Changes in bone turnover after parathyroidectomy in dialysis patients: role of calcitriol administration

Sandro Mazzaferro1,, Silvana Chicca1, Marzia Pasquali1, Francesco Zaraca2, Paola Ballanti3, Franco Taggi4, Giorgio Coen1, Giulio Alberto Cinotti1 and Manlio Carboni2

1 Department of Clinical Science, 2 2nd Surgical Clinic, 3 Department of Experimental Medicine and Pathology, University ‘La Sapienza’ and 4 Istituto Superiore di Sanità, Rome, Italy



   Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background. Available data on changes in serum levels of bone markers after parathyroidectomy (PTx) in dialysis patients are not uniform. Changes are thought to be due to either a reduction in PTH activity per se or to a direct effect of vitamin D therapy on bone cells. We aimed to verify whether treatment with vitamin D modifies serum levels of markers of bone synthesis (alkaline phosphatase (AP), osteocalcin (BGP), procollagen type I C-terminal peptide (PICP)) and resorption (collagen type I C-terminal peptide (ICTP)) within a period of 15 days in haemodialysis patients with severe secondary hyperparathyroidism following PTx.

Methods. We randomized two groups (A, treatment and B, placebo, 10 patients each) with comparable basal PTH values and measured bone markers 3, 7 and 15 days after surgery. All patients were treated with calcium supplements (i.v. and p.o.), and group A also received calcitriol (2.4±1.0 µg/day, p.o.).

Results. In both groups, PTx induced significant changes in all the markers evaluated, except for BGP in group B. Compared to basal values, ICTP decreased from 481±152 ng/ml in group A and 277±126 ng/ml in group B to 267±94 and 185±71 ng/ml (M±SD) respectively, and PICP increased from 307±139 ng/ml in group A and 309±200 ng/ml in group B to 1129±725 and 1231±1267 ng/ml (M±SD) respectively, within 3 days of surgery. AP values increased after 15 days from 1115±734 mU/ml in group A and 1419±1225 mU/ml in group B to 1917±1225 and 1867±1295 mU/ml (M±SD) respectively. On the contrary, mean values of BGP were never different from basal levels after PTx in either group. In the two groups, the pattern of changes of all the bone markers after PTx was almost identical. Group A patients predictably required lower doses of oral calcium supplements to correct hypocalcaemia (16.9±5.7 vs 22.1±5.0 g/10 days; M±SD, P<0.04).

Conclusions. The opposite behaviour of serum PICP and ICTP after PTx, in both the treated and untreated groups suggests that quantitative uncoupling between bone synthesis and resorption is responsible for hypocalcaemia. This phenomenon, as reflected by the evaluated bone markers, is unaffected by calcitriol. Based on our data we conclude that immediately after parathyroid surgery, vitamin D therapy does not influence bone cell activity, but improves hypocalcaemia mainly through its known effect on intestinal calcium absorption.

Keywords: alkaline phosphatase; bone markers; calcitriol, effects of; collagen type I C-terminal peptide (ICTP); parathyroidectomy; secondary hyperparathyroidism



   Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
After parathyroidectomy (PTx) performed in dialysis patients with severe secondary hyperparathyroidism, acute functional changes are predictable in bone cells. In fact, after surgery, the largely increased pool of osteoblasts and osteoclasts immediately loses the stimulus of PTH and this can presumably result in a substantial modification of bone cell proliferation rate and activity. Changes in the plasma levels of circulating bone markers have been described [1–6].

Elevations in serum levels of markers of bone synthesis such as osteocalcin (BGP) [6], alkaline phosphatase (AP) [1,3–6] and procollagen type I C-terminal peptide (PICP) [6] following PTx have already been described, and in some studies these increases have been tentatively related to the stimulation of osteoblasts by vitamin D, which is administered to improve hypocalcaemia [4–6]. However, vitamin D therapy has been variably associated with increments of BGP [4] or AP [1,3], and none of the published reports was specifically designed to clarify the effect of vitamin D on serum levels of these markers of bone synthesis.

Serum levels of collagen type I C-terminal peptide (ICTP), a bone collagen degradation product that has been shown to correlate with bone resorption in dialysis patients [7], has been recently reported to fall immediately after PTx [8]. However, in this study, information regarding vitamin D therapy was not given. In vitro [9] and in vivo [10] experimental studies suggest that the sterol, in addition to osteoblasts, [11] may actually stimulate osteoclasts [12] and theoretically induce an increment of serum ICTP.

