0021-972X/91/7202-0462$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 72, No. 2 Printed in U.S.A.
Correlation between Serum Osteocalcin and 24,25Dihydroxyvitamin D Levels in Paget's Disease of Bone* N. CASTRO-ERRECABORDE, C. DE LA PIEDRA, A. RAPADO, M. V. ALVAREZARROYO, R. TORRES, AND M. L. TRABA Unidad Metabolica, Fundacion Jimenez Diaz, 28040 Madrid, Spain
ABSTRACT. We have studied the possible correlation between serum 24,25-dihydroxyvitamin D [24,25-(OH)2D] and osteocalcin levels (sBGP) in Paget's disease of bone. We measured serum calcium, phosphate, PTH, 25-hydroxyvitamin D, 1,25(OH)2D, 24,25-(OH)2D, alkaline phosphatase (sAP), and the urinary hydroxyproline/creatinine ratio (UOH prol/creat) in 19 patients with Paget's disease of bone and 16 age- and sexmatched controls. As expected, sAP, UOH prol/creat, and sBGP levels were significantly elevated, and there was a tendency to a decrease in serum levels of 24,25-(OH)2D in Pagetic patients with respect to the control group. There was no significant difference between patients and controls in serum calcium,
phosphate, PTH, 25-hydroxyvitamin D, and 1,25-(OH)2D. The Pagetic patients were subdivided into two subgroups; subgroup A had normal sBGP levels (5 ng/mL). Serum 24,25-(OH)2D levels in subgroup B were significantly lower than those in controls, while subgroup A showed leve.s similar to those in the control group. We also found a positive linear correlation between sAP and sBGP and between sAP £nd UOH prol/creat as well as a negative linear correlation between sBGP and 24,25-(OH)2D and between 24,25-(OH)2D ami UOH prol/creat in Pagetic patients. These results point to a possible role of 24,25-(OH)2D in disease activity. (J Clin Endocrinol Metab 72: 462-466, 1991)
P
AGET'S disease of bone is a disorder characterized by excessive bone resorption coupled with massive bone formation, giving rise to characteristic histological, radiographical, biochemical, and clinical changes (1, 2). This excessive bone turnover causes an increase in the levels of the classic biochemical markers of bone formation and resorption: serum alkaline phosphatase (sAP) and urinary excretion of hydroxyproline. There are also other markers that could reflect bone remodeling. The one most commonly used recently is serum osteocalcin [serum bone Gla protein (sBGP)]. There is general agreement that sBGP levels are a marker of bone formation, and that they are elevated in bone diseases characterized by high bone turnover. It is generally accepted that 1,25-dihydroxyvitamin D [1,25-(OH)2D], the most active vitamin D metabolite, is the main activator of sBGP synthesis (3), but a recent study performed in rats (4) showed that 24,25-(OH)2D could be implicated in sBGP metabolism, increasing the synthesis and binding of BGP to bone. This finding suggests that sBGP synthesis could be regulated by both 1,25-(OH)2D and 24,25-(OH)2D. In spite of the high remodeling rate in Paget's disease
of bone, not all patients with active disease present with high sBGP levels. Two subgroups of patients have been described with high and normal sBGP levels (5, 6). With respect to vitamin D metabolism in Paget's disease of bone, several researchers (7-9) have found that the Pagetic patients with a great increase in bone activity show low serum levels of 24,25-(OH)2D, while serum levels of 1,25-(OH)2D are normal. With respect to serum 25-hydroxyvitamin D (25OHD) levels, Guillard-Gumming (7), Devlin (8), and their co-workers found low levels of this metabolite, but Traba and co-workers (9) reported normal serum levels of 25OHD in patients with Paget's disease of bone. Using two subgroups of Pagetic patients, one with normal sBGP levels and the other with increased levels and considering the 24,25-(0H)2D levels of the two subpopulations and the possible influence of 24,25-(OH)2D on sBGP levels, we have; investigated the possible correlation between these last two parameters. This area of study has not, to our knowledge, been previously described in Paget's disease of bone. In addition, it has been suggested that there may be some degree of hyperparathyroidism in the most severely affected patients with this disease (10). For this reason we also decided to study serum PTH levels.
