Poster
# 55
Poster |
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6th Internet World Congress for Biomedical Sciences |
Bartolomé Quintero(1), María del Carmen Cabeza(2)
(1)(2)Dpt. Physical Chemistry. Faculty of Pharmacy. University of Granada - Granada. Spain
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[Biophysics] |
[Cell Biology & Cytology] |
The diethylenetriaminpentaacetic acid (DTPA) in combination with different ions such as In, Tc or Gd is used in several diagnosis/imaging techniques. Moreover, DTPA ability as a chelator is also used in the study of the arenediazonium ions reduction to prevent the action of adventitious reducing metal ions such as Fe2+.
The arenediazonium ions are widely employed in chemical synthesis. Apart from the diazocopulation reactions, these ions can undergo either thermal or photochemical heterolytic dediazoniation yielding the aryl cation. Likewise, the arenediazonium ions can be dediazoniated in a homolytic process via monoelectronic reduction which originates the appearance of the aryl radical. The arenediazonium ions are believed to be genotoxic. The electronic structure [Glaser et al., 1999], the dediazoniation mechanism [Pazo Llorente et al., 1999] and the identification of the ultimate genotoxic agent derivated from these ions [Gannett et al., 1999] are aspects currently under investigation.
Some phenolic compounds present in foodstuff, beverage and therapeutical drugs are substrates which can be transformed into the corresponding hydroxybenzenediazonium ions. We have studied the dediazoniation of p-hydroxybenzenediazonium ion (PDQ) in a neutral aqueous medium [Quintero et al., unpublished results]. The dediazoniation process rate decreases in the presence of DTPA. In this paper we report the results obtained in the study of the influence of DTPA in the dediazoniation of PDQ.
Chemicals of the highest available purity (Merck and Aldrich) were used to obtain PDQ tetrafluoroborate. DTPA, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), N-t-butyl- a -phenylnitrone (PBN) and a -(4-pyridil-1-oxide)-N-t-butylnitrone (4-POBN) were purchase from Sigma. Hydroquinone (Merck) was used to synthethize quinone by causing it to react with potassium bromide in an acid medium at 80ºC. The solid obtained had a melting point of 115ºC. Aqueous solutions of quinone showed an absorption maximum at 245 nm ( e = 21500 ± 400 M-1 cm-1). PDQ tetrafluoroborate was synthesized by us. The solid obtained was stored in darkness at below -18ºC. The synthesized solid was subject to elemental analysis in a Fisons-Carlo Erba EA 1108CHNS-0 Elemental Analyzer (Scientific Instrument Center [SIC], University of Granada). IR spectra were registered at room temperature with an FT-IR Nicolet 20SXB spectrophotometer (SIC, University of Granada). For ESR measurements a Bruker ESP 300E spectrometer (SIC, University of Granada) was used. Perkin-Elmer Lambda 5 and Lambda 16 spectrophotometers were used for the spectrophotometric analysis of PDQ tetrafluoroborate. Merck-Hitachi equipment for HPLC, including a Merck L-6220 biocompatible pump and a Merck L-4500 diode array detector, were used. Aqueous media were filtered by Millipore HA filters with a pore size of around 0.45 m m. The column was a Spherisorb ODS-2 (4.6 mm x 200 mm) with a particle size of 5 m m. The mobile phases were deaereated with a Selecta P 150 W ultrasound generator, producing waves of 40 kHz. Mobile phase methanol/ammonium formiate 0.1 M (1:39) with a flow of 0.7 mL/min was routinely used. The PDQ solutions were in buffer phosphate (0.05 M, pH 6.97) with concentrations of between 12 mM and 10 m M. The possible appearance of hydroquinone was monitored by steady-state fluorescence using a Shimadzu RF-5001 PC spectrofluorimeter. A Radiometer pH M64 potentiometer with a GK2401C mixed electrode was used whenever called for. The calibrations were carried out with Crison buffer references (pH 4 and pH 7).
We checked for any effects caused by either laboratory environmental light or apparatus light sources on PDQ tetrafluoroborate degradation. The results were taken into account when designing the methodology for the spectrophotometric and spectrofluorimetric measurements. All measurements were routinely made with aliquots taken from a stock solution kept in darkness.The influence of oxygen dissolved in the solution upon the PDQ decomposition rate was noted at an early stage in our experiments and thus whenever deaereated samples were required the oxygen was purged by bubbling with argon for at least 10 min.
