F. Petrat

The chelatable iron pool in living cells : A methodically defined quantity

Abstract A very small, predominantly cytosolic pool of iron ions plays the central role in the cellular iron metabolism. It links the cellular iron uptake with the insertion of the metal in iron storage proteins and other essential ironcontaining molecules. Furthermore, this transit (labile) pool is essentially involved in the pathogenesis of a number of diseases. Due to its high physiological and pathophysiological significance, numerous methods for its characterization have been developed during the last five decades. Most of these methods, however, influence the size and nature of the transit iron pool artificially, as they are not applicable to viable biological material. Recently, fluorescence spectroscopic methods for measurements within viable cells have become available. Although these methods avoid the artifacts of previous methods, studies using fluorescent iron indicators revealed that the intracellular transit iron pool, which is methodically assessed as chelatable iron, is substantially defined by the method and/or the ironchelating indicator applied for its detection, since the iron ions are bound to a large number of different ligands in different metabolic compartments. A more comprehensive characterization of the nature and the role of the thus not uniform cellular transit iron pool therefore requires parallel employment of different indicator molecules, which clearly differ in their intracellular distribution and their physicochemical characteristics.

Friedreich's Ataxia, No Changes in Mitochondrial Labile Iron in Human Lymphoblasts and Fibroblasts A DECREASE IN ANTIOXIDATIVE CAPACITY?

Friedreichs ataxia (FRDA) is caused by low expression of frataxin, a small mitochondrial protein. Studies with both yeast and mammals have suggested that decreased frataxin levels lead to elevated intramitochondrial concentrations of labile (chelatable) iron, and consequently to oxidative mitochondrial damage. Here, we used the mitochondrion-selective fluorescent iron indicator/chelator rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzylester (RPA) to determine the mitochondrial chelatable iron of FRDA patient lymphoblast and fibroblast cell lines, in comparison with age- and sex-matched control cells. No alteration in the concentration of mitochondrial chelatable iron could be observed in patient cells, despite strongly decreased frataxin levels. Uptake studies with 55Fe-transferrin and iron loading with ferric ammonium citrate revealed no significant differences in transferrin receptor density and iron responsive protein/iron regulatory element binding activity between patients and controls. However, sensitivity to H2O2 was significantly increased in patient cells, and H2O2 toxicity could be completely inhibited by the ubiquitously distributing iron chelator 2,2′-dipyridyl, but not by the mitochondrion-selective chelator RPA. Our data strongly suggest that frataxin deficiency does not affect the mitochondrial labile iron pool or other parameters of cellular iron metabolism and suggest a decreased antioxidative defense against extramitochondrial iron-derived radicals in patient cells. These results challenge current concepts favoring the use of mitochondrion-specific iron chelators and antioxidants to treat FRDA.

Cell type-dependent release of nitric oxide and/or reactive nitrogenoxide species from intracellular SIN-1: effects on cellular NAD(P)H

Abstract SIN-1 is frequently used in cell culture studies as an extracellularly operating generator of peroxynitrite. However, little is known about the nature of the reactive species produced intracellulary from SIN-1. SIN-1 can easily penetrate cells as exemplified for both L-929 mouse fibroblasts and bovine aortic endothelial cells (BAECs) by utilizing capillary zone electrophoresis. In L-929 cells, SIN-1 produced nitric oxide (NO) as monitored by the fluorescent NO scavenger FNOCT-1 and by means of a NO electrode, as well as reactive nitrogenoxide species (RNOS, e.g. peroxynitrite, nitrogen dioxide, dinitrogen trioxide), as detected with the fluorescent indicator DAF-2. Laser scanning microscopy revealed that in L-929 cells SIN-1-derived species initially oxidized the major fraction of the NAD(P)H within the cytosol and the nuclei, whereas the mitochondrial NAD(P)H level was somewhat increased. In marked contrast to this, in BAECs no evidence for NO formation was found although the intracellular amount of SIN-1 was fourfold higher than in L-929 cells. In BAECs, the level of NAD(P)H was slightly decreased within the first 10 min after administration of SIN-1 in both the cytosol/nuclei and mitochondria. These observations reflect the capability of SIN-1 to generate intracellularly either almost exclusively RNOS as in BAECs, or RNOS and freely diffusing NO as in L-929 cells. Nitric oxide as well as RNOS may decisively affect cellular metabolism as indicated by the alterations in the NAD(P)H level. Hence, care should be taken when applying SIN-1 as an exclusively peroxynitrite-generating compound in cell culture systems.

