Fish Physiology and Biochemistry

, Volume 38, Issue 6, pp 1807–1813 | Cite as

NADH-dependent cytochrome b5 reductase and NADPH methemoglobin reductase activity in the erythrocytes of Oncorhynchus mykiss

  • M. C. Saleh
  • S. McConkeyEmail author


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.


Methemoglobin 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.


  1. Beutler E (1984) NADH methemoglobin reductase (NADH-ferricyanide reductase). In: Red cell metabolism, 3rd edn. Grune and Straton Inc., Orlando, pp 81–82Google Scholar
  2. Board PG (1981) NADH-ferricyanide reductase, a convenient approach to the evaluation of NADH-methaemoglobin reductase in human erythrocytes. Clin Chim Acta 109:233–237CrossRefGoogle Scholar
  3. Board PG, Agar NS, Gruca M, Shine R (1977) Methaemoglobin and its reduction in nucleated erythrocytes from reptiles and birds. Comp Biochem Physiol B 57:265–267CrossRefGoogle Scholar
  4. Bulbarelli A, Valentini A, DeSilvestris M, Cappellini MD, Borgese N (1998) An erythroid-specific transcript generates the soluble form of NADH-cytochrome b5 reductase in humans. Blood 92:310–319PubMedGoogle Scholar
  5. do Nascimento TS, Pereira RO, de Mello HL, Costa J (2008) Methemoglobinemia: from diagnosis to treatment. Rev Bras Anestesiol 58:651–664CrossRefGoogle Scholar
  6. Du M, Shirabe K, Takeshita M (1997) Identification of alternative first exons of NADH-cytochrome b5 reductase gene expressed ubiquitously in human cells. Biochem Biophys Res Commun 235:779–783CrossRefGoogle Scholar
  7. Freeman L, Beitinger TL, Huey DW (1983) Methemoglobin reductase activity in phylogenetically diverse piscine species. Comp Biochem Physiol B 75:27–30CrossRefGoogle Scholar
  8. 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
  9. Hegesh E, Calmanovici N, Avron M (1968) New method for determining ferrihemoglobin reductase (NADH-methemoglobin reductase) in erythrocytes. J Lab Clin Med 72:339–344PubMedGoogle Scholar
  10. Ito T, Mezawa K, Okazaki T, Shukuya R (1984) NADH- and NADPH-dependent reduction of methemoglobin in the nucleated erythrocytes from hen and bullfrog. Comp Biochem Physiol B 78:683–686CrossRefGoogle Scholar
  11. Kiyoshi ABE, Yoshiki S (1979) Properties of cytochrome b5 and methemoglobin reductase in human erythrocytes. Eur J Biochem 101:423–428CrossRefGoogle Scholar
  12. Lacey JA, Rodnick KJ (2001) Important considerations for methaemoglobin measurement in fish blood: assay choice and storage conditions. J Fish Biol 60:1155–1169CrossRefGoogle Scholar
  13. Mohr A, Wolf W, Bohl M, Hoffman R (1986) Quantification of methaemoglobin reduction in red blood cells of the rainbow trout Salmo gairdneri. J Fish Biol 29:483–487CrossRefGoogle Scholar
  14. Percy MJ, McFerran NV, Lappin TR (2005) Disorders of oxidised haemoglobin. Blood Rev 19:61–68CrossRefGoogle Scholar
  15. Power GG, Bragg SL, Oshiro BT, Dejam A, Hunter CJ, Blood AB (2007) A novel method of measuring reduction of nitrite-induced methemoglobin applied to fetal and adult blood of humans and sheep. J Appl Physiol 103:1359–1365CrossRefGoogle Scholar
  16. Roma GW, Crowley LJ, Barber MJ (2006) Expression and characterization of a functional canine variant of cytochrome b5 reductase. Arch Biochem Biophys 452:69–82CrossRefGoogle Scholar
  17. 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
  18. 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
  19. Stolk JM, Smith RP (1966) Species differences in methemoglobin reductase activity. Biochem Pharmacol 15:343–351CrossRefGoogle Scholar
  20. 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
  21. Wells RMG, Baldwin J, Seymour RS (1997) Low concentrations of methaemoglobin in marine fishes of the Great Barrier Reef, Australia. Mar Freshw Res 48:303–309CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  1. 1.Biomedical Sciences, Atlantic Veterinary CollegeUniversity of Prince Edward IslandCharlottetownCanada

Personalised recommendations