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1,25-Dihydroxyvitamin D3 receptors in peripheral blood mononuclear cells from patients with postmenopausal osteoporosis
ELSEVIER SCIENTIFIC PUBLISHERS IRELAND
Bone and Mineral 23 (1993) 207-212
1,25-Dihydroxyvitamin D 3 receptors in peripheral
blood mononuclear cells from patients with postmenopausal osteoporosis Josefina Martinez, Jos6 M. Olmos, Jos6 A. Amado, Jos6 A. Riancho, Julio Freijanes, Jesfs Gonzfilez-Maclas* Departamento de Medicina Interna, Hospital Marquis de Valdecilla, Universidad de Cantabria, Santander, Spain
(Received 17 March 1993; revision received 10 June 1993; accepted 2 July 1993)
Abstract
A decrease in intestinal calcium absorption, in spite of normal serum calcitriol levels, has been reported in postmenopausal osteoporotic women, raising the possibility of an intestinal resistance to the hormone. The mechanism responsible for it could lie at the receptor or postreceptor level. Intestinal receptors are difficult to study on clinical settings, but calcitriol receptors have been found in peripheral blood mononuclear cells (PBMC). We have studied the PBMC calcitriol receptors by means of Scatchard analysis in l l postmenopausal osteoporotic women without any treatment and in 12 normal postmenopausal women of similar age. No differences were found in the dissociation constant (Kd) or the concentration of binding sites (Nmax) (Kd in patients: 0.90 • 0.75 • 10-l~ M; Kd in controls: 0.85 4- 0.40 X lO -10 M; Nmax in patients: 2.4 • 1.2 fmol/107 cells; Nmax in controls: 2.1 4- 0.6 fmol/107 cells), supporting the contention that the disorder responsible for the resistance to calcitriol in postmenopausal osteoporotic women is located at the postreceptor level. In addition, our study included five postmenopausal osteoporotic women treated with calcitriol (0.5 #g/day). The number of calcitriol receptors was increased in this group (Nmax: 3.9 4- 2.0 fmol/107 cells vs. 2.1 4- 0.6 fmol/107 cells; P = 0.02). Key words." 1,25-Dihydroxyvitamin D3; Receptors; Peripheral blood mononuclear cells; Osteoporosis
* Corresponding author, Departamento de Medicina Interna, Hospital Marqu6s de Valdecilla, Avda. Valdeeilla, s/n. 39008 Santander, Spain. 0169-6009/93/$06.00 9 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0169-6009(93)00624-C
208
J. Martinez et al. / Bone Miner. 23 (1993) 207-212
1. Introduction
A decrease in intestinal calcium absorption, in spite of normal calcitriol levels, has been reported in postmenopausal osteoporotic women [1-3]. This finding provides evidence in support of an intestinal resistance to 1,25-dihydroxyvitamin D 3 a s a contributory factor in the development of postmenopausal osteoporosis [2,3]. Whether the basic disturbance is located at the receptor or at the postreceptor level is not known. Intestinal receptors for calcitriol are difficult to study on a clinical setting [4]. However, calcitriol receptors have been found in peripheral blood mononuclear cells (PBMC) [5,6]. Therefore, we decided to study the PBMC calcitriol receptors by means of Scatchard analysis in postmenopausal osteoporotic women, and to compare the results with those of normal women of similar age. 2. Patients and methods
2.2. Subjects Eleven postmenopausal osteoporotic women aged 57-73 years (mean • S.D. = 64 • 5) have been studied. All of them had one or more atraumatic vertebral fracture, and vertebral bone mineral density (L2-L4) lower than 0.90 g/cm 2 measured by dual-energy X-ray absortiometry (Lunar DPX, Lunar Radiation Co., Madison, WI, USA). No patients with previous treatment with vitamin D or its metabolites were included. A second group of five patients aged 61-73 years (69 • 4 years) satisfying the same criteria regarding vertebral fractures and bone mineral density, but treated with calcitriol (0.5/zg/day) for at least 3 months, was studied for comparison. Twelve healthy women, aged 51-64 years (57 • 4) were also studied and considered as a normal control group. None of them were taking any drug. 2.3. Biochemical measurements Serum total calcium, phosphate, creatinine, albumin, alkaline phosphatase, as well as 24-h urine calcium, phosphate, and creatinine, were measured by standard automated methods (Hitachi 737). Serum ionized calcium was determined by a calcium-sensitive electrocie (Nova - - 7, Nova Instruments, Boston, USA). Serum tartrate-resistant acid phosphatase (TRAP) was measured with the method of Lau et al. [7]. Plasma levels of calcitriol were determined as previously reported [8]. The interassay coefficient of variation (CV) was 14%. 