Saunders Janet, Speare David J, McConkey Sandra
Department of Biomedical Sciences, Atlantic Veterinary College, Charlottetown, Prince Edward Island, Canada.
Vet Clin Pathol. 2012 Dec;41(4):471-7. doi: 10.1111/j.1939-165X.2012.00466.x. Epub 2012 Sep 12.
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.
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.
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).
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.
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.
高铁血红蛋白（metHb）是一种不能可逆结合氧气的氧化型血红蛋白，据报道，健康鱼类体内的高铁血红蛋白浓度为0.6% - 24.8%，而健康哺乳动物体内的浓度为0% - 3%。在鱼类中，高铁血红蛋白是使用氰化钾（KCN）分光光度法进行测量的，但未使用对人体样本更为适用的多波长血氧计法。
本研究旨在评估多波长血氧计法作为测量虹鳟鱼高铁血红蛋白的一种方法，将多波长血氧计法与KCN分光光度法进行比较，并确定使用多波长血氧计法测量虹鳟鱼、大西洋鲑和溪红点鲑体内高铁血红蛋白浓度的参考值。
从健康的雌性虹鳟鱼、雌性大西洋鲑以及雌性和雄性溪红点鲑采集血样，先在Tris/NaCl/EDTA缓冲液中分离并洗涤红细胞，然后在Tris/EDTA缓冲液中裂解。使用IL - 682多波长血氧计测量高铁血红蛋白浓度。在体外使用亚硝酸钠（NaNO₂）产生中度和高度高铁血红蛋白浓度。
在低浓度高铁血红蛋白时，多波长血氧计法日内精密度的变异系数（CV）为10.3%，KCN分光光度法为53.9%。多波长血氧计法日间精密度的CV为11.9%。高铁血红蛋白浓度在4℃储存7天的全血中保持稳定。使用多波长血氧计法时，高铁血红蛋白浓度在高达58.2%时呈线性（r = 0.99），使用KCN法时在27.5%时呈线性（r = 0.94）。使用多波长血氧计法时高铁血红蛋白的检测下限为0.02 g/dL。使用多波长血氧计法测量虹鳟鱼、大西洋鲑和溪红点鲑（每个物种n = 40）体内高铁血红蛋白浓度的参考值分别为0.6% - 1.8%、1.1% - 1.9%和1.1% - 4.0%。
多波长血氧计法可用于测量鱼类血液中的高铁血红蛋白，特别是虹鳟鱼，且优于KCN分光光度法。虹鳟鱼、大西洋鲑和溪红点鲑体内高铁血红蛋白浓度的参考值与哺乳动物相似。
