| Abstract: | 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. IntroductionBone 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 methodsFour 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 ResultsAverage 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).Fig. 1. Serum osteocalcin (OC) over time (mean ± standard error). A, Changes over time between breeds. B, Changes over time between gender. P < .05. The intraassay coefficient of variation (CV) was 2.3%, 4.4%, and 10.3% for 4.7, 20.7, and 159.4 ng/mL osteocalcin serum pools. Interassay CV was 5.4%, 4.8%, and 6.1% for 2.3, 6.4, and 24.1 ng/mL osteocalcin serum pools.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). Daily serum BALP breed differences were detected only on days 12 and 112, with Thoroughbred foals having higher values than QH foals at 240.4 ± 18.4 U/L versus 168.6 ± 24.2 U/L (P = .05) and 172.3 ± 14.3 U/L versus 107.6 ± 23.2 U/L (P = .05; Fig 2).
Fig. 2. Serum BALP over time (mean ± standard error). A, Changes over time between breeds. B, Changes over time between genders. P < .05. No gender differences were seen (P = .48; Fig 2). The intraassay CV was 3.6%, 2.8%, and 4.7% for the 51.9, 139.4, and 401.8 U/L BALP serum pools. Interassay CV was 5.78%, 11.8%, and 13.7% for the 15.8, 71.3, and 145.6 U/L BALP serum pools.The Prolagen-C immunoassay procedure for the determination of PICP used in this study could not be validated. Linearity for serial dilutions of serum samples could not be shown. Therefore, no PICP data are shown. DiscussionEarly age-related changes in plasma osteocalcin have been previously reported for the foal with RIA.14 Plasma osteocalcin levels were low at birth, increased to day 8, and then dropped to day 15. This study supports those trends in plasma levels of osteocalcin. However, as Hoyt and Siciliano16 observed, serum osteocalcin values determined with the immunoassay (Metra Biosystems) were higher than those observed with RIA. The antibody specificities may be different between the 2 assays. Thoroughbred foals had higher average serum osteocalcin than QH foals, which supports findings that serum osteocalcin differs among breeds.19 No gender differences had previously been reported for serum osteocalcin in horses of different ages, but when foals are stressed by weaning or exercise, gender differences were seen.12 and 20 Although no overall gender differences were seen in this study, on day 1, colts had higher serum osteocalcin than fillies. Plasma cortisol is high in the newborn foal.21 Although no serum cortisol was measured in this study, there may be different levels of cortisol or different responses to cortisol between the colts and fillies as a result of foaling. Because glucocorticoid administration results in suppressed serum osteocalcin in the horse,22 it would be of great value to understand the relationship between gender and cortisol in the neonatal foal.Serum BALP (making up most of serum ALP) values in the neonatal foal, extensively documented in this study, are in agreement with the measurements of serum ALP established in earlier reports.5, 6 and 7 Serum BALP is high at birth and decreases to a lower level by 2 months of age. Average serum BALP is higher in Thoroughbred foals than QH foals, and there are no gender serum BALP differences. No other reports for breed or gender differences concerning serum BALP in the horse have been documented. Likewise, in newborn humans, no serum BALP gender differences have been observed for the first 10 weeks of life.23Price24 and Jackson et al9 used the radioimmunoassay provided by Orion Diagnostica to determine PICP concentrations.4 and 9 Because the Metra Biosystems PICP procedure could not be validated in this study, no comparison with reported data could be made. ConclusionSerum BALP and osteocalcin concentrations were measured during the first 112 days of age with age, gender, and breed ranges for the foals being established when ELISA assays are used (Metra Biosystems). Because the total number of animals was small and variability of the data was large, the statistical power to detect meaningful differences for gender and breed was small. However, the data presented show trends of serum bone formation markers, some statistical differences for gender and breed, and variability of the foal during the first 112 days of age. In agreement with Price,24 a single measurement of a serum bone metabolism marker is of little clinical value, especially for the young foal where the variability is high. Because RIA ranges are typically lower than with ELISA assays for serum osteocalcin, the type of assay used should be considered when comparing serum osteocalcin levels between experiments. For use of serum markers to assess bone metabolism in the foal, the relationship of these markers with foal maturation, endocrinology, and skeletal growth needs to be resolved. |