2.4. Cell isolation Peripheral blood mononuclear cells (PBMC) were obtained at 09:00 h from blood samples by Ficoll-Hypaque density gradient [9]. The percentage of monocytes was estimated in every assay by non-specific esterase staining. 2.5. Binding studies Binding of [3H]I,25(OH)2D3 by intact cell preparations was determined as
J. Martinez et al./ Bone Miner. 23 (1993) 207-212
209
described by Freake et al. [10] with modifications [11]. Briefly, mononuclear cells (15-20 • 106/ml) were suspended for 3 h at 37~ with increasing amounts (0.4-6.4 nM) of [3HI1,25(OH)2D 3 (90-100 Ci/mmol; Amersham, UK). Non-specific binding was determined in parallel incubation mixtures containing 200-fold molar excess of radioinert 1,25(OH)2D3 (Hoffman-La Roche, N J, U S A ) . After incubation, cells were centrifugated, rinsed in phosphate-buffered saline, and then dissolved in 1 M NaOH. Apparent equilibrium dissociation constant (Kd) and maximal binding capacity (Nmax) were estimated by Scatchard analysis. The results were expressed as the mean • S.D. Statistical analysis was carried out with Student's t-test and Pearson correlation coefficient using an IBM PS-2 computer with the 'Stata II' package of software. 3. Results
Biochemical data from patients are shown in Table 1. Recovery of monocytes from mononuclear cells was similar in untreated patients (21 q- 3%), in patients treated with oral calcitriol (20 • 5%), and in controls (19 • 7%). No difference was found either in the Kd or the Nma x between untreated patients and controls (Table 2). Nor was any difference found when the results were expressed in fmol/107 monocytes instead of fmol/107 mononuclear cells (untreated patients 11.4 • 5.6 fmol/107monocytes; controls 10.8 • 3.9 fmol/107 monocytes). Patients on calcitriol treatment showed a significantly higher concentration of binding sites than controls (Table 2). The difference persisted when the results were expressed as f m o l / 1 0 7 monocytes (20.6 • 8.6 vs. 10.8 • 3.9 fmol/107 monocytes; P = 0.02). However, differences between both groups of patients did not reach statistical significance (P = 0.07). No significant difference between calcitriol-treated patients and controls was found in the mean dissociation constant, although there was a trend for it (P = 0.07).
Table 1 Biochemical features of patients with postmenopausal osteoporosis (Mean • S.D.) Parameter
Untreated patients
Patients treated with calcitriol
Normal range
Serum calcium (mg/dl) Serum phosphate(mg/dl) Serum creatinine(mg/dl) Serum albumin (g/dl) Serum Ca 2§ (mM) Serum A. Phosphatase (U/l) Serum TRAP(U/1) Urine calcium (mg/24 h) U Ca/U Cr ratio (mg/mg) 1,25(OH)2D (pg/ml)
9.6 3.9 0.9 4.4 1.25 192 14.8 167 0.21 31
9.7 • 3.7 • 0.8 • 4.5 • 1.25 • 227 • 14.5 • 183 • 0.34 • 37 +
8.5-10.4 2.5-4.5 0.4-1.2 3.5-5.2 1.15-1.35 70-289 7-18 95-200 <0.22 16-56
• 0.6 + 0.4 • 0.1 • 0.5 • 0.05 • 24 • 4.0 • 127 • 0.08 • 14
0.4 0.6 0.1 0.2 0.08 46 2.9 87 0.19 14
Serum Ca 2+, serum ionized calcium; Serum A phosphatase, serum alkaline phosphatase; Serum TRAP, serum tartrate-resistant acid phosphatase; U Ca/U Cr ratio, urine calcium/urine treat• ratio.
J. Martinez et al./Bone Miner. 23 (1993) 207-212
210
Table 2 Dissociation constant (Ka) and maximal binding capacity (Nmax) in patients with postmenopausal osteoporosis and controls
Kd (10 -l~ M) Nmax (fmol/107 cells)
Untreated patients
Patients with calcitriol
Controls
0.90 • 0.75
1.80 a- 1.18
0.85 q- 0.40
2.4 • 1.2
3.9 "4" 2.0 a
2.1
+ 0.6
apatients with calcitriol treatment vs. controls; P = 0.02.
