V. Naidoo and others Toxicity of ketoprofen to Gyps vultures
Ketoprofen Toxicity - Supplementary Information 2
Estimating MLE and analysis of cattle tissue residues and blood parameters
The estimation of Maximum Levels of Exposure (MLE) was based upon the methods outlined
in Appendix II of Swan et al. (2006) estimated for a 4.75 kg Oriental white-backed vulture (G.
bengalensis) consuming a 1.023 kg meal (sufficient to provide a birds’ energetic requirements
for three days; Swan et al 2006) of kidney tissues from cattle slaughtered 1 hour after a threeday course of ketoprofen. The choice of slaughter at 1 hour was based on the rapid
elimination of ketoprofen in cattle (plasma half-life of 0.42 to 0.49 hours; De Graves et al 1996;
Landoli et al 1995) and information on ketoprofen tissue residues indicating highest levels
were in kidney tissues (EMEA 1995). Following the protocol of Swan et al (2006) and Swarup
et al (2007) for meloxicam, ketoprofen was administered to cattle at 6.0 mg kg -1, which is twice
the normal veterinary dose of 3.0 mg kg-1. Higher than recommended doses were
administered as the analysis of livestock carcass samples available to vultures in India
indicates that animals are sometimes administered doses of NSAIDs far in excess of the
recommended guidelines (Taggart et al 2007). Five cattle (Bos taurus) were dosed by attending
veterinarians at the Faculty of Veterinary Science, University of Pretoria and were
slaughtered 1 hour after the final injection at the Veterinary Pathology Department by means
of captive bolt to the brain and subsequent pithing. After slaughter, three samples of each
kidney and two of liver, each composed of two sub-samples from different areas of the organ
(e.g. 12 (since 2 kidneys) and 4 samples in total respectively) were collected from each animal
and placed in sealed glass vials and frozen at -20 C. Frozen tissue samples were couriered
on dry ice to the University of Aberdeen and remained frozen throughout transportation.
Analysis of ketoprofen residues was initially undertaken by LC-MS at the University of
Pretoria Medical School. Samples were homogenised and extracted in to acetonitrile and
analysed following a standard methodology. The estimated residue levels from this analysis
resulted in an estimated MLE of 5.11 mg kg-1 and a dose of 5.0 mg kg-1 was therefore
V. Naidoo and others Toxicity of ketoprofen to Gyps vultures
administered in Phase 3. A subsequent analysis of ketoprofen residues in cattle tissues was
undertaken using a validated LC-ESI/MS methodology (Taggart et al., 2009). Samples of
tissue (0.5g) were weighed to an accuracy of ± 0.001g, homogenised for 45 seconds with 2ml
of HPLC grade acetonitrile, and then centrifuged at 1000 g for five minutes. Quantification
was undertaken at m/z of 253.1 in negative ion mode (with confirmation at m/z of 197.1)
following compound separation on an Agilent ZORBAX 300SB-C18 column (4.6mm x
150mm, 5μm). Limits of quantification were equivalent to 12 ug kg -1 in liver tissue (see
Taggart et al. 2009 for a full description of the methodology). Repeat analysis of additional
tissue samples and known dilutions of ketoprofen dosing solutions (analysed blind) at both
Aberdeen and Pretoria revealed that the initial estimation of residue levels at Pretoria was
erroneous and too high. Based on the re-analysis of 30 kidney samples and 20 liver samples (6
kidney and 4 replicate liver samples per cow) the LC-ESI/MS analysis indicated that 1 hour
after the final dose average tissue concentrations were 7.13 mg kg -1 in kidney tissues and 0.16
mg kg-1in liver tissues. Based on these validated results the correct MLE was 1.54 mg kg -1 and
a dose of 1.5 mg kg-1 was administered in Phase 4.
Plasma levels of blood parameters were analysed with standard laboratory procedures. Uric
acid concentration was measured using an ACE TM Uric Acid reagent, albumin concentration
using the NExTTM Albumin reagent, ALT activity using the Alfa Wasserman ALT reagent,
and CK using the Alfa Wasserman CK reagent e ACE TM clinical chemistry system (Alfa
Wassermann, Bayer Health). The analyses were performed using the ACE TM and NExTTM
Clinical Chemistry Systems (Alfa Wassermann, Bayer Health Care, SA). The electrolytes
sodium (Na+), potassium (K+) and calcium (Ca2+) were measured with a blood gas analyser
(Rapidlab 34E Chiron diagnostics, Bayer SA).
V. Naidoo and others Toxicity of ketoprofen to Gyps vultures
De Graves, F.J., Riddell M.G. & Schumacher J. 1996 Ketoprofen concentrations in plasma and
milk after intravenous administration in dairy cattle. American Journal of Veterinary
Research 57, 1031-1033.
EMEA 1995 Committee for Veterinary Medicinal Products: Ketoprofen Summary Report.
EMEA/MRL/020/95. Accessed online at: http://www.emea.europa.eu/pdfs/vet/mrls
/002095en.pdf
Landoli, M.F., Cunningham, F.M. & Lees, P. 1995 Pharmacokinetics and pharmacodynamics
of ketoprofen in calves applying PK/PD modelling. J. Vet. Pharm. Thera. 18, 315-324.
Swan, G., Naidoo, V., Cuthbert, R., Green, R., Pain, D., Swarup, D., Prakash, V., Taggart,
M.A., Bekker, L., Das, D., Diekmann, J., Diekmann, M., Killian, E., Meharg, Patel, R.,
Mohini, S. & Wolter, K. 2006 Removing the Threat of Diclofenac to Critically
Endangered Asian Vultures. PLoS Biology 4, 395-402.
Taggart, M.A., Senacha, K., Green, R.E., Cuthbert, R., Pain, D.J. Randagan, B., Jhala, Y.,
Rahmani, A., Meharg, A.A. 2007 Diclofenac residues in carcasses of domestic ungulates
available to vultures in India. Environment International 33, 759-765.
Taggart, M.A., Senacha, K., Green, R.E., Cuthbert, R., Jhala, Y., Rahmani, A., Meharg, A.A.
Mateo, R. & Pain, D.J. 2009 Analysis of nine NSAIDs in ungulate tissues available to
Critically Endangered vultures in India. Environ. Sci. Technol. In press