Received December 8,1989. Address all correspondence and requests for reprints to: C. de la Piedra, Laboratorio de la Unidad Metabolica, Fundacion Jimenez Diaz, Avda Reyes Catolicos 2, 28040 Madrid, Spain. * This work was supported in part by a grant from the Spanish Institute of Health (FISss 88/1536).
Materials and Methods Subjects We studied 19 randomly selected patients with Paget's disease of bone (11 women and 8 men; age range, 40-70 yr). The 462
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 21 November 2015. at 21:36 For personal use only. No other uses without permission. . All rights reserved.
sBGP AND 24,25-(OH)2D IN PAGET'S DISEASE diagnosis of Paget's disease of the bone was established by increased levels of sAP, high values of the urinary hydroxyproline/creatinine ratio (UOH prol/creat), and characteristic radiographical features. In 2 Pagetic patients with normal biochemical values, at the moment of the study, but with an active disease in past years, the diagnosis was made by bone biopsy. All patients had been untreated for a minimum of 6 months, and for a minimum of 1 yr in the case of bisphosphonate therapy. All of the Pagetic patients had normal renal function, as quantified by serum creatinine and creatinine clearance. The results in the Pagetic patients were compared with those in 16 age- and sex-matched control subjects, except in the case of 24,25-(OH)2D, where 21 control subjects were used. After an overnight fast, blood samples were obtained from the patients. Serum was separated and stored at 4 C before determination of creatinine, calcium, phosphate, and sAP. Serum aliquots for determination of sBGP, PTH, and vitamin D metabolites were separated and stored at -20 C until analysis. Twenty-four-hour urine specimens were collected from the Pagetic and control subjects to determine UOH prol/creat levels. Methods Determination of serum phosphate and sAP were performed using methods previously described (11, 12). Calcium was assessed by atomic absorptiometry. UOH prol was determined by the Kivirikko method (13) and expressed as a ratio with respect to the urinary creatinine concentration. Measurements of sBGP and PTH by midmolecule assay were performed by RIA (catalog no. 1500 and 15065, respectively, Incstar, Stillwater, MN). 25OHD and 24,25-(OH)2D were extracted from serum using acetonitrile, purified through Sep-Pak C18 cartridges (octadecylsilane), and separated by high performance liquid chromatography on an abnormal phase column (Radial Pak silica gel cartridge) using hexane-ethanol (95:5, vol/vol) as solvent. The fractions containing 25OHD and 24,25-(OH)2D were collected after calibration of the column using standards of these vitamin D metabolites. Both metabolites were quantified by competitive protein-binding assays, using the vitamin D-binding protein obtained from normal human serum (1416). To calculate the exact serum concentrations of 25OHD and 24,25-(OH)2D, we took into account the analytical recovery after the extraction, purification, and separation procedures. The 1,25-(OH)2D determination was performed by RRA (Incstar catalog no. 60065), based on the method of Reinhardt et al. (17), after the metabolite was extracted using acetonitrile, and purified, and separated through a cartridge of Sep-Fak Cl8(OH) (octadecylsilane and silica). Comparisons of the values of control subjects and the Pagetic patients were carried out using the Mann-Whitney test. Comparisons of values among control subjects, patients with high sBGP values, and patients with normal sBGP values were performed by one-way analysis of variance. Correlations between variables were calculated by linear regression analysis.
Results Table 1 shows the biochemical values determined in the Pagetic patients and controls. As expected, sAP and UOH prol/creat values were significantly elevated in
463
TABLE 1. Biochemical values in Paget's disease of bone Parameters
Controls (n = 16)
Serum Calcium (mmol/L) 2.45 ± 0.10 Phosphate (mmol/L) 1.17 ±0.17 Alkaline phosphatase (jtkat/L) 0.45 ± 0.03 BGP (ng/mL) 3.2 ± 0.9 PTH (ng/L) 571 ± 128 25OHD (nmol/L) 39 ±10 24,25-(OH)2D (nmol/L)* 7.2 ± 4.3 1,25-(OH)2D (pmol/L) 84 ±26 UOH prol/creatc 0.021 ± 0.008
Pagetic patients (n = 19)
pa
2.46 ± 0.11 NS 1.16 ±0.13 NS 3.0 ± 2.0
Vol. 72, No. 2 Printed in U.S.A.