No correlation was seen between the Kd o r Nma x and the BMD or the biochemical data, either considering both groups of patients together or separately. 4. Discussion
We have not found any difference between postmenopausal osteoporotic patients and normal women of similar age, either in the equilibrium dissociation constant or the maximal binding capacity, of calcitriol receptors of peripheral blood mononuclear cells as measured by Scatchard analysis. A decreased intestinal calcium absorption, in spite of normal serum calcitriol levels in postmenopausal osteoporotic women has been reported, suggesting the occurrence of an intestinal resistance to 1,25-dihydroxyvitamin D 3 in these patients [3]. In agreement with it are the results of Koren et al. [12], who have shown that PBMC from postmenopausal osteoporotic women display a reduced responsiveness to calcitriol on lymphocyte mitogenesis. The mechanism responsible for the resistance could lie at the receptor or the postreceptor level. The normality of our results suggests that the abnormality has to be placed at the postreceptor level. A lack of involvement of calcitriol receptors on the pathogenesis of osteoporosis is consistent with the fact that neither our own results nor those of Yu et al. [13] have found any relationship between the equilibrium dissociation constant or the maximal binding capacity and a defining parameter of osteoporosis like BMD. Neither our own results, nor those o f Y u et al. [13] have found any relation between both parameters of calcitriol receptors and bone turnover markers. The possibility remains that results from peripheral blood mononuclear studies can not be extrapolated to intestinal cells. Nevertheless, there is evidence that in other situations of end organ resistance to calcitriol, PBMC have been a suitable model to investigate the 1,25(OH)2D3 receptor status [14-17]. A similar consideration could be done regarding calcitriol receptors in parathyroid cells. Korkor [18] has reported a higher number of calcitriol receptors in parathyroid glands from patients with primary hyperparathyroidism than in parathyroid glands from patients with hyperparathyroidism secondary to chronic renal failure; and we have shown that calcitriol receptors of PBMC show the same changes in both cases [19,20].
J. Martinez et al. / Bone Miner. 23 (1993) 207-212
211
Osteoporotic women on treatment with calcitriol showed a higher concentration of binding sites than controls. This was an expected finding, since in cultured mammalian cells, 1,25(OH)2D3receptor expression increases after exposure to physiological concentrations of calcitriol [21]. Similar results have been reported by several authors [22,231 in kidney and intestine from rats treated with calcitriol. In humans, Merke et al. [24] have demonstrated the existence of a homologous in vivo up-regulation of calcitriol receptors in human monocytes. This up-regulation suggests that the calcitriol treatment is reinforced by the fact that the drug makes the organism more sensitive to its own action. Our negative results could rise the comment that PBMCs are not suitable to study the monocyte receptors. Peripheral blood mononuclear cells contain lymphocytes too, and even if they do not exhibit calcitriol receptors at rest, they could show them when activated. However, the 3-h incubation period in our study is shorter than the 24-h period required for activation of lymphocytes [6]. Finally, there are no reasons to think that there was an activation of these cells before blood was drawn. 5. References
1 Gallagher JC, Riggs BL, Eisman J, Hamstra A, Arnaud SB, De Luca HF. Intestinal calcium absorption and serum vitamin D metabolites in normal subjects and osteoporotic patients. Effect of age and dietary calcium. J Clin Invest 1979;64:729-736. 2 Nordin BEC, Robertson A, Seamark RF, et al. The relation between calcium absorption, serum dehydroepiandrosterone and vertebral mineral density in postmenopausal women. J Clin Endocrinol Metab 1985;60:651-657. 3 Morris HA, Need AG, Horowitz M, O'Loughlin PD, Nordin BEC. Calcium absorption in normal and osteoporotic postmenopausal women. Calcif Tissue Int 1991;49:240-243. 