Boletín de la Sociedad Chilena de Química
versión impresa ISSN 0366-1644
Bol. Soc. Chil. Quím. v.47 n.2 Concepción jun. 2002
Bol. Soc. Chil. Quím., 47, 145-149 (2002)
URIC ACID REACTION WITH DPPH RADICALS
AT THE MICELLAR INTERFACE
E. ABUIN*, E. LISSI, P. ORTIZ AND C. HENRIQUEZ
Facultad de Química y Biología, Universidad de Santiago de Chile
Casilla 40-Correo 33. Santiago- Chile
(Receveid: January 11, 2002 Accepted: February 28, 2002)
Uric acid, a water soluble antioxidant, is able to react with micelle incorporated diphenylpicrylhydracyl radicals. This indicates that uric acid can react at the lipid/water interface. The rate of the process follows the order
cationic micelles >buffer/etanol > neutral micelles anionic micelles
These results can be explained in terms of the compartmentalization of the reactants in different environments and the effect of electrostatic interactions in modulating the access of the urate anion to the micellar interface. Experiments aimed to determine if urate is able to react with a-tocopheroxyl radicals regenerating a-tocopherol were uncesessful, indicating that urate is not able to delay vitamin E consumption in the presence of lipid - soluble free radicals.
KEY WORDS : DPPH, uric acid, micelles, sodium dodecylsulfate, Brij-35, dodecyltrimethylammonium bromide
El ácido úrico es un importante antioxidante hidrosoluble. En este trabajo se muestra que es capaz de reaccionar con radicales liposolubles (difenilpicrilhidracil, DPPH) en la interface de soluciones micelares. La velocidad del proceso sigue el orden
micelas cationicas > mezcla etanol:buffer > micelas neutras micelas anionicas
Estos resultados pueden racionalizarse en términos de la compartimentalización de ambos reactantes en diferentes microfases y del efecto que ejercen las interacciones electrostáticas sobre el acceso del anión urato a la interface micelar. Experimentos diseñados para evaluar una posible regeneración del radical derivado del a-tocoferol por el anión urate entregaron resultados negativos, indicando que este anión no es capaz de regenerar la vitamina E en este tipo de sistemas.
PALABRAS CLAVES: DPPH, ácido úrico, micelas, dodecilsulfato de sodio, Brij-35, bromuro de dodeciltrimetilamonio.
Uric acid has been proposed as an important antioxidant and/or free radical scavenger in biological systems .1-5 It constitutes the main antioxidant titrated in techniques aimed to establish the total charge of antioxidants in biological fluids, where, depending on the employed methodology, it can constitute up to nearly 80 % of the measured value.6,7 It is important then to estimate its reactivity towards free radicals and to determine if it is able to react at lipid-water interfaces with hydrophobic radicals. In particular, it is relevant to establish if it can regenerate vitamin E by hydrogen (and/or electron) transfer to an a-tocopheroxyl radical. In the present communication we report kinetic data on the reaction of urate with the 2,2'-diphenyl- 1-1-picrylhydrazyl radical (DPPH*) in homogeneous and in micellar solutions. Furthermore, we show that urate is not able to prolong the life expectancy of vitamin E in micellar solutions in the presence of DPPH*.
Uric acid (Sigma Ultra 99%), a-tocopherol (Fluka), 2,2'-diphenyl-1-picrylhydrazyl, DPPH* (Aldrich), diphenyl pycrylhydrazine DPPH (H) (Aldrich), sodium dodecylsulfate, SDS (Merck), dodecyltrimethylammonium bromide, DTAB (Sigma) and polyoxyethylene 23 lauryl ether, Brij-35 (Sigma) were used as received.
The rate of the reaction was followed by registering the absorbance of DPPH* at 517 nm (e = 1,05 x 103 L/mol cm) as a function of time using a Hewlett Packard 8452A spectrophotometer. Homogeneous or micellar solutions containing DPPH* at the desired working concentration were prepared and poured into a cuvette (1 cm path length) fitting the spectrophotometer cell holder. An appropiate aliquot of a stock solution of uric acid in buffer was added to the cuvette and, after hand shaking for ca. 10 seconds, the register of the absorbance of DDPH* was started. All experiments were carried out in air equilibrated solutions at room temperature.
