Changes in serum osteocalcin levels in the follow-up of kidney transplantation

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1 Original Article Ann Clin Biochem 1997; 34: Changes in serum osteocalcin levels in the follow-up of kidney transplantation M R Bonnin", M T Gonzalez-, J M GriIi6 2, J M Cruzado", J Bover-, J M Martinez! and M A Navarro! From the 'Llormone Unit (Biochemistry Service) and 2Nephrology Service, Ciudad Sanitaria y Universitaria de Bellvitge, Cl Feixa Llarga, s.n, 08907, L'Hospitalet de Llobregat, Barcelona, Spain SUMMARY. Serum osteocalcin, total alkaline phosphatase, intact parathyroid hormone (PTH), creatinine, calcium, and phosphate were determined in 23 kidney cadaveric allograft recipients, immediately before and 0 5, I, 3 and 6 months after surgery. Immunosuppressive treatment was based on low doses of corticosteroids and cyclosporin combined with antilymphoblast globulin. The decrease in serum creatinine was accompanied by falling PTH concentrations. Serum osteocalcin levels were higher than normal before kidney transplantation and diminished at 0 5 and I month after surgery. Significant increases in serum osteocalcin concentrations were observed 3 and 6 months after kidney transplantation with a significant correlation with alkaline phosphatase levels. The increase in serum osteocalcin levels observed in our transplanted patients is not related with a parallel increase in serum creatinine levels nor with an increment in PTH levels; it seems to reflect an increase in the osteoblastic activity, which is not altered by steroid therapy. Additional key phrases: parathyroid hormone; markers of bone turnover; steroid treatment Osteocalcin, bone Gla protein or y-carboxyglutamic acid containing protein is a vitamin K-dependent protein synthesized by the osteoblasts.' Although its significance is not completely known, it has been reported to be a marker of bone turnover, and its concentration seems to indicate the extent of bone formation whenever resorption and formation are dissociated.? It has been observed to be a specific marker of bone formation in patients on maintenance haemodialysis with renal osteodystrophy.' Nevertheless, it is also increased due to reduced renal clearance of the protein. In patients with chronic renal failure and different degrees of secondary hyperparathyroidism the intact molecule and some immunoreactive fragments of unknown significance have been detected." These fragments tend to accumulate, thus contributing to the high levels of circulating immunoreactivity detected by most currently available methods. The recovery of renal Correspondence: Dr M R Bonnin. function after kidney transplantation is usually accompanied by a significant decrease in osteocalcin concentrations as well as by a marked improvement of bone lesions." Osteocalcin may also be useful to differentiate patients with high and low turnover lesions." In patients on chronic dialysis programmes a good correlation between intact parathyroid hormone (PTH) and osteocalcin has been reported. However, some discrepancies have arisen from the results obtained by different groups regarding the evolution of osteocalcin after kidney transplantation. Some authors have reported a decrease in serum osteocalcin during the first six months following a successful kidney transplant." Previous results reported by our group have shown increased osteocalcin levels in 56 renal transplanted patients." However, the wide variations in osteocalcin concentrations reported in the literature can also be attributed to the different specificity of currently available methods," which are directly dependent on the origin of the standards, tracers and antisera. Furthermore, corticosteroids can reduce bone 651

