Biochemical markers of bone cell activity can be classified into those which are markers of bone formation or resorption. Generally, suitable markers are enzymes expressed by osteoblasts or osteoclasts, or they are organic components released during bone synthesis or resorption. There are several reviews detailing markers of bone metabolism (Allen 2003; Looker et al. 2000; Risteli and Risteli 1993; Seibel 2000, 2005; Watts 1999). Table 3.1 provides details of the main markers of bone resorption and formation. There are some limitations associated with the use of biochemical markers to measure bone turnover in vivo, including variability associated with age (Lepage et al. 1990; Mora et al. 1998; Nishimoto et al. 1985; Wishart et al. 1995), diurnal rhythms (Gertz et al. 1998; Gundberg et al. 1985; Karlsson et al. 1992) and, in females, reproductive status (pregnant, menopausal etc.) (Karlsson et al. 1992; Rosenbrock et al. 2002). In addition, tissue specificity can be a problem as biomarkers are only relatively specific for bone. For example, alkaline phosphatase is derived from a wide variety of non‐skeletal sources (Crofton 1982; Meyer‐ Sabellek et al. 1988). Therefore, it is preferable to assay a combination of markers to allow more detailed information on bone remodelling rates to be collected as each marker may reflect a different physiological process within bone. Biochemical markers can also be used to evaluate the composition of the bone matrix.
Table 3.1: Summary of biochemical markers of bone formation and resorption.
Bio‐marker Major source Sample References
Bone formation
BALP Osteoblasts Serum/Plasma Gomez et al. (1995), Mohamadnia et al. (2007)
Osteocalcin Osteoblasts Serum/Plasma Lee et al. (2000), Brown et al. (1984)
PICP Osteoblasts Serum Eriksen et al. (1993), Franke et al. (1998), Melkko et al. (1990) PINP Osteoblasts Serum Melkko et al.(1996)
BSP Osteoblasts Serum Seibel et al. (1996)
Bone resorption
Hydroxyproline Collagen Urine Dull and Henneman (1963) PYD Collagen Urine Robins et al. (1996), Seyedin et al.
(1993), Uebelhart et al. (1990) DPD Collagen Urine Robins et al.(1994), Robins et al.
(1996)
ICTP / CTX‐MMP Collagen Serum/Urine Eriksen et al. (1993), Risteli et al. (1993)
NTX‐I Collagen Serum/Urine Franke et al. (1998)
TRAP Osteoclasts Serum/Plasma Minkin (1982), Janckila et al. (2001)
Abbreviations: Carboxy‐terminal propeptide of type I procollagen (PICP), amino‐terminal propeptide of type I procollagen (PINP), bone sialoprotein (BSP), pyridinoline (PYD), deoxypyridinoline (DPD), carboxy‐terminal cross‐linked telopeptide of type I collagen (ICTP or CTX‐MMP), amino‐terminal cross‐linked telopeptide of type I collagen (NTX‐I) and tartrate resistant alkaline phosphatase (TRAP).
Matrix metalloproteinases
Matrix metalloproteinases (MMPs), also known as matrixins, are a family of zinc‐dependent proteases that play a major role in the proteolytic degradation of structural components of the extracellular matrix (ECM) including collagen (Nagase and Woessner 1999; Sternlicht and Werb 2001). Most are secreted as inactive zymogens: these pro‐MMPs are activated in vitro by proteinases and by non‐proteolytic agents and once activated they degrade collagens and other ECM proteins (Nagase 1997; Nagase and Woessner 1999). Based on their structural and functional characteristics, human MMPs can be classified into several subfamilies, which include type IV collagenases, also known as gelatinases (Matrisian 1992; Vu and Werb 2000; Woessner 1991). There are two types of type IV collagenase; 72‐kDa gelatinase (gelatinase A) or MMP‐2 and 92‐kDa gelatinase (gelatinase B) or MMP‐9. MMP‐2 and MMP‐9 are responsible for the degradation of type IV and V collagen, and of denatured collagens such as type I collagen (Matrisian 1992). MMP‐2 is expressed by osteoblasts (Meikle et al. 1992; Rifas et al. 1994; Rifas et al. 1989), while MMP‐9 is highly expressed by osteoclasts (Tezuka et al. 1994; Wucherpfennig et al. 1994). In addition to being expressed by bone cells, both MMP‐2 and MMP‐9 are produced by immune cells such as macrophages and monocytes (Campbell et al. 1991; Garbisa et al. 1986). Inhibition of the gelatinases prevents bone resorption in vitro (Hill et al. 1995; Hill et al. 1994b). Approximately 95 % of the total collagen content of bone is type I collagen, with types III, V and VI present at low levels (Keene et al. 1991; Niyibizi and Eyre 1994). Therefore, increased expression of MMP‐ 2 and MMP‐9 indicates increased levels of collagen degradation and hence increased bone resorption. Hydroxyproline
Hydroxyproline is formed by post‐translational hydroxylation of the amino acid proline (Udenfriend 1966). It represents about 14% of the amino acid content of collagen (Eastoe
1955) and quantification of hydroxyproline can be used to estimate the total collagen content of bone.
Bone specific alkaline phosphatase
Alkaline phosphastase (ALP) is a zinc‐containing glycoprotein enzyme synthesized by cells in a wide variety of tissues including bone (Crofton 1982; Meyer‐Sabellek et al. 1988). Bone‐ specific ALP (BALP) is synthesised by, and expressed on the external surface of, osteoblasts during bone formation (Clarke 2008). BALP hydrolyses pyrophosphate, thereby removing an osteogenesis inhibitor and allowing bone mineralisation to proceed (Balcerzak et al. 2003). As such, BALP is one of the most frequently used non‐collagenous markers of osteogenesis (Allen 2003; Magnusson et al. 1999; Watts 1999).
3.2.9 Study aim and hypothesis
We hypothesised that the asymmetric cyclic loading experienced by Greyhounds during racing would lead to left‐to‐right differences in the BMD and in the markers of bone resorption and new bone formation. In comparison, as they are not subjected to asymmetric stresses, we believed there would be no such left‐to‐right differences in SBTs. To test this we examined all of the carpal, metacarpal, tarsal and metatarsal bones, with the exceptions of the first tarsal (T1) and first metatarsal (MT1). These two bones are vestigial in the dog: the degree to which they are present vary greatly between individual dogs, in some they can be fused together, in others they may be absent (Miller et al. 1964). Therefore, any left‐to‐right differences observed in T1 and MT1 would more than likely be false.