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Serum 25(OH)D2 and 25(OH)D3 were measured separately by application to a second Zorbax-Sil Column eluted with hexane:propan-2-ol (98:2) and quantified by UV absorbance at 265 nm and corrected for recovery (sensitivity 5 nmol/L, intra- and inter-assay coefficients of variation 3��0% and 4��2%, respectively) (Berry and others 2000). Results are expressed as total 25(OH)D. Following separation by HPLC, 1,25(OH)2D (1,25(OH)2D2+1,25(OH)2D3) was quantified by radioimmunoassay as described in detail (sensitivity 3 pmol per assay tube, intra- and interassay coefficients of variation 7��8% and 10��5%, selleck products respectively) (Mawer and others 1990). The assay laboratory is accredited to ISO 9001:2000 and ISO 13485:2003 and participates successfully in the Vitamin D quality assurance scheme (DEQAS). The biochemical and hormone concentrations were compared between the groups by a Kruskal-Wallis test with post-test and Dunn��s multiple comparison test. A Mann-Whitney U test was used to compare CIBDAI scores between the PLE and IBD groups. A Spearman Rank correlation test was used to investigate for the presence of a correlation between albumin and CIBDAI scores and hormone concentrations. A Fisher��s exact test was used to compare the proportion of dogs with normal appetite between the PLE and IBD groups. Statistical analysis was performed with a commercial software package (GraphPad Prism 5) and PFlavoprotein approved by the University of Edinburgh Veterinary Ethical Review Committee. Thirty-six healthy dogs (17 different breeds Rapamycin manufacturer and 6 cross-breeds, median age 2��7 years, range 5 months to 10 years), 49 dogs in the hospital group (28 different breeds and 5 cross-breeds, median age 2��1 years, range 3 months to 13 years), 21 dogs in the IBD group (13 different breeds, 1 cross-breed, median age 4��9 years, range 6 months to 10 years) and 12 dogs in the PLE group (7 different breeds, 4 cross-breeds, median age 7 years, range 3 years to 12 years) were enrolled in the study. There was a significant difference in the ages between the four groups (P