title: Evaluation of co-oximetry for the measurement of methemoglobin in rainbow trout (Oncorhynchus mykiss) and values in 3 salmonid species.
abstract: BACKGROUND
  Methemoglobin (metHb) is oxidized hemoglobin that cannot reversibly bind oxygen, and concentrations in healthy fish have been reported to be 0.6-24.8% compared with 0-3% in healthy mammals. In fish, metHb has been measured using spectrophotometric methods using potassium cyanide (KCN), but not using co-oximetry, which is the preferred method for human samples.
  
  
  OBJECTIVE
  The aims of this study were to evaluate co-oximetry as a method for measuring metHb in Oncorhynchus mykiss, compare co-oximetry with a KCN spectrophotometric method, and establish reference values for metHb concentrations as measured using co-oximetry in O mykiss, Salmo salar, and Salvelinus fontinalis.
  
  
  METHODS
  Blood samples from healthy female O mykiss, female S salar, and female and male S fontinalis were prepared by separation and washing of erythrocytes in Tris/NaCl/EDTA buffer followed by lysis in Tris/EDTA buffer. MetHb concentrations were measured using an IL-682 co-oximeter. Moderate and high metHb concentrations were produced in vitro using NaNO(2).
  
  
  RESULTS
  At low concentrations of methemoglobin, CVs for intraday precision were 10.3% and 53.9% using co-oximetry and the KCN spectrophotometric method, respectively. The CV for interday precision using co-oximetry was 11.9%. MetHb concentrations were stable in whole blood stored at 4°C for 7 days. MetHb concentrations were linear up to 58.2% (r = .99) using co-oximetry and 27.5% (r = .94) using the KCN method. The lower limit of detection for metHb was 0.02 g/dL using co-oximetry. Reference values for metHb concentrations using co-oximetry in O mykiss, S salar, and S fontinalis (n = 40 of each species) were 0.6-1.8%, 1.1-1.9%, and 1.1-4.0%, respectively.
  
  
  CONCLUSIONS
  Co-oximetry can be used to measure methemoglobin in blood from fish, in particular in O mykiss, and is better than the KCN spectrophotometric method. Reference values for methemoglobin concentrations in O mykiss, S salar, and S fontinalis are similar to those in mammals.
authors.affiliation: Department of Biomedical Sciences, Atlantic Veterinary College, Charlottetown, Prince Edward Island, Canada. jsaunders@upei.ca
authors.forename: Janet
authors.initials: J
authors.lastname: Saunders
authors.forename: David J
authors.initials: DJ
authors.lastname: Speare
authors.forename: Sandra
authors.initials: S
authors.lastname: McConkey
mesh_terms: D000818:Animals; D004912:Erythrocytes / Q000378:metabolism; D005260:Female; D057230:Limit of Detection; D008297:Male; D008706:Methemoglobin / Q000032:analysis* / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D010092:Oximetry / Q000379:methods / Q000662:veterinary*; D010100:Oxygen / Q000097:blood; D011190:Potassium Cyanide; D055550:Protein Stability; D012016:Reference Values; D015203:Reproducibility of Results; D020317:Salmo salar / Q000378:metabolism*; D013053:Spectrophotometry / Q000662:veterinary; D014337:Trout / Q000378:metabolism*
chemical_list: D008706:Methemoglobin; D011190:Potassium Cyanide; D010100:Oxygen