Use of biochemical markers of bone metabolism in veterinary medicine

Effective, non-invasive bone assessment methods for screening, diagnosis and follow-up of the skeleton are more and more requested in veterinary medicine. In contrast to clinical parameters, invasive methods and imaging techniques, indices of bone turnover is a tool for bone metabolism evaluation of the whole skeleton. Biochemical bone markers therefore provide a more real-time assessment of the bone status with simple blood- or urine-analysis. This article surveys currently available biochemical marker of bone metabolism used in veterinary medicine. Additionally, information is provided about physiological and pathological, as well as therapeutic variations of biochemical bone marker concentrations in various species.     

Evaluation of serum biochemical markers of bone metabolism for early diagnosis of nonunion and infected nonunion fractures in rabbits

OBJECTIVE: To evaluate the use of serum concentrations of biochemical markers of bone metabolism (osteocalcin [OC], bone-specific alkaline phosphatase [BS-ALP], and deoxypyridinoline [DPYR]) to compare healing in infected versus noninfected fractures and in fractures with normal repair versus delayed (nonunion) repair in rabbits. ANIMALS: 32 female 9- to 10-month-old New Zealand White rabbits. PROCEDURE: A femoral fracture defect was made in each rabbit. Rabbits were assigned to the following groups: the bone morphogenetic-2 gene treatment group with either noninfected nonunion or infected (ie, inoculation of defects with Staphylococcus aureus) nonunion fractures or the luciferase (control) gene treatment group with either noninfected nonunion or infected nonunion fractures. Serum samples were obtained before surgery (time 0) and 4, 8, 12, and 16 weeks after surgery. Callus formation and lysis grades were evaluated radiographically at 16 weeks. RESULTS: Serum OC and BS-ALP concentrations decreased from time 0 at 4 weeks, peaked at 8 weeks, and then decreased. Serum DPYR concentration peaked at 4 weeks and then decreased, independent of gene treatment group or fracture infection status. Compared with rabbits with noninfected fractures, those with infected fractures had lower serum OC and BS-ALP concentrations at 4 weeks, higher serum OC concentrations at 16 weeks, and higher serum DPYR concentrations at 4, 8, and 16 weeks. Combined serum OC, BS-ALP, and DPYR concentrations provided an accuracy of 96% for prediction of fracture infection status at 4 weeks. CONCLUSIONS AND CLINICAL RELEVANCE: Measurement of multiple serum biochemical markers of bone metabolism could be useful for clinical evaluation of fracture healing and early diagnosis of osteomyelitis.     

Evaluation of serum concentrations of biochemical markers of bone metabolism and insulin-like growth factor I associated with treadmill exercise in young horses

OBJECTIVE: To evaluate changes in serum concentrations of biochemical markers of bone metabolism and insulin-like growth factor I (IGF-I) associated with treadmill exercise in young horses. ANIMALS: 12 two-year-old Thoroughbred mares. PROCEDURE: During a 20-week study period, 6 horses were exercised on a treadmill 3 times a week (exercise group) and 6 horses received walking exercise 6 days a week (controls). Serum concentrations or activity of biochemical markers and IGF-I were assessed biweekly. Bone mineral density and content of the first phalanx were measured by dual-energy X-ray absorbiometry (DEXA) on completion of the study. RESULTS: Compared with values in controls, bone mineral density and content were higher and serum concentrations of osteocalcin (a marker of bone formation) and the carboxy-terminal telopeptide of type I collagen (a marker of bone resorption; ICTP) were lower in exercised horses. Serum concentration and activity of the bone formation markers carboxy-terminal propeptide of type I collagen and bone-specific alkaline phosphatase (BAP) were not different between the 2 groups. Serum IGF-I concentration was lower in the exercise group, compared with control values; there was a significant correlation between change in IGF-I values and changes in osteocalcin, ICTP, and BAP values at the end of the study. CONCLUSIONS AND CLINICAL RELEVANCE: Treadmill exercise over 20 weeks induced adaptive changes in bones of 2-year-old Thoroughbreds; training appears to increase bone mineral density, thereby enhancing mechanical strength of bone, but decreases bone turnover. Results indicated an association between changes in serum IGF-I concentration and bone cell activity in horses.     