4 Ebeling PR, Sandgren ME, DiMagno EP, Lane AW, DeLuca HF, Riggs BL. Evidence of an age-related decrease in intestinal responsiveness to vitamin D: relationship between serum 1,25-dihydroxyvitamin D3 and intestinal Vitamin D receptor concentration in normal women. J Clin Endocrinol Metab 1992;75:176-182. 5 Provvedini DM, Tsoukas CD, Deftos L J, Manolagas SC. 1,25-Dihydroxyvitamin D3 receptors in human leukocytes. Science 1983;221:1181-1183. 6 Bhalla AK, Amento EP, Clemens TL, Holick MF, Krane SM. Specific high-affinity receptors for 1,25-dihydroxyvitamin D3 in human peripheral blood mononuclear cells: presence in monocytes and induction in T lymphocytes following activation. J Clin Endocrinol Metab 1983;57:1308-1310. 7 Lau KHW, Onishi T, Wergedal JE, Singer FR, Baylink DJ. Characterization and assay of tartrateresistant acid phosphatase activity in serum: potential use to assess bone resorption. Clin Chem 1987;32:458-462. 8 Riancho JA, De Francisco ALM, Del Arco C, et al. Serum levels of 1,25-dihydroxyvitamin D after renal transplantation. Miner Elect Metab 1988;14:332-337. 9 Bfyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 1968;21(Suppl 97):77-89. 10 Freake HC, Iwasaki J, McCarthy DM. Specific uptake of 1,25-Dihydroxycholecalciferol by human chronic myeloid leukemia cells. Cancer Res 1984;44:3627-3631. 11 Olmos JM, Amado JA, Riancho JA, Alb/tjar M, Gonz~ilez-Macias J. Sex and age distribution of 1,25(OH)2D3 receptor in peripheral blood mononuclear cells from normal human subjects. Bone 1990;11:407-409. 12 Koren R, Ravid A, Liberman UA, et al. Responsiveness to 1,25-Dihydroxyvitamin D3 is reduced in lymphocytes from osteoporotic women. J Bone Miner Res 1992;7:1057-1061. 13 Yu XP, Hustmyer FG, Benninger L, Peacock M, Manolagas SC. 1,25(OH)2D3 receptor levels in
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peripheral blood mononuclear cells of patients with postmenopausal osteoporosis. In: Norman AW, Bouillon R, Thomaset M, eds. Vitamin D: gene regulation, structure-function analysis, and clinical application. Berlin: Walter de Gruiter, 1991;840-841. Koren R, Ravid A, Liberman UA, Hochberg Z, Weisman Y, Novogrodsky A. Defective binding and function of 1,25-dihydroxyvitamin D3 receptors in peripheral blood mononuclear cells of patients with end organ resistance to 1,25-dihydroxyvitamin D. J Clin Invest 1985,76:2012-2015. Bollerslev J, Nielsen HK, Storm T, Mosekilde L. Serum vitamin D metabolites and nuclear uptake of (3H)-l,25-dihydroxyvitamin D3 in monocytes from patients with autosomal dominant osteopetrosis. A study of two radiological types. Bone 1988;9:355-359. Koeffier HP, Bishop JE, Reichel H, et al. Lymphocyte cell lines from vitamin D-dependent rickets type II show functional defects in the 1,25-dihydroxyvitamin D3 receptor. Mol Cell Endocrinol 1990;70:1-11. Horen R, Ravid A, Liberman UA. Peripheral blood mononuclear cells: a model for the human vitamin D endocrine system in health and disease. Mol Cell Endocrinol 1992;83:C9-C12. Korkor AB. Reduced binding of (3H)l,25-dihydroxyvitamin D3 in the parathyroid glands of patients with renal failure. N Engl J Med 1987;316:1573-1577. Olmos JM, Martinez J, Amado JA, Riancho JA, Gonzfilez-Macias J. Increased 1,25-dihydroxyvitamin D3 receptors in peripheral blood mononuclear cells from patients with primary hyperparathyroidism. In: Norman AW, Bouillon R, Thomaset M, eds. Vitamin D: gene regulation, structure-function analysis, and clinical application. Berlin: Walter de Gruiter, 1991;100-101. Martinez J, Olmos JM, de Francisco ALM, Riancho JA, Amado JA, Gonzfilez-Macias J. Reduced binding of (3H)-l,25-dihydroxyvitamin D3 in peripheral blood mononuclear cells from patients with chronic renal failure. Bone Miner 1992;17 (Suppl 1):128. Costa EM, Feldman D. Measurement of 1,25 dihydroxyvitamin D3 receptor turnover by dense amino acid labeling. Endocrinology 1987;120:1173-1178. Costa EM, Feldman D. Homologous up-regulation of the 1,25(OH)2D3 receptor in rat. Biochem Biophys Res Commun 1984;137:742-747. Goff JP, Reinhardt TA, Beckman M J, Horst RL. Contrasting effects of exogenous 1,25-dihydroxyvitamin D (1,25(OH)2D) versus endogenous 1,25(OH)2D, induced by dietary calcium restriction, on vitamin D receptors. Endocrinology 1990;126:1031-1035. Merke J, Nawrot M, Hiigel U, Szabo A, Ritz E. Evidence for in vivo up-regulation of 1,25(OH)2D3 receptor in human monoeytes. Calcif Tissue Int 1989;45:255-256.