Perfluorocarbon-filled poly(lactide-co-gylcolide) nano- and microcapsules as artificial oxygen carriers for blood substitutes: a physico-chemical assessment.

The physico-chemical suitability of perfluorocarbon-filled capsules as artificial oxygen carriers for blood substitutes is assessed on the example of biodegradable poly(lactide-co-gylcolide) micro- and nanocapsules with a liquid content of perfluorodecalin. The morphology of the capsules is studied by confocal laser scanning microscopy using Nile red as a fluorescent marker. The mechanical stability and the wall flexibility of the capsules are examined by atomic force microscopy. The permeability of the capsule walls in connection with the oxygen uptake is detected by nuclear magnetic resonance. It is shown that the preparation in fact leads to nanocapsules with a mechanical stability which compares well with the one of red blood cells. The capsule walls exhibit sufficient permeability to allow for the exchange of oxygen in aqueous environment. In the fully saturated state, the amount of oxygen dissolved within the encapsulated perfluorodecalin in aqueous dispersion is as large as for bulk perfluorodecalin. Simple kinetic studies are presently restricted to the time scale of minutes, but so far indicate that the permeability of the capsule walls could be sufficient to allow for rapid gas exchange.

Critical O2 and NO concentrations in NO-induced cell death in a rat liver sinusoidal endothelial cell line

Abstract Nitric oxide (NO) plus oxygen (O2) are known to cause cell damage via formation of reactive nitrogen species. NO itself directly inhibits cytochrome oxidase of the mitochondrial respiratory chain in competition with O2, thus inducing a hypoxic-like injury. To assess the critical NO and O2 concentrations for both mechanisms of NO-induced cell injury, cells of a rat liver sinusoidal endothelial cell line were incubated in the presence of the NO donor spermineNONOate at different O2 concentrations, and their loss of viability was determined by the release of lactate dehydrogenase. Protection by ascorbic acid was used as indication for the involvement of reactive nitrogen species, whereas a hypoxiclike injury was indicated by the protective effects of glycine and glucose and the increase in NAD(P)H fluorescence. High concentrations of NO (approx. 10 uM NO) and O2 (21% O2) were required to induce endothelial cell death mediated by formation of reactive nitrogen species. On the other hand, pathophysiologically relevant NO concentrations at low but physiological O2 concentrations (ca. 2 uM NO at 5% O2 and about 1 uM NO at 2% O2) induced hypoxiclike cell death in the endothelial cells that was prevented by the presence of glucose.

The necessity for the coating of perfluorodecalin-filled poly(lactide-co-glycolide) microcapsules in the presence of physiological cholate concentrations: Tetronic-908 as an exemplary polymeric surfactant

Recently, we demonstrated that biodegradable poly(lactide-co-glycolide) (PLGA) micro- and nanocapsules with a liquid content of perfluorodecalin are principally useful for the development of artificial oxygen carriers. In order to solve a decisive and well-known problem with PLGA microcapsules, i.e. the spontaneous agglomeration of the capsules after depletion of the emulsifying agent (i.e. cholate), coating with the ABA block copolymer, Tetronic-908 was studied. After Tetronic-908 treatment at concentrations that were harmless to cultured cells, the clustering of the microcapsules was prevented, the adsorption of opsonins was decreased and the attachment to cells was inhibited, but the oxygen transport capacity of PLGA microcapsules was even increased. The present data clearly show that perfluorodecalin-filled PLGA microcapsules must be coated before decreasing the emulsifying agent cholate to physiological concentrations, in order to develop a solution that has the capabilities to function as a potential artificial oxygen carrier suspension.