Age-related changes for serum bone metabolism markers in thoroughbred and quarter horse foals

Serum bone specific alkaline phosphatase (BALP) and osteocalcin were measured in 9 Thoroughbred and 4 Quarter Horse (QH) foals. Eight were colts, and 5 were fillies. The first blood sample was collected from foals between 10 and 14 hours after birth on day 1. Blood then was collected on days 3, 6, 9, 12, 15, 18, 21, 28, 35, 42, 49, 56, 70, 84, 98, and 112 between 7:00 and 9:00 am. Serum bone metabolism marker raw data were analyzed with analysis of variance with repeated measures over time with gender and breed in the model. Average serum osteocalcin concentrations were higher for Thoroughbred than QH foals: 152.1 ± 4.6 ng/mL and 131.3 ± 6.3 ng/mL (mean ± standard error), respectively (P = .01). No overall differences were seen for gender (P = .10). However, on day 1, colts had higher osteocalcin than did fillies at 199.6 ± 30.2 ng/mL and 93.8 ± 32.4 ng/mL, respectively (P = .04). Thoroughbred foals had higher average serum BALP concentrations than did QH foals, with average values of 260.8 ± 13.4 U/L and 205.1 ± 18.5 U/L, respectively (P = .02). No gender differences were seen for serum BALP (P = .48). Serum carboxy-terminal propeptide of Type I procollagen (PICP) concentrations could not be measured in this study because the Metra Biosystems assay for PICP could not be validated.

Introduction

Bone synthesis by the osteoblast can be divided into 3 phases: proliferation, matrix development and maturation, and mineralization.¹ Gene expression of type I collagen takes place during the proliferation of the osteoblast cells. The expression of bone specific alkaline phosphatase (BALP) reaches its maximum during matrix maturation and declines as matrix mineralization starts. The osteocalcin gene is expressed during matrix mineralization.When type I collagen is produced as procollagen and released into the extracellular space, the amino and carboxyterminal propeptides of type I procollagen (PINP and PICP, respectively) are cleaved off.² Serum PICP has been shown to be a good marker for bone formation in metabolic bone diseases.³ In Thoroughbred fillies, PICP has an inverse relationship with age, with highest values found in animals less than 1 year of age.⁴ Serum alkaline phosphatase (ALP) has been measured in the young foal and is highest at birth, decreasing to a constant level by 2 months of age.^{5, 6 and 7} Serum BALP constitutes 60% to 92% of the total serum ALP in the horse and is highest in the foal.^{4 and 8} As the foal matures, there is an inverse relationship between age and serum BALP.^{4 and 9} Serum osteocalcin in foals less than 6 months of age has not been reported as having the same age-related pattern as serum BALP.¹⁰However, younger horses have higher serum osteocalcin values than mature horses.^{11, 12 and 13} Davicco et al¹⁴ showed plasma osteocalcin age-related changes for Thoroughbred foals with radioimmunoassay (RIA). Plasma osteocalcin was low at birth, increased to day 8, and then dropped to day 15. The objective of this study was to establish normal ranges and age-related changes in serum BALP, PICP, and osteocalcin in the foal with enzyme-linked immunospecific assays (ELISAs).

Materials and methods

Four Quarter Horse (QH; 2 fillies and 2 colts) and 9 Thoroughbred (3 fillies and 6 colts) foals were included in the study from birth through 112 days of age. Foals were born from February 5 to May 13, 1998. Mares and foals were housed on 40 acres of Bahiagrass (Paspalum notatum) pasture and fed a 15% crude protein (as fed) sweet feed. Body scores were recorded every 28 days on a scale from 1 to 9.¹⁵ Concentrate was fed to each mare at 1.5 kg/100 kg body weight daily and was increased by 20% for each body condition score below 5 and decreased by 20% for each body condition score above 5. Mares were individually fed in 3.6 × 3.6—m stalls twice daily, with foals allowed access to the mares feed. Trace mineral salt blocks were available in the pastures. Water was available at all times.Blood was collected from foals between 10 and 14 hours after birth on day 1. Blood then was collected on days 3, 6, 9, 12, 15, 18, 21, 28, 35, 42, 49, 56, 70, 84, 98, and 112 after morning feedings. Except for day 1, all blood samples were collected between 7:00 and 9:00 am. All blood samples were collected with jugular venipuncture into a glass vacutainer containing no additives or anticoagulants and were allowed to clot. Serum was separated and frozen at −20°C within 4 hours of collection. All samples were analyzed within 6 months of collection.The Alkphase-B immunoassay for the determination of BALP (Metra Biosystems, Mountainview, Calif) and the NovoCalcin immunoassay for determination of osteocalcin (Metra Biosystems), used in this study, have been previously validated in the horse.^{16 and 17} The Prolagen-C immunoassy for determination of the PICP (Metra Biosystems) has not been previously validated in the horse.¹⁷Serum bone metabolism marker raw data were analyzed with analysis of variance with repeated measures over time with gender and breed in the model. Analyses were performed with Statistical Analysis System with proc glm for the analysis of variances.¹⁸