The aim of our study was to rule out acute effects of calcitriol administration on bone cells in dialysis patients undergoing PTx because of severe secondary hyperparathyroidism. Therefore the acute changes in serum levels of bone markers of synthesis and resorption occurring after PTx have been evaluated in patients with or without calcitriol treatment.



   Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Study design
Of 115 dialysis patients (56 female/59 male, age 47±13 years) referred to our outpatient unit between 1993 and 1997 for the evaluation of renal osteodystrophy, 20 (8 male/12 female, age 45.2±10.6, on dialysis for 8.8±4.4 years) were diagnosed as having a severe form of secondary hyperparathyroidism. Based on previous medical records, they were considered unresponsive to medical treatment with calcitriol and then underwent PTx. In all patients vitamin D treatment was withdrawn for at least 1 month before surgery. During this month the clinical records were examined and a blood sample and bone biopsy were obtained. A fasting predialysis blood sample was drawn for the assay of calcium, phosphorus, PTH, AP, BGP, PICP and ICTP. AP, BGP and PICP are markers of bone synthesis, and ICTP is a marker of bone resorption. Transiliac bone biopsy was invariably performed for histomorphometric evaluation and to exclude aluminium accumulation. Informed consent to the study was obtained from every patient.

On the basis of basal PTH levels and with the aim of obtaining two comparable groups, patients were randomly assigned either to a treatment (A) or to a control (B) group.

Complete PTx followed by transplantation of 20 small pieces of parathyroid tissue to the forearm was performed. For the purposes of the present study, efficacy of surgery was verified according to the following criteria: PTH reduction >30% of basal value; serum calcium <7.2 mg/dl and/or calcium decrease >2.5 mg/dl after PTx [13].

After surgery, patients of group A received calcium supplements p.o. and i.v. at doses established daily as clinically required, plus calcitriol p.o. (starting with 2 µg/day and increasing to 4 µg/day if necessary). Patients in group B were treated as necessary with calcium supplements p.o. and i.v. and placebo. In all patients, serum samples for humoral parameters were obtained 3, 7 and 15 days after surgery. A 2-week period was judged suitable to evaluate the effects of calcitriol on bone markers because available data [4–6] demonstrate that the major biochemical changes after PTx occur within this time interval after surgery. After collection, serum samples were immediately frozen and stored at -30°C until assay. To reduce interassay variation, all samples of the same patients were assayed together.

Assay procedures
Details on serum assays have been already reported [6]. Serum calcium was assayed by atomic absorption spectrophotometry (Perkin Elmer, model 2380, Norwalk, CT, USA). Normal values were 8.8–10.5 mg/dl. Serum phosphate and alkaline phosphatase were measured spectrophotometrically by an autoanalyser (DU-65 Beckman, Fullerton, USA) using the substrates molybdate and p-nitrophenylphosphate respectively. Normal ranges are 3.0–4.5 mg/dl and 35–125 mU/ml respectively.

Serum intact iPTH was assayed with a commercial kit (INCSTAR Stillwater, USA) which employs an immunoradiometric technique based on a double polyclonal antibody. Intra- and inter-assay variations are 6.5 and 9.8% respectively. Normal values are 20.8±7.4 pg/ml (M±SD) with a range of 10–55 pg/ml.

Serum osteocalcin was measured by a radioimmunoassay (CIS Biointernational, France). The intra- and inter-assay variations are less than 5 and 8% respectively. Normal values are 6.7±2.6 ng/ml (M±SD).

Serum PICP was assayed by a RIA method (Orion Diagnostica, Finland) employing a rabbit antibody against the propeptide obtained by enzymatic cleavage of the procollagen type I molecule from culture medium of human fibroblasts. Intra- and inter-assay variations are 3 and 5% respectively. Our normal values are 125±30 ng/ml.

Serum ICTP was assayed by an RIA method employing a rabbit antibody directed against human ICTP obtained after bacterial collagenase digestion of femoral-derived bone collagen. Intra- and inter-assay coefficient of variation are 4.9 and 7.1% respectively. Normal values are between 1.8 and 5.0 ng/ml.

Bone biopsy was performed and handled as previously described [7]. The presence of aluminium was checked by the Aluminon histochemical staining technique.

