Circadian Rhythms of Osteocalcin in Equine Serum. Correlation with Alkaline Phosphatase, Calcium, Phosphate and Total Protein Levels Olivier M. Lepage, Luc DesCoteaux, Marcel Marcoux and Armand Tremblay
ABSTRACT The purpose of the study was to determine whether there were circadian variations in serum osteocalcin in normal horses and to determine whether it was important to regulate the time of blood sampling in clinical investigations. Osteocalcin or bone Gla-protein (BGP), alkaline phosphatase, total calcium, phosphate and total protein were studied over a 24 h period. Blood samples were taken every 60 min from nine adult Standardbred horses. There was a correlation between serum levels of alkaline phosphatase (r = 0.3, p < 0.01), phosphate (r = 0.42, p < 0.01) and serum osteocalcin levels. There was a very marked invididual effect on serum levels of osteocalcin and alkaline phosphatase (p < 0.01). This effect was present for phosphate levels but not significant for total calcium. The individual effect was lower and time effect was higher for serum osteocalcin if the subjects were divided into two age groups, one of horses of five years or less (n = 4) and a second group older than five years (n = 5). In both groups a circadian rhythmicity was observed. Serum osteocalcin showed a biphasic pattern. Levels were constant during daytime (light period) and underwent significant variations during the night (dark period), going through a nadir at 2000 h and through a maximum peak at 0500 h. It was concluded that in
normal horses the blood osteocalcin level follows a circadian variation. Also daytime (light period) seems to be the more appropriate period for blood sampling.
RESUME
Le but du travail consistait a verifier l'existence d'une variation circadienne des niveaux seriques d'osteocalcine et ainsi de determiner le moment le plus opportun pour effectuer un prelevement sanguin dans le cas d'une investigation clinique. Les dosages seriques d'osteocalcine ou "bone Glaprotein (BGP)", la phosphatase alcaline, le calcium total, le phosphate et les proteines totales ont ete effectuees aux 60 minutes pendant 24 heures chez neuf chevaux adultes de race Standardbred. Une correlation a ete retrouvee entre le taux serique de phosphatase alcaline (r = 0.3, p > 0.01) et le taux d'osteocalcine serique. D'importantes variations individuelles des niveaux seriques d'osteocalcine et de phosphatase alcaline (p < 0.01) ont ete decelees. Cet effet etait modere pour le phosphate et nonsignificatif pour le calcium. Suivant la creation de deux groupes selon l'age, ceux de cinq ans et moins (n = 4) et ceux de plus de cinq ans (n = 5), on a observe une diminution de l'effet individuel et une augmentation de l'effet temps. Dans les deux cas, un rythme circadien biphasique a ete
observe pour l'osteocalcine: Les niveaux d'osteocalcine etaient stables le jour (periode claire) tandis que des variations significatives (p < 0.05) etaient observees la nuit (periode sombre passant par un minimum aux environs de 2000 h et un maximum aux environs de 0500 h). Les resultats de cette etude demontrent qu"il existe un rythme circadien pour l'osteocalcine et que le jour (periode claire) semble la meilleure periode de prelevement dans le cas d'investigations cliniques afin de determiner les taux seriques de cette proteine.
INTRODUCTION It has been established that collagen synthesis (1) and new bone formation are diurnally regulated (2). Biorhythmic profiles in the rat skeleton were studied (3). At the same time circadian variation of serum albumin, phosphate, growth hormone, parathyroid hormone, calcium (4) and ionized calcium (5,6) has been well documented in normal human adults or in adolescent subjects (7). Osteocalcin or bone Gla-protein (BGP) is a small abundant noncollagenous calcium binding protein, indiginous to the organic matrix of bone dentin and prossibly other mineralized tissues (8), which circulates in the blood (9). This protein is synthesized by osteoblasts and odontoblasts (10,11) and several reports
Departement de Medecine (Lepage, DesCoteaux, Marcoux) and Laboratoire de Biochimie (Tremblay), Faculte de Medecine veterinaire, Universite de Montreal, C.P. 5000, Saint-Hyacinthe (Quebec) J2S 7C6. Reprint requests to Dr M. Marcoux. The research was supported by the Groupe de Recherche en Medecine Equine du Quebec (GREMEQ) and by the Fonds du Centenaire. Submitted April 6, 1990.
