For a long time, superoxide generation by an NADPH oxidase was considered as an oddity only found in professional phagocytes. Over the last years, six homologs of the cytochrome subunit of the phag...

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The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology

Acute kidney injury (AKI) is a complex disorder for which currently there is no accepted definition. Having a uniform standard for diagnosing and classifying AKI would enhance our ability to manage these patients. Future clinical and translational research in AKI will require collaborative networks of investigators drawn from various disciplines, dissemination of information via multidisciplinary joint conferences and publications, and improved translation of knowledge from pre-clinical research. We describe an initiative to develop uniform standards for defining and classifying AKI and to establish a forum for multidisciplinary interaction to improve care for patients with or at risk for AKI. Members representing key societies in critical care and nephrology along with additional experts in adult and pediatric AKI participated in a two day conference in Amsterdam, The Netherlands, in September 2005 and were assigned to one of three workgroups. Each group's discussions formed the basis for draft recommendations that were later refined and improved during discussion with the larger group. Dissenting opinions were also noted. The final draft recommendations were circulated to all participants and subsequently agreed upon as the consensus recommendations for this report. Participating societies endorsed the recommendations and agreed to help disseminate the results. The term AKI is proposed to represent the entire spectrum of acute renal failure. Diagnostic criteria for AKI are proposed based on acute alterations in serum creatinine or urine output. A staging system for AKI which reflects quantitative changes in serum creatinine and urine output has been developed. We describe the formation of a multidisciplinary collaborative network focused on AKI. We have proposed uniform standards for diagnosing and classifying AKI which will need to be validated in future studies. The Acute Kidney Injury Network offers a mechanism for proceeding with efforts to improve patient outcomes.

Acute Kidney Injury Network: report of an initiative to improve outcomes in acute kidney injury

The goal of this review is to present a comprehensive survey of the many intriguing facets of creatine (Cr) and creatinine metabolism, encompassing the pathways and regulation of Cr biosynthesis an...

http://www.afboard.com/library/creatinemetabolism.pdf

Creatine and Creatinine Metabolism

Free radicals and other reactive species (RS) are thought to play an important role in many human diseases. Establishing their precise role requires the ability to measure them and the oxidative damage that they cause.


This article first reviews what is meant by the terms free radical, RS, antioxidant, oxidative damage and oxidative stress.


It then critically examines methods used to trap RS, including spin trapping and aromatic hydroxylation, with a particular emphasis on those methods applicable to human studies.


Methods used to measure oxidative damage to DNA, lipids and proteins and methods used to detect RS in cell culture, especially the various fluorescent ‘probes' of RS, are also critically reviewed.


The emphasis throughout is on the caution that is needed in applying these methods in view of possible errors and artifacts in interpreting the results.





Keywords: Cell culture, free radical, reactive species, antioxidant, oxidative stress, oxidative damage, fluorescent probe, lipid peroxidation, superoxide


Introduction
Free radicals and other ‘reactive oxygen (ROS)/nitrogen/chlorine species' (for an explanation of these terms see Table 1) are widely believed to contribute to the development of several age-related diseases, and perhaps, even to the aging process itself (Halliwell & Gutteridge, 1999; Sohal et al., 2002) by causing ‘oxidative stress' and ‘oxidative damage' (terms explained in Table 2). For example, many studies have shown increased oxidative damage to all the major classes of biomolecules in the brains of Alzheimer's patients (Halliwell, 2001; Butterfield, 2002; Liu et al., 2003). Other diseases in which oxidative damage has been implicated include cancer, atherosclerosis, other neurodegenerative diseases and diabetes (Hagen et al., 1994; Chowienczyk et al., 2000; Halliwell, 2000a, 2001, 2002a, 2002b; Parthasarathy et al., 2000). If oxidative damage contributes significantly to disease pathology (Table 3 lists the criteria needed to establish this), then actions that decrease it should be therapeutically beneficial (Halliwell, 2001; Lee et al., 2002a; Liu et al., 2003). If the oxidative damage is involved in the origin of a disease, then successful antioxidant treatment should delay or prevent the onset of that disease (Halliwell, 1991, 2002a, 2002b; Galli et al., 2002; Steinberg & Witztum, 2002). To establish the role of oxidative damage (Table 3), it is therefore essential to be able to measure it accurately. For example, the failure of interventions with antioxidants such as vitamin E, β-carotene or ascorbate to decrease disease incidence in several human intervention trials may have simply been due to the failure of these compounds to decrease oxidative damage in the subjects tested (Halliwell, 1999a, 2000c; Levine et al., 2001; Meagher et al., 2001). In this review, we will examine the methods available to measure reactive species (RS) and oxidative damage, with a particular emphasis on those applicable to human studies.



Table 1

Nomenclature of reactive species





Table 2

Some key definitions





Table 3

Criteria for implicating RS as a significant mechanism of tissue injury in human disease




Measuring RS in vivo: basic principles

Some fascinating techniques such as L-band electron spin resonance (ESR) with nitroxyl probes and magnetic resonance imaging spin trapping are under development to measure RS directly in whole animals (e.g. Berliner et al., 2001; Han et al., 2001; Utsumi & Yamada, 2003), but no probes are currently suitable for human use. Most RS persist for only a short time in vivo and cannot be measured directly. There are a few exceptions: examples include H2O2 (discussed below), and perhaps, NO•, in the sense that serum levels of NO2− have been claimed to measure vascular endothelial NO• synthesis (Kelm et al., 1999), despite the fact that NO2− is quickly oxidized to NO3− in vivo (Kelm et al., 1999; Oldreive & Rice-Evans, 2001). Essentially, there are two approaches to detecting transient RS: 


attempting to trap these species and measure the levels of the trapped molecules and


measuring the levels of the damage done by RS, that is, the amount of oxidative damage.




