Individual direct biomarkers of bone metabolism: Biomarkers of anabolic processes
During typical Type-I collagen synthesis, as with Type-II collagen, cleavage of carboxy-and amino-terminal propeptides (PICP
and PINP, respectively) of the procollagen molecule occurs, and these cleaved propeptide fragments can be exploited as markers
reflective of bone formation.
In a preliminary study in the United Kingdom, PICP was shown to have potential value as a molecular marker for monitoring
changes in matrix turnover after tendon injury, and increases in PICP with age and exercise also have been demonstrated.
Osteocalcin is a small, non-collagenous protein associated with bone assembly and turnover. Concentrations in the horse appear
to vary with age, administration of corticosteroids and general anesthesia.
In a study in our laboratory where various serum markers were used to differentiate changes with exercise from pathologic
change in joints, concentrations of osteocalcin and CS-846 provided the best correlation to the modified Mankin score (r2
= 0.72) and clinical degree of pain (r2 = 0.70) using multivariate linear regression (step-wise model selection).
Bone-specific alkaline phosphatase (BAP) is expressed at high concentrations on the cell surface in bone, forming osteoblasts.
In a study with treadmill exercise in young horses, serum BAP amounts were not different between exercise and control groups,
although previously there had been a suggestion that there was a correlation between amounts of BAP and the amount of arthroscopically
defined joint damage.
Biomarkers of catabolic processes
The release of a fragment of the Type-I collagen non-helical telopeptide (ICTP), which includes the collagen cross-linking
region, has been evaluated as a marker of bone resorption in humans (Garnero et al., 1999). Concentrations of ICTP in the
horse have not been shown of value in detecting pathological processes in another study conducted in the United Kingdom.
Although Type-I collagen C-telopeptide (CTX-1) has been useful in identifying bone resorption in humans, in the CSU horse
OA model, CTX was less useful than CS846, CPII and GAG biomarker concentrations in predicting whether a horse was from a control,
exercised or an osteoarthritic horse.
Other work in our laboratory has identified CPII and CTX-1 as potential serum indicators of the exercise effects on the developing
skeletal system in young horses. Higher serum concentrations of CTX-1 and lower concentrations of CPII were found in trained
foals compared to other groups, but these differences later disappeared during an additional six months of identical exercise
(Billinghurst et al., 2003).
One of the latest principal aims of biomarker research at CSU has been to diagnose early subchondral bone disease and thereby
potentially predict fracture. This was the basis of a study funded by the Grayson-Jockey Club Foundation and carried out with
racing Thoroughbreds in Southern California.
Monthly lameness examinations and serum collections were performed in 238 2- and 3-year old Thoroughbred racehorses. Exit
criteria were lack of training for greater than 30 days or completion of 10 study months. Horses with solitary musculoskeletal
injuries and completion of greater than two months were analyzed and compared to aged matches, sex-matched controls.
Seven biomarkers were used for examination, including serum glycosaminoglycans (GAG), Type-I and II collagen degradation (C1,
2C), Type II collagen synthesis (CPII), Type II collagen degradation (ColCEQ), aggrecan synthesis (CS846), osteocalcin (OC)
as a marker of bone formation and CTX-1 of bone degradation.
Fifty-nine horses sustained a single musculoskeletal injury, and 71 acted as uninjured controls. Based on the injured horses,
16 (27 percent) sustained an intra-articular fracture, 17 (29 percent) a tendon or ligamentous injury, 7 (12 percent) a stress
fracture and 19 (32 percent) were diagnosed with dorsal metacarpal disease (DMD).
Control population showed a longitudinal increase in GAG and a decrease in OC. Horses with intra-articular fractures showed
a decrease in CS846 and an increase in OC and CTX compared to the control population. Horses with tendon or ligamentous injuries
demonstrated a decrease in GAG and an increase in CTX1 when compared to the control population. Horses diagnosed with stress
fractures showed an increase in CTX when compared to the control population, and horses with dorsometacarpal disease demonstrated
a decrease in CS846 and GAG, as well as an increase in OC and CTX1.
The greatest change in biomarker levels appeared to be four to six months prior to injury.
This study is the first step toward development of a biomarker screening platform that can be used to identify horses at risk
New forms of biomarkers
Gene-chip microarray is the latest advance in biomarkers and represents a molecular approach to defining a disease process.
The principle is to have an array of a large number of gene sequences (cDNAs) on a computer chip. The entire equine genome
is currently available, and gene chips for microarray have been developed. The production of such chips facilitates the simultaneous
relative quantitation of multiple mRNAs and allows for comprehensive assessment of expression amounts.
In recent work from our laboratory, the potential usefulness of gene-chip microarray as a diagnostic tool in OA has been explored.
Blood samples were taken during the development of experimental OA (using the carpal chip fragment exercise model) in the
horse, and we were able to identify notable upregulation of 18 different genes in the OA group compared to the controls.
This change in gene expression started early in the development of the osteoarthritic disease process. It is envisioned that
this ability will be combined with conventional immunologic biomarkers (previously discussed) to provide a diagnostic platform
for OA and other diseases.
Other options being explored as biomarkers include proteonomics and metabolomics.
Dr. McIlwraith is University Distinguished Professor of Surgery, Barbara Cox Anthony University Chair and director of the Orthopaedic Research at
Colorado State University.