Can J Vet Res 1991; 55: 5-10
indicate that measurements of circulating osteocalcin may reflect osteoid production (12,13) or bone turnover in humans (14-16). The measurement of serum osteocalcin has also been applied to several metabolic bone diseases (17). Since diurnal rhythms in blood mineral concentrations and in the metabolic activity of bone tissue have been demonstrated, the circadian rhythm of osteocalcin in the serum and its correlation with mineral variation in normal (18), in pathological (19) and in treated human subjects (20) have been studied. A normative model of the circadian rhythm in osteocalcin levels was described by a third order polynomial equation (18). The purpose of this research was to determine whether there were circadian variations in serum osteocalcin values in normal horses and to determine whether it was important to regulate the time of blood sampling for osteocalcin determinations in clinical investigations of metabolic disease. Circadian variations of the concentrations of alkaline phosphatase, calcium, phosphate and total protein were also described and correlations between those biochemical variables and osteocalcin were verified. MATERIALS AND METHODS
Experiments followed guidelines equivalent to those in "Guide to the Care and Use of Experimental Animals", Volumes 1 and 2, Canadian Council on Animal Care. ANIMALS
Nine healthy adult Standardbred horses aged between 2 and 17 yr as listed in Table I were used. One stallion, six pregnant mares and two nonpregnant mares came from the same farm. All pregnant mares were in their fourth or fifth month of gestation. The horses were not on a training program during the month before the experiment and activity was restricted to movements in the box stall during the experimental phase. None of the animals were under 6
medication and no bone related disease had been diagnosed during the previous year. The subjects were adapted to their box stall for two days. SAMPLES
The jugular vein of each subject was catheterized using a 14 G 2-¼/4 inch teflon catheter (Cathlon IV, catheter placement unit, Jelco Laboratories, Raritan, New Jersey) at noon before the first sampling. Blood was sampled every 60 min from 0800 h to 0800 h on the next day. Water was given ad lib and meals were given at 0400 h, 100 h and 1600 h. The lights in the box stall were turned off during the time of the experiment and the animals were subjected to sunlight variations. The sun rose at 0652 h and set at 1830 h. During the night we approached the horses with a small hand light to identify the catheter. All samples were collected into glass tubes (Vacutainer tubes, Becton, Dickinson & Co., Rutherford, New Jersey). Blood samples were allowed to clot, and the serum was then obtained by centrifugation 1 to 2 h after sampling and stored frozen at -25° C for 3 wk before being analyzed. ANALYSIS
Serum concentrations of osteocalcin were measured by radioimmunoassay (Osteocalcin Radioimmunoassay Kit, Cat. #15065, Incstar Corporation, Stillwater, Minnesota) according to the manufacturer's instructions. More details have been previously described (21). The intraassay coefficient of variation was less than 5%. Serum alkaline phosphatase (ALP) activity was also determined (Automated Analysis, Boehringer Mannheim Phosphatase alcaline opt., Mannheim, West Germany). In each serum sample, total calcium (Cat. # 857825, Boehringer Mannheim), phosphate (Cat. # 836281, Boehringer Mannheim) and total protein (Cat. # 704105, Boehringer Mannheim) concentrations were measured. The method of Jaffe (kinetic) without deproteinization was used for creatinine determination (Automated Analysis, Boehringer Mannheim, West Germany)).
DATA ANALYSIS
The horses were divided in two age groups, animals of five years or less and those older than five years. Pearson's correlation was used to examine the relationship between total calcium, phosphate, alkaline phosphatase, oesteocalcin and total
protein. A general linear model with repeated measures (22,23) was used to study the individual effect and time effect between two consecutive hours during the 24 h period for the biochemical variables.
