Short Communication An automated method for the measurement of methaemoglobin in avian blood Mo ´ nica Martı ´nez-Haro, Rafael Mateo * Instituto de Investigacio ´ n en Recursos Cinege ´ticos, IREC (CSIC, UCLM, JCCM), Ronda de Toledo s/n, 13071 Ciudad Real, Spain Accepted 26 February 2007 Abstract An automated method has been developed for the measurement of methaemoglobin (metHb) in blood using a biochemistry analyser. The method was validated using blood collected from red-legged partridges exposed in vitro to increasing concentrations of nitrite in order to obtain different percentages of metHb. Results obtained using the original manual method and those using the new automated technique were compared and no significant differences were found. Intra-day and inter-day variabilities (8.8% and 2.6%, respectively) were acceptable for samples containing high levels (63–81%) of metHb. Methaemoglobin measured in blood samples stored in liquid nitrogen was stable for 10 days, but increased significantly by day 20 in nitrite-treated samples. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Nitrate; Nitrite; Methaemoglobinaemia; Wildlife; Birds Methaemoglobin (metHb) is haemoglobin (Hb) in which heme iron is oxidized to iron (III) and therefore can- not function as an effective oxygen transporting protein. The percentage of metHb in blood is used as a biomarker of exposure to oxidising toxicants such as nitrates, nitrites and N-nitroso compounds. Signs of poisoning in different species appear at 10% metHb and death occurs when the percentage exceeds 60–80% (Bruning-Fann and Kaneene, 1993; Fewtrell, 2004). The analysis of metHb in blood has usually been per- formed using manual spectrophotometric assays based on the observation that metHb has an absorption peak of 635 nm at pH 6.6, which disappears after the conversion of metHb to cyan-metHb by neutralized cyanide (Evelyn and Malloy, 1938). This method has been modified by other authors (Horecker and Brackett, 1944; Leahy and Smith, 1960; Hegesh et al., 1970; Rodkey et al., 1979; Lacey and Rodnick, 2002) to decrease the sample volume and the test time, and also to achieve a better accuracy. Here we describe an automated assay for metHb in blood based on the Evelyn and Malloy (1938) method but adapted using a biochemistry analyser. The reagents for the automated assay were phosphate buffer (9.85 mM KH 2 PO 4 , 5.26 mM Na 2 HPO 4 , pH 6.6, in water); KCN 4.07% w/v, in water; acetic acid 4.88% v/v, in water; and K 3 Fe(CN) 6 0.06% w/v, in phosphate buffer. A neutralized cyanide solution was prepared 1 h before use with equal volumes of acetic acid and KCN solutions at pH 5.4. The phosphate buffer for the manual method was the same as in the automated technique; the other reagents were prepared as described by Lacey and Rodnick (2002). A solution of NaNO 2 1% w/v in phosphate buffer was prepared to oxidize Hb in blood samples in vitro. Whole blood (1–2 mL) was collected from red-legged partridges (Alectoris rufa) using sterile syringes and hepa- rinised needles by puncture of the brachial vein. The sam- ples were immediately chilled in wet ice until assay, which was performed within 3 h. To compare the manual and automated methods (Experiment 1), aliquots of 500 lL of blood from a pool of four partridges were treated with 0, 10, 20, 40, 60 and 80 lL of 1% sodium nitrite. Buffer was 1090-0233/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2007.02.021 * Corresponding author. Tel.: +34 926295450; fax: +34 926 295451. E-mail address: rafael.mateo@uclm.es (R. Mateo). www.elsevier.com/locate/tvjl The Veterinary Journal 176 (2008) 405–407 The Veterinary Journal