Fish Physiol Biochem (2012) 38:1807–1813 DOI 10.1007/s10695-012-9677-2
NADH-dependent cytochrome b5 reductase and NADPH methemoglobin reductase activity in the erythrocytes of Oncorhynchus mykiss M. C. Saleh • S. McConkey
Received: 17 February 2012 / Accepted: 11 June 2012 / Published online: 26 June 2012 Ó Springer Science+Business Media B.V. 2012
Abstract 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
M. C. Saleh S. McConkey (&) Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PEI, Canada e-mail:
[email protected]
(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. Keywords Methemoglobin Fish Cytochrome b5 reductase
Introduction Living cells require oxygen for the generation of energy. Oxygen is transported from lungs or gills to tissues by hemoglobin containing reduced (ferrous) iron that reversibly binds oxygen (Roma et al. 2006; Telen 2009). Methemoglobin is hemoglobin containing oxidized (ferric) iron (Steinberg 2009). This form of hemoglobin is unable to bind to or dissociate from oxygen (Steinberg 2009). Small amounts of methemoglobin are produced daily by autoxidation, and larger quantities can occur following exposure to an oxidizing chemical or drug such as nitrite or benzocaine (Steinberg 2009). Increased methemoglobin concentrations in
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humans and other mammals cause dose-related clinical signs, such as headaches, decreased exercise tolerance, coma, or death (Steinberg 2009). Elevated methemoglobin in fish is called brown fish disease and is most commonly caused by excess nitrites in the water, due to either malfunctioning recirculation systems in tanks or excess nitrogen-rich waste such as fertilizer or sewage in wild habitats. Brown fish disease can be associated with decreased growth, abnormal swimming, respiratory difficulties, lethargy, or even death due to hypoxia (do Nascimento et al. 2008; Lacey and Rodnick 2001; Wells et al. 1997). The quantity of methemoglobin is kept in balance by an efficient redox system within erythrocytes. The primary methemoglobin-reducing enzyme in mammalian red blood cells (RBCs) is NADH-dependent cytochrome b5 reductase (CB5R), which catalyzes the reduction of ferric iron to ferrous iron (Steinberg 2009). The enzyme CB5R is well conserved between species (Roma et al. 2006) and in humans is encoded by genes on chromosome 22 (Roma et al. 2006). There are two isoforms of CB5R with identical catalytic domains (Bulbarelli et al. 1998): a membrane-bound isoform on the inner side of plasma, microsomal, and mitochondrial membranes and a soluble, cytosolic isoform derived from the same gene (Du et al. 1997; Percy et al. 2005; Power et al. 2007). In mammals, soluble CB5R is only found in erythrocytes, where it catalyzes methemoglobin reduction; in contrast, membranous CB5R is an ubiquitous enzyme that participates in the desaturation of fatty acids, biosynthesis of cholesterol, and some P450-mediated drug metabolism (Kiyoshi and Yoshiki 1979; Percy et al. 2005; Roma et al. 2006). A second enzyme within RBCs, NADPH-dependent methemoglobin reductase, can also reduce methemoglobin but requires an intermediary electron acceptor and therefore under normal circumstances contributes to \ 5 % of methemoglobin reduction (Harvey 2000; Telen 2009). Clinical methemoglobinemia in mammals is treated with methylene blue because this chemical can act as an electron acceptor for NADPH-dependent methemoglobin reductase. Glutathione and ascorbate also make small contributions to methemoglobin reduction (Telen 2009). Fish, avian, amphibian, and reptile erythrocytes are nucleated and contain organelles, whereas mammalian red blood cells (RBCs) are anuclear and have no organelles. Fish erythrocytes are known to have CB5R
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activity, but the activity has not been well characterized (Freeman et al. 1983; Mohr et al. 1986). CB5R activity in the nucleated RBCs of birds, reptiles, and amphibians is associated with the membrane-bound isoform of CB5R as opposed to the soluble cytosolic CB5R activity that predominates in mammalian anuclear RBCs (Board et al. 1977; Ito et al. 1984). The location of the CB5R activity in the nucleated RBCs of fish has not been investigated. Board and Ito also identified NADPH-dependent methemoglobin reduction in the nucleated RBCs of birds, reptiles, and amphibians (Board et al. 1977; Ito et al. 1984). This is in contrast to Mohr et al. (1986) who found no NADPH-dependent reduction in the nucleated erythrocytes of rainbow trout. The studies described in this paper were conducted to determine the location of CB5R activity in rainbow trout erythrocytes and whether there is NADPH-dependent methemoglobin reductase activity in rainbow trout RBCs. Finally, we wished to compare the activity of fish erythrocyte CB5R to that of mammalian species.