RESULTS The mean concentration values for each individual and for each biochemical variable throughout the 24 h study are listed in Table I. Serum creatinine levels were always in the normal range, between 90 and 150 ,umol/L. Osteocalcin showed an age dependent level, being higher in the youngest horses as described previously (21). Every 60 min for a 24 h period, mean concentrations for the nine horses were determined for each biochemical variable as shown in Fig. 1. Serum osteocalcin levels were not significantly different (p > 0.05) (Table II) during the day from 0700 to 1900 h. The levels fell in the early evening between 1900 and 2000 h, gradually rose during the night, reached a peak at 0500 h and finally dropped to a constant daytime level around 0700 h. A marked individual effect on the serum osteocalcin level was observed (p < 0.01). If the subjects were divided into two age groups (I and II) (Table I), this individual effect was less marked, but the effect of time on serum osteocalcin levels was higher. Group I, horses of five years or less (n = 4) had mean serum osteocalcin levels equal to or higher than 14 ng/ mL, and group II, horses older than five years (n = 5) had serum osteocalcin levels lower than 14 ng/ mL. The circadian pattern was the same (Fig. 2) in both groups. If we compare variations between two consecutive hours during the 24 h period for both groups of age no
TABLE I. Mean values ± SD of 24 hour biochemical data and characteristics of each subject Age Groupa No. Sex (yr) Pregnantb I 1 2.5 F NP I 2 4 F NP I 3 4 P F I 4 5 F P II 5 P 6 F II 6 7 P F II 7 M 7 II 8 9 F P 11 9 17 P F aGroup 1: less than 5 yr; group II: more than 5 yr bp: pregnant; NP: nonpregnant cOsteocalcin dAlkaline phosphatase eTotal calcium fPhosphate
significant variations were observed between 0700 h and 1900 h (p > 0.05) (Table II). This period corresponds approximately to the light period which extended from 0700 to 1830 h. Significant fluctuations (p < 0.05) were observed during darkness from 2000 to 0600 h except between 0300 and 0400 h (Fig. 2). No effect of the time of feeding and the circadian rhythmicity of osteocalcin levels (Fig. 2) was noted. Marked fluctuations were observed during the 24 h period for total serum alkaline phosphatase levels, which showed a minimum at 1400 and 0200 h and maximum concentration levels at 1600 and 0500 h (Fig. 1). A significant correlation (r = 0.3, p < 0.01) was observed between osteocalcin concentration and serum levels of alkaline phosphatase. A marked individual effect on alkaline phosphatase concentrations was also observed (p 0.05) when values were compared at concurrent time points. No significant individual effect was observed for total calcium level (p > 0.05).
BGPc (ng/mL ± SD) 20.3+2.0 17.4±3.0 15.7±2.1 14.5 ± 2.4 6.5 ± 0.6 10.7 ± 1.0 13.8± 1.4 6.8±0.9 9.1 ± 1.5
ALPd (U/L ± SD) 297±29 385±25 286±9 247 ± 12 226 ± 12 377 ± 15 255±33 201 ±9 361 ± 34
Serum phosphate showed the lowest levels at noon and at 2000 h. Two peaks were reached, one at 1600 h and another at 0400 h. A correlation with osteocalcin was present (r = 0.42; p < 0.01) but no significant correlation was observed between total calcium and phosphate values (p > 0.05).
DISCUSSION In 1979, Simmons and collaborators (3) studied the biorhythmic profiles of the rat skeleton and concluded that the metabolic activities of the cells in the linear bone growth and appositional bone growth apparatus were not in phase. The growth apparatus appeared to be day active and the cells in the articular cartilage and diaphyseal bone seemed metabolically most active during the first 4 h of darkness although mineralization proceeded most actively at night in both tissues. The current study indicates that serum osteocalcin, a specific product of osteoblasts, the circulating levels of which reflect the rate of bone turnover
(24), undergoes circadian rhythmicity in normal adult horses of different ages. This was already well documented in humans (18). The creatinine level was determined to reflect renal function of the horses. All the values collected were in the normal range. A defect in the renal function would be responsible for an increase in serum
CaTe (mmol/L ± SD) 3.1 ±0.1 3.1 ±0.1 3.1 ±0.1 3.1 ± 0.1 3.0 ± 0.1 3.1 ± 0.2 3.1 ±0.1 3.1 ±0.1 3.0 ± 0.2
pf
(mmol/L ± SD) 1.1 0.1 1.1 0.1 1.1 0.1 1.3 0.2 1.0 0.1 1.0 0.1 0.9±0.1 0.9±0.1 0.9 0.1
