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
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Abstract |
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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
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Introduction |
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Elevations in serum levels of markers of bone synthesis such as osteocalcin (BGP) [6], alkaline phosphatase (AP) [1,36] 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 [46]. 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.
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Patients and methods |
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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 [46] 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 8.810.5 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 3.04.5 mg/dl and 35125 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 1055 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.
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Results |
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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 1), after 3 days.
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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 2) 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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Mean values of serum AP (Figure 2) 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 2) 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.
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Discussion |
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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 715 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.
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Notes |
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References |
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