Bone and Mineral 23 (1993) 207-212
1,25-Dihydroxyvitamin D 3 receptors in peripheral
blood mononuclear cells from patients with postmenopausal osteoporosis Josefina Martinez, Jos6 M. Olmos, Jos6 A. Amado, Jos6 A. Riancho, Julio Freijanes, Jesfs Gonzfilez-Maclas* Departamento de Medicina Interna, Hospital Marquis de Valdecilla, Universidad de Cantabria, Santander, Spain
(Received 17 March 1993; revision received 10 June 1993; accepted 2 July 1993)
Abstract
A decrease in intestinal calcium absorption, in spite of normal serum calcitriol levels, has been reported in postmenopausal osteoporotic women, raising the possibility of an intestinal resistance to the hormone. The mechanism responsible for it could lie at the receptor or postreceptor level. Intestinal receptors are difficult to study on clinical settings, but calcitriol receptors have been found in peripheral blood mononuclear cells (PBMC). We have studied the PBMC calcitriol receptors by means of Scatchard analysis in l l postmenopausal osteoporotic women without any treatment and in 12 normal postmenopausal women of similar age. No differences were found in the dissociation constant (Kd) or the concentration of binding sites (Nmax) (Kd in patients: 0.90 • 0.75 • 10-l~ M; Kd in controls: 0.85 4- 0.40 X lO -10 M; Nmax in patients: 2.4 • 1.2 fmol/107 cells; Nmax in controls: 2.1 4- 0.6 fmol/107 cells), supporting the contention that the disorder responsible for the resistance to calcitriol in postmenopausal osteoporotic women is located at the postreceptor level. In addition, our study included five postmenopausal osteoporotic women treated with calcitriol (0.5 #g/day). The number of calcitriol receptors was increased in this group (Nmax: 3.9 4- 2.0 fmol/107 cells vs. 2.1 4- 0.6 fmol/107 cells; P = 0.02). Key words." 1,25-Dihydroxyvitamin D3; Receptors; Peripheral blood mononuclear cells; Osteoporosis
* Corresponding author, Departamento de Medicina Interna, Hospital Marqu6s de Valdecilla, Avda. Valdeeilla, s/n. 39008 Santander, Spain. 0169-6009/93/$06.00 9 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0169-6009(93)00624-C
208
J. Martinez et al. / Bone Miner. 23 (1993) 207-212
1. Introduction
A decrease in intestinal calcium absorption, in spite of normal calcitriol levels, has been reported in postmenopausal osteoporotic women [1-3]. This finding provides evidence in support of an intestinal resistance to 1,25-dihydroxyvitamin D 3 a s a contributory factor in the development of postmenopausal osteoporosis [2,3]. Whether the basic disturbance is located at the receptor or at the postreceptor level is not known. Intestinal receptors for calcitriol are difficult to study on a clinical setting [4]. However, calcitriol receptors have been found in peripheral blood mononuclear cells (PBMC) [5,6]. Therefore, we decided to study the PBMC calcitriol receptors by means of Scatchard analysis in postmenopausal osteoporotic women, and to compare the results with those of normal women of similar age. 2. Patients and methods
2.2. Subjects Eleven postmenopausal osteoporotic women aged 57-73 years (mean • S.D. = 64 • 5) have been studied. All of them had one or more atraumatic vertebral fracture, and vertebral bone mineral density (L2-L4) lower than 0.90 g/cm 2 measured by dual-energy X-ray absortiometry (Lunar DPX, Lunar Radiation Co., Madison, WI, USA). No patients with previous treatment with vitamin D or its metabolites were included. A second group of five patients aged 61-73 years (69 • 4 years) satisfying the same criteria regarding vertebral fractures and bone mineral density, but treated with calcitriol (0.5/zg/day) for at least 3 months, was studied for comparison. Twelve healthy women, aged 51-64 years (57 • 4) were also studied and considered as a normal control group. None of them were taking any drug. 2.3. Biochemical measurements Serum total calcium, phosphate, creatinine, albumin, alkaline phosphatase, as well as 24-h urine calcium, phosphate, and creatinine, were measured by standard automated methods (Hitachi 737). Serum ionized calcium was determined by a calcium-sensitive electrocie (Nova - - 7, Nova Instruments, Boston, USA). Serum tartrate-resistant acid phosphatase (TRAP) was measured with the method of Lau et al. [7]. Plasma levels of calcitriol were determined as previously reported [8]. The interassay coefficient of variation (CV) was 14%. 