osteocalcin level as the primary mode of clearance is kidney filtration(25). Serum osteocalcin decreased with age. This observation was previously described in female Standardbred horses (21) and in humans in which the normal range of osteocalcin is high in children and declines to adult levels with completion of puberty (26). We interpret these data in horses as indicating a significant slowdown in the rate of bone formation in adults compared to foals. To decrease the effect of age in the interpretation of our data the horses were divided into two age groups, young horses of five years or less and adults older than five years. Blood samples were taken every 60 min in the nine Standardbred horses to determine if circadian variations in serum osteocalcin values are present in normal horses and if correlations existed with serum calcium, phosphate and alkaline phosphatase. Mean serum concentrations of osteocalcin showed a biphasic time pattern (Figs. 1 and 2). The light or day time pattern seemed to correspond to a relatively constant level of osteocalcin and the dark or night time pattern revealed important variations of osteocalcin levels, with lowest levels in early evening between 1900 and 2000 h and a peak at 0500 h. On the day of the experiment the sun rose at 0652 h and set at 1830 h. It was interesting to note that serum osteocalcin levels began to decline at 1700 to 1900 h. The decline was then followed by a progressive rise
7
OSTEOCALCIN (ng/mL'
16r 15
14
13 12 11
101 8
340 r
12
individual differences observed on serum osteocalcin levels as mainly due to the influence of age (21). If the subjects are divided into two age groups (I and II) (Fig. 2), no significant differences (p > 0.05) between subjects of the same group could be observed but the older group had a lower mean level as described in the literature (21). A circadian rhythm for osteocalcin has been described already in male or female humans (18), in which osteocalcin levels fell during the morning, rose in the afternoon and early evening and reached a peak between 0400 and 0800 h, as was observed in this study. A pulsatile pattern of osteocalcin concentration was revealed by an increased frequency of blood sampling and this may be related to secretion or clearance of the protein. The heterogeneity of our group, one male, two nonpregnant mares and six pregnant mares might also have influenced the osteocalcin levels. For example, the male which was seven years old (Table I) had a higher mean level of osteocalcin compared to the other members of the group. Differences with sex were observed in humans (28) and an influence of pregnancy and lactation (29) was also described. Diurnal variations in bone metabolism in the rat (1) were well documented and since circulating levels of osteocalcin reflect bone turnover (30) it has been proposed that variations in the circulating pool may be linked to
TABLE II. Significance of variations in serum osteocalcin levels between consecutive hours during the 24 hour period in age groups
16
20
24
8
4
ALKALINE PHOSPHATASE (U/L)
330 320
Sampling No. 1-12 13 14 15 16 17 18 19 20 21 22 23 24
Hour 08:00-19:00 20:00 21:00 22:00 23:00 24:00 01:00 02:00 03:00 04:00 05:00 06:00 07:00
Significance NS p
ABSTRACT The purpose of the study was to determine whether there were circadian variations in serum osteocalcin in normal horses and to determine whether it was important to regulate the time of blood sampling in clinical investigations. Osteocalcin or bone Gla-protein (BGP), alkaline phosphatase, total calcium, phosphate and total protein were studied over a 24 h period. Blood samples were taken every 60 min from nine adult Standardbred horses. There was a correlation between serum levels of alkaline phosphatase (r = 0.3, p < 0.01), phosphate (r = 0.42, p < 0.01) and serum osteocalcin levels. There was a very marked invididual effect on serum levels of osteocalcin and alkaline phosphatase (p < 0.01). This effect was present for phosphate levels but not significant for total calcium. The individual effect was lower and time effect was higher for serum osteocalcin if the subjects were divided into two age groups, one of horses of five years or less (n = 4) and a second group older than five years (n = 5). In both groups a circadian rhythmicity was observed. Serum osteocalcin showed a biphasic pattern. Levels were constant during daytime (light period) and underwent significant variations during the night (dark period), going through a nadir at 2000 h and through a maximum peak at 0500 h. It was concluded that in
normal horses the blood osteocalcin level follows a circadian variation. Also daytime (light period) seems to be the more appropriate period for blood sampling.