Correlation between Serum Osteocalcin and 24,25Dihydroxyvitamin D Levels in Paget's Disease of Bone* N. CASTRO-ERRECABORDE, C. DE LA PIEDRA, A. RAPADO, M. V. ALVAREZARROYO, R. TORRES, AND M. L. TRABA Unidad Metabolica, Fundacion Jimenez Diaz, 28040 Madrid, Spain
ABSTRACT. We have studied the possible correlation between serum 24,25-dihydroxyvitamin D [24,25-(OH)2D] and osteocalcin levels (sBGP) in Paget's disease of bone. We measured serum calcium, phosphate, PTH, 25-hydroxyvitamin D, 1,25(OH)2D, 24,25-(OH)2D, alkaline phosphatase (sAP), and the urinary hydroxyproline/creatinine ratio (UOH prol/creat) in 19 patients with Paget's disease of bone and 16 age- and sexmatched controls. As expected, sAP, UOH prol/creat, and sBGP levels were significantly elevated, and there was a tendency to a decrease in serum levels of 24,25-(OH)2D in Pagetic patients with respect to the control group. There was no significant difference between patients and controls in serum calcium,
phosphate, PTH, 25-hydroxyvitamin D, and 1,25-(OH)2D. The Pagetic patients were subdivided into two subgroups; subgroup A had normal sBGP levels (5 ng/mL). Serum 24,25-(OH)2D levels in subgroup B were significantly lower than those in controls, while subgroup A showed leve.s similar to those in the control group. We also found a positive linear correlation between sAP and sBGP and between sAP £nd UOH prol/creat as well as a negative linear correlation between sBGP and 24,25-(OH)2D and between 24,25-(OH)2D ami UOH prol/creat in Pagetic patients. These results point to a possible role of 24,25-(OH)2D in disease activity. (J Clin Endocrinol Metab 72: 462-466, 1991)
P
AGET'S disease of bone is a disorder characterized by excessive bone resorption coupled with massive bone formation, giving rise to characteristic histological, radiographical, biochemical, and clinical changes (1, 2). This excessive bone turnover causes an increase in the levels of the classic biochemical markers of bone formation and resorption: serum alkaline phosphatase (sAP) and urinary excretion of hydroxyproline. There are also other markers that could reflect bone remodeling. The one most commonly used recently is serum osteocalcin [serum bone Gla protein (sBGP)]. There is general agreement that sBGP levels are a marker of bone formation, and that they are elevated in bone diseases characterized by high bone turnover. It is generally accepted that 1,25-dihydroxyvitamin D [1,25-(OH)2D], the most active vitamin D metabolite, is the main activator of sBGP synthesis (3), but a recent study performed in rats (4) showed that 24,25-(OH)2D could be implicated in sBGP metabolism, increasing the synthesis and binding of BGP to bone. This finding suggests that sBGP synthesis could be regulated by both 1,25-(OH)2D and 24,25-(OH)2D. In spite of the high remodeling rate in Paget's disease
of bone, not all patients with active disease present with high sBGP levels. Two subgroups of patients have been described with high and normal sBGP levels (5, 6). With respect to vitamin D metabolism in Paget's disease of bone, several researchers (7-9) have found that the Pagetic patients with a great increase in bone activity show low serum levels of 24,25-(OH)2D, while serum levels of 1,25-(OH)2D are normal. With respect to serum 25-hydroxyvitamin D (25OHD) levels, Guillard-Gumming (7), Devlin (8), and their co-workers found low levels of this metabolite, but Traba and co-workers (9) reported normal serum levels of 25OHD in patients with Paget's disease of bone. Using two subgroups of Pagetic patients, one with normal sBGP levels and the other with increased levels and considering the 24,25-(0H)2D levels of the two subpopulations and the possible influence of 24,25-(OH)2D on sBGP levels, we have; investigated the possible correlation between these last two parameters. This area of study has not, to our knowledge, been previously described in Paget's disease of bone. In addition, it has been suggested that there may be some degree of hyperparathyroidism in the most severely affected patients with this disease (10). For this reason we also decided to study serum PTH levels.