Statistics
Statistical evaluation was performed using SPSS statistics program. Data were evaluated by ANOVA (repeated measurements), and to validate differences between means, Bonferroni'stest was applied. Student'st-test has also been used to compare results of the two allotted groups. The accepted level for a significant difference was P<0.05.



   Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
As shown in Table 1Go, basal clinical and humoral data were not different in the two groups of patients, except for ICTP, which averaged 481±152 ng/ml in group A and 277±126 ng/ml in group B (P<0.009).


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Table 1. Basal clinical, biochemical and bone histological data (M±SD) in the two groups of patients

 
Bone histology invariably revealed a condition of severe, pure hyperparathyroidism and no case of positive staining for aluminium was found. As shown by the histomorphometric results, the severity of bone lesions was comparable in the two groups (Table 1Go).

Based on the criteria suggested by Gagné et al. [13], surgical procedure was considered effective in all patients whose serum levels of PTH dropped to mean values of 43±58 (ANOVA, P<0.00001) and 125±114 pg/ml (ANOVA, P<0.00001) respectively in groups A and B (Figure 1Go), after 3 days.



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Fig. 1. Mean values (±SEM) of serum PTH and calcium in group A (calcitriol+calcium) and group B (calcium) following parathyroidectomy. Mean values after 3, 7 and 15 days were always significantly different from basal values in both groups for both parameters (Bonferroni test, P<0.05). *P<0.02 compared to the corresponding mean value of group B.

 
Hypocalcaemia (Figure 1Go) required intensive treatment and surveillance mostly within the first 10 days, but no patient experienced cramps or tetany. In this period, the total administered dose of i.v. calcium gluconate was 558.6±485 mg in group A and 634±298 mg in group B (n.s.), while total oral calcium salts supplements averaged 16.9±5.7 and 22.1±5.0 g (P<0.04) respectively in the two groups. In the treated group, the average administered daily dose of calcitriol was 2.4±1.0 µg. Group A had significantly higher serum calcium levels at day 15 than group B (6.9±0.7 vs 7.7±0.7 mg/dl, P<0.02, Figure 1Go).

As with calcium levels, serum levels of phosphate promptly fell in both groups from 6.4±1.5 to 3.4±0.9 and 3.0±0.48 mg/dl in group A and from 5.6±1.5 to 3.1±1.0 and 2.7±1.0 mg/dl in group B after 3 and 15 days respectively, P<0.0001).

Despite different basal values, a decrease in ICTP serum levels was recorded in both groups (ANOVA, P<0.0001, Figure 2Go) with values significantly different from basal levels after 3, 7 and 15 days (Bonferroni test, P<0.05). The difference between mean values of the two groups persisted until day 7.



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Fig. 2. Mean values (±SEM) of serum markers of bone resorption (ICTP) and synthesis (PICP, AP, BGP) in group A (calcitriol+calcium) and group B (calcium) following parathyroidectomy. *Significantly different from the basal value of the same group (Bonferroni test, P<0.05). Mean values of ICTP were found to be significantly different between the two groups at baseline (P<0.009), at 3 days (P<0.05), and at 7 days (P<0.02) after surgery.

 
Among the bone markers indicating synthesis, we found an early transient increase in serum levels of PICP (Figure 2Go), peaking in both groups at 3 and 7 days (Bonferroni test, P<0.05 as compared to the respective basal value). Mean changes of PICP after PTx were almost identical in the two groups.

Mean values of serum AP (Figure 2Go) also increased; however, this occurred after 15 days. In addition, for this parameter mean changes after surgery were not different between the two groups.

Finally, the mean values of serum BGP (Figure 2Go) obtained on days 0, 3, 7 and 15 were different in group A (ANOVA, P<0.09); however, the values obtained 3, 7 and 15 days after PTx were not statistically different from the basal levels.



   Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The described time-course changes of bone markers after PTx are not uniform [1–6], and for AP and BGP both increases [1,3–6] and stability [2–5] have been reported. Because in vitro studies [9,11,12] demonstrate a direct action of vitamin D on bone, increases have been generally attributed to a direct stimulation of osteoblasts by vitamin D therapy. This hypothesis seems to be confirmed indirectly by the available data in osteoporotic women, which showed an increase in serum levels of BGP [14–16] and AP [16] after 1 week of treatment with vitamin D. In addition an increase in serum BGP has been described in haemodialysis patients after 2 weeks of calcitriol therapy [17].

