Circadian variation in biochemical markers of bone cell activity and insulin-like growth factor-I in two-year-old horses

Studies in humans have found circadian 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 concentrations of three biochemical markers of bone cell activity 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 technique, which fits a 24-h cycle to each dataset. A significant 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 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 concentrations 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. Further studies are now required to determine the potential association between circadian changes in IGF-I and osteocalcin in the horse. Although no significant circadian variation was found in concentrations of the car-boxy-terminal propeptide of type-I collagen and the carboxy-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.     

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.     

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.     

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 modulation and sources of variation affecting ventricular repolarization in Warmblood horses

ObjectivesIrregularities in cardiac repolarization are known to predispose for arrhythmias and sudden cardiac death in humans. The QT interval is a quantitative measurement of repolarization, and clinically, the QT_c (QT interval corrected for heart rate) and T_peak to T_end intervals (T_pT_e) are used as repolarization markers. To support the use of these markers in horses, we sought to describe the possible influence of the environment, time of day, day-to-day effects, T wave conformation, age, body weight (BW), and horse-to-horse variation on repolarization measurements.Animals12 Warmblood geldings, age 10.8 ± 4.8 years.MethodsHolter ECGs were performed on days 0, 7 and 14. Measures of RR, QT, QT_p, QT_c and T_pT_e intervals and T wave conformation were obtained each hour during the recordings. An ANCOVA analysis was performed to estimate diurnal variation and the sources of variation affecting these intervals.ResultsDifferences between individual horses were the largest source of repolarization variability although the environment had a significant effect on repolarization as well. Diurnal variation affected both the RR interval and the repolarization markers. The QT, QT_c and T_pT_e intervals were prolonged on day 0. Biphasic T waves shortened the T_pT_e interval approximately 10 ms. Age and BW did not appear to affect repolarization.ConclusionsEquine repolarization markers exhibit significant variation. Factors affecting repolarization measurements include horse-to-horse variation, diurnal variation, the environment, and T wave conformation. These factors must be considered if markers of equine repolarization are used diagnostically.     

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.     

Diurnal variation of dynamic compliance, resistance and viscous work of breathing in normal horses and horses with lung disorders

Dynamic compliance, airway resistance, viscous work and respiratory frequency were measured at intervals in horses over 12 h periods. Variation, daily averages and circadian patterns were recorded. Examinations were performed on 24 horses. They were divided into three groups: Normal horses, horses with chronic latent bronchitis and horses with manifest chronic obstructive pulmonary disease (COPD). Significant differences were found between the daily averages and the fluctuations when the normal group and the COPD group were compared. In most cases differences between all three groups were significant. A significant circadian rhythm for airway resistance was detected in diseased horses. It was also established that single measurements give an incomplete picture of lung function. As has been established in humans, analysis of fluctuations in respiratory mechanics may be of diagnostic value. The above results should be taken into account particularly in trials with bronchodilators in horses with COPD.     

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.     

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.     

Biochemical markers of bone turnover in the dairy cow during lactation and the dry period

We measured a bone-formation marker recognizing osteocalcin, and a bone-resorption marker recognizing C-telopeptide (CT(x)) fragments of collagen type I, in a longitudinal study. The levels of these markers in the plasma of dairy cows (n=11) were recorded over a 12 month postpartum period, including a full lactation and a dry period. The plasma concentration of CT(x) was highest in the first week after parturition. It then declined slowly over the next 33 weeks and remained low until the next parturition. Osteocalcin concentration was lowest around parturition, reached a plateau during mid-lactation, then fell again towards term. There were large variations in bone metabolism during a lactation, that were not directly related to milk production. These results may be used to facilitate appropriate adjustments to calcium and phosphorous concentrations in the diet, reflecting the specific needs of each stage of the reproductive cycle.     

Response of serum biochemical markers of bone metabolism totraining in the juvenile racehorse

Sixteen Quarter Horse-type geldings were used to examine the response of biochemical markers of bone metabolism to forced exercise prior to and during race training. The study began when the average age of the horses was 15 months. Horses were exercised on a high-speed treadmill for 14 weeks, and were subsequently placed into race training. Serum was collected and assayed for concentrations of osteocalcin (BGP), the carboxyterminal telopeptide of type I collagen (ICTP) and the carboxyterminal propeptide of type I procollagen (PICP). When data were normalized from the onset of race training, ICTP and PICP concentrations were higher in the pre-exercised horses (P<.05 and P<.1, respectively) indicating higher rates of bone turnover. Overall, bone turnover appeared to be decreased during race training, as concentrations of PICP and ICTP were lower when compared to values seen during the pre-training Phase.     

Diurnal and episodic variations of plasma hydrocortisone concentrations in horses

Using a specific high-performance liquid chromatographic technique, plasma hydrocortisone values were measured hourly in 6 horses and every 10 minutes in 4 horses over 24 hours. Both circadian and episodic variation was observed. The mean plasma hydrocortisone concentration was a maximum of 58.8 ± 9.54 ng/ml at 9.19 ± 0.59 hr and a minimum of 27.85 ± 6.85 g/ml at 21.19 ± 0.59 hr. The number of episodes of secretion was 10.0 ± 1.41; the mean amplitude and duration of a peak were 26.21 ± 3.71 ng/ml and 105.25 ± 21.24 min respectively.     

