Direct detection of reactive oxygen species ex vivo

Kidney International, Vol. 67 (2005), pp. 1662–1664

Direct detection of reactive oxygen species ex vivo RALF P. BRANDES and MARIANO JANISZEWSKI ¨ Kardiovaskul¨are Physiologie, J.W. Goethe-Universit¨at, Frankfurt, Germany Institut fur

Direct detection of reactive oxygen species ex vivo. Oxidative stress is thought to play an important role in the initiation and progression of renal, cardiovascular, neoplastic, and neurodegenerative diseases. It is also widely believed that oxidative stress is a main cause of aging. Although considerable progress has been made in the understanding of the sources and actions of oxidative stress, the true role of oxygen-derived free radicals in the pathology of most human diseases largely remains to be determined. One major obstacle for radical research is the lack of specific and sensitive methods to quantify oxidative stress in vivo and in vitro. Although a multitude of different assays is available to assess free radical generation, each of these methods has substantial limitations. This article will provide a brief review on the most frequently used techniques to assess oxygenderived free radical generation in isolated tissue preparations and cells. Emphasis will be put on most recent technical innovations and the shortcomings associated with current techniques.

nitric oxide yields the highly aggressive oxidant peroxynitrite (ONOO− ) which largely limits nitric oxide bioavailability. In aqueous solution, O 2 − slowly dismutates to hydrogen peroxide (H 2 O 2 ), and this reaction is catalyzed by superoxide dismutases (SOD) in the body. In the presence of trace metals, particularly copper and iron, H 2 O 2 and O 2 − can generate hydroxyl radicals (OH•), which react with every biologic molecule at high rate [2]. MEASUREMENT OF RADICAL FORMATION The short half-life and the high reactivity of ROS limit the direct detection of these compounds in vivo. Research of the past years, however, led to the identification of certain molecules particularly prone to react with ROS, which can therefore be used as footprints of these radicals. Consequently, these biomarkers, which include isoprostanes and oxidative modifications of proteins, are currently the most reliable indicators for oxidative stress in vivo [3]. Measurements of the activities of antioxidative and pro-oxidative enzymes may also provide some direct information about the redox balance. In contrast to this, ex vivo measurements using cultured cells and preparations from isolated organs allow a direct determination of oxygen-derived free radicals. Enzyme activity assays, colorimetic, fluorimetric, and luminescence-based assays are used to measure radicals. The basic principle is the reaction between the radical and a tracer generating a measurable product [1]. As radicals have an unpaired electron, direct detection of radicals is possible by electron spin resonance (ESR) technique, but the concentrations of radicals reached in living tissue are below the detection limit of this method. The sensitivity of ESR can be greatly enhanced by the use of stable spin traps, and some of these traps even allow the identification of the radical species. The complex chemistry involving ROS, spin traps or spin labels, together with the restricted practicability of ESR, so far prevents its more frequent use.

Research on the role of oxidative stress for the initiation and progression of various diseases encompasses a large part of the current basic science. The view that almost every disease is associated with some form of oxidative stress is contrasted by the lack of specific and sensitive methods to quantify oxygen-derived free radical generation in vivo and ex vivo and by the inability to exactly identify the type, the localization, and the true rate of radical formation. Over the recent years, several methods to assess oxidative stress have been advocated superior and were later disapproved. In fact, several concepts on the enzymatic sources of reactive oxygen species (ROS) formation have to be queried as they were based solely on some artifacts of the radical assay used. The most prominent example of this is the NADH-induced redox cycling of lucigenin [1]. THE CHEMISTRY Oxygen-derived free radicals generated enzymatically in the body result from electron transfer from a reductant to molecular oxygen. Single electron transfer leads to the formation of superoxide anions (O 2 − ), which are the precursors of most other ROS. The reaction of O 2 − with

Key words: radicals, superoxide anion, redox balance.
C

2005 by the International Society of Nephrology

DETECTION OF SUPEROXIDE ANIONS Reduction of ferricytochrome C to ferrocytochrome C has been used very frequently to measure O 2 − formation photometrically. At 550 nm reduced cytochrome C has a different extinction coefficient than 1662