2.4. Cell isolation Peripheral blood mononuclear cells (PBMC) were obtained at 09:00 h from blood samples by Ficoll-Hypaque density gradient [9]. The percentage of monocytes was estimated in every assay by non-specific esterase staining. 2.5. Binding studies Binding of [3H]I,25(OH)2D3 by intact cell preparations was determined as
J. Martinez et al./ Bone Miner. 23 (1993) 207-212
209
described by Freake et al. [10] with modifications [11]. Briefly, mononuclear cells (15-20 • 106/ml) were suspended for 3 h at 37~ with increasing amounts (0.4-6.4 nM) of [3HI1,25(OH)2D 3 (90-100 Ci/mmol; Amersham, UK). Non-specific binding was determined in parallel incubation mixtures containing 200-fold molar excess of radioinert 1,25(OH)2D3 (Hoffman-La Roche, N J, U S A ) . After incubation, cells were centrifugated, rinsed in phosphate-buffered saline, and then dissolved in 1 M NaOH. Apparent equilibrium dissociation constant (Kd) and maximal binding capacity (Nmax) were estimated by Scatchard analysis. The results were expressed as the mean • S.D. Statistical analysis was carried out with Student's t-test and Pearson correlation coefficient using an IBM PS-2 computer with the 'Stata II' package of software. 3. Results
Biochemical data from patients are shown in Table 1. Recovery of monocytes from mononuclear cells was similar in untreated patients (21 q- 3%), in patients treated with oral calcitriol (20 • 5%), and in controls (19 • 7%). No difference was found either in the Kd or the Nma x between untreated patients and controls (Table 2). Nor was any difference found when the results were expressed in fmol/107 monocytes instead of fmol/107 mononuclear cells (untreated patients 11.4 • 5.6 fmol/107monocytes; controls 10.8 • 3.9 fmol/107 monocytes). Patients on calcitriol treatment showed a significantly higher concentration of binding sites than controls (Table 2). The difference persisted when the results were expressed as f m o l / 1 0 7 monocytes (20.6 • 8.6 vs. 10.8 • 3.9 fmol/107 monocytes; P = 0.02). However, differences between both groups of patients did not reach statistical significance (P = 0.07). No significant difference between calcitriol-treated patients and controls was found in the mean dissociation constant, although there was a trend for it (P = 0.07).
Table 1 Biochemical features of patients with postmenopausal osteoporosis (Mean • S.D.) Parameter
Untreated patients
Patients treated with calcitriol
Normal range
Serum calcium (mg/dl) Serum phosphate(mg/dl) Serum creatinine(mg/dl) Serum albumin (g/dl) Serum Ca 2§ (mM) Serum A. Phosphatase (U/l) Serum TRAP(U/1) Urine calcium (mg/24 h) U Ca/U Cr ratio (mg/mg) 1,25(OH)2D (pg/ml)
9.6 3.9 0.9 4.4 1.25 192 14.8 167 0.21 31
9.7 • 3.7 • 0.8 • 4.5 • 1.25 • 227 • 14.5 • 183 • 0.34 • 37 +
8.5-10.4 2.5-4.5 0.4-1.2 3.5-5.2 1.15-1.35 70-289 7-18 95-200 <0.22 16-56
• 0.6 + 0.4 • 0.1 • 0.5 • 0.05 • 24 • 4.0 • 127 • 0.08 • 14
0.4 0.6 0.1 0.2 0.08 46 2.9 87 0.19 14
Serum Ca 2+, serum ionized calcium; Serum A phosphatase, serum alkaline phosphatase; Serum TRAP, serum tartrate-resistant acid phosphatase; U Ca/U Cr ratio, urine calcium/urine treat• ratio.
J. Martinez et al./Bone Miner. 23 (1993) 207-212
210
Table 2 Dissociation constant (Ka) and maximal binding capacity (Nmax) in patients with postmenopausal osteoporosis and controls
Kd (10 -l~ M) Nmax (fmol/107 cells)
Untreated patients
Patients with calcitriol
Controls
0.90 • 0.75
1.80 a- 1.18
0.85 q- 0.40
2.4 • 1.2
3.9 "4" 2.0 a
2.1
+ 0.6
apatients with calcitriol treatment vs. controls; P = 0.02.
