Cobalt accumulation in horses following repeated administration of cobalt chloride RG Wenzel, a,b * D Major, c KF Hesp, a E Hall d and P Doble b Objective To monitor cobalt concentrations in urine, red blood cells and plasma after chronic parenteral administration of cobalt chloride evaluate these results against the current International Federation of Horseracing Authorities thresholds for detecting cobalt misuse. Design Eight mares were randomly assigned to four treatment groups, with two mares in each group: Group 1 – control group, Group 2 – 25 milligrams cobalt intravenously as CoCl 2 weekly, Group 3 – 50 milligrams cobalt intravenously as CoCl 2 weekly, and Group 4 – 25 milligrams cobalt intravenously mid-week and at the end of the week. Urine and blood samples were collected before each weekly administration so that trough levels were assessed. In the group receiving two doses per week, urine and blood were collected prior to the dose given at the end of each week. Samples were initially collected at time zero then weekly for 10 weeks. Three further collections of urine and blood were made at days 81, 106 and 127. Methods Urine creatinine measurements to assess horse hydra- tion status were performed by the Jaffe reaction method. Cobalt determinations in plasma, blood and urine were by inductively coupled plasma—mass spectrometry. Haematocrit concentra- tions, used to calculate red cell cobalt levels, were performed using a microhematocrit centrifuge. Statistical analyses were con- ducted in Genstat (v17, VSNi). Results Marked cobalt accumulation was evident with increas- ing cobalt concentrations for all sample matrices in specimens collected immediately prior to cobalt administration. Correlation between the sample matrices improved when urine cobalt con- centration was adjusted for creatinine level. Red cell cobalt levels remained elevated for at least 12 weeks after cessation of admin- istration, consistent with the lifespan of the red cell. There was no significant change in haematocrit concentrations for the duration of the study. Conclusion The current urine cobalt threshold was only effec- tive at detecting acute cobalt exposure while the plasma cobalt threshold was able to consistently identify chronic high-level cobalt exposure and potential cobalt misuse. The threshold values legislated for urine cobalt do not correlate with those set for plasma. The acute nature of urinary cobalt excretion provides a relatively small window through which cobalt administration is detected. Plasma and red cell cobalt concentrations can provide a clearer picture of potential cobalt misuse. Keywords accumulation; blood; cobalt; haematocrit; horse; urine Abbreviations IFHA, International Federation of Horseracing Authorities; REML, restricted maximum likelihood Aust Vet J 2019 doi: 10.1111/avj.12872 C obalt is an essential trace element required by the intestinal bacteria of horses for the synthesis of vitamin B12 (cobala- min). In a 500 kg working horse consuming 10–12.5 kg dry mass feed per day, a minimum daily dietary intake of 0.1–0.15 mg cobalt per kg of dry feed is recommended with signs of cobalt toxic- ity unlikely to occur up to a daily dietary intake of 20 mg cobalt per kg dry feed. 1 The uptake and distribution of cobalt have principally been studied in laboratory animals and humans. 2 With prolonged exposure, increasing amounts of cobalt enter the red blood cells via transfer proteins. Once sequestered within red cells, cobalt remains for the life of the cell, approximately 120 days in humans. 3 Carter et al 4 estimated the lifespan of the equine erythrocyte at approxi- mately 150 days. Kinobe 5 conducted a comprehensive review of cobalt in horses and emphasised the need for further work to clarify the pharmacody- namics of long-term exposure of horses to cobalt. He noted that there was no evidence of either performance enhancing or toxic effects of cobalt in horses and emphasised that with a compartmental excretion pattern and a long elimination half-life, the cumulative effect of repeated dosing was unknown. Most human studies consider whole blood, plasma or serum cobalt levels, although some studies have compared all the three samples. Van der Straeten, in Estey et al, 6 noted that blood or plasma was pre- ferred and advised that 24-h elimination samples are more reliable for urine cobalt measurement. Daniel et al 7 states ‘Taking account of only the concentration of metal in urine is unsatisfactory and subject to error from differential urinary dilution’. Likewise, Krug et al, 8 in proposing a human athletic regulatory threshold, noted that urine levels would need correction for creatinine. These authors also stated that further studies were required to elucidate other factors which may elevate cobalt levels. They further proposed measurement of accumulation of cobalt in erythrocytes as a method of determining long-term exposure. 8 The International Federation of Horseracing Authorities (IFHA) set maximum permissible raceday thresholds of 0.1 microgram total cobalt per millilitre in urine and 0.025 microgram total cobalt (free and protein bound) per millilitre in plasma. 9 The urine threshold *Corresponding author. a NSW Health Pathology, Trace Elements Laboratory, Royal North Shore Hospital, Level 5, Acute Services Building, Pacific Highway, St Leonards, New South Wales 2065, Australia; ross.wenzel@health.nsw.gov.au b Centre for Forensic Science, University of Technology Sydney, Broadway, New South Wales 2001, Australia c Derek Major Consulting Pty Ltd, Richmond, New South Wales 2753, Australia d Veterinary Biostatistics, University of Sydney, Camden, New South Wales 2570, Australia © 2019 Australian Veterinary Association Australian Veterinary Journal 1 EQUINE AUSTRALIA’S PREMIER VETERINARY SCIENCE TEXT EQUINE