Materials and methods Materials HEPES, KCl, MgSO4, glucose, NADH, NADPH, K3Fe(CN)6, 2-mercaptoethanol, TRIS, methylene blue, and NaNO2 were purchased from Sigma–Aldrich (St. Louis, MO, USA). NaCl and Na2HPO4 were obtained from EMD Chemicals Inc. (Gibbstown, NJ, USA). CaCl2, EDTA, and NaH2PO4 were obtained from Fisher Scientific Limited (Ottawa, ON, Canada). Fish Rainbow trout (Oncorhynchus mykiss) (1.5–2 kg) were purchased from Ocean Trout Farm (Brookvale, PE, Canada) and housed in the Atlantic Veterinary College (AVC) aquatics teaching facility (Charlottetown, PE, Canada) in a 1,700-L high-density polyethylene tank (Aquamerik, QC, Canada) with a flowthrough freshwater system originating from devoted facility wells. Water temperature was maintained at 10.1–10.4 °C and a pH of 7.9. Fish were fed a commercial 4-mm trout pellet diet (OptimumÒ, Corey Feeds Ltd., Fredericton, NB, Canada). All procedures were performed in accordance with the guidelines of
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the Canadian Council on Animal Care and with the approval of the University of Prince Edward Island Animal Care Committee.
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Methemoglobin measurement
Fish were anesthetized for 3–5 min in a water bath containing 10 % benzocaine solution (50 g benzocaine in 500 mL of 90–92 % denatured ethanol) at a dose of 65 mg/L. Blood was collected from caudal vasculature into heparinized syringes. Whole blood was stored at 4 °C in plastic tubes for \24 h prior to incubation with nitrite solutions. Samples for CB5R measurement were centrifuged and washed, and aliquots stored at -80 °C for up to 4 weeks.
Whole blood was centrifuged at 3,000g for 3 min at 4 °C. The plasma and buffy coat were removed and replaced by two volumes of Tris wash buffer (290 mM NaCl, 10 lM EDTA, and 1 mM Tris, pH 8.2). Centrifugation and wash were repeated twice. Following a third centrifugation, one volume of lysing buffer (10 lM EDTA and 1 mM Tris, pH 8.2) was added, and samples were vortexed for 15 s before a final 3-min centrifugation at 12,000g and 4 °C (Mohr et al. 1986; Wells et al. 1997). The supernatant/hemolysate was removed and analyzed for methemoglobin using an IL-682 co-oximeter (Instrumentation Laboratory, Lexington, MA, USA) (Saunders et al. 2012).
Erythrocyte fractionation
CB5R measurement
Whole blood from fish (n = 6) was prepared by centrifugation at 3,000g for 3 min and then washed with 10 mM PBS (1.9 mM NaH2PO4, 8.1 mM Na2HPO4, 150 mM NaCl) to remove the plasma and buffy coat. RBCs were resuspended in 1 volume of 15 mM HEPES buffer, pH 7.4 (15 mM Hepes, 125 mM NaCl, 6 mM KCl, 1.2 mM MgSO4, 1 mM NaH2PO4, 1 mM CaCl2, 10 mM glucose) and vortexed to lyse the samples. The resuspended sample was centrifuged at 12,000g for 20 min at 4 °C to remove the pellet consisting of mitochondria, plasma membranes, and nuclei. The supernatant was centrifuged at 100,000g for 1 h to isolate the microsomes from the cytosol. The pellets from each fraction were washed in PBS, resuspended in cold 15 mM HEPES buffer, and frozen at -80 °C for CB5R measurement at a later time.
Aliquots of RBC fractions stored at -80 °C were thawed on ice and diluted 1:10 with 2.7 mM EDTA (pH 7) and 0.7 mM 2-mercaptoethanol and then vortexed (Beutler 1984; Board et al. 1981; Hegesh et al. 1968). Cytosolic fractions with/without microsomes and reconstituted microsomal pellets were assayed undiluted. The CB5R activity was assayed using a ferricyanide method (Board et al. 1981; Hegesh et al. 1968). The absorbance at 340 nm was measured every minute for 10 min. (BioTek Synergy HT Microplate Reader, BioTek Instruments Inc., Winooski, VT, USA).
