms perferrylmyoglobin when exposed to peroxides, and this hypervalent iron pigment radical may abstract hydrogen atoms from proteins and unsaturated lipids, in effect initiating oxidative damage. Ferrylmyoglobin, the nonradical iron(IV) heme pigment formed by one-electron reduction of perferrylmyoglobin, further cleaves preformed protein and lipid hydroperoxides oxidatively to generate peroxyl radicals as reactive intermediates, boosting oxidative damaging processes.12 Current dietary recommendations include a higher intake of vegetables and fruits together with meat, as the presence of polyphenols and carotenoids may yield protection against the oxidative damage to tissue otherwise initiated by the hypervalent heme pigments.13−15 The beneficial effects of many dietary polyphenols in disease prevention have thus been © 2014 American Chemical Society

assigned to their activity in the gut rather than after absorption.16 However, under the reducing conditions in the gut, compounds such as CO naturally formed inside or outside cellular structures may also be involved in such protection and may even act synergistically with meal components. The reactions of CO with activated myoglobin have not been studied so far for conditions relevant for meat digestion, and as a continuation of previous studies of natural antioxidants,17,18 herein we now report results from an investigation of the radical chemistry of interaction between carbon monoxide and activated myoglobin.

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MATERIALS AND METHODS

Chemicals and Materials. Deuterium oxide (99.0% D), FeSO4 and FeCl3 salts, formic acid mass spectrometry grade, horse heart myoglobin, K2HPO4 and KH2PO4, Rose Bengal sodium salt, Sephadex-G25, L-tryptophan, sodium 3-trimethylsilyl-2,2,3,3-d4-propionate (TMSP-d4), tetra(4-sulfonatophenyl)-porphyrinate sodium salt, and xylenol orange tetrasodium salt were purchased from SigmaAldrich (St. Louis, MO, USA). Myoglobin was further purified according to the procedure described in the literature.7 Tetra(4sulfonatophenyl)-porphyrinate iron(III) (Fe(III)TPPS) was prepared according to the procedure in the literature.19 Hydrogen peroxide 30% was from Merck (Darmstadt, Germany). Acetonitrile HPLC grade was obtained from Mallinckrodt (Phillipsburg, NJ, USA). Water was purified (18 MΩ cm) by means of a Milli-Q purification system from Millipore (Billerica, MA, USA). Synthesis and Isolation of TrpOOH Derivatives. A 1.6 mg/L oxygen saturated solution of L-tryptophan in D2O containing 1.0 × 10−4 mol L−1 Rose Bengal was irradiated with 532 nm light from a green LED (5 mW) for 2 h at 0 °C under continuous flux of oxygen.20 Received: Revised: Accepted: Published: 1950

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Figure 1. EPR spectra at 77 K of perferrylmyoglobin (•MbFe(IV)O) during reduction to ferrylmyoglobin (MbFe(IV)O) in aqueous solution of pH 7.4 at 25 °C saturated with CO (A) or in the absence of CO (B) at different aging times, as indicated using a stopped-flow freeze-quench technique. Each spectrum is from an independent experiment, and the decay of the tryptophan peroxyl radical (gx = 2.036) is normalized relative to the tyrosine carbon-centered radical (g = 2.005). The ratio of the integrated signals A1/A2 is probing the tryptophan peroxyl radical decay. After irradiation, the Rose Bengal was removed by filtering the solution through a Teflon membrane filter of 0.2 μM (Chromafil, MachareyNagel GmbH & Co., Dueren, Germany). The TrpOOH, tryptophan hydroperoxide derivatives, were further isolated from the reaction mixture by semipreparative liquid chromatography (HPLC20A Prominence system equipped with a DAD detector SPDM20A, Shimadzu Co., Kyoto, Japan) employing a Luna C18 reverse-phase column (250 mm × 7 mm × 10 μm; Phenomenex Inc., North Hill, CA, USA) with aqueous formic acid 0.05% containing 4% acetonitrile as isocratic mobile phase at a flow rate of 1 mL/min. The product peaks were collected in an ice bath protected from light and immediately lyophilized using a ModulyoD freeze-dryer (Thermo Electron Corp., São Paulo, Brazil). The hydroperoxide concentration was evaluated by the ferric-xylenol orange method (FOX) using H2O2 as a standard.21 Typically, a yield of 10% for the high-purity TrpOOH derivative was obtained after the purification step. NMR Spectroscopy. NMR analyses (1H, COSY, and HSQC) were recorded at 25.0 °C on a Bruker AVANCE III, 14.1 T instrument (600 MHz for hydrogen frequency) equipped with an automatic sample changer, a cryo-probe TCI (1H/13C/15N) of 5 mm with ATMA (automatic tuning matching) and z-field gradient (Bruker BioSpin, Rheinstetten, Germany). Aliquots of each isolated product were dissolved in D2O containing 0.050% w/w TMSP-d4 for NMR analysis. The 1H NMR experiment was based on the 1D version of the NOESY sequence, using water suppression during the relaxation delay (2.40 s). The spectrum was acquired using 64K data points in a 12019.23 Hz spectral width, giving an acquisition time of 2.73 s. Processing was performed using exponential multiplication, applying a line broadening factor of 0.3 Hz. 1H−1H COSY experiments were performed using a spectral width of 12019.23 Hz, an acquisition time of 0.17 s, a relaxation delay of 1 s, and 16 scans for each increment (256 increments). The 1H−13C HSQC experiment employed spectral widths of 12019.23 Hz for the F2 (1H) dimension and 36057.69 Hz

