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...

/pdf/the-nox-family-of-ros-generating-nadph-oxidases-physiology-zjc88zvh59.pdf

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).

/pdf/measuring-reactive-species-and-oxidative-damage-in-vivo-and-5a9iiwcjlt.pdf

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

Isolated rat hearts were prelabeled with 3H-norepinephrine (NE), submitted to coronary artery ligation, and perfused through the left atrium with a modified Krebs-Henseleit solution containing 3 mM potassium and four different substrates: 5.5 mM glucose, 5.5 mM glucose plus 0.15 or 0.5 mM palmitate bound to albumin in a molar ratio of 6:1, and 11 mM glucose. The coronary artery ligature was removed after 30 min of perfusion of the ischemic working heart. With all substrates the release of radioactivity in the coronary effluent remained relatively constant during the ischemic period. Reperfusion was associated with a sudden release of radioactivity and of 3H-NE, but the intensity of the efflux was influenced by the nature of the perfusion substrate. The highest release was observed with 5.5 mM glucose and the lowest release in the presence of 0.15 mM palmitate. Intermediate and similar releases were seen with the two other substrates. On reperfusion of the ischemic heart, ventricular arrhythmias (tachycardia and fibrillation) were very marked with 5.5 mM glucose and in the presence of 0.5 mM palmitate. They were significantly delayed in the presence of 0.15 mM palmitate and almost absent with 11 mM glucose. These results do not show a relationship between the amount of NE liberated during the post-ischemic period and the extent of ventricular reperfusion arrhythmias. We conclude that either myocardial NE is not implicated in the genesis of reperfusion arrhythmias or that cardiac vulnerability to the arrhythmogenic effect of NE is influenced by the metabolic state of the myocardium, which is dependent on the nature of the perfusion substrate.

Effect of substrate on release of myocardial norepinephrine and ventricular arrhythmias following reperfusion of the ischemic isolated working rat heart.

Asymmetric dimethylarginine (ADMA) and hyperhomocysteinemia in patients with acute myocardial infarction.

Free radicals species have been implicated as important agents involved in myocardium ischemia and reperfusion injuries (Mac Cord, 1985). The major cytotoxic effect of oxygen-derivated free radicals may be peroxidation of lipid components of cellular membranes which may be an important process for cell damage during myocardial infarction. In this study, Electron Paramagnetic Resonance Spectroscopy (EPR) was used to directly mesure free radical generation in isolated perfused rat hearts (Maupoil and Rochette, 1988). We also investigated the formation of thiobarbituric acid reactants as an index of lipid peroxidation. The effect of TMZ in the postischemic heart was determined.

Direct measurement of free radical generation in isolated rat heart by electron paramagnetic resonance spectroscopy: effect of trimetazidine.

To evaluate the early effect of low doses of adriamycin (ADR) on cardiac parameters, male Wistar rats were injected with ADR (1 mg∙kg−1∙day−1) or saline for 10 days. Seven days later, T1 and T2 relaxation times were determined in left ventricular (LV) free wall, septum, and thigh muscle samples. In another experiment, performed on isolated working hearts of rats pretreated with ADR, LV performance was determined along with an index of myocardial lipid peroxidation in this tissue. Lipid peroxidation was enhanced (p < 0.05). This change was not associated with a reduced LV performance, since both aortic and cardiac flows measured in working heart preparations were similar between control and treated rats. However, the coronary flow was significantly reduced (control group, 21 ± 1 mL∙min−1∙g−1, ADR group, 15 ± 1 mL∙min−1∙g−1; p < 0.001). T1 increased in the LV free wall (665 ± 3 to 696 ± 5 ms, p < 0.001) and in the septum (657 ± 3 to 696 ± 5 ms, p < 0.01), while T2 increased only in the LV free wall (50.8 ± ...

Early incidence of adriamycin treatment on cardiac parameters in the rat.

The objective of this study was to verify the presence of oxidative stress in type 1 and type 2 diabetic patients and to provide evidences for an use of ascorbyl free radical (AFR)/vitamin C ratios as tools exploring the level of oxidative stress in diabetic patients. TBARS levels, oxygen-radical absorbing capacity assay and AFR release assessed by electron paramagnetic resonance (EPR) were used to explore the existence of oxidative stress in diabetes. Endogenous antioxidants (alpha-tocopherol, vitamin C and uric acid) were also measured. Scavenging capacities of plasma were decreased in diabetic patients. A significant decrease of plasma vitamin C and an increase of AFR/vitamin C ratios were noted in type 2 diabetic patients. Uricemia was decreased in type 1 diabetic patients. Our results suggest a possible use of AFR/vitamin C ratios as indicators of oxidative stress in diabetes mellitus.

Luc Rochette

Papers

Effect of substrate on release of myocardial norepinephrine and ventricular arrhythmias following reperfusion of the ischemic isolated working rat heart.

Asymmetric dimethylarginine (ADMA) and hyperhomocysteinemia in patients with acute myocardial infarction.

Direct measurement of free radical generation in isolated rat heart by electron paramagnetic resonance spectroscopy: effect of trimetazidine.

Early incidence of adriamycin treatment on cardiac parameters in the rat.

Ascorbyl free radical release in diabetic patients.