Semi-quantitative estimation of heme/hemoprotein with dichlorodihydrofluorescin diacetate

Ohashi, Takao; Kakimoto, Kazuhiro; Sokawa, Yoshihiro; Taketani, Shigeru

doi:10.1016/s0003-2697(02)00248-8

Cited by 10 publications

(8 citation statements)

References 10 publications

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“…Total ROS in C2C12 cells was measured by a modified method of Ohashi et al [23]. Briefly, the medium from treated or untreated C2C12 cells was replaced with 1 ml Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 5 mM HEPES, 2 mM CaCl2 and 10 mM Glucose, pH 7.4).…”

Section: Measurement Of Reactive Oxygen Species (Ros) In Cellsmentioning

confidence: 99%

Oxidative Damage in Muscular Dystrophy Correlates with the Severity of the Pathology: Role of Glutathione Metabolism

et al. 2012

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Muscular dystrophies (MDs) such as Duchenne muscular dystrophy (DMD), sarcoglycanopathy (Sgpy) and dysferlinopathy (Dysfy) are recessive genetic neuromuscular diseases that display muscle degeneration. Although these MDs have comparable endpoints of muscle pathology, the onset, severity and the course of these diseases are diverse. Different mechanisms downstream of genetic mutations might underlie the disparity in these pathologies. We surmised that oxidative damage and altered antioxidant function might contribute to these differences. The oxidant and antioxidant markers in the muscle biopsies from patients with DMD (n = 15), Sgpy (n = 15) and Dysfy (n = 15) were compared to controls (n = 10). Protein oxidation and lipid peroxidation was evident in all MDs and correlated with the severity of pathology, with DMD, the most severe dystrophic condition showing maximum damage, followed by Sgpy and Dysfy. Oxidative damage in DMD and Sgpy was attributed to the depletion of glutathione (GSH) and lowered antioxidant activities while loss of GSH peroxidase and GSH-S-transferase activities was observed in Dysfy. Lower GSH level in DMD was due to lowered activity of gamma-glutamyl cysteine ligase, the rate limiting enzyme in GSH synthesis. Similar analysis in cardiotoxin (CTX) mouse model of MD showed that the dystrophic muscle pathology correlated with GSH depletion and lipid peroxidation. Depletion of GSH prior to CTX exposure in C2C12 myoblasts exacerbated oxidative damage and myotoxicity. We deduce that the pro and anti-oxidant mechanisms could be correlated to the severity of MD and might influence the dystrophic pathology to a different extent in various MDs. On a therapeutic note, this could help in evolving novel therapies that offer myoprotection in MD.

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Section: Measurement Of Reactive Oxygen Species (Ros) In Cellsmentioning

confidence: 99%

Oxidative Damage in Muscular Dystrophy Correlates with the Severity of the Pathology: Role of Glutathione Metabolism

et al. 2012

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“…After incubation with 20 lM of H 2 DCFDA at 37°C for 30 min, the cells were washed twice with phosphate buffer saline (PBS). H 2 DCFDA level was analyzed by FACS Aria flow cytometer [12][13][14].…”

Section: Analysis Of Intracellular Reactive Oxygen Species (Ros)mentioning

confidence: 99%

The effect of iron overload and chelation on erythroid differentiation

et al. 2011

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We investigated the mechanisms of hematopoietic disorders caused by iron overload and chelation, in particular, the inhibition of erythroblast differentiation. Murine c-kit(+) progenitor cells or human CD34(+) peripheral blood hematopoietic progenitors were differentiated in vitro to the erythroid lineage with free iron and/or an iron chelator. Under iron overload, formation of erythroid burst-forming unit colonies and differentiation to mature erythroblasts were significantly suppressed; these effects were canceled by iron chelation with deferoxamine (DFO). Moreover, excessive iron burden promoted apoptosis in immature erythroblasts by elevating intracellular reactive oxygen species (ROS). Interestingly, both DFO and a potent anti-oxidant agent reduced intracellular ROS levels and suppressed apoptosis, thus restoring differentiation to mature erythroblasts. Accordingly, intracellular ROS may represent a new therapeutic target in the treatment of iron overload.

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“…This method is, however, disadvantageous because of the toxicity of pyridine and the low inherent sensitivity of hemochromogen. Although several other sensitive methods have been developed using colorimetric measurements [15,16] as well as fluorescent-and chemiluminescent-based techniques [17][18][19][20][21], discrimination of free heme from protein-bound heme and/or heme relative compounds and determination of the exact concentrations of free heme in living cells and organisms are issues that remain unresolved. In addition, some assays based on the peroxidase properties of heme are subject to interference by contamination with oxidizing species [17,18].…”

Section: Introductionmentioning

confidence: 99%