Results

Average serum osteocalcin concentration for the testing period was higher for Thoroughbred than QH foals: 152.1 ± 4.6 and 131.3 ± 6.3 ng/mL (mean ± standard error), respectively (P = .01). No overall differences were seen for gender (P = .10). However, on day 1, colts had higher osteocalcin concentrations than did fillies at 199.6 ± 30.2 ng/mL and 93.8 ± 32.4 ng/mL (P = .04; Fig 1).

A comparison of methods for measuring serum and urinary markers of bone metabolism in cats

Biochemical markers of bone cell activity have recently been shown to be useful for monitoring skeletal health in domestic animals, including dogs and horses. The aim of this study was to evaluate a number of biochemical assays, originally developed for use in humans, for their ability to measure indicators of bone cell activity in serum and urine of normal cats over a range of ages. Bone alkaline phosphatase (BAP), a marker of bone formation, was measured in serum using wheatgerm lectin precipitation (WGL) and by ELISA. The curve derived from serial dilution of feline serum was parallel with the ELISA standard curve, indicating species cross-reactivity, and there was a significant relationship between assays (rs = 0.97, P < 0.001). Deoxypyridinoline (DPD), a marker of bone resorption, was measured in its total form in urine by HPLC and ELISA, and in its free form in serum and urine by ELISA. The dilution curve for free DPD in urine showed parallelism with the assay standard curve; however, the curves for total DPD in urine and serum did not. A significant relationship was established between total urinary DPD (HPLC) with total serum DPD (rs = 0.69, P < 0.001), and with free urinary DPD (rs = 0.95, P < 0.001) concentrations. Carboxy-terminal telopeptide of type I collagen (CTX) concentration, another marker of bone resorption, was measured in serum and urine by ELISA, and there was a significant relationship between assays (rs = 0.82, P < 0.001). CTX could not be measured reliably using an auto-analysis method. A significant relationship was established between total urinary DPD (HPLC) with serum CTX (rs = 0.59, P < 0.05), and urinary CTX (rs = 0.65, P < 0.001) concentrations. BAP (ELISA and WGL), total urinary DPD (HPLC), urinary CTX (ELISA), and serum CTX (ELISA) concentrations were significantly inversely correlated with age (rs = -0.66, -0.88, -0.61, -0.70, and -0.51, P < 0.05 respectively). Cats under two years of age had significantly higher BAP, total urinary DPD (HPLC), and urinary CTX concentrations compared to older cats. In conclusion, this study has shown that a number of commercially available assays provide reliable methods for non-invasively monitoring bone cell activity in cats and has shown that bone turnover decreases within the first two years of life, until complete skeletal maturity is attained. Future studies can now be directed at evaluating the potential clinical application of these methods.     

Biochemical markers of bone metabolism in draught andwarmblood horses

Concentrations of the cross-linked carboxyterminal telopeptide of type I collagen (ICTP) and osteocalcin (OC) have been determined in the serum of one hundred clinically healthy adult Draught or Warmblood horses. The correlation between these two markers has been evaluated and the influence of gender, age and type of horse described. No significant variations were observed between animals of different sex, but a significant inverse correlation (P<0.0001) with age was observed for both measured parameters. After correction for age, serum levels of OC were found to be lower in Draught [adjusted least square mean (LSM)=6.612μg. L⁻¹] than in Warmblood horses (adjusted LSM=8.596μg.L⁻¹), whereas levels of ICTP were higher in Draughts (adjusted LSM=8.035pg.L⁻¹) than in Warmbloods (adjusted LSM=6.643μg.L⁻¹). A significant correlation (P<0.0001) was observed between OC and ICTP. This correlation was stronger if the type of horse was taken into account in the statistical model. The ratio OC:ICTP was independent of gender and age. A higher OC:ICTP ratio in Warmbloods compared to the Draught horses might reflect a higher bone remodelling level of horses submitted to regular daily work. It was concluded that ICTP and OC are influenced by the type of horse, and probably reflect a physiological difference in bone remodelling between these animals.     