Received December 8,1989. Address all correspondence and requests for reprints to: C. de la Piedra, Laboratorio de la Unidad Metabolica, Fundacion Jimenez Diaz, Avda Reyes Catolicos 2, 28040 Madrid, Spain. * This work was supported in part by a grant from the Spanish Institute of Health (FISss 88/1536).
Materials and Methods Subjects We studied 19 randomly selected patients with Paget's disease of bone (11 women and 8 men; age range, 40-70 yr). The 462
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 21 November 2015. at 21:36 For personal use only. No other uses without permission. . All rights reserved.
sBGP AND 24,25-(OH)2D IN PAGET'S DISEASE diagnosis of Paget's disease of the bone was established by increased levels of sAP, high values of the urinary hydroxyproline/creatinine ratio (UOH prol/creat), and characteristic radiographical features. In 2 Pagetic patients with normal biochemical values, at the moment of the study, but with an active disease in past years, the diagnosis was made by bone biopsy. All patients had been untreated for a minimum of 6 months, and for a minimum of 1 yr in the case of bisphosphonate therapy. All of the Pagetic patients had normal renal function, as quantified by serum creatinine and creatinine clearance. The results in the Pagetic patients were compared with those in 16 age- and sex-matched control subjects, except in the case of 24,25-(OH)2D, where 21 control subjects were used. After an overnight fast, blood samples were obtained from the patients. Serum was separated and stored at 4 C before determination of creatinine, calcium, phosphate, and sAP. Serum aliquots for determination of sBGP, PTH, and vitamin D metabolites were separated and stored at -20 C until analysis. Twenty-four-hour urine specimens were collected from the Pagetic and control subjects to determine UOH prol/creat levels. Methods Determination of serum phosphate and sAP were performed using methods previously described (11, 12). Calcium was assessed by atomic absorptiometry. UOH prol was determined by the Kivirikko method (13) and expressed as a ratio with respect to the urinary creatinine concentration. Measurements of sBGP and PTH by midmolecule assay were performed by RIA (catalog no. 1500 and 15065, respectively, Incstar, Stillwater, MN). 25OHD and 24,25-(OH)2D were extracted from serum using acetonitrile, purified through Sep-Pak C18 cartridges (octadecylsilane), and separated by high performance liquid chromatography on an abnormal phase column (Radial Pak silica gel cartridge) using hexane-ethanol (95:5, vol/vol) as solvent. The fractions containing 25OHD and 24,25-(OH)2D were collected after calibration of the column using standards of these vitamin D metabolites. Both metabolites were quantified by competitive protein-binding assays, using the vitamin D-binding protein obtained from normal human serum (1416). To calculate the exact serum concentrations of 25OHD and 24,25-(OH)2D, we took into account the analytical recovery after the extraction, purification, and separation procedures. The 1,25-(OH)2D determination was performed by RRA (Incstar catalog no. 60065), based on the method of Reinhardt et al. (17), after the metabolite was extracted using acetonitrile, and purified, and separated through a cartridge of Sep-Fak Cl8(OH) (octadecylsilane and silica). Comparisons of the values of control subjects and the Pagetic patients were carried out using the Mann-Whitney test. Comparisons of values among control subjects, patients with high sBGP values, and patients with normal sBGP values were performed by one-way analysis of variance. Correlations between variables were calculated by linear regression analysis.
Results Table 1 shows the biochemical values determined in the Pagetic patients and controls. As expected, sAP and UOH prol/creat values were significantly elevated in
463
TABLE 1. Biochemical values in Paget's disease of bone Parameters
Controls (n = 16)
Serum Calcium (mmol/L) 2.45 ± 0.10 Phosphate (mmol/L) 1.17 ±0.17 Alkaline phosphatase (jtkat/L) 0.45 ± 0.03 BGP (ng/mL) 3.2 ± 0.9 PTH (ng/L) 571 ± 128 25OHD (nmol/L) 39 ±10 24,25-(OH)2D (nmol/L)* 7.2 ± 4.3 1,25-(OH)2D (pmol/L) 84 ±26 UOH prol/creatc 0.021 ± 0.008
Pagetic patients (n = 19)
pa
2.46 ± 0.11 NS 1.16 ±0.13 NS 3.0 ± 2.0