Methemoglobin production
RBCs from rainbow trout (n = 8) were centrifuged and washed two times with Tris wash buffer and then resuspended in 1 volume of cold 15 mM HEPES buffer. Aliquots were incubated with 1 mM NaNO2 (except T = 0) and equal volumes of either 15 mM HEPES (controls), 2 mM NADH, 2 mM NADPH or 2 mM NADPH ? 10-5 M methylene blue at 30 °C. The methemoglobin was measured at 0, 20, 40, 60, and 90 min.
Blood collection
Whole-blood samples (n = 6) were centrifuged and washed with PBS to remove the plasma and buffy coat. Washed RBCs were resuspended in cold 15 mM HEPES buffer, pH 7.4, and divided into aliquots. To each aliquot was added an equivolume of either 15 mM HEPES buffer (control), cytosolic fraction with or without microsomes, or diluted mitochondrial/ membrane fraction. Zero time samples were processed immediately for methemoglobin measurement. All other aliquots were incubated at room temperature with a final concentration of 1 mM NaNO2 for 30, 60, or 90 min prior to methemoglobin measurement.
Methemoglobin production and comparison of NADH and NADPH reductase dependent reactions
Species comparison of CB5R activity Fresh whole blood from healthy dogs (n = 8), cats (n = 9), horses (n = 10), and rainbow trout (n = 14)
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Statistical evaluation GraphPad Prism 5, version 5.02 (2008, GraphPad Software Inc., La Jolla, CA, USA) was used for statistical data analysis. In vitro studies comparing the reduction of NaNO2–induced methemoglobin were analyzed with two-way ANOVAs followed by Bonferroni post-tests. The comparison of CB5R activity between species and between cellular fractions was by one-way ANOVAs and Bonferroni post-tests. The reduction of methemoglobin by CB5R was compared to the reduction by NADPH-dependent methemoglobin reductase using paired t-tests to compare the area under the curve (AUC) of samples containing NADH versus the AUC of samples containing NADPH plus methylene blue. Statistical significance was defined as P \ 0.05.
Results There was no significant difference in the reduction of NaNO2-induced methemoglobin in control rainbow trout RBCs compared to rainbow trout RBCs incubated with NADH or NADPH except for a significant difference (P \ 0.01) in the reduction of samples incubated with NADPH after 20 min. There was a significant (P \ 0.001) increase in methemoglobin reduction in aliquots incubated with NADPH and methylene blue after 20, 40, 60, and 90 min (Fig. 1). The peak methemoglobin level in NADH samples was 25.5 ± 1.4 % (mean ± SD) after 40 min, and the peak methemoglobin level in NADPH ? methylene blue samples was 17.4 ± 1.7 % after 20 min. The total methemoglobin produced in samples incubated with NADH as represented by the AUC was significantly greater than the AUC of samples incubated with NADPH ? methylene blue (P \ 0.0001). The ratio of the AUC for NADH samples versus the AUC of the NADPH ? methylene blue samples was 1.8:1. The peak of samples incubated with NADH was significantly greater than the peak of samples incubated with NADPH and methylene blue (P \ 0.0001). The CB5R activity in lysed RBCs, cytosol fraction, cytosol plus microsome fraction, mitochondria plus
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was centrifuged and washed with PBS two times to remove the buffy coat and plasma and then frozen at -80 °C until measurement.