Synthetized PDQ tetrafluoroborate is a yellowish crystalline solid which melts in the range of 135ºC to 140ºC accompanied by a noticeable change of colour and the formation of gas. The results of the elemental analysis were C: 42.8%; H:2.76% and N:17.05%. These results agree very well with the formation of the tetrafluoroborate of the dimer of PDQ. In the IR spectrum are noticeable bands at 2189 cm-1 (N º N stretching) and 1591 cm-1 (aromatic group). From the spectrophotometric measurements a value of e = 41990 ± 220 M-1 cm-1 was obtained at the absorption maximum (348 nm) using solutions in neutral aqueous medium (phosphate buffer, pH7). Likewise, spectrophotometric measurements were used to detemine the value of the pKa correspondig to the hydroxyl dissociation in PDQ. The result obtained was 3.35 ± 0.02 at 25 ºC. Chromatographic analysis by HPLC of newly prepared PDQ aqueous solutions (phosphate buffer, pH7) showed only one signal, with a retention time of 6.66 min, associated to a spectrum with a maximum at 348 nm.
The kinetic analysis performed in the present work were made by incubating 0.4 mM PDQ solutions either in the presence or in the absence of other chemicals (DTPA, ferrous ion) kept in darkness at 37 ºC. From this solutions aliquots were taken to made PDQ 0.01 mM solutions which were used for the spectrophotometric measurements.
The absorption spectra registered with aliquots taken from samples of acid aqueous solutions of 10 mM PDQ (37 ºC, acetate buffer pH5) kept in darkness in the presence of oxygen, did not show significant changes. On the contrary, the spectra from neutral aqueous solution of PDQ (37 ºC, phosphate buffer pH7) showed a noticeable decrease in the absorption band located at 348 nm 24 h after preparing the solution.
Degradation of aerated PDQ solutions (aerobic conditions) involves the appearance of of hydroquinone and quinone. So, the fluorescent emission from hydroquinone (lexc = 280 nm; lem = 327 nm) is observed with 10 mM PDQ solutions (37 ºC, phosphate buffer pH7) kept for 24 h in darkness. Moreover, HPLC analysis of PDQ in an aqueous solution (phosphate buffer, pH7), kept for 24 hours in darkness at 25ºC under aerobic conditions, reveals the appearance of signals belonging to hydroquinone (10.8 min) and quinone (30.1 min). The chromatogram of the sample kept under similar conditions but previously purged with argon (anaerobic conditions) shows no signal for either hydroquinone or quinone.
The ESR spectra registered using 80 mM PDQ solutions in phosphate buffer pH7 in the presence of the spin-trap DMPO (110 mM) either in aerobic or in anaerobic conditions, gave only one stable adduct which can be put down to a p-hydroxyphenyl radical (aH = 24.5 G; aN = 15.9 G). This result can be also obtained using as spin trap PBN (aH = 4.1 G; aN = 15.9 G) o 4-POBN (aH = 3.2 G; aN = 15.6 G) [conc = 50 mM] even in the presence of hydroxyl radical scavengers such as DMSO (1.4 M) or ethanol (2.0 M).
DTPA does not change the features of the PDQ absorption spectrum (Fig. 1) but decreases the intensity of the signals registered in RSE as well as the PDQ degradation rate. Figure 2 shows the variation as a function of the time of the absorbance measured at 348 nm with aerated 0.01 mM PDQ solutions (37º C, phosphate buffer pH7) kept in darkness. As it can be observed by adding DTPA (1.5 mM) practically the reaction is stopped in the time interval studied. In addition, the degradation rate does not change as the Fe2+ is present in concentration 1 mM or 10 mM
The oxygen consumption measured in the first step of the dediazoniation process (up to 180 min) indicates (Fig. 3) that the consumption decreases in the presence of DTPA (0.5 mM)
The absorption spectrum of 0.44 mM hydroquinone (H2Q) solution in phosphate buffer pH7 changes 24 h after preparing the solution. The band located at 288 nm decreases and a new band appears centered at 245 nm. In the presence of DTPA (2.8 mM) there is no lowering in the band at 288 nm and the band at 245 nm is significantly less intense (Fig.4)
In basic medium (borate buffer pH9) the hydroquinone degradation rate increases. The spectra shown in Figure 5 were obtained just after preparing the solution. It can be seen that the spectrum registered with the 0.35 mM H2Q presents an absorbance at 288 nm lower than that measured with a solution containing the same concentration of H2Q added with DTPA (2.8 mM). Likewise, it can be checked that the absorption about 311 nm is higher in the case of the solution containing only H2Q
PDQ degradates in a neutral aqueous medium. By keeping the samples in darkness, the process originates the appearance of the p-hydroxyphenyl radical both in aerobic and anaerobic conditions. However, the appearance of hydroquinone and quinone is observed after 24 hours only when the reaction is carried out in aerobic conditions. These data appears to point out a homolytic reaction induced by the solvent wherein oxygen plays a catalytic role since the heterolytic process leading to the aryl cation should have produce the appearance of hydroquinone in anaerobic conditions. Taking into account the ability of the aryl radicals to react with oxygen affording the corresponding peroxyl radicals, we have proposed the pathways outlined in Figure 6 in order to explain the results found in the analysis of the dediazoniation of PDQ. So, the primary reaction could be induced by hydroxyl anion giving the non-protonated p-hydroxyphenyl radical (-Oph·). Subsequent reaction with oxygen would lead to the corresponding peroxyl radical (-OphO2·). The transformation of this latter radical into the anion semiquinone (SQ-·) could take place via tetroxide as an intermediate. The dediazoniation rate would increase because of the presence of this reducing species since two additional pathways in the reduction of PDQ can be considered. One of them involving PDQ direct reduction to form quinone (Q) and the other pathway starting from the disproportionation of semiquinone to give quinone (Q) and hydroquinone (H2Q). We have checked that hydroquinone can efectively reduce PDQ.
