NADH-dependent cytochrome b5 reductase and NADPH methemoglobin reductase activity in the erythrocytes of Oncorhynchus mykiss
- 316 Downloads
Methemoglobin is oxidized hemoglobin that cannot bind to or dissociate from oxygen. In fish, it is most commonly caused by exposure to excess nitrites and can lead to abnormal swimming, buoyancy, or death. The methemoglobin concentration in mammals is determined by the balance of oxidizing agents versus reducing enzymes in erythrocytes. The objective of our studies was to characterize the enzymes that reduce methemoglobin in fish erythrocytes. Whole blood was collected from healthy rainbow trout. Methemoglobin was induced in vitro by NaNO2 exposure. Methemoglobin reduction in controls was compared to reduction in samples with added NADH, NADPH, or NADPH and methylene blue. Rainbow trout whole blood was also fractionated into cytosol, microsomal, and mitochondria/plasma membranes/nuclei fractions. The fractions were compared for NADH-dependent cytochrome b5 reductase (CB5R) activity and for nitrite induction of methemoglobin. The CB5R activity in rainbow trout erythrocytes was compared to the CB5R activity in equine, feline, and canine erythrocytes. Rainbow trout erythrocytes had significant NADPH methemoglobin reductase activity in the presence of methylene blue (P < 0.001). The CB5R activity was greatest (P < 0.001) in the plasma membrane/mitochondria/nuclei fraction. The CB5R activity in rainbow trout erythrocytes was not significantly different than canine or equine activity but was significantly lower than feline CB5R activity (P < 0.0001). Methemoglobin in rainbow trout erythrocytes can be reduced by CB5R or NADPH-dependent methemoglobin reductase. Unlike mammalian anuclear erythrocytes, which are dependent on soluble CB5R, the nucleated RBCs of rainbow trout use membrane-bound CB5R to reduce methemoglobin.
KeywordsMethemoglobin Fish Cytochrome b5 reductase
The authors would like to thank the Atlantic Veterinary College, University of Prince Edward Island, for funding these studies and Nicole Guselle for her assistance with fish sampling.
- Beutler E (1984) NADH methemoglobin reductase (NADH-ferricyanide reductase). In: Red cell metabolism, 3rd edn. Grune and Straton Inc., Orlando, pp 81–82Google Scholar
- Harvey J (2000) Erythrocyte metabolism. In: Feldman BF, Zinkl JG, Jain NC (eds) Schalm’s veterinary hematology, 5th edn. Lippincott Williams & Wilkins, Philadelphia, pp 125–128Google Scholar
- Saunders J, Speare DJ, McConkey S (2012) Validation of co-oximetry for the measurement of methemoglobin in rainbow trout (Oncorhynchis mykiss). Vet Clin Pathol (in press)Google Scholar
- Steinberg MH (2009) Hemoglobins with altered oxygen affinity, unstable hemoglobins, M-Hemoglobins, and dyshemoglobinemias. In: Greer JP et al (eds) Wintrobe’s clinical hematology, 12th edn. Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia, pp 1132–1142Google Scholar
- Telen MJ (2009) The mature erythrocyte. In: Greer JP et al (eds) Wintrobe’s clinical hematology, 12th edn. Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia, pp 126–155Google Scholar