RESUME
Le but du travail consistait a verifier l'existence d'une variation circadienne des niveaux seriques d'osteocalcine et ainsi de determiner le moment le plus opportun pour effectuer un prelevement sanguin dans le cas d'une investigation clinique. Les dosages seriques d'osteocalcine ou "bone Glaprotein (BGP)", la phosphatase alcaline, le calcium total, le phosphate et les proteines totales ont ete effectuees aux 60 minutes pendant 24 heures chez neuf chevaux adultes de race Standardbred. Une correlation a ete retrouvee entre le taux serique de phosphatase alcaline (r = 0.3, p > 0.01) et le taux d'osteocalcine serique. D'importantes variations individuelles des niveaux seriques d'osteocalcine et de phosphatase alcaline (p < 0.01) ont ete decelees. Cet effet etait modere pour le phosphate et nonsignificatif pour le calcium. Suivant la creation de deux groupes selon l'age, ceux de cinq ans et moins (n = 4) et ceux de plus de cinq ans (n = 5), on a observe une diminution de l'effet individuel et une augmentation de l'effet temps. Dans les deux cas, un rythme circadien biphasique a ete
observe pour l'osteocalcine: Les niveaux d'osteocalcine etaient stables le jour (periode claire) tandis que des variations significatives (p < 0.05) etaient observees la nuit (periode sombre passant par un minimum aux environs de 2000 h et un maximum aux environs de 0500 h). Les resultats de cette etude demontrent qu"il existe un rythme circadien pour l'osteocalcine et que le jour (periode claire) semble la meilleure periode de prelevement dans le cas d'investigations cliniques afin de determiner les taux seriques de cette proteine.
INTRODUCTION It has been established that collagen synthesis (1) and new bone formation are diurnally regulated (2). Biorhythmic profiles in the rat skeleton were studied (3). At the same time circadian variation of serum albumin, phosphate, growth hormone, parathyroid hormone, calcium (4) and ionized calcium (5,6) has been well documented in normal human adults or in adolescent subjects (7). Osteocalcin or bone Gla-protein (BGP) is a small abundant noncollagenous calcium binding protein, indiginous to the organic matrix of bone dentin and prossibly other mineralized tissues (8), which circulates in the blood (9). This protein is synthesized by osteoblasts and odontoblasts (10,11) and several reports
Departement de Medecine (Lepage, DesCoteaux, Marcoux) and Laboratoire de Biochimie (Tremblay), Faculte de Medecine veterinaire, Universite de Montreal, C.P. 5000, Saint-Hyacinthe (Quebec) J2S 7C6. Reprint requests to Dr M. Marcoux. The research was supported by the Groupe de Recherche en Medecine Equine du Quebec (GREMEQ) and by the Fonds du Centenaire. Submitted April 6, 1990.
Can J Vet Res 1991; 55: 5-10
indicate that measurements of circulating osteocalcin may reflect osteoid production (12,13) or bone turnover in humans (14-16). The measurement of serum osteocalcin has also been applied to several metabolic bone diseases (17). Since diurnal rhythms in blood mineral concentrations and in the metabolic activity of bone tissue have been demonstrated, the circadian rhythm of osteocalcin in the serum and its correlation with mineral variation in normal (18), in pathological (19) and in treated human subjects (20) have been studied. A normative model of the circadian rhythm in osteocalcin levels was described by a third order polynomial equation (18). The purpose of this research was to determine whether there were circadian variations in serum osteocalcin values in normal horses and to determine whether it was important to regulate the time of blood sampling for osteocalcin determinations in clinical investigations of metabolic disease. Circadian variations of the concentrations of alkaline phosphatase, calcium, phosphate and total protein were also described and correlations between those biochemical variables and osteocalcin were verified. MATERIALS AND METHODS
Experiments followed guidelines equivalent to those in "Guide to the Care and Use of Experimental Animals", Volumes 1 and 2, Canadian Council on Animal Care. ANIMALS
Nine healthy adult Standardbred horses aged between 2 and 17 yr as listed in Table I were used. One stallion, six pregnant mares and two nonpregnant mares came from the same farm. All pregnant mares were in their fourth or fifth month of gestation. The horses were not on a training program during the month before the experiment and activity was restricted to movements in the box stall during the experimental phase. None of the animals were under 6
medication and no bone related disease had been diagnosed during the previous year. The subjects were adapted to their box stall for two days. SAMPLES
The jugular vein of each subject was catheterized using a 14 G 2-¼/4 inch teflon catheter (Cathlon IV, catheter placement unit, Jelco Laboratories, Raritan, New Jersey) at noon before the first sampling. Blood was sampled every 60 min from 0800 h to 0800 h on the next day. Water was given ad lib and meals were given at 0400 h, 100 h and 1600 h. The lights in the box stall were turned off during the time of the experiment and the animals were subjected to sunlight variations. The sun rose at 0652 h and set at 1830 h. During the night we approached the horses with a small hand light to identify the catheter. All samples were collected into glass tubes (Vacutainer tubes, Becton, Dickinson & Co., Rutherford, New Jersey). Blood samples were allowed to clot, and the serum was then obtained by centrifugation 1 to 2 h after sampling and stored frozen at -25° C for 3 wk before being analyzed. ANALYSIS
Serum concentrations of osteocalcin were measured by radioimmunoassay (Osteocalcin Radioimmunoassay Kit, Cat. #15065, Incstar Corporation, Stillwater, Minnesota) according to the manufacturer's instructions. More details have been previously described (21). The intraassay coefficient of variation was less than 5%. Serum alkaline phosphatase (ALP) activity was also determined (Automated Analysis, Boehringer Mannheim Phosphatase alcaline opt., Mannheim, West Germany). In each serum sample, total calcium (Cat. # 857825, Boehringer Mannheim), phosphate (Cat. # 836281, Boehringer Mannheim) and total protein (Cat. # 704105, Boehringer Mannheim) concentrations were measured. The method of Jaffe (kinetic) without deproteinization was used for creatinine determination (Automated Analysis, Boehringer Mannheim, West Germany)).
