Available online at www.sciencedirect.com InorganicJOURNAL OF Biochemistry Journal of Inorganic Biochemistry 102 (2008) 1329–1333 www.elsevier.com/locate/jinorgbio The detection of hydroxyl radicals in vivo Wolfhardt Freinbichler b, Loria Bianchi a,1, M. Alessandra Colivicchi a, Chiara Ballini a, Keith F. Tipton a,2, Wolfgang Linert b, Laura Della Corte a,* a Dipartimento di Farmacologia Preclinica e Clinica M. Aiazzi Mancini, Universita` degli Studi di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy b Institute for Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163-AC, A-1060 Vienna, Austria Received 13 September 2007; received in revised form 12 November 2007; accepted 14 December 2007 Available online 28 December 2007 Abstract Several indirect methods have been developed for the detection and quantification of highly reactive oxygen species (hROS), which may exist either as free hydroxyl radicals, bound ‘‘crypto” radicals or Fe(IV)-oxo species, in vivo. This review discusses the strengths and weaknesses associated with those most commonly used, which determine the hydroxylation of salicylate or phenylalanine. Chemical as well as biological arguments indicate that neither the hydroxylation of salicylate nor that of phenylalanine can guarantee an accurate hydroxyl radical quantitation in vivo. This is because not all hydroxylated product-species can be used for detection and the ratio of these species strongly depends on the chemical environment and on the reaction time. Furthermore, at least in the case of salicylate, the high concentrations of the chemical trap required (mM) are known to influence biological processes associated with oxidative stress. Two, newer, alternative methods described, the 4-hydroxy benzoic acid (4-HBA) and the terephthalate (TA) assays, do not have these drawbacks. In each case reaction with hROS leads to only one hydroxylated product. Thus, from a chemical viewpoint, they should pro- vide a better hROS quantitation. Further work is needed to assess any possible biological effects of the required millimolar (4-HBA) and micromolar (TA) concentrations of the chemical traps. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Hydroxyl radicals; Salicylate; Phenylalanine; 4-Hydroxybenzoic acid; Terephthalate 1. Introduction neurodegenerative diseases, ischemic or traumatic brain injuries, cancer, diabetes liver injury and AIDS [4–6]. Oxidative stress is defined as an imbalance between the Moreover, the possibility that oxidative stress plays an production of reactive oxygen species (ROS) and a biolog- important role in the ageing process is under discussion ical system’s ability to readily detoxify the reactive interme- [7–9]. However, it has been difficult to distinguish whether diates and/or easily repair the resulting damage. The term oxidative stress causes the pathologies or is itself a conse- ROS covers several substances, ranging from the rather quence of them. The problematic nature of investigating À unreactive, H2O2 through O2 and singlet O2 to the highly this question may be illustrated by taking the example of reactive oxygen species (hROS), which may exist as free ageing. There is no doubt that if an organism gets older hydroxyl radicals (HOÅ), as bound (‘‘crypto”) radicals or its ability to repair DNA and protein damage decreases, as Fe(IV)-oxo species [1–3]. The occurrence of oxidative whereas the concentration of ROS increases. This observa- stress correlates with numerous pathologies including tion led to the formulation of the ‘‘radical theory of aging”, which predicts that increasing the concentrations of antiox- idants or blocking ROS generation should result in * Corresponding author. Tel.: +39 055 4271226; fax: +39 055 410778. increased lifespan. Most studies have, necessarily, involved E-mail address: laura.dellacorte@unifi.it (L.D. Corte). 1 Permanent address: Azienda USL 3 di Pistoia, 51100 Pistoia, Italy. laboratory models and uncertainties in extrapolation to 2 Permanent address: Department of Biochemistry, Trinity College, human aging compound the problems. However, although Dublin 2, Ireland. there have been several reports, mostly with invertebrates, 0162-0134/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2007.12.017 1330 W. Freinbichler et al. / Journal of Inorganic Biochemistry 102 (2008) 1329–1333 that an enhanced production of antioxidants by genetic the derivatives to be reliable. However, as shown in Table manipulation significantly increases lifespan [10–12], the 1 and Fig. 1, this requirement is far from being fulfilled [20– results of similar studies with mice have been largely disap- 22]. The product ratio shows time dependence and there is pointing, and did not show a significant correlation also a strong influence of the chemical environment. As the between an enhanced production of ROS and ageing [13– ratio between 2,3-DHBA and 2,5-DHBA varies from 5:1 to 16]. Similarly, unclear results can also be found in research 1:1, the resulting quantitation error is far from being neg- reports on the possible relation between oxidative stress ligible. 