No correlation was seen between the Kd o r Nma x and the BMD or the biochemical data, either considering both groups of patients together or separately. 4. Discussion
We have not found any difference between postmenopausal osteoporotic patients and normal women of similar age, either in the equilibrium dissociation constant or the maximal binding capacity, of calcitriol receptors of peripheral blood mononuclear cells as measured by Scatchard analysis. A decreased intestinal calcium absorption, in spite of normal serum calcitriol levels in postmenopausal osteoporotic women has been reported, suggesting the occurrence of an intestinal resistance to 1,25-dihydroxyvitamin D 3 in these patients [3]. In agreement with it are the results of Koren et al. [12], who have shown that PBMC from postmenopausal osteoporotic women display a reduced responsiveness to calcitriol on lymphocyte mitogenesis. The mechanism responsible for the resistance could lie at the receptor or the postreceptor level. The normality of our results suggests that the abnormality has to be placed at the postreceptor level. A lack of involvement of calcitriol receptors on the pathogenesis of osteoporosis is consistent with the fact that neither our own results nor those of Yu et al. [13] have found any relationship between the equilibrium dissociation constant or the maximal binding capacity and a defining parameter of osteoporosis like BMD. Neither our own results, nor those o f Y u et al. [13] have found any relation between both parameters of calcitriol receptors and bone turnover markers. The possibility remains that results from peripheral blood mononuclear studies can not be extrapolated to intestinal cells. Nevertheless, there is evidence that in other situations of end organ resistance to calcitriol, PBMC have been a suitable model to investigate the 1,25(OH)2D3 receptor status [14-17]. A similar consideration could be done regarding calcitriol receptors in parathyroid cells. Korkor [18] has reported a higher number of calcitriol receptors in parathyroid glands from patients with primary hyperparathyroidism than in parathyroid glands from patients with hyperparathyroidism secondary to chronic renal failure; and we have shown that calcitriol receptors of PBMC show the same changes in both cases [19,20].
J. Martinez et al. / Bone Miner. 23 (1993) 207-212
211
Osteoporotic women on treatment with calcitriol showed a higher concentration of binding sites than controls. This was an expected finding, since in cultured mammalian cells, 1,25(OH)2D3receptor expression increases after exposure to physiological concentrations of calcitriol [21]. Similar results have been reported by several authors [22,231 in kidney and intestine from rats treated with calcitriol. In humans, Merke et al. [24] have demonstrated the existence of a homologous in vivo up-regulation of calcitriol receptors in human monocytes. This up-regulation suggests that the calcitriol treatment is reinforced by the fact that the drug makes the organism more sensitive to its own action. Our negative results could rise the comment that PBMCs are not suitable to study the monocyte receptors. Peripheral blood mononuclear cells contain lymphocytes too, and even if they do not exhibit calcitriol receptors at rest, they could show them when activated. However, the 3-h incubation period in our study is shorter than the 24-h period required for activation of lymphocytes [6]. Finally, there are no reasons to think that there was an activation of these cells before blood was drawn. 5. References
1 Gallagher JC, Riggs BL, Eisman J, Hamstra A, Arnaud SB, De Luca HF. Intestinal calcium absorption and serum vitamin D metabolites in normal subjects and osteoporotic patients. Effect of age and dietary calcium. J Clin Invest 1979;64:729-736. 2 Nordin BEC, Robertson A, Seamark RF, et al. The relation between calcium absorption, serum dehydroepiandrosterone and vertebral mineral density in postmenopausal women. J Clin Endocrinol Metab 1985;60:651-657. 3 Morris HA, Need AG, Horowitz M, O'Loughlin PD, Nordin BEC. Calcium absorption in normal and osteoporotic postmenopausal women. Calcif Tissue Int 1991;49:240-243. 4 Ebeling PR, Sandgren ME, DiMagno EP, Lane AW, DeLuca HF, Riggs BL. Evidence of an age-related decrease in intestinal responsiveness to vitamin D: relationship between serum 1,25-dihydroxyvitamin D3 and intestinal Vitamin D receptor concentration in normal women. J Clin Endocrinol Metab 1992;75:176-182. 