DATA ANALYSIS
The horses were divided in two age groups, animals of five years or less and those older than five years. Pearson's correlation was used to examine the relationship between total calcium, phosphate, alkaline phosphatase, oesteocalcin and total
protein. A general linear model with repeated measures (22,23) was used to study the individual effect and time effect between two consecutive hours during the 24 h period for the biochemical variables.
RESULTS The mean concentration values for each individual and for each biochemical variable throughout the 24 h study are listed in Table I. Serum creatinine levels were always in the normal range, between 90 and 150 ,umol/L. Osteocalcin showed an age dependent level, being higher in the youngest horses as described previously (21). Every 60 min for a 24 h period, mean concentrations for the nine horses were determined for each biochemical variable as shown in Fig. 1. Serum osteocalcin levels were not significantly different (p > 0.05) (Table II) during the day from 0700 to 1900 h. The levels fell in the early evening between 1900 and 2000 h, gradually rose during the night, reached a peak at 0500 h and finally dropped to a constant daytime level around 0700 h. A marked individual effect on the serum osteocalcin level was observed (p < 0.01). If the subjects were divided into two age groups (I and II) (Table I), this individual effect was less marked, but the effect of time on serum osteocalcin levels was higher. Group I, horses of five years or less (n = 4) had mean serum osteocalcin levels equal to or higher than 14 ng/ mL, and group II, horses older than five years (n = 5) had serum osteocalcin levels lower than 14 ng/ mL. The circadian pattern was the same (Fig. 2) in both groups. If we compare variations between two consecutive hours during the 24 h period for both groups of age no
TABLE I. Mean values ± SD of 24 hour biochemical data and characteristics of each subject Age Groupa No. Sex (yr) Pregnantb I 1 2.5 F NP I 2 4 F NP I 3 4 P F I 4 5 F P II 5 P 6 F II 6 7 P F II 7 M 7 II 8 9 F P 11 9 17 P F aGroup 1: less than 5 yr; group II: more than 5 yr bp: pregnant; NP: nonpregnant cOsteocalcin dAlkaline phosphatase eTotal calcium fPhosphate
significant variations were observed between 0700 h and 1900 h (p > 0.05) (Table II). This period corresponds approximately to the light period which extended from 0700 to 1830 h. Significant fluctuations (p < 0.05) were observed during darkness from 2000 to 0600 h except between 0300 and 0400 h (Fig. 2). No effect of the time of feeding and the circadian rhythmicity of osteocalcin levels (Fig. 2) was noted. Marked fluctuations were observed during the 24 h period for total serum alkaline phosphatase levels, which showed a minimum at 1400 and 0200 h and maximum concentration levels at 1600 and 0500 h (Fig. 1). A significant correlation (r = 0.3, p < 0.01) was observed between osteocalcin concentration and serum levels of alkaline phosphatase. A marked individual effect on alkaline phosphatase concentrations was also observed (p 0.05) when values were compared at concurrent time points. No significant individual effect was observed for total calcium level (p > 0.05).
BGPc (ng/mL ± SD) 20.3+2.0 17.4±3.0 15.7±2.1 14.5 ± 2.4 6.5 ± 0.6 10.7 ± 1.0 13.8± 1.4 6.8±0.9 9.1 ± 1.5
ALPd (U/L ± SD) 297±29 385±25 286±9 247 ± 12 226 ± 12 377 ± 15 255±33 201 ±9 361 ± 34
Serum phosphate showed the lowest levels at noon and at 2000 h. Two peaks were reached, one at 1600 h and another at 0400 h. A correlation with osteocalcin was present (r = 0.42; p < 0.01) but no significant correlation was observed between total calcium and phosphate values (p > 0.05).