for the F1 (13C) dimension. The acquisition time was 0.17 s, the relaxation delay was 1 s, and 64 scans were performed for each increment (256 increments). TopSpin 3.0 software (Bruker BioSpin) was used for data acquisition and processing. FT-IR Spectroscopy. Experiment type A: 2 mL of a solution of 2 × 10−4 mol L−1 of MbFe(III), or lactoperoxidase, or Fe(III)TPPS in aqueous phosphate buffer, pH 7.4 (0.16 M), was saturated with CO for 20 min in a half-filled cylindrical quartz cuvette (5 cm optical path), and the reaction was initiated by the addition of an equimolar quantity of H2O2. A blank solution without heme iron or iron compound was also investigated. The equilibrated gas-phase spectra were recorded after 1 h using a FT-IR IRAffinity-1 instrument (Shimadzu Co., Kyoto, Japan). Experiment type B: 2 mL of a solution of 2 × 10−4 mol L−1 of Fe(III)TPPS or Fe(II) or Fe(III) ions in aqueous formate buffer, pH 4.7 (0.16 M), was saturated with CO for 20 min in the half-filled cylindrical quartz cuvette (5 cm optical path), and the reaction was initiated by the addition of an equimolar quantity of TrpOOH. A blank solution without Fe(III)TPPS or iron salt was also investigated. The equilibrated gas-phase spectra were recorded after 1 h using a FTIR IRAffinity-1 instrument. Experiment type C: 2 mL of a solution of 2 × 10−4 mol L−1 of preformed MbFe(IV)O (H2O2 plus MbFe(III) after 10 min of reaction) in aqueous phosphate buffer pH 7.4 (0.16 M) was saturated with CO for 20 min in the half-filled cylindrical quartz cuvette (5 cm optical path), and the reaction was initiated by the addition of an equimolar quantity of TrpOOH. A blank solution without TrpOOH was also investigated. The equilibrated gas-phase spectra were recorded after 1 h using a FT-IR IRAffinity-1 instrument. Freeze-Quench EPR Spectroscopy. An SFM-20 freeze-quench apparatus (Biologic Scientific Instruments, Claix, France) with a minimal aging time of 9 ms and an EMXplus X-band EPR instrument (Bruker Biospin GmbH, Karlsruhe, Germany) using a TM101 cylindrical cavity were used for freeze-quench kinetics. For this experiment, a solution containing 1.7 × 10−4 mol L−1 MbFe(III) 1951

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saturated or not with CO was loaded in syringe A and a solution of 7.7 × 10−4 mol L−1 of H2O2 was loaded in the opposite syringe B. The solutions of syringes A and B were mechanically pushed to the reaction chamber at a ratio of 1:1 at 25 °C and aged inside the calibrated aging loop for different reaction times from 5 to 40 s before being sprayed by the ejection nozzle into a cryogenic bath at 77 K. EPR spectrometer parameters: center field, 3395.58 G; sweep width, 200 G; MW power, 2.00 mW; MW frequency, ∼9.52 GHz; modulation frequency, 100.00 kHz; modulation amplitude, 3.00 G; time constant, 81.92 ms; conversion time, 81.92 ms; temperature, 77 K. Each spectrum is an average of eight scans.

