Mitoferrofluor - A new approach to determine mitochondrial chelatable iron

Description:

Inventors have created a fluorescent marker, mitoferrofluor (MFF) for measuring mitochondrial chelatable iron in living cells and tissues.  It is an improvement on existing markers, as it covalently bonds to the mitochondria once it has entered, thus it will remain there through any subsequent reactions. Iron is an essential nutrient. Incorporation of iron into prosthetic groups (e.g., heme, sulfur-iron clusters) occurs exclusively in the mitochondrial matrix. This fluorescence probe measures chelatable iron in the mitochondrial matrix and will be useful to researchers studying iron metabolism and also the role of iron in pathophysiology.

 

MFF fluorescence shows excitation and emission peaks at 554 and 598 nm, respectively. In cell free medium, MFF fluorescence is strongly and stoichiometrically quenched by ferrous (Fe2+) but not by ferric (Fe3+) iron. In cultured rat hepatocytes, MFF selectively accumulates into mitochondria. Unlike the membrane potential (ΔΨ) indicator rhodamine 123, MFF is retained by mitochondria after collapsing ΔΨ by uncoupler (10 μM CCCP) in the presence of inhibitors of the mitochondrial ATP synthase (10 μg/ml oligomycin) and respiratory Complex III (10 μM myxothiazol). In MFF-loaded hepatocytes, intramitochondrial MFF fluorescence decreased by ~80% when excess extracellular Fe2+ was added. In conclusion, MFF retention by mitochondria is independent of mitochondrial ΔΨ unlike earlier mitochondrial iron indicators, such as rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester (RPA). Thus, MFF can be used to determine mitochondrial chelatable iron in normal hepatocytes with polarized mitochondria as well as in cells undergoing loss of mitochondrial membrane potential as in case of ischemia.

 

Overview: Iron is an essential nutrient. Incorporation of iron into prosthetic groups (e.g., heme, sulfur-iron clusters) occurs exclusively in the mitochondrial matrix. Mitochondrial chelatable iron contributes to reactive oxygen species (ROS) formation in several pathophysiological settings, including ischemia/reperfusion and acetaminophen hepatotoxicity. Previous studies show that bafilomycin, ischemia and other stresses cause lysosomes to release Fe2+ and that this iron is subsequently taken up into mitochondria to promote ROS formation. A need, therefore, exists in the art for fluorescence probes to measures chelatable iron in the mitochondrial matrix useful to researchers studying iron metabolism, for example to assess the mitochondrial chelatable iron pool when mitochondrial membrane potential (ΔΨ) is compromised as during cardiovascular events such as ischemia.

 

Applications: Determining subcellular iron translocation in acetaminophen hepatotoxicity, ischemia reperfusion injury, as well as normal iron metabolism.

Advantages:  MFF fluorescence is strongly and stoichiometrically quenched by Fe2+ but not by Fe3+, MFF retention by mitochondria is independent of mitochondrial ΔΨ unlike earlier mitochondrial iron indicators, such as rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester (RPA)

Key Words: mitochondria, iron, membrane potential

 

Publications: Hu, Jiangting, et al. "Translocation of Iron from Lysosomes to Mitochondria during Acetaminophen-Induced Hepatocellular Injury: Protection by Starch-Desferal and Minocycline." Free Radical Biology and Medicine (2016).

 

Inventors: Andaleb Kholmukhamedov, Christopher Lindsey, Craig Beeson, John Lemasters

Patent Status: US Application Filed 03/29/2017

MUSC-FRD Technology ID: P1531

 

Patent Information:
Category(s):
Other
For Information, Contact:
Scott Davis
Sr Licensing Manager
MUSC Foundation for Research Development
843-876-1900
davissco@musc.edu
Inventors:
John Lemasters
Andaleb Kholmukhamedov
Christopher Lindsey
Craig Beeson
Keywords:
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