Evaluation of co-oximetry for the measurement of methemoglobin in rainbow trout (Oncorhynchus mykiss) and values in 3 salmonid species

• Published in: Veterinary clinical pathology 2012-12, Vol.41 (4), p.471-477
• Main Authors: Saunders, Janet, Speare, David J., McConkey, Sandra
• Format: Article
• Language: English
• Subjects: Animals; Erythrocytes - metabolism; Female; Fish; Limit of Detection; Male; Methemoglobin - analysis; Methemoglobin - metabolism; Oncorhynchus mykiss - metabolism; Oximetry - methods; Oximetry - veterinary; Oxygen - blood; Potassium Cyanide; Protein Stability; Reference Values; Reproducibility of Results; Salmo salar; Salmo salar - metabolism; Salvelinus fontinalis; spectrophotometry; Spectrophotometry - veterinary; trout; Trout - metabolism; validation
• ISSN: 0275-6382; 1939-165X
• DOI: 10.1111/j.1939-165X.2012.00466.x

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. Objectives 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 NaNO2. 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.