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RESULTS AND DISCUSSION Purified metmyoglobin (MbFe(III)) when reacting with hydrogen peroxide (H2O2), in neutral aqueous solution, forms perferrylmyoglobin (dot-MbFe(IV)O), a protein radical of the iron(IV) heme pigment.22 Perferrylmyoglobin decays to ferrylmyoglobin (MbFe(IV)O), the iron(IV) pigment for ambient conditions with a half-life of 30 s at 25 °C and neutral pH.23 The EPR spectrum of perferrylmyoglobin shows under aerobic conditions two features (see Figure 1), one assigned to a tryptophan peroxyl radical (gx = 2.036, gy = 2.008, gz = 2.0028) and another septet EPR signal with g = 2.005 assigned to a carbon-centered tyrosyl radical.23,24 The tryptophan peroxyl radical of perferrylmyoglobin decays by reaction with external reductants or by slower intramolecular electron transfer.15,16,21 The ratio between the two integrated signals, as shown in Figure 1, was used to follow the decay of the tryptophan peroxyl radical in aqueous solution at 25 °C in the absence of CO (Figure 1B) or for aqueous solution saturated with CO (Figure 1A), using a freeze-quench stoppedflow technique. The decay of the tryptophan peroxyl radical was found both in the absence and in the presence of CO to follow first-order kinetics (see Figure 2), but clearly the presence of CO accelerated the decay, as is seen for the values of the rate constant in the absence of CO, k = (5.3 ± 0.1) × 10−2 s−1, and for water saturated with CO, k = (2.8 ± 0.1) × 10 −1 s −1. Formation of CO2 was detected by FT-IR spectroscopy of the gas phase in equilibrium with aqueous solution (see Figure 3). The absorption maximum for CO2 at 2335 and 2361 cm−1 increased for the reaction mixture as the one illustrated in Figure 3, for which the FT-IR spectrum is presented for a reaction time of 60 min.25,26 Tryptophan hydroperoxide, as a mixture of cis and trans isomers, was synthesized photochemically using Rose Bengal as a sensitizer for singlet-excited oxygen generation and isolated by HPLC employing a semipreparative C18 reverse-phase column (see Figure 4) and included as reactant with CO for comparison with activated myoglobin. The photolysis of an oxygen-saturated solution of 30 mM tryptophan in D2O containing 8 μM Rose Bengal using 532 nm light from a green LED diode (5 mW) for 2 h at 0 °C resulted in the formation of mainly the tryptophan-derived hydroperoxides as cis and trans isomers, as may be seen in Figure 4. The identity of the compounds seen as peaks P1 and P2 was confirmed by 1D and 2D NMR analysis (1H/COSY/HSQC) after isolation as the HSQC spectrum collected for peak P2, the cis-TrpOOH isomer (Figure S1 in the Supporting Information). The cis- and transTrpOOH, after the purification step, were quantified using the ferric-xylenol orange method (FOX) and fully characterized by 1 H, COSY, and HSQC NMR experiments (Table 1); the obtained data were consistent with literature values for the proposed structures.20,21 The most remarkable difference in the 1 H NMR spectrum for the cis- and trans-TrpOOH derivatives is

Figure 2. Decay of tryptophan peroxyl radical of perferrylmyoglobin (•MbFe(IV)O) in aqueous solution of pH 7.4 at 25 °C saturated with CO (A) or in the absence of CO (B) as probed by the A1/A2 ratio from EPR spectra obtained by a stopped-flow freeze-quench technique (see Figure 1). Curves are exponential fits corresponding to first-order decay kinetics with observed rate constants of k1 = 0.28 ± 0.04 s−1 (A) and k1 = 0.05 ± 0.01 s−1 (B) to yield k1 = 0.23 ± 0.04 s−1 for CO reduction of •MbFe(IV)O.

associated with the C3 methylene hydrogens. As may be seen from Table 1, for the cis-TrpOOH derivative, the C3 methylene hydrogens are magnetically equivalent and appear as one doublet at 2.66 ppm. On the other hand, the C3 methylene hydrogens for the trans-TrpOOH derivative show a double− doublet coupling pattern for nonequivalent hydrogens in an ABX system, the nonequivalent hydrogens being the AB part and the H2 hydrogen the X part. Tryptophan hydroperoxide did not react with CO to form CO2 unless catalyzed by tetra(4-sulfonatophenyl)-porphyrinate iron(III) (Fe(III)TPPS) or by FeSO4 or FeCl3 as simple iron salts. The efficiency by which these and other reactant mixtures were oxidizing CO to CO2 for comparable reaction conditions and reaction time is depicted in Figure 5 as the integrated FTIR signal to provide a relative concentration of CO2. From the results presented in Figure 5, it is evident that H2O2 and CO do not produce CO2 in buffered aqueous solution of pH 7.4 at 25 °C in the absence of iron salts or in the presence of Fe(III)TPPS. Solutions of tryptophan incubated with CO in the presence of hydrogen peroxide and in the absence of heme iron or iron salts do not produce CO2. Tryptophan hydroperoxide plus CO likewise does not produce CO2 in the absence of iron salts, but CO2 is clearly produced when iron(II) or iron(III) is present and especially when 1952