Brandes and Janiszewski: Measurement of oxidative stress

oxidized cytochrome C (ferricytochrome C 0.89 × 104 L/ mol/cm, ferrocytochrome C 2.99 × 104 L/mol/cm,
E therefore is 2.1 × 104 L/mol/cm [4]). As reduction of cytochrome C is not specific for O 2 − , measurements have to be performed in the presence and absence of SOD to allow the determination of the SOD-sensitive portion of cytochrome C reduction, which is specifically mediated by O 2 − . Several molecules, including H 2 O 2 and ONOO− on the other hand have been shown to oxidize reduced cytochrome C, leading to an underestimation of the O 2 − formation. In the case of H 2 O 2, this problem can be overcome by the addition of catalase, ONOO− can be scavenged by urate. Aspects of practicability limit the use of cytochrome C, as the changes in optical density are often very small and thus prone to artifacts and require relatively long incubation intervals up to 60 minutes to become reliably detectable [1]. Reduction of nitro blue tetrazolium (NBT) is a second photometric assay based on the same principle as cytochrome C reduction. An interesting aspect of NBT is that the reaction product, formazan, is hardly water soluble and thus precipitates, allowing some limited identification of radical-generating cells in tissue assays. As also NBT reduction is occurring O 2 − independently, only the SOD-sensitive part can be utilized as measure for O 2 − . NBT has been criticized for its capacity to generate O 2 − under aerobic conditions [5], and consequently, NBT should not be considered as a first choice to detect superoxide. Luminometric assays are a frequently used alternative to photometric assays. Luminometric assays, in which an enhancer emits light during the reaction with the radical of interest, are considered more sensitive and allow temporal resolution of the signal. Nevertheless, using nonleukocyte cells or tissue preparations, the light signals obtained are still relatively low, requiring high sensitive luminometers. Most microplate systems, even when they are equipped with a luminometer mode, are not sensitive enough to detect the signal. Lucigenin is currently the most frequently used enhancer, and this compound is relatively specific for O 2 − . Unfortunately, lucigenin exhibits a fatal tendency to undergo redox cycling at concentrations higher than 5 lmol/L and in the present of NADH [6]. Many previous observations obtained with high concentrations of lucigenin and in the presence of NADH, therefore, have to be queried. It is crucial to realize that NADH- or NADPH-driven lucigenin chemiluminescence, regardless of whether it is performed in intact organs or homogenates does not reflect only radical generation but rather the reaction of the enhancer with the reductant, which is by some means catalyzed by unknown mechanism activated by the sample studied [7]. The light signal obtained with 5 lmol/L lucigenin in the absence of NADH or NADPH is very low and therefore several other enhancers have been evaluated recently. Derivates

1663

of CLA and coelenterazine were advocated being superior to lucigenin, as they do not exhibit any redox cycling [8, 9]. Nevertheless, autooxidation and unfavorable signal to background ratio limit the use of these enhancers. L-012 is an enhancer which appears to have the capacity to replace lucigenin as the primary system to detect O 2 − [10, 11]. As L-012 does not undergo redox cycling, it can be used in concentrations up to 100 lmol/L, yielding a robust signal [12]. L-012 is not specific for O 2 − and also detects ONOO− . Moreover, NADPH and NADH are quenching the L-012 signal [13]. Certainly, more work is needed to characterize this compound but without a doubt the strong signals obtained with L-012 will make it the enhancer of choice in many studies. Fluorimetric techniques, different to luminescence measurements, allow the topolocalization of ROS. Although there are several ROS-sensitive fluorescence probes, the only one considered being specific for O 2 − is dihydroethidium (DHE) [14]. The compound has become widely used to image ROS formation [15]. Nevertheless, there are several problems associated with the use of DHE. Ethidium, the oxidation product of DHE, intercalates with DNA and therefore differences in the chromatin density in heterocellular organs complicate imaging with DHE. Recently, it became clear that unspecific oxidation of DHE can be discriminated from the O 2 − –mediated reaction of DHE forming oxyethidium, as ethidium and oxyethidium have different spectral characteristics [16]. Assays are currently being developed to take advantage of this fact either using specific emission wave lengths of the two dyes [16] or using high-performance liquid chromatography (HPLC) [17] to separate ethidium from oxyethidium. The specificity of DHE for O 2 − will certainly increase with the introduction of these techniques and the influence of the observer in the interpretation of the microscopy images may be reduced. DETECTION OF PEROXYNITRITE, HYDROXYL RADICALS, AND HYDROGEN PEROXIDE ONOO− and OH• oxidize most biologic material [6]. Consequently, a wide variety of fluorimetric assays, using dihydrofluorescein derivates, amplex red, and several other compounds, can be used to detect these two radicals [1]. Scavengers for ONOO− and OH• are used to discriminate the specific signal from unspecific oxidation. Frequently used scavengers are mannitol, urate, and ebselen. Because of the extremely high reactivity of these radical species and the lack of specificity of the scavengers, it remains difficult to ascertain the type of radical generated, the localization of generation, and the rate of formation. H 2 O 2 is not a radical and thus its reactivity is much lower than that of OH• or ONOO− . In fact, most assays for H 2 O 2 involve the use of peroxidase, which reduces

