Circadian variation in biochemical markers of bone cell activity and insulin-like growth factor-I in two-year-old horses 1 B. F. Jackson*, A. Blumsohn†, A. E. Goodship*, A. M. Wilson*, J. S. Price* 2 *Department of Veterinary Basic Sciences, The Royal Veterinary College, London NW1 0TU, U.K. and †Bone Metabolism Group, Northern General Hospital, University of Sheffield, Sheffield S5 7AU, U.K. ABSTRACT: Studies in humans have found circa- dian changes to be one of the most important sources of controllable preanalytical variability when evaluating bone cell activity using biochemical markers. It remains unclear whether similar circadian changes influence bone marker concentrations in the horse. The aim of this study was to characterize changes in serum concen- trations of three biochemical markers of bone cell activ- ity over a 24-h period in six 2-yr-old Thoroughbred mares, and to determine circadian variability in IGF- I, which regulates bone turnover. Three bone markers were measured in serum: osteocalcin, a marker of bone formation, the carboxy-terminal propeptide of type-I collagen (a marker of bone formation), and the carboxy- terminal telopeptide of type-I collagen (a marker of bone resorption). Data were analyzed using the cosinor tech- nique, which fits a 24-h cycle to each dataset. A signifi- cant circadian rhythm was observed for osteocalcin (P = 0.028), with an estimated amplitude of 7.6% of the mean (95% confidence interval 1.3% to 16.3%), and an estimated peak time of 0900. However, the observed Key Words: Bone Tissue, Circadian Rhythm, Horses, Insulin-Like Growth Factor, Osteocalcin 2003 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2003. 81:2804–2810 Introduction Biochemical markers can be used to noninvasively monitor bone cell activity, and a number of studies have explored the potential applications of bone markers in horses (Price 1999; Lepage et al., 2001). Markers stud- ied include osteocalcin, a noncollagenous protein pro- duced by osteoblasts and a marker of bone formation; the carboxy-terminal propeptide of type-I collagen (PICP), a marker of type-I collagen formation; and the carboxy-terminal telopeptide of type-I collagen (ICTP), a marker of type-I collagen degradation. 1 Supported by the Horserace Betting Levy Board. 2 Correspondence—phone: 0207-468-5238; E-mail: jprice@rvc.ac. uk. Received January 21, 2003. Accepted July 9, 2003. 2804 rhythm for the carboxy-terminal telopeptide of type-I collagen (amplitude = 7.4%) was not significant (P = 0.067), and there were no significant changes in concen- trations of the carboxy-terminal propeptide of type-I collagen over the 24-h study period (P = 0.44). There was a small but significant circadian rhythm for IGF- I(P = 0.04), with an estimated amplitude of 3.4% (95% confidence interval 0.2 to 7.1%) and peak at 1730. Fur- ther studies are now required to determine the poten- tial association between circadian changes in IGF-I and osteocalcin in the horse. Although no significant circa- dian variation was found in concentrations of the car- boxy-terminal propeptide of type-I collagen and the car- boxy-terminal telopeptide of type-I collagen, this may in part be a result of the age of the animals that were still skeletally immature. Future studies should aim to determine whether these markers develop a circadian rhythm at a later age when growth is complete. In the meantime, consistency in time of sampling should continue to be considered best practice when measuring biochemical markers of bone turnover in the horse. Studies in humans (reviewed by Hannon and Eastell 2000) and animals (Liesegang et al., 1999; Srivastava et al., 2001; Ladlow et al., 2002) have identified circadian rhythms as an important source of preanalytical vari- ability in these markers. However, whereas some stud- ies have reported a circadian rhythm in osteocalcin in horses (Lepage et al., 1991; Black et al., 1999), others have found no change (Hope et al., 1993; Geor et al., 1995). To date, no studies have established whether PICP and ICTP show circadian variability in the horse as they do in humans (Hassager et al., 1992; Heshmati et al., 1998). If a bone marker displays circadian vari- ability it is important that samples are collected at precise times for results to be meaningful. Studies in humans and animals have suggested en- dogenous factors, including hormones, may play an im- portant role in regulating daily rhythms in bone metab- olism (Nielsen et al., 1991; Ostrowska et al., 2002). In