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TrpO• + CO → Trp• + CO2

(4)

•

of which the reaction of ROO with CO has been reported to be faster than the reaction of TrpO• with CO by a factor of 102.27 For perferrylmyoglobin, a similar reactivity is recognized, as activation of MbFe(III) by hydrogen peroxide prior to addition of CO yields no reaction compared to activation of MbFe(III) by hydrogen peroxide in the presence of CO, indicating that when •MbFe(IV)O has decayed to MbFe(IV)O, the reactivity toward CO will disappear. The presence of tryptophan hydroperoxide in experiments with preformed ferrylmyoglobin enhances the reaction moderately, showing that the heme iron may cleave the peroxide oxidatively corresponding to the reaction of eq 1 to yield reactive peroxyl radicals or that the tryptophan hydroperoxide may have reactivated ferrylmyoglobin to perferrylmyoglobin. Lactoperoxidase, as heme enzyme also with solvent-exposed tryptophan, included for comparison in the resting iron(III) form, showed less activity than MbFe(III) when both heme compounds were activated by H2O2 in the presence of CO.28 The reactivity of the two tryptophan radicals, TrpOO• and TrpO•, in oxygen transfer reaction to CO finds a parallel in the activated MbFe(III). The solvent-exposed tryptophan of MbFe(III) becomes oxidized to TrpOO• during the oxidation of MbFe(III) by H2O2 in aerated aqueous solution:

Figure 3. FT-IR spectra of equilibrated gas phase of aqueous solution of pH 7.4 with reactants shown in sketch of quartz cylindrical cuvette at 25 °C for metmyoglobin following 60 min of reaction and equilibration. [MbFe(III)] = 1 × 10−4 mol L−1; [H2O2] = 1 × 10−4 mol L−1; [CO] = 7 × 10−4 mol L−1; [TrpOOH] = 1 × 10−4 mol L−1; [Fe(III)TPPS] = 1 × 10−4 mol L−1.

MbFe(III) + H 2O2 → •+MbFe(IV)O + H 2O → •MbFe(IV)O + H+ + H 2O

(5)

•

MbFe(IV)O is a tryptophan peroxyl radical, as shown in eq 5 and will react with CO in a reaction similar to the reaction of eq 3 to yield CO2, and the tryptophan alkoxyl radical still capable of transferring an oxygen atom to CO will react as in the reaction of eq 4. The carbon-centered tryptophan radical thus formed will decay by intramolecular reaction to form a carbon-centered tyrosine radical of higher stability, which may undergo an intermolecular reaction to form heme dimers. This reaction sequence as depicted in eq 5 accounts for the experimental observations related to CO2 formation from the different reaction mixtures described in Figure 5 and also the EPR spectra of Figure 1. Notably, only •MbFe(IV)O and not MbFe(IV)O is capable of oxidizing CO to CO2. From the observed rate of disappearance of •MbFe(IV)O in the presence of CO (saturated water corresponding to 0.7 mM CO in the reaction chamber), it becomes possible to estimate the rate constant for the bimolecular reaction between • MbFe(IV)O and CO to have the value of 330 ± 60 L mol−1 −1 s . This seems to be the first value available for the rate constant for deactivation of perferrylmyoglobin by an external reductant. For the reaction of perferrylmyoglobin with the water-soluble carotenoid crocin, a lower limit for the rate constant was estimated to be 4 × 105 L mol−1 s−1,17 and for the reaction with 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) to form the ABTS•+ radical the lower limit was 6 × 105 L mol−1 s−1.18 The reaction with crocin is an electrontransfer reaction

Figure 4. Separation and characterization of tryptophan hydroperoxide trans/cis isomers as formed by singlet oxygen addition to tryptophan in oxygen-saturated D2O with Rose Bengal added as photosensitizer and exposed to green light (see Materials and Methods). HPLC separation of trans isomer (P1) and cis isomer (P2) is marked to indicate collection range.