AP values have been reported to increase within 2 weeks of PTx either during [4] or in the absence of [2] vitamin D administration, while Clair et al. [3] described no change in treated patients and an increase in untreated patients. Serum levels of BGP have been reported to be stable after PTx both with [4,5] and without [2] calcitriol administration; however, in at least two studies [4,5] the presence of bone aluminium accumulation could have played an inhibitory role on BGP production [18]. In fact in the absence of aluminium accumulation, we reported an increase of BGP within 7–15 days of PTx in dialysis patients treated with vitamin D [6]. Several potential variable factors (type of bone lesion, aluminium toxicity etc.) do not allow comparison of studies, and may well explain the observed discrepancies in the literature. Moreover, none of the available clinical studies performed in CRF patients after PTx is specifically designed to unravel the possible direct effect of vitamin D on bone markers and on bone cells. Moreover, only one paper [8] reports on the levels of ICTP, a potential marker of resorption.

In the present study all patients had biopsy-proven severe renal osteodystrophy with prevailing hyperparathyroidism and absence of aluminium accumulation. In both groups the abrupt decrease in PTH resulted in a prompt reduction of ICTP beginning after the third day after PTx (due to reduction of bone resorption) associated with an increment of PICP (suggesting increased bone formation). AP increased after 15 days, while basal serum BGP values were not different from those obtained in the follow-up period in both groups. Of note is the fact that the pattern of change in serum levels of all the evaluated bone markers was almost identical in the two groups, independently of calcitriol administration. In particular, the reduction of ICTP serum levels, comparable in both groups both with and without vitamin D, suggests that the sterol has no direct significant stimulus on bone resorption, which is contrary to what has been reported in normal subjects [10]. On the other hand, the transient increase in serum PICP, similarly found in both groups, confirms the specific inhibitory effect of PTH on osteoblastic collagen synthesis [19] and suggests that this action too is unaffected by vitamin D administration.

We are aware, nonetheless, that the action of vitamin D on bone could have been reflected by other bone markers such as bone-specific AP or tartrate-resistant acid phosphatase. These were not included in our study because when we began our prospective trial their assays were not as reliable as they now are.

The fall in ICTP serum levels, together with the increment of PICP, mirror the presence of a transient quantitative uncoupling between bone formation and resorption, a phenomenon currently invoked to explain hypocalcaemia after PTx [20]. Based on our data, we can estimate that this phenomenon is substantially unaffected by vitamin D administration. On the other hand, taking into account the lower oral dose of calcium supplements employed in patients treated with calcitriol, we would favour the hypothesis that administration of vitamin D after PTx improves hypocalcaemia mainly through stimulation of the intestinal absorption of calcium. Alternatively, we could hypothesize that the sterol enhances calcium efflux across the quiescent layer of bone cells from the rapidly exchangeable pool.

The humoral changes observed in this study could theoretically be regarded as changes in bone cell numbers rather than activity. In fact, experimental animal studies demonstrate that within 60 h of PTH administration or withdrawal, the numbers of osteoclasts in bone really do change [21]. Therefore, in our opinion, the humoral modifications after PTx could be ascribed both to numerical and functional changes in bone cells.

Finally, in the present study we have not confirmed the increase in BGP following PTx, described in a previous study [6]. Although the employed assay procedures were identical in the two studies, it must be underlined that in contrast to PICP and ICTP, which showed uniform changes after surgery, BGP modifications are much more variable in the individual patient. In fact, while most patients showed increasing serum BGP levels after PTx, some cases showed stable or even declining values. In our opinion this variability helps explain the disagreement of published results pertaining to BGP after PTx.

In conclusion, at the doses employed herein, vitamin D administration seems to be almost irrelevant to osteoblastic and osteoclastic cells after PTx, as reflected by serum levels of PICP and ICTP. Such cells appear rather to be regulated mostly by PTH.



   Notes
 
Correspondence and offprint requests to: Dr Sandro Mazzaferro, Department of Clinical Science, c/o Policlinico Umberto I, 2 Clinica Medica, Viale del Policlinico, 151, I-00161 Rome, Italy. Back



   References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Received for publication: 29. 4.99
Revision received 9. 2.00.



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