Sometimes other approaches are used. They include measurements of erythrocyte antioxidant defences and of total antioxidant activity of body fluids; falls in these parameters are often taken as evidence of oxidative stress. Erythrocytes cannot synthesize proteins, however, and their antioxidant enzyme levels may drop as they ‘age' in the circulation (Denton et al., 1975). Thus changes in their levels are more likely to reflect changes in the rates of red blood cell turnover: if this slows down, the circulating erythrocytes will be older on average and so levels of antioxidant enzymes in them will appear to fall. Vice versa, if an intervention accelerates red cell removal or increases erythropoiesis, levels of antioxidants in red cells will seem to rise. Hence, such data should be interpreted with caution.

Depending on the method that is used to measure it, the plasma or serum ‘total antioxidant capacity' (TAC) usually involves major contributions from urate, ascorbate and sometimes albumin −SH groups (Benzie & Strain, 1996; Halliwell & Gutteridge, 1999; Prior & Cao, 1999; Rice-Evans, 2000; Bartosz, 2003), although different methods measure different things (Schlesier et al., 2002; Bartosz, 2003). Thus, for example, if plasma albumin levels fall, TAC will fall. If urate levels rise, TAC will rise. The multiple changes in blood chemistry that occur in sick people mean that TAC changes should be interpreted with caution. TAC is also influenced by diet, often because consumption of certain foods may produce changes in plasma ascorbate and/or urate levels (Halliwell, 2003b).

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Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?

Free radical production occurs continuously in all cells as part of normal cellular function. However, excess free radical production originating from endogenous or exogenous sources might play a role in many diseases. Antioxidants prevent free radical induced tissue damage by preventing the formation of radicals, scavenging them, or by promoting their decomposition. This article reviews the basic chemistry of free radical formation in the body, the consequences of free radical induced tissue damage, and the function of antioxidant defence systems, with particular reference to the development of atherosclerosis.

https://jcp.bmj.com/content/jclinpath/54/3/176.full.pdf

Antioxidants in health and disease

Dissociation between vascular oxidative stress and cardiovascular function in Wistar Kyoto and spontaneously hypertensive rats.

Non-genomic actions of thyroid hormones: Molecular aspects

Purpose. To determine the variations of vitamin E in rat retinas after transient ischemia and reperfusion. Methods. Male Wistar rats 11 to 12 weeks of age were used in all experiments. Ischemia was induced by ligation of the optic nerve and vessels, then reperfusion was obtained by removing the ligature. Retinas were dissected, and vitamin E was analyzed by a modified high-performance liquid chromatography assay. Results. There was an insignificant rise in vitamin E during ischemia and a dramatic decrease after reperfusion. No changes were dependent on the period of ischemia (30, 60 or 90 minutes) or reperfusion (15 or 120 minutes). Conclusion. Vitamine E decreased only in the reperfused groups, demonstrating a depletion of defenses against free radical production occurring during reperfusion. Vitamin E decreased only in the reperfused groups, demonstrating a depletion of defenses against free radical production occurring during reperfusion. Vitamin E changes may represent an indirect measure of lipoperoxidation during ischemia-reperfusion and, thus, a potential parameter to evaluate drugs in vitro

Modification of vitamin E during ischemia-reperfusion in rat retina.

The effects of a number of interventions influencing sympathetic nervous activity on the severity of coronary artery reperfusion-induced arrhythmias in anaesthetised rats have been examined. Noradrenaline (0.1 microgram kg-1 min-1) reduced the mortality that usually occurred as a consequence of ventricular fibrillation. Isoprenaline (5 micrograms kg-1) did not significantly affect the severity of reperfusion-induced arrhythmias, although arrhythmias occurring during the 5-min period of ischaemia were exacerbated. The alpha-adrenoceptor antagonist nicergoline (0.25 and 0.5 mg kg-1 min-1) markedly suppressed both the ventricular tachycardia and fibrillation occurring upon release of the occlusion, whereas prazosin (1.0 mg kg-1) only slightly reduced the incidence of ventricular tachycardia. The beta-adrenoceptor antagonists atenolol and timolol did not significantly modify the severity of these reperfusion-induced arrhythmias. Pretreatment with reserpine (0.1 mg kg-1) or 6-hydroxydopamine (20 mg kg-1), which depleted myocardial catecholamine concentrations by 90%, had no effect on the indices of arrhythmic activity. Similarly, administration of L-thyroxine (1 mg kg-1) or propylthiouracil (50 mg kg-1) on 7 consecutive days prior to coronary artery occlusion did not alter the incidence of arrhythmias occurring upon reperfusion. Taken as a whole, these results do not suggest an important role for sympathetic nervous activity in the genesis of reperfusion-induced arrhythmias in anaesthetised rats.

Luc Rochette

Papers

Dissociation between vascular oxidative stress and cardiovascular function in Wistar Kyoto and spontaneously hypertensive rats.

Non-genomic actions of thyroid hormones: Molecular aspects

Modification of vitamin E during ischemia-reperfusion in rat retina.

Effects of alterations in sympathetic nervous activity on the severity of reperfusion-induced arrhythmias in anaesthetised rats.

Assessment of the cytoprotective role of adenosine in an in vitro cellular model of myocardial ischemia.