1664

Brandes and Janiszewski: Measurement of oxidative stress

H 2 O 2 to H 2 O and simultaneously oxidizes the radical tracer [1]. Therefore, the same tracers to study ONOO− and OH• are used to measure H 2 O 2 . Consequently, it is difficult to discriminate between the radical species in living cells, as cells contain peroxidases and simultaneously generate ONOO, OH•, and H 2 O 2 [1]. Luminol is a chemiluminescence enhancer which exhibits low sensitivity of H 2 O 2 , O 2 − , and nitric oxide and yields light with OH• and ONOO− . Luminol has been used to directly detect the formation of ONOO− . Inhibition of nitric oxide synthase (NOS) can be used to block ONOO− formation. Mannitol may be used to determine the amount of chemiluminescence arising from OH•-mediated oxidation of luminol. Finally, peroxidase can be added to measure H 2 O 2 formation with luminol [1]. CONCLUSION A wide spectrum of different techniques has been developed to measure radical formation ex vivo. Recent developments like L-012 and DHE will further improve these assays. Nevertheless, each single method has specific limitations and drawbacks and therefore measurements should be always performed using two independent methods. ACKNOWLEDGMENTS This manuscript was supported by a grant from the Deutsche Forschungsgemeinschaft to R.P.B. (BR1839/2–1) and the DAAD to M.J. M.J. is a recipient of the Georg Forster research stipend of the Alexander von Humboldt Foundation. ¨ Kardiovaskul¨are Reprint requests to Ralf P. Brandes, Institut fur Physiologie, Klinkum der J.W. Goethe-Universit¨at, Theodor-Stern-Kai 7, D-60596 Frankfurt am Main, Germany. E-mail: [email protected]

REFERENCES 1. TARPEY MM, FRIDOVICH I: Methods of detection of vascular reactive species: Nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite. Circ Res 89:224–236, 2001

2. FRIDOVICH I: Fundamental aspects of reactive oxygen species, or what’s the matter with oxygen? Ann NY Acad Sci 893:13–18, 1999 3. MAYNE ST: Antioxidant nutrients and chronic disease: Use of biomarkers of exposure and oxidative stress status in epidemiologic research. J Nutr 133 (Suppl 3):933S–940S, 2003 4. MASSEY V: The microestimation of succinate and the extinction coefficient of cytochrome c. Biochim Biophys Acta 34:255–256, 1959 5. AUCLAIR C, TORRES M, HAKIM J: Superoxide anion involvement in NBT reduction catalyzed by NADPH-cytochrome P-450 reductase: a pitfall. FEBS Lett 89:26–28, 1978 6. JANISZEWSKI M, SOUZA HP, LIU X, et al: Overestimation of NADHdriven vascular oxidase activity due to lucigenin artifacts. Free Radic Biol Med 32:446–453, 2002 7. BAKER MA, KRUTSKIKH A, CURRY BJ, et al: Identification of cytochrome P450-reductase as the enzyme responsible for NADPHdependent lucigenin and tetrazolium salt reduction in rat epididymal sperm preparations. Biol Reprod 71:307–318, 2004 8. MUNZEL T, AFANAS’EV IB, KLESCHYOV AL, et al: Detection of superoxide in vascular tissue. Arterioscler Thromb Vasc Biol 22:1761– 1768, 2002 9. TARPEY MM, WHITE CR, SUAREZ E, et al: Chemiluminescent detection of oxidants in vascular tissue. Lucigenin but not coelenterazine enhances superoxide formation. Circ Res 84:1203–1211, 1999 10. NISHINAKA Y, ARAMAKI Y, YOSHIDA H, et al: A new sensitive chemiluminescence probe, L-012, for measuring the production of superoxide anion by cells. Biochem Biophys Res Commun 193:554–559, 1993 11. IMADA I, SATO EF, MIYAMOTO M, et al: Analysis of reactive oxygen species generated by neutrophils using a chemiluminescence probe L-012. Anal Biochem 271:53–58, 1999 12. SOHN HY, GLOE T, KELLER M, et al: Sensitive superoxide detection in vascular cells by the new chemiluminescence dye L-012. J Vasc Res 36:456–464, 1999 13. DAIBER A, OELZE M, AUGUST M, et al: Detection of superoxide and peroxynitrite in model systems and mitochondria by the luminol analogue L-012. Free Radic Res 38:259–269, 2004 14. KOBZIK L, GODLESKI JJ, BRAIN JD: Oxidative metabolism in the alveolar macrophage: analysis by flow cytometry. J Leukoc Biol 47:295–303, 1990 15. MILLER FJ, JR., GUTTERMAN DD, RIOS CD et al: Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res 82:1298–1305, 1998 16. ZHAO H, KALIVENDI S, ZHANG H, et al: Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic Biol Med 34:1359–1368, 2003 17. FINK B, LAUDE K, MCCANN L, et al: Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay. Am J Physiol Cell Physiol 287:C895–C902, 2004