DISCUSSION In 1979, Simmons and collaborators (3) studied the biorhythmic profiles of the rat skeleton and concluded that the metabolic activities of the cells in the linear bone growth and appositional bone growth apparatus were not in phase. The growth apparatus appeared to be day active and the cells in the articular cartilage and diaphyseal bone seemed metabolically most active during the first 4 h of darkness although mineralization proceeded most actively at night in both tissues. The current study indicates that serum osteocalcin, a specific product of osteoblasts, the circulating levels of which reflect the rate of bone turnover
(24), undergoes circadian rhythmicity in normal adult horses of different ages. This was already well documented in humans (18). The creatinine level was determined to reflect renal function of the horses. All the values collected were in the normal range. A defect in the renal function would be responsible for an increase in serum
CaTe (mmol/L ± SD) 3.1 ±0.1 3.1 ±0.1 3.1 ±0.1 3.1 ± 0.1 3.0 ± 0.1 3.1 ± 0.2 3.1 ±0.1 3.1 ±0.1 3.0 ± 0.2
pf
(mmol/L ± SD) 1.1 0.1 1.1 0.1 1.1 0.1 1.3 0.2 1.0 0.1 1.0 0.1 0.9±0.1 0.9±0.1 0.9 0.1
osteocalcin level as the primary mode of clearance is kidney filtration(25). Serum osteocalcin decreased with age. This observation was previously described in female Standardbred horses (21) and in humans in which the normal range of osteocalcin is high in children and declines to adult levels with completion of puberty (26). We interpret these data in horses as indicating a significant slowdown in the rate of bone formation in adults compared to foals. To decrease the effect of age in the interpretation of our data the horses were divided into two age groups, young horses of five years or less and adults older than five years. Blood samples were taken every 60 min in the nine Standardbred horses to determine if circadian variations in serum osteocalcin values are present in normal horses and if correlations existed with serum calcium, phosphate and alkaline phosphatase. Mean serum concentrations of osteocalcin showed a biphasic time pattern (Figs. 1 and 2). The light or day time pattern seemed to correspond to a relatively constant level of osteocalcin and the dark or night time pattern revealed important variations of osteocalcin levels, with lowest levels in early evening between 1900 and 2000 h and a peak at 0500 h. On the day of the experiment the sun rose at 0652 h and set at 1830 h. It was interesting to note that serum osteocalcin levels began to decline at 1700 to 1900 h. The decline was then followed by a progressive rise
7
OSTEOCALCIN (ng/mL'
16r 15
14
13 12 11
101 8
340 r
12
individual differences observed on serum osteocalcin levels as mainly due to the influence of age (21). If the subjects are divided into two age groups (I and II) (Fig. 2), no significant differences (p > 0.05) between subjects of the same group could be observed but the older group had a lower mean level as described in the literature (21). A circadian rhythm for osteocalcin has been described already in male or female humans (18), in which osteocalcin levels fell during the morning, rose in the afternoon and early evening and reached a peak between 0400 and 0800 h, as was observed in this study. A pulsatile pattern of osteocalcin concentration was revealed by an increased frequency of blood sampling and this may be related to secretion or clearance of the protein. The heterogeneity of our group, one male, two nonpregnant mares and six pregnant mares might also have influenced the osteocalcin levels. For example, the male which was seven years old (Table I) had a higher mean level of osteocalcin compared to the other members of the group. Differences with sex were observed in humans (28) and an influence of pregnancy and lactation (29) was also described. Diurnal variations in bone metabolism in the rat (1) were well documented and since circulating levels of osteocalcin reflect bone turnover (30) it has been proposed that variations in the circulating pool may be linked to
TABLE II. Significance of variations in serum osteocalcin levels between consecutive hours during the 24 hour period in age groups
16
20
24
8
4
ALKALINE PHOSPHATASE (U/L)
330 320
Sampling No. 1-12 13 14 15 16 17 18 19 20 21 22 23 24
Hour 08:00-19:00 20:00 21:00 22:00 23:00 24:00 01:00 02:00 03:00 04:00 05:00 06:00 07:00
Significance NS p