Bone density in the juvenile racehorse treated with exogenous somatotropin (eST)

Thirty juvenile horses were paired by age within sex, and one horse from each pair was randomly assigned to either eST treatment or to a control group. The horses were “broke” to ride and trained in a regimen typical for young racehorses. Radiographs were taken on days 0, 32, 50, 64, 82, 96 and 128 of the study. Increase in total radiographic bone aluminum equivalence (RBAE) was significantly greater in eST treated horses than in the control group (P<.003). Increase in medial RBAE was also significantly greater due to eST treatment (P<.0001). Trends were apparent for increases due to eST treatment in dorsal RBAE (P=.07) and lateral RBAE (P=.07). Bone density was selectively increased in the dorsal, lateral and medial vs. the palmar cortex in the eST treated horses.     

Serum osteocalcin or bone Gla-protein, a biochemical marker for bone metabolism in horses: differences in serum levels with age.

Levels of alkaline phosphatase and osteocalcin or bone Gla-protein, a new marker of bone metabolism, were analyzed in blood samples of 50 clinically normal female Standardbred horses between four months and twenty years of age. Samples were collected in the morning before exercise. Serum osteocalcin was measured by radioimmunoassay using bovine antibodies. There was a significant inverse correlation between alkaline phosphatase, osteocalcin and the age of the animals up to 48 months. The decrease in osteocalcin levels in serum was very marked during the first 30 months of life. The mean osteocalcin concentration was respectively 47.3, 35.7 and 6.7 ng/mL for animals less than one year, between 1.5 and 2.5 years of age and older than 3.5 years. Alkaline phosphatase serum activity was higher in foals less than one year of age (means = 856 U/L) than in the two older groups (meansII = 339, meansIII = 351 U/L). We believe that osteocalcin is a useful parameter for the evaluation of bone metabolism in growing animals and in adults and is probably more specific than alkaline phosphatase.     

Diurnal variation and age differences in the biochemical markers of bone turnover in horses

Biochemical markers of bone turnover provide sensitive, rapid, and noninvasive monitoring of bone resorption and formation. Serum concentrations of osteocalcin (OC) reflect rates of bone formation, and urinary concentrations of the pyridinium crosslinks pyridinoline (Pyd) and deoxypyridinoline (Dpd) are specific and sensitive markers of bone resorption. These markers are age-dependent and are used to detect and monitor changes in the rates of bone turnover in a variety of orthopedic diseases in humans and may prove to have similar application in horses. This study examined age differences and diurnal variation in OC, Pyd, and Dpd in eight adult geldings and seven weanling colts. Blood and urine were collected at regular intervals over 24 h. Serum OC and cortisol, and urinary Pyd and Dpd were analyzed. Mean 24-h concentrations of cortisol and all three markers were higher (P<.003) in weanlings than adults. Significant 24-h variation was observed in adult gelding OC, Pyd, and Dpd concentrations (P< .02). Adult OC concentrations were highest between 2400 and 0900; Pyd and Dpd peaked between 0200 and 0800. Similar patterns of bone turnover were observed in weanling values, but they were not significant (P>.17) owing to greater variability between individuals. Cortisol secretion varied (P<.001) over 24 h in both adults and weanlings and, thus, did not seem to be responsible for greater variability in markers of bone turnover between weanlings. These data demonstrate that diurnal rhythms exist for serum OC and urinary Pyd and Dpd in adult horses, as reported in humans, and that sample timing is an important consideration in future equine studies using these markers.     