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Time (min) Fig. 1 In vitro incubation of rainbow trout red blood cells with 1 mM NaNO2 for 90 min. Observed methemoglobin percentage (mean ± SEM) at 0, 20, 40, 60, and 90 min. Samples containing equivolumes of washed rainbow trout RBCs and one of filled circle HEPES (control), circle HEPES containing 2 mM NADH, dotted inverted triangle HEPES containing 2 mM NADPH or solid inverted triangle HEPES containing 2 mM NADPH and methylene blue were incubated with NaNO2 at 30 °C (n = 5 for each group). Samples containing NADPH and the electron acceptor methylene blue had the greatest rate of methemoglobin reduction (n = 5) (P \ 0.001)
plasma membrane and nuclei fraction, and microsomal fraction was as follows: 4.16 ± 1.67, 0.10 ± 0.08, 0.09 ± 0.05, 3.11 ± 0.64 and 0.29 ± 0.5 lmol/min/lL RBCs, respectively (mean ± SD). There was significantly greater CB5R activity in the lysed RBCs (P \ 0.001) than in the cytosol, cytosol plus microsomes, or microsomal fractions. There was no significant difference in the CB5R activity in lysed RBC samples and the mitochondria/plasma membranes and nuclei fraction. There was significantly greater CB5R activity in the mitochondria/plasma membrane and nuclei fraction than the cytosol and cytosol plus microsomes (P \ 0.001), and microsomal fractions (P \ 0.01). The reduction of NaNO2-induced methemoglobin by washed RBCs resuspended in HEPES (controls) was compared to the reduction by washed RBCs resuspended in HEPES with cytosol, microsomes, or mitochondria/plasma membranes/nuclei. The reduction was significantly greater (P \ 0.001) in samples with added mitochondria/plasma membranes/nuclei at 60 and 90 min (Fig. 2). The CB5R activity of rainbow trout was compared to the CB5R activity in dogs, cats, and horses. The CB5R activity was significantly greater in feline erythrocytes than in equine (P \ 0.01), canine, or
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Fig. 2 Comparison of the reduction of NaNO2-induced methemoglobin (mean ± SEM) by different cellular fractions. Filled circle controls (washed RBCs with HEPES), triangle washed RBCs with added cytosol, circle microsomes, or diamond mitochondria/plasma membranes were incubated with 1 mM NaNO2 at 30 °C. The reduction of methemoglobin was followed for 90 min. There was significantly greater methemoglobin reduction in samples containing mitochondria/plasma membranes/nuclei at 60 and 90 min (P \ 0.001) than in controls or samples containing cytosol or microsomes
trout erythrocytes (P \ 0.001) (Fig. 3). There was no significant difference in the CB5R activity of canine, equine, or trout erythrocytes.
Discussion The increasing use of land-based tanks with recirculation systems and high stocking densities in aquaculture, as well as the continued exposure of fish in the wild to nitrogen-rich pollutants, suggests that both clinical and subclinical methemoglobinemia in fish will continue to be an economical and environmental concern. Our studies aimed at characterizing the enzymes that form the redox system that reduces methemoglobin in rainbow trout erythrocytes. Previous studies have shown that the membranous isoform of CB5R is responsible for methemoglobin reduction in the nucleated RBCs of reptiles, amphibians, and birds (Board et al. 1977; Ito et al. 1984). This is contrary to mammalian erythrocytes, which depend on a cytosolic isoform of CBR5. In our studies, the CB5R activity in rainbow trout erythrocytes was concentrated in the cellular fractions containing plasma membranes/ mitochondria and nuclei, and there was minimal CB5R activity in fish RBC cytosol. This suggests that the nucleated RBCs of fish, like those of amphibians,
Fig. 3 Species comparison of cytochrome b5 reductase activity (mean ± SEM). The CB5R activity in equine (n = 10), canine (n = 8), feline (n = 9), and rainbow trout blood (n = 14) was compared. There was significantly greater CB5R activity in feline erythrocytes versus equine erythrocytes (P \ 0.01) or canine and trout RBCs (P \ 0.001). There was no significant difference in CB5R activity between trout, equine, and canine erythrocytes
reptiles and birds, use a membranous isoform of CB5R to reduce methemoglobin. The anuclear RBCs of mammals evolved later than the nucleated RBCs of fish, reptiles, birds, and amphibians. The loss of erythrocyte nuclei during evolution may have required an alternative methemoglobin reduction pathway and subsequently led to the development of a soluble isoform of CB5R. During the production of RBCs in mammalian bone marrow, erythrocytes start out as nucleated cells but lose their nuclei prior to release into circulation. Soluble CB5R does not appear in the nucleated RBC progenitors in mammalian bone marrow until just prior to nuclear extrusion (Bulbarelli et al. 1998). Therefore, the signal to erythrocyte nuclei to produce the soluble isoform of CB5R may be linked to the signal to extrude the nucleus. The total CB5R activity of all the erythrocyte fractions was slightly lower than the activity of the lysed samples. This may have been due to some enzyme loss from the repeated washing and separation during the fractionating process. The minimal CB5R activity within the cytosol may reflect a true small quantity of soluble CB5R, or it may have been due to contamination by other cell fractions. In mammals, the enzyme NADPH methemoglobin reductase requires an electron acceptor to act as an intermediary to reduce methemoglobin (Steinberg 2009).