Changes in some biochemical indicators of bone turnover after ultraviolet irradiation of dairy cows

The effect of ultraviolet irradiation on some biochemical indicators of bone turnover in dairy cows was determined. The irradiation was performed using a stationary system for two months and comprised a regimen of 10 days irradiation followed by 10 days rest. After ultraviolet irradiation, significant differences in the activities of serum alkaline phosphatase (P < 0.001) and bone alkaline phosphatase isoenzyme (P < 0.05) and concentration of osteocalcin (P < 0.01) were demonstrated. The results suggest that supplementary ultraviolet irradiation during winter could be used as a simple but reliable method of preventing the development of generalised metabolic bone disorders in dairy cows.     

Breed, age, and gender differences in plasma antithrombin-III activity in clinically normal young horses

Plasma antithrombin-III activity was quantitated in plasma samples obtained from 165 clinically normal horses 3 years old or younger. In the horses as a group, antithrombin-III activity ranged from 63 to 131% of a species-specific reference plasma. Thoroughbred horses had significantly higher antithrombin-III activity (103.3 +/- 18.3; mean +/- SD) than did Standardbred horses (92.3 +/- 14.2). The plasma antithrombin-III activities were significantly lower in horses younger than 16 months old when compared with those in more mature horses (3 years old). There was no statistically significant gender-related effect on plasma antithrombin-III activity. Breed and age factors should therefore be taken into consideration when interpreting plasma antithrombin-III activity in horses.     

Investigation of selected biochemical indicators of Equine Rhabdomyolysis in Arabian horses: pro-inflammatory cytokines and oxidative stress markers

A total of 30 horses were divided into two groups, one served as a control whereas other was rhabdomyolysis diseased horses. After blood collection, the resulted sera were used for estimation of the activities of creatin kinase (CK), aspartate transaminase (AST), lactate dehydrogenase (LDH), lactic acid, triacylglycerol (TAG), glucose, total protein, albumin, globulin, urea, creatinine, Triiodothyronine (T₃), calcium, sodium, potassium, phosphorus, chloride, vitamin E, interleukin-6 (IL-6) and tumor necrosis-α (TNF-α). In addition, whole blood was used for determination of selenium, reduced glutathione (G-SH) and prostaglandin F2-α (PGF2α). The erythrocyte hemolysates were used for the determination of the activities of super oxide dismutase (SOD), catalase (CAT), total antioxidant capacity (TAC), nitric oxide (NO) and malondialdehyde (MDA). The present findings revealed a significant (p ≤ 0.05) increase in the values of CK, AST, LDH, glucose, lactate, TAG, urea, creatinine, phosphorus, MDA, TNF- α, IL6 and PGF2- α in diseased horses when compared with the control. Furthermore, the values of calcium, SOD, CAT, TAC, NO and GSH in diseased horses were significantly (p ≤ 0.05) lower than the control. The other examined parameters were not statistically significant. In conclusion, the examined pro-inflammatory cytokines were useful biomarkers for the diagnosis of Equine rhabdomyolysis (ER) in Arabian horses beside the old examined biomarkers. In the future, efforts should be made to confirm this in other breed. If this could be achieved, it would open up new perspectives in research fields dealing with ER.     

Biochemical markers of bone activity in young standardbred horses during different types of exercise and training

Seven untrained Standardbred horses were used in a training programme of 6 weeks to evaluate the effects of exercise and training on bone metabolism. The horses were exercised on a treadmill according to a standardized exercise test (SET 1: six incremental steps, 5 min duration each; start 5 m/s, increase 1 m/s). SET 1 was followed by a training programme of 6 weeks. In alternating order: high-speed exercise (HSE): 15 min duration, start at VLa4, continuous increase in speed every 60 s by 0.3 m/s (14 incremental steps); low-speed exercise (LSE): constant velocity at VLa2.5, duration: approximately 60-90 min (total training programme: eight HSE and eight LSE sessions). SET 2 finished the training programme and a deconditioning period of 12 weeks followed. Blood samples for lactate, total plasma protein (TPP), osteocalcin, and ICTP (cross-linked C-telopeptide of type I collagen) were collected. ICTP increased during SET 1 and SET 2, whereas osteocalcin decreased to below resting concentration 24 h after SET 1. A rise in ICTP was observed during LSE 1 and LSE 8, which was followed by a drop 24 h after exercise. No changes in osteocalcin were noted during LSE 1, but 24 h after LSE 1 osteocalcin dropped to below pre-exercise levels. LSE 8 resulted in an increase in osteocalcin, followed by a drop 24 h after LSE 8. Osteocalcin and ICTP were not affected by HSE. Baseline osteocalcin levels dropped during the course of training. The acute response of biochemical bone markers indicates a direct influence of a single bout of exercise on bone metabolism.