2,3-DHBA is also quite unstable and, in common and neurodegenerative diseases, such as Alzheimer’s and with the other species, is quickly metabolised [18,23]. Parkinson’s diseases. Although some ROS species are believed to have specific Table 1 biological roles, many studies have been based on the Yields of 2,3-DHBA and 2,5-DHBA, and ratios of 2,3-DHBA:2,5-DHBA reported for different conditions of hROS generation extrapolation of superoxide or H2O2 data to hROS behav- iour. This may have led to contradictory results. One Method 2,3-DHBA 2,5-DHBA Ratio Reference important reason for the use of such experimental proce- (lM) (lM) dures is the lack of a simple and reliable in vivo method Radiolysisa 105 20 5.25 [22] for hROS quantitation. This account presents a brief over- Radiolysisa (1 mM 100 75 1.33 [22] view of the most common and also some newer methods Fe(III)EDTA added) Fenton systemb 22 4 5.5 [22] and their advantages and disadvantages. Fenton systemb (100 lM 25 9.5 2.6 [22] Fe(III)EDTA added) b 2. Methods for hROS quantitation Fenton system (1.3 mM 52 44 1.2 [22] Fe(III)EDTA added) Photochemical reduction of 1 [20] The only direct method of detecting hROS is electron c Fe(III) in O2 sat. solution spin resonance (ESR) spectroscopy, including the spin (0.3 mM K2C2O4) trapping technique. However, its application to in vivo Photochemical reduction of 1.7 [20] c experiments, in particular in freely moving animals, is Fe(III) in O2 sat. solution impracticable because of technical difficulties, which (3 mM K2C2O4) a include high disturbing noise levels and low sensitivity The yields were measured after a radiation dose of 600 Gy, buffered [17]. All the common methods used for in vivo experiments with 5 mM phosphate, pH 7.5, under N2O; initial concentration of sali- cylic acid = 1 mM. are indirect and based on the hydroxylation of aromatic b De-aerated solutions of 200 lM Fe(II)EDTA were mixed with 200 lM compounds. H2O2, buffered at pH 7.5 with 5 mM phosphate; initial concentration of salicylic acid = 1 mM. c Initial concentrations: 0.8 mM salicylic acid; 0.06 mM; Fe(III). 2.1. Salicylate The most commonly used procedure is based on the hydroxylation of salicylic acid, which is based on the chem- ical reaction shown in Scheme 1. This yields three reaction products, I catechol, II 2,5-dihydroxybenzoic acid (2,5- DHBA) and III 2,3-dihydroxybenzoic acid (2,3-DHBA). By measuring all three hydroxylation products the assay may allow a simple and accurate quantitative detection of hROS by either electrochemical or photometric methods. As catechol is formed only in small amounts, only species 2,5-DHBA and 2,3-DHBA are normally used for determin- ing the amount of hROS. However, several problems arise under in vivo conditions. 2,5-DHBA is also produced enzy- Fig. 1. Dependence of the ratio of 2,3-DHBA:2,5-DHBA on the reaction matically [18,19], leaving only 2,3-DHBA available for time during the photo-hydroxylation of 0.7 mM salicylic acid by 10 mM detection as a simple hROS product. A constant product hydrogen peroxide. The reaction was sensitised with methylene blue ratio is essential for determinations based on only one of (0.01 mM) in the presence of ferric acetate (adapted from 21). COOH OH COOH COOH OH OH OH OH + OH + + HO OH III III Scheme 1. The hydroxylation of salicylic acid by radical oxidation. W. Freinbichler et al. / Journal of Inorganic Biochemistry 102 (2008) 1329–1333 1331 Furthermore, salicylic acid inhibits cyclooxygenase (EC photometric), it seems unlikely that a coeluted unknown 1.14.99.1), a key enzyme in the pathway of prostaglandin compound was the sole explanation for these results. An synthesis. Products of this pathway have many physiologi- alternative explanation would be that, as in the case of sal- cal functions in both normal and disease conditions [24,25] icylic acid, there is a change in the ratio of hydroxylated and are known to influence inflammatory processes associ- products due to a change in the chemical environment, ated with oxidative stress [26–28]. Thus the biochemical which favours m-tyrosine formation in comparison to o- actions of salicylate itself may perturb the hROS results. tyrosine. Since p-tyrosine cannot be considered for hROS All these considerations raise serious questions about the detection and the above considerations suggest that it is validity of in vivo results obtained using this method [17– doubtful whether if the sum of o-andm-tyrosine can be 19]. taken for an artefact-free hROS quantitation in vivo see [29,34,35], results obtained with this procedure should be 2.2. Phenylalanine treated with caution.