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When an electron acceptor is present, NADPH methemoglobin reductase is able to reduce methemoglobin at a faster rate than CB5R. Our studies indicate that NADPH-dependent methemoglobin reductase is also present in fish erythrocytes and similarly requires an electron acceptor to catalyze methemoglobin reduction. We also found that NADPH-dependent methemoglobin reductase in fish reduces methemoglobin at a faster rate than CB5R when the electron acceptor methylene blue is present. Our results are contrary to those of Mohr et al. (1986) who found no NADPHdependent methemoglobin reductase activity in rainbow trout erythrocytes. Mohr’s group, however, did not use an electron acceptor in their experiments, which would account for the apparent lack of reduction by NADPH-dependent methemoglobin reductase activity. Our findings have clinical implications. Methylene blue is the primary treatment for humans with methemoglobinemia and is frequently advocated for use in fish with methemoglobinemia despite never, to best of our knowledge, having been tested for efficacy in fish. On the basis of Mohr’s earlier findings of there being no NADPH-dependent methemoglobin reductase, it should be pointless to treat fish with brown fish disease with methylene blue as there would be no enzyme to catalyze the reduction of methemoglobin by the NADPH and methylene blue. Our studies, however, support that at least in vitro, methylene blue would seem to be a logical treatment option for brown fish disease. Further studies investigating the use of methylene blue in fish with methemoglobinemia are needed to confirm its efficacy and provide a dosage regimen. In mammals, excess methylene blue can lead to hemolysis due to overconsumption of NADPH, but we have not found any reports of hemolysis in fish linked to methylene blue (Harvey 2000). The rate-limiting factor for CB5R reduction of methemoglobin in mammals is NADH (Kiyoshi and Yoshiki 1979; Roma et al. 2006). The addition of NADH to RBCs did not increase methemoglobin reduction significantly in our studies. This is in contrast to previous studies that have shown an increase in methemoglobin reduction in rainbow trout RBCs following the addition of NADH in vitro (Mohr et al. 1986). The lack of a significant increase in activity when NADH was added in our studies could indicate that there was no CB5R activity or that the reaction was already proceeding at its maximum
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velocity. We know from our ferricyanide-based CB5R measurements that CB5R activity was present. The lack of increase in activity subsequent to the addition of NADH in our studies may have been because we used a HEPES buffer containing glucose. NADH is produced via glycolysis. Older studies often used the addition of glucose to provide NADH for CB5R studies, and our glucose-containing buffer may have provided sufficient NADH for the reactions to proceed at maximum velocity (Stolk and Smith 1966). It could also indicate that NADH is not the rate-limiting factor for this reaction in fish. Our laboratory has done several similar studies using mammalian blood and has found that the addition of NADH to mammalian blood does increase the rate of reduction of methemoglobin by CB5R, even when a buffer of HEPES with glucose is used. The CB5R activity in rainbow trout erythrocytes was not significantly different than equine or canine activity, suggesting that the membranous isoform of CB5R is capable of reducing methemoglobin as efficiently as the soluble isoform in some mammals. We have demonstrated that the nucleated RBCs of rainbow trout, like the nucleated RBCs of birds, reptiles, and amphibians, use a membranous isoform of CB5R for methemoglobin reduction rather than a soluble isoform that is used by mammals. Our studies have also shown that rainbow trout erythrocytes, like mammalian erythrocytes, can reduce methemoglobin with either CB5R or NADPH-dependent methemoglobin reductase. The NADPH-dependent methemoglobin reductase enzyme, similar to mammals, requires an electron acceptor such as methylene blue to reduce methemoglobin. This supports the use of methylene blue to treat fish with brown fish disease. Further studies are required to confirm the in vivo efficacy of methylene blue for the treatment of brown fish disease in fish and to establish a safe dosing regimen. Acknowledgments 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.
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1813 Lacey JA, Rodnick KJ (2001) Important considerations for methaemoglobin measurement in fish blood: assay choice and storage conditions. J Fish Biol 60:1155–1169 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 29:483–487 Percy MJ, McFerran NV, Lappin TR (2005) Disorders of oxidised haemoglobin. Blood Rev 19:61–68 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 103:1359–1365 Roma GW, Crowley LJ, Barber MJ (2006) Expression and characterization of a functional canine variant of cytochrome b5 reductase. Arch Biochem Biophys 452:69–82 Saunders J, Speare DJ, McConkey S (2012) Validation of cooximetry for the measurement of methemoglobin in rainbow trout (Oncorhynchis mykiss). Vet Clin Pathol (in press) 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 1132–1142 Stolk JM, Smith RP (1966) Species differences in methemoglobin reductase activity. Biochem Pharmacol 15:343–351 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 126–155 Wells RMG, Baldwin J, Seymour RS (1997) Low concentrations of methaemoglobin in marine fishes of the Great Barrier Reef, Australia. Mar Freshw Res 48:303–309
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