The influence of sex on biochemical markers of bone turnover in dogs

Biochemical markers of bone turnover have been shown to be useful as inexpensive and noninvasive tools for monitoring skeletal health. The reference range for bone markers in dogs has been set by different age groups. However, other sources of biological variations were not fully investigated in dogs. To explore whether sex influences the interpretation of bone marker data we examined serum bone markers in 33 male and 25 female dogs. The bone markers selected for this study were: bone alkaline phosphatase (BALP) and osteocalcin (OC) as indicators of bone formation, and C-terminal telopeptide (CTx) of type I collagen as marker of bone resorption. All concentrations of bone markers were lower, but still within the reference range reported for dogs. We found statistically significant differences of the median OC and CTx serum concentrations between males and females. The results of this study suggest that there are sex differences in biochemical markers of bone turnover in dogs which should be considered in interpretation of bone marker data.     

Effect of longeing and glucosamine supplementation on serum markers of bone and joint metabolism in yearling quarter horses.

The effect of longeing and glucosamine supplementation on known biological markers of joint disease was studied in yearling quarter horses. Twenty-one yearling quarter horses were randomly assigned to one of 4 treatments: 1) longeing (longeing 20 min daily) supplement control (LN); 2) longeing/glucosamine (LG); 3) walking (mechanical walker for 120 min daily (WN)); and 4) walking/glucosamine (WG). Oral glucosamine was administered at 5.5 g b.i.d. weeks 1-4, 3.5 g b.i.d. during weeks 5-6, and 2.0 g b.i.d. during weeks 7-8. Serum was obtained weekly for 8 wk and analyzed for keratan sulfate and osteocalcin concentrations. Walked horses receiving glucosamine showed slight elevation in serum keratan sulfate compared to controls (P = 0.04). Glucosamine or longeing exercise had no significant effect (6 > or = 0.08) on serum osteocalcin concentrations. Under these conditions, longeing and/or glucosamine supplementation did not significantly alter serum concentrations of keratan sulfate or osteocalcin.     

Biochemical markers of bone metabolism in animals: uses and limitations

Routine assessment of the skeleton's response to disease and injury traditionally has consisted of plain-film radiography supplemented with advanced imaging techniques, such as computed tomography, magnetic resonance imaging, nuclear scintigraphy, and bone densitometry. Although these techniques provide increased sensitivity as compared with radiography, they still are limited by the fact that they can only document the net results of past skeletal activity. In contrast, serum and urinary biomarkers of bone formation and resorption provide near real-time information about bone cell activity. In this review, I describe the scientific rationale behind the use of these markers in humans and detail the efforts that have been made to adapt this technology to veterinary medicine and animal research. Commercial assay kits that are applicable to different animal species are described, and the potential limitations of the technology are discussed. The goal of this review is to provide clinical pathologists and researchers with the information needed to decide whether the use of bone markers is likely to be helpful and to select the most appropriate marker (or panel of markers) to answer a particular question.     

Relationship between plasma vitamin C and serum diagnostic biochemical markers in lactating cows

We investigated the relationship between plasma vitamin C concentration and serum levels of some diagnostic biochemical markers in 118 lactating Holstein cows. Blood sample was collected once from each cow and we measured the plasma vitamin C concentration and the serum levels of glucose, beta-hydroxybutyrate, free fatty acids, triacylglycerol, total cholesterol, albumin, total bilirubin, alkaline phosphatase, aspartate aminotransferase and gamma-glutamyltransferase. The regression of plasma vitamin C with each serum diagnostic biochemical marker indicated that the vitamin C concentration significantly decreased as glucose, alkaline phosphatase or aspartate aminotransferase level increased and as total cholesterol or albumin concentration decreased. Furthermore, the plasma vitamin C concentration was significantly lower in the cows showing that each of these marker levels was out of its reference interval than in the cows showing that the marker level was within its reference interval. The significant correlations were observed among total cholesterol, albumin, alkaline phosphatase and aspartate aminotransferase levels, to which the glucose concentration was not related. These results showed that the plasma vitamin C concentration was low in the cows that had concurrently low levels of total cholesterol and albumin, and high levels of alkaline phosphatase and aspartate aminotransferase. Therefore, a hepatic malfunction possibly decreases plasma vitamin C concentration through suppressing vitamin C production. On the other hand, the high level of glucose possibly decreases plasma vitamin C concentration through suppressing vitamin C recycling.     