RESULTS AND DISCUSSION
Uric acid ( 24 mM) was added to a DPPH* solution ( 70 mM) in an homogeneous solvent (ethanol: buffer, 1:1) and to micellar solutions of Brij-35, SDS and DTAB. The reaction in DTAB was too fast to be followed by conventional kinetics, being complete in less than 3 seconds. In the other solutions, the reaction was slow enough to allow estimation of the initial reaction rate. This was obtained by fitting the absorbance decay, measured at 517 nm, to a bi-exponential function. The data obtained were corrected by the interference of the reaction products. The correction was estimated by determining the absorbance of the sample after the reaction was completed in presence of an excess of uric acid. This procedure was justified by the presence of a clear isosbestic point in the reacting sample (see Fig. 1).The relevance of the correction strongly depends on the reaction medium, being almost negligible in SDS and Brij - 35 solutions but highly significant in homogeneous and DTAB solutions. In fact, the remaining absorbance in homogeneous solution was also dependent on the employed buffer, being larger in phosphate buffer (0,05 M, pH = 7 ) that in Tris buffer (0,05 M, pH = 7), implying that the buffer components are involved in secondary reactions of the produced radicals. The reaction rates obtained in the different media are collected in Table I. The data given in this Table indicate that DPPH solubilized into the micelles is able to react with uric acid present in the external medium, and that the reaction rate follows the order
DTAB > homogeneous solution > Brij-35 SDS
The very fast reaction observed in the cationic micelles (DTAB) can be explained in terms of an increased urate local concentration at the micellar border. In this regard, although exchange constants are not available for this counter-ion, it can be expected a strong association of urate due to hydrophobic interactions.8-10 The slower rate observed in the presence of Brij-35 micelles than in the homogenous solution can be due to a "solvent effect" and/or to a lower urate concentration at the average locus of the DPPH* molecules.11
Fig.1 Temporal dependence of DPPH* absorption spectra. Time increases from A to B.
|(DPPH*) = 70 mM in buffer:ethanol (1:1) |
(Uric acid) = 24 mM
The fact that the rate in the homogeneous solution is very similar in the ethanol:buffer (1:1) solution than in a solvent mixture comprising 75 % ethanol (data not shown) would favor the second explanation. This would imply that the interface of the micelle (the most probable locus of the DPPH* molecule) is little accessible to the urate anions. On the other hand, it is noticeable that the rate in the negative micelle (SDS) is very close to that measured in the neutral micelles (Brij-35). This is a rather unexpected result if electrostatic repulsion considerations are taken into account. In an attempt to explain the reason of this apparent anomaly we evaluated the dependence of the reaction rate with the surfactant concentration in both micellar solutions. The results obtained in Brij-35 solutions were independent of the surfactant concentration in the 35 to 100 mM range. This is compatible with a reaction occurring at the interphase with the DPPH* totally incorporated to the micelles and urate anions predominantly dissolved in the external solvent. On the other hand, the rate in SDS solutions increased when the surfactant concentration decreases from 1.35 x 10-7 (SDS 0.1 M) to 3.2 x 10-7 mM/min (0.025 M SDS). This result could suggest that part of the reaction observed is due to a fast reaction of traces of DPPH* present in the dispersium media. A different locus of the reaction carried out in Brij-35 and SDS micelles is suggested by the stoichiometry of the reaction. In excess of DPPH*, the number of radicals consumed per added urate were 2.2 in homogeneous solution, 1.8 in SDS (0.1M) and 1.3 in Brij-35 (0.1M) micelles. These stoichiometries can be explained in terms of the following reaction scheme:
Step (1) can involve hydrogen abstraction from the fraction of uric acid present in the protonated form, or electron transfer between DPPH* and urate coupled to a proton capture. The first possibility can be disregarded since changes in the pH from 7.0 to 8.0 barely modifies the rate of the process (data not shown obtained in the homogeneous solution). This result also indicates that the proton capture is posterior to the rate limiting step or, more likely, that the proton is taken from the solvent, which would be acting as a general acid. Step (1) should then be written as
DPPH* + urate- + H2O Æ DPPH(H) + R* + HO-
that, at pH's well above the uric acid pKa (3.89), should be pH independent.
Predominance of reaction channels (3) and/or (5) for the uric derived radical could lead to a stoichiometric factor of two, as observed in homogenous solution and SDS micelles. On the other hand, predominance of a reaction such as (4) for the decay of the uric derived radical would lead to an stoichiometric factor of one. Intermediate stoichiometries, as that observed in Brij-35 micelles, can be understood in terms of a competition between the different decay channels for the uric acid derived radicals.
Process (2) takes into account the reversibility of the initial step. This can take place between the geminate pair produced in the micelle were has taken place the initial step or involve non-geminate reactants. In the first case, it should reduce the observed rate, which will be independent of the total DPPH(H) concentration. In the second case, it will be dependent upon the total DPPH(H) concentration. A moderate contribution of this second process was observed in SDS micelles, were the addition of DPPH(H) (43 mM) reduces the initial rate of the proces by nearly 33 %.
Attempts were carried out in order to determine if in Brij-35 micelles uric acid was able to recycle a - tocopheroxyl (TO*) radicals generated in the reaction between DPPH* and micelle incorporated a-tocopherol (TOH). The reaction between these reagents is almost instantaneously. It was speculated that if a process such represented in Eq. (7)
TO* + urate- + H+ ®TOH + R*
takes place in the time scale of the experiment, the rate in the presence of TOH and urate should be faster than that expected from the reactivities of both compounds, as measured in separate experiments. The results obtained in a mixture comprising Brij-35 (0.1 M), DPPH* (30 mM), TOH (10 mM) and uric acid (20 mM) in Tris buffer were exactly those expected from the reactivities measured in individual experiments. This implies that under the present conditions, uric acid is not able to recycle TO* radicals. This could be partly due to the high concentration of radicals, which favours radical-radical reactions of the TO* radical.
Thanks are given to Dicyt (USACH) and Fondecyt (Grant # 1980211) for financial support of this work.
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