Fe(III)TPPS is present. This identifies a tryptophan radical rather than tryptophan peroxide as the reactant for oxidation of CO to CO2, as iron(III) and iron(II) may cleave hydroperoxides by oxidation or by reduction, respectively: TrpOOH + Fe3 + → TrpOO• + H+ + Fe 2 +

(1)

TrpOOH + Fe2 + → TrpO• + OH− + Fe3 +

(2)

•

MbFe(IV) = O + crocin

The tryptophan radicals will react with CO by oxygen atom transfer27 according to TrpOO• + CO → TrpO• + CO2

→ −MbFe(IV) = O + crocin•+

(3) 1953

(6)

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Table 1. 1H and 13C NMR Chemical Shifts of the cis- and trans-Tryptophan Hydroperoxides in D2O

been shown to be deactivated by reaction with CO. The reaction of CO involves oxygen atom transfer to form CO2 in principle in two-reaction steps, eventually forming a heme dimer through a dityrosine linkage. The scavenging of activated myoglobin by CO may protect sensitive structures against oxidative damage involving proteins and unsaturated lipids. CO may in this respect function as an inherent antioxidant. Future experiments are required to investigate whether CO may act synergistically with dietary antioxidants such as polyphenols and carotenoids through binding to ferrous myoglobin or through a subsequent reduction of MbFe(IV)O following the initial reduction of perferrylmyoglobin by CO.

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ASSOCIATED CONTENT

* Supporting Information S

1 H−13C HSQC spectrum for the isolated cis-TrpOOH derivative. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

Figure 5. CO2 production from reaction mixtures as indicated for experiments shown in Figure 3 using integrated absorption from 2280 to 2400 cm−1 as a relative measure of CO2 concentration.

*(L.H.S.) E-mail: [email protected]. Phone: + 45 35 33 32 21. *(D.R.C.) E-mail: [email protected]. Phone: +55 16 33 73 99 76. Funding

as in the reduction with ABTS, explaining the higher rate compared to the oxygen atom transfer for the reaction of eqs 3 and 4. Activated myoglobin, which will be formed during digestion of red meat through reaction of MbFe(III) with peroxides, has

This research is part of the bilateral Brazilian/Danish Food Science Research Program “BEAM − Bread and Meat for the Future” supported by FAPESP (Grant 2011/51555-7) to D.R.C. and by the Danish Research Council for Strategic 1954

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(19) Barley, M. H.; Takeuchi, K. J.; Meyer, T. J. Electrocatalytic reduction of nitrite to ammonia based on a water-soluble iron porphyrin. J. Am. Chem. Soc. 1986, 108, 5876−5885. (20) Ronsein, G. E.; Oliveira, M. C. B.; Miyamoto, S.; Medeiros, M. H. G.; Di Mascio, P. Tryptophan oxidation by singlet molecular oxygen O-2 (1Δg): mechanistic studies using O-18-labeled hydroperoxides, mass spectrometry, and light emission measurements. Chem. Res. Toxicol. 2008, 21, 1271−1283. (21) Gay, C.; Collins, J.; Gebicki, J. M. Hydroperoxide assay with the ferric-xylenol orange complex. Anal. Biochem. 1999, 273, 149−155. (22) King, N. K.; Winfield, M. E. Mechanism of metmyoglobin oxidation. J. Biol. Chem. 1963, 238, 1520−1528. (23) Svistunenko, D. A. Reaction of haem containing proteins and enzymes with hydroperoxides: the radical view. Biochim. Biophys. Acta: Bioenerg. 2005, 1707, 127−155. (24) Irwin, J. A.; Ostdal, H.; Davies, M. J. Myoglobin-induced oxidative damage: evidence for radical transfer from oxidized myoglobin to other proteins and antioxidants. Arch. Biochem. Biophys. 1999, 362, 94−104. (25) Seichter, F.; Wilk, A.; Woerle, K.; Kim, S.-S.; Vogt, J. A.; Wachter, U.; Radermacher, P.; Mizaikoff, B. Multivariate determination of (CO2)-C-13/(CO2)-C-12 ratios in exhaled mouse breath with mid-infrared hollow waveguide gas sensors. Anal. Bioanal. Chem. 2013, 405, 4945−4951. (26) Young, L. J.; Caughey, W. S. Mitochondrial oxygenation of carbon-monoxide. Biochem. J. 1986, 239, 225−227. (27) Denisov, E. T.; Shestakov, A. F. Reactions of alkoxy and peroxy radicals with carbon monoxide. Kinet. Catal. 2008, 49, 1−10. (28) Fielding, A. J.; Singh, R.; Boscolo, B.; Loewen, P. C.; Ghibaudi, E. M.; Ivancich, A. Intramolecular electron transfer versus substrate oxidation in lactoperoxidase: investigation of radical intermediates by stopped-flow absorption spectrophotometry and (9−285 GHz) electron paramagnetic resonance spectroscopy. Biochemistry 2008, 47, 9781−9792.

Research (Grant 11-116064) to L.H.S. D.R.C. thanks CNPq for the research fellowship. Notes

The authors declare no competing financial interest.

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