Electrocardiographic anomalies in the racehorse

Extract

The veterinarian is often asked to examine the heart of the horse when its performance is below the standard that its owner knows — or hopes — that it should be. The usual complaint is that the horse has become deficient in stamina — it races well up to a point in the race, but when the gap comes and the jockey or driver tries to urge the horse through it, he finds that the gap is going faster than the horse and by the time he gets to the post there is nothing behind him but the ambulance. The clinician is usually under some pressure to provide a diagnosis and it is tempting to point to a prolonged PR interval or dropped beat. Of the 20 years I spent in practice, a good deal of my time was devoted to investigating horses with reduced exercise capacity and I must admit that the heart was only rarely involved. Perhaps this is not surprising since racehorses are not exposed to factors that cause heart disease in other species — e.g., high cholesterol-producing diets, persistent emotional tension, high circulating androgen levels, degenerative aging changes, genetic predisposition — horses are selected to eliminate this — and insufficient exercise.     

Biochemical markers of bone metabolism in growing thoroughbreds: a longitudinal study

This study describes longitudinal changes in serum levels of biochemical markers of bone cell activity in a group of 24 thoroughbred foals from birth to 18 months of age. The markers of bone formation included the type I collagen carboxy-terminal propeptide (PICP), the bone-specific isoenzyme of alkaline phosphatase (BAP), and osteocalcin (OC). Levels of the cross-linked telopeptide of type I collagen (ICTP), a marker of bone resorption, and the N-terminal propeptide of type III collagen (PNIIIP), a marker of soft tissue turnover, were also measured. Levels of all markers fell significantly between birth and 18 months of age (70-80 per cent); this decrease being most marked between 0 and 6 months. However, a transient increase in levels of the markers then occurred between 6 and 14 months of age. The timing of this increase was specific for each parameter. ICTP and OC concentrations increased between October and December. PICP concentrations increased between December and April whereas the increase in PIIINP was coincident with the peak in weight gain between April and June. Changes in BAP concentration were less distinct at this time. Season was shown to have significant effects on the biochemical markers independent from the effect of age. Concentrations of all markers decreased with increasing body weight and at any given age heavier horses had lower marker levels. These results show that biochemical markers of bone cell activity and soft tissue turnover follow characteristic patterns of change in growing thoroughbreds influenced by age, season and bodyweight. The demonstration that the reference ranges for the biochemical markers change from month to month means that single samples from individuals are of little value for monitoring bone cell activity in growing thoroughbreds.     

Diurnal variation in concentrations of various markers of bone metabolism in dogs.

OBJECTIVE: To evaluate diurnal variation in concentrations of selected markers of bone metabolism in dogs. ANIMALS: Ten 3- to 4-year-old ovariectomized Beagles. PROCEDURE: Blood and urine samples were obtained in the morning before dogs were fed (8 AM) and then at 2-hour intervals for 24 hours. This procedure was repeated 2 weeks later. Concentrations of osteocalcin (OC) and carboxy terminal telopeptide of type-I collagen (ICTP) were measured in serum, using a radioimmunoassay; concentrations of hydroxyproline (HYP), pyridinoline (PYD), and deoxypyridinoline (DPD) were analyzed in urine. Hydroxyproline concentration was measured by means of a colorimetric test, whereas PYD and DPD concentrations were quantified by use of high-performance liquid chromatography. RESULTS: In both parts of the study, HYP concentrations increased significantly, compared with values before feeding, until 8 hours after feeding; HYP concentrations then returned to prefeeding values. Concentrations of DPD and PYD decreased from before feeding until 2 PM and then increased until 8 PM. The ICTP concentrations slowly decreased until 4 PM but returned to prefeeding values thereafter. In both parts of the study, concentrations of OC decreased during the day and then increased to reach values similar to those obtained before feeding. CONCLUSIONS: Changes in the concentrations of bone markers were detected throughout the day in the dogs of this study. Increase in HYP concentration most likely was related to feeding. As documented for bone resorption and formation in other species, circadian rhythms were evident for concentrations of DPD, PYD, and OC. Investigators should consider the time of sample collection when measuring these markers.     

Orthopedic surgery in the racehorse

This article attempts to address what the authors consider to be the major orthopedic problems in the racehorse, with particular attention to their treatment and prognosis. These problems include fractures, osteochondral fragments, synovitis, degenerative joint disease, tendinitis, desmitis, osteochondritis dissecans, and subchondral cystic lesions of the joints.