The chelating agent DTPA appears to decrease the PDQ degradation process rate. In principle, chelation of reducing metal cation by DTPA would prevent a possible direct reduction of PDQ. This is mainly the expected effect in adding DTPA to the system, however no significant difference in the degradation rate was observed as a result of either the presence or the absence of DTPA in anaerobic conditions. The possibility of another involvement of the reducing metal ions in the redox cycles shown in Figure 6 appears apparentely ruled out. In fact, concentrations of ferrous ion up to 0.01 mM do not modify the PDQ degradation rate although a clear increase is observed following the addition of hydroquinone. Influence of other ions apart from Fe2+ has not been analyzed. Nevertheless, it would seem rather improbable that PDQ should be reduced by means of these ions via an independent degradation pathway since the quantity of reducing ions, mainly Fe2+, in the buffers used is very low [around 0.17 ppm for phosphate buffer (0.05 M), 0.35 ppm for acetate buffer (0.2 M) and 0.003 ppm for HCl solution at pH 1] and the DTPA concentration required to stop the degradation reaction in our experimental conditions is about 1.5 mM. Moreover, the presence of DTPA appears to be associated to a decrease in the oxygen consumption. The possible formation of a complex such as DTPA-Fe(II)-O2 observed in other system could reduce the PDQ degradation rate by disminishing the amount of the molecular oxygen avalaible for the formation of peroxyl radical. However, the formation of the above mentioned complex should be accompanied by an increase in the oxygen consumption. In addition, a possible interaction between DTPA and PDQ in the ground state blocking the reduction of this latter compound, appear also ruled out because of the absence of significant changes in the absorption spectrum of PDQ in the presence of DTPA.
It is known that hydroquinone can undergo an autooxidation reaction in aqueous medium. It has been reported that 2,3-dimethyl-1,4-naphthohydroquinone undergoes autoxidation to the corresponding quinone at pH 7.4, with stoichiometric consumption of oxygen and formation of hydrogen peroxide. In the presence of trace metals in the buffer the rate of oxidation was low, but it increased when trace metals were removed from the buffer by treatment with Chelex resin, DTPA or bathophenanthroline sulfonate [Munday, 1999]. Likewise it has been pointed out that DTPA does not affect the autooxidation of tetrachlorhydroquinone [Zhu et al., 1998]. Our results indicate the p-hydroquinone protolytic equilibria could be altered (and subsequently the autooxidation reaction) by the presence of DTPA. In Figure 5 is noticeable how in the presence of DTPA the absorption at 288 increases and simultaneously decreases about 311 nm. In addition, the spectrum registered with aqueous solution of hydroquinone 24 h after preparing the solution and in the absence of DTPA (Fig. 4) shows the appearance of an absorption band located about 244 nm likely originates by quinone. This band is clearly less intense as DTPA is present. Bearing in mind those results and the equilibria shown in Figure 7 a tentative explanation is proposed. .At pH 9 hydroquinone is partially dissociated (pK = 9.9) and the absorption about 311 nm could be attributed in part to HQ- (absorption maximum at 307 nm). The reaction of H2Q with molecular oxygen is very slow at neutral pH [Roginsky et al., 1999] therefore the increase in the reaction rate at pH 9 seems to be related with the faster oxidation of HQ-. We propose that the spectral changes observed in basic medium in the presence of DTPA are a consequence of the protonation of HQ- to give H2Q. So that, apparentely the influence of relatively high concentrations of DTPA appears to be related with the shift of the equilibrium in the system hydroquinone/semiquinone/quinone. Somehow, DTPA interfers as well at neutral pH decreasing the rate of appearance of quinone (Fig. 4) probably by decreasing the concentration of anion semiquinone. Admitting that the concentration of anion semiquinone is a critical factor in the analyzed processes, a decrease in the concentration of anion semiquinone would justify the observed decrease in the H2Q degradation rate in a neutral medium as well as the decrease in the PDQ degradation rate wherein is also assumed that semiquinone plays an essential role. We are currently making other experiences in order to check the points included in our hypothesis.
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[Biophysics] |
[Cell Biology & Cytology] |