5 Provvedini DM, Tsoukas CD, Deftos L J, Manolagas SC. 1,25-Dihydroxyvitamin D3 receptors in human leukocytes. Science 1983;221:1181-1183. 6 Bhalla AK, Amento EP, Clemens TL, Holick MF, Krane SM. Specific high-affinity receptors for 1,25-dihydroxyvitamin D3 in human peripheral blood mononuclear cells: presence in monocytes and induction in T lymphocytes following activation. J Clin Endocrinol Metab 1983;57:1308-1310. 7 Lau KHW, Onishi T, Wergedal JE, Singer FR, Baylink DJ. Characterization and assay of tartrateresistant acid phosphatase activity in serum: potential use to assess bone resorption. Clin Chem 1987;32:458-462. 8 Riancho JA, De Francisco ALM, Del Arco C, et al. Serum levels of 1,25-dihydroxyvitamin D after renal transplantation. Miner Elect Metab 1988;14:332-337. 9 Bfyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 1968;21(Suppl 97):77-89. 10 Freake HC, Iwasaki J, McCarthy DM. Specific uptake of 1,25-Dihydroxycholecalciferol by human chronic myeloid leukemia cells. Cancer Res 1984;44:3627-3631. 11 Olmos JM, Amado JA, Riancho JA, Alb/tjar M, Gonz~ilez-Macias J. Sex and age distribution of 1,25(OH)2D3 receptor in peripheral blood mononuclear cells from normal human subjects. Bone 1990;11:407-409. 12 Koren R, Ravid A, Liberman UA, et al. Responsiveness to 1,25-Dihydroxyvitamin D3 is reduced in lymphocytes from osteoporotic women. J Bone Miner Res 1992;7:1057-1061. 13 Yu XP, Hustmyer FG, Benninger L, Peacock M, Manolagas SC. 1,25(OH)2D3 receptor levels in
212
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16
17 18 19
20
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J. Martinez et al./Bone Miner. 23 (1993) 207-212
peripheral blood mononuclear cells of patients with postmenopausal osteoporosis. In: Norman AW, Bouillon R, Thomaset M, eds. Vitamin D: gene regulation, structure-function analysis, and clinical application. Berlin: Walter de Gruiter, 1991;840-841. Koren R, Ravid A, Liberman UA, Hochberg Z, Weisman Y, Novogrodsky A. Defective binding and function of 1,25-dihydroxyvitamin D3 receptors in peripheral blood mononuclear cells of patients with end organ resistance to 1,25-dihydroxyvitamin D. J Clin Invest 1985,76:2012-2015. Bollerslev J, Nielsen HK, Storm T, Mosekilde L. Serum vitamin D metabolites and nuclear uptake of (3H)-l,25-dihydroxyvitamin D3 in monocytes from patients with autosomal dominant osteopetrosis. A study of two radiological types. Bone 1988;9:355-359. Koeffier HP, Bishop JE, Reichel H, et al. Lymphocyte cell lines from vitamin D-dependent rickets type II show functional defects in the 1,25-dihydroxyvitamin D3 receptor. Mol Cell Endocrinol 1990;70:1-11. Horen R, Ravid A, Liberman UA. Peripheral blood mononuclear cells: a model for the human vitamin D endocrine system in health and disease. Mol Cell Endocrinol 1992;83:C9-C12. Korkor AB. Reduced binding of (3H)l,25-dihydroxyvitamin D3 in the parathyroid glands of patients with renal failure. N Engl J Med 1987;316:1573-1577. Olmos JM, Martinez J, Amado JA, Riancho JA, Gonzfilez-Macias J. Increased 1,25-dihydroxyvitamin D3 receptors in peripheral blood mononuclear cells from patients with primary hyperparathyroidism. In: Norman AW, Bouillon R, Thomaset M, eds. Vitamin D: gene regulation, structure-function analysis, and clinical application. Berlin: Walter de Gruiter, 1991;100-101. Martinez J, Olmos JM, de Francisco ALM, Riancho JA, Amado JA, Gonzfilez-Macias J. Reduced binding of (3H)-l,25-dihydroxyvitamin D3 in peripheral blood mononuclear cells from patients with chronic renal failure. Bone Miner 1992;17 (Suppl 1):128. Costa EM, Feldman D. Measurement of 1,25 dihydroxyvitamin D3 receptor turnover by dense amino acid labeling. Endocrinology 1987;120:1173-1178. Costa EM, Feldman D. Homologous up-regulation of the 1,25(OH)2D3 receptor in rat. Biochem Biophys Res Commun 1984;137:742-747. Goff JP, Reinhardt TA, Beckman M J, Horst RL. Contrasting effects of exogenous 1,25-dihydroxyvitamin D (1,25(OH)2D) versus endogenous 1,25(OH)2D, induced by dietary calcium restriction, on vitamin D receptors. Endocrinology 1990;126:1031-1035. Merke J, Nawrot M, Hiigel U, Szabo A, Ritz E. Evidence for in vivo up-regulation of 1,25(OH)2D3 receptor in human monoeytes. Calcif Tissue Int 1989;45:255-256.