Rocket fuel for the quantification of S-nitrosothiols. Highly specific reduction of S-nitrosothiols to thiols by methylhydrazine

Abstract Reduction of S-nitrosothiols to the corresponding thiol function is the key step in analyzing S-nitrosocysteinyl residues in proteins. Though it has been shown to give low yields, ascorbate-dependent reduction is commonly performed in the frequently used biotin-switch technique. We demonstrate that the compound methylhydrazine can act as a specific and efficient reducing agent for S-nitrosothiols. The corresponding thiol function is exclusively generated from low molecular weight and proteinaceous S-nitrosothiols while methylhydrazine failed to reduce disulfides. It was possible to optimize the experimental conditions so that thiol autoxidation is excluded, and high reaction yields (> 90%) are obtained for the thiol function. The biotin-switch technique performed with methylhydrazine-dependent reduction shows remarkably improved sensitivity compared to the ascorbate-dependent procedure.

Enzymatic reduction of labile iron by organelles of the rat liver. Superior role of an NADH-dependent activity in the outer mitochondrial membrane

The enzymatic system mainly responsible for the reduction of labile iron ions in mammalian cells is still unknown. Using isolated organelles of the rat liver, i.e. mitochondria, microsomes, nuclei and the cytosol, we here demonstrate that Fe(III), added as Fe(III)-ATP complex, is predominantly reduced by an NADH-dependent enzyme system associated with mitochondria (65% of the overall enzymatic Fe(III) reduction capacity within liver cells). Microsomes showed a significantly smaller Fe(III) reduction capacity, whereas the cytosol and nuclei hardly reduced Fe(III). Studying the mitochondrial iron reduction, this NADH-dependent process was not mediated by superoxide, ascorbic acid, or NADH itself, excluding low-molecular-weight reductants. No evidence was found for the involvement of complex I and III of the respiratory chain. Submitochondrial preparations revealed the highest specific activity reducing Fe(III) in the outer membrane fraction. In conclusion, an NADH-dependent mitochondrial enzyme system, most likely the NADH-cytochrome c reductase system, located at the outer membrane, should decisively contribute to the enzymatic reduction of labile iron within liver cells, especially under pathological conditions.

Early Detection of Patients at Risk for Laparotomy by Serum Markers

Objective: Mesenteric ischemia (MESI) is a rare but often fatal complication for patients after cardiac surgery. Non-specific clinical symptoms and lack of specific laboratory parameters complicate diagnosis. We evaluated potential serum markers for MESI in cardiac surgery patients. Methods: Between 03/2011 and 10/2012 serial serum samples of cardiothoracic patients were collected. In patients receiving laparotomy for suspected MESI serum concentrations of potential markers (α-glutathione-S-transferase: αGST; intestinal-fatty-acid-binding-protein-2: iFABP2; D-lactate) were measured retrospectively in samples taken 1, 24 and 48 h after the initial cardiac operation and compared with an collective of 12 uncomplicated CABG patients. Results: Laparotomy was performed in 18 patients 11 ± 7 days after cardiac surgery. MESI was found in 9/18 patients. Already 1h after cardiac surgery D-lactate (37 ± 4 versus 16 ± 2 nmol/µl, p = 0.0005) and iFABP2 (1 ± 0.2 versus 0.06 ± 0.05 ng/ml, p = 0.0013) serum concentrations of patients experiencing laparotomy were significantly increased compared with the control group. These differences remained highly significant in the 24 hour and 48 hour serum samples. In the subgroup analysis of patients with and without MESI a significant difference was only found for iFABP2 after 24 h (1.1 ± 0.4 vs 2.9 ± 0.6 ng/ml, p = 0.04). No significant differences were found for αGST. Conclusions: D-Lactat and iFABP2 are increased significantly in the first postoperative hours in patients experiencing laparotomy within their postoperative course. Systematically used these markers might help to identify patients at risk for laparotomy.