2 652 Bonnin et al. formation!' expressed by a marked decrease in osteocalcin levels.l? The aim of this study was to assess osteocalcin as a marker of bone metabolism in patients treated with low doses of corticosteroids. MATERIALS AND METHODS Subjects Twenty-three patients (10 men and 13 women; mean age 42 2 years, range 18-66) with different degrees of secondary hyperparathyroidism who received a kidney cadaveric allograft were included in the study. None of the patients had undergone parathyroidectomy, and all had received calcitriol supplements prior to transplantation. Liver function was assessed in all patients through y-glutamyl transferase (GGT) levels; patients with impaired liver function were excluded. None of the females were receiving oestrogen treatment. The post-transplant immunosuppressive therapy was prednisone (0' 5 mg/kg/day during the first 10 days, 0 25 mgjkg/day until day 90 and 0 1 mg/kg/day thereafter), in combination with cyclosporin and lymphoglobulin.l' The total cumulative doses of steroids were similar to those described in previous reports." No rejection episodes were detected in any of the patients during the study, so the dose of corticosteroids was decreased progressively after kidney transplantation. Basal serum samples were obtained prior to transplantation and at 15, 30, 90 and 180 days after kidney transplantation to measure the serum biochemistry. Analytical methods Serum creatinine, calcium, phosphate, total alkaline phosphatase and GGT were routinely measured by a Hitachi analyser (Boehringer Mannheim, Lewes, UK) in the day of venipuncture. Serum PTH concentrations were determined by an immunoradiometric assay (lncstar Corporation, Stillwater, MN, USA), with a reference interval of 1 1 to 4 6 pmol/l, as calculated by our laboratory.p The inter-assay coefficient of variation (CY) was 7 1% at 4 1 pmol/l, Serum osteocalcin concentrations were determined with a commercial immunoradiometric assay (ELSA-OSTEO, Cis biointernational) by means of two monoclonal antibodies against human osteocalcin. With this procedure both the intact molecule and a big fragment (1-43) were assessed. The reference interval, mean (SD) as established in our laboratory from 81 healthy individuals (43 men and 38 women; age range years, mean 41) was 19 8 (5'7) Jl.g/Lin men and 18 3 (7'5) Jl.g/L in women. No significant differences were found between sexes. Serum for PTH and osteocalcin was separated by centrifugation within I h of venipuncture and stored at - 80 nc until analysis. In both assays, samples were assayed in duplicate and the longitudinal samples of one patient were performed within the same assay. Statistical analysis Between and within subject differences were checked through the analysis of variance (ANOYA) for repeated measures and the Friedman test. Subgroup differences measured by Scheffe test were neglected if the overall test was not significant. Correlation coefficients were obtained from the non-parametric Spearman's rank test. The level of significance was defined in all tests as P < RESULTS After kidney transplantation, individual serum creatinine concentrations showed a progressive decrease when compared with basal values (P=0 0004; ANOVA), being in most patients below 200 Jl.mol/L at day 180. Prior to transplantation serum phosphate levels were high and decreased to normal levels after kidney transplantation (P= ; ANOVA). Serum calcium concentration remained in the normal range throughout the study. Serum GGT levels were always within the reference range. Serum alkaline phosphatase levels did not change significantly during the first months after surgery, however a significant increase was observed 3 months after kidney transplantation (P = 0'03; Friedman). Although seven patients (30%) had basal concentrations of ipth in the normal range, the mean value was higher than the normal population (20'5 pmol/l versus 4 6 prnol/l). In patients with high serum PTH levels a progressive decrease was observed during follow-up (P = ), however some patients retained abnormally high PTH values. The patients with low serum PTH levels before kidney transplantation did not change significantly during follow-up.

3 Serum osteocalcin levels after kidney transplantation 653 Serum osteocalcin concentrations were about eight-fold higher than normal before kidney transplantation, showing a marked decrease during the first 2 weeks after transplantation. Within the first month osteocalcin levels remained just above the upper reference interval and subsequently increased (P=O'OOOOI; Friedman). Mean serum levels of PTH, osteocalcin and alkaline phosphatase are given in Table I. A significant positive correlation was obtained between alkaline phosphatase and osteocalcin values after I month (r = 0 49; P = 0,04), 3 months (r = 0 49; P = 0,03) and 6 months after a transplant (r = 0 60; P = 0'006). Although a significant correlation was found between PTH and osteocalcin values prior to kidney transplantation (r = 0,49, P = 0,02), these two parameters were not correlated at any time after surgery. DISCUSSION Secondary hyperparathyroidism is a common complication of chronic renal failure" resulting in bone disease." In the first weeks after kidney transplantation metabolic and endocrine adaptations usually result in the overactivity of the parathyroid gland diminishing. 18 However, there is considerable variation between patients in the time taken for these falls in PTH to OCCUr. 19 Parathyroidectomy may still be required if hyperparathyroidism is severe. Furthermore, immunosuppressive therapy may influence the recovery of bone disease in kidney transplant recipients. Corticosteroids induce a reduction of bone mass probably due to their effect on the osteoblasts-? in association with increased bone resorption." This is particularly relevant in patients with previous bone lesions due to secondary hyperparathyroidism. There are conflicting results with respect to whether cyclosporin can affect osteocalcin concentration and bone remodelling. In a previous study by our group it was demonstrated that immunosuppressive treatment employed after kidney transplantation does not appear to influence the development of hyperparathyroidism.f Bone biopsy is by far the most accurate technique to study these abnormalities, but is considered to be too invasive for routine use. Therefore, biochemical markers of bone turnover could be very helpful in the follow-up of transplant patients with potential metabolic bone disorders. Osteocalcin is considered a marker of bone remodelling, in which changes in renal disease have been previously studied. 23,24 Assays for serum osteocalcin measure both the whole, native molecule and some of its immunoreactive fragments (products of osteocalcin breakdown). The high serum osteocalcin concentrations described in patients with chronic renal failure reflect both the elevated bone turnover and the glomerular retention of the fragments. Following successful renal transplantation serum osteocalcin levels fall rapidly, partly due to the improvement in renal function, leading to the increased clearance from the circulation. In addition, serum osteocalcin concentrations seem to decrease with high doses of steroids.p In a cross-sectional study? we have previously shown decreased osteocalcin concentrations after kidney transplantation in 56 patients examined at various times between I and 70 months after transplantation. Other reports have shown decreased osteocalcin levels 3, 6 and 8 months after kidney allografting.! In our patients serum PTH and creatinine levels decreased significantly with time, however some patients did not normalize PTH levels at 3 TABLE 1. Serum parathyroid hormone (PTH). osteocalcin and alkaline phosphatase (ALP) concentrations before and after kidney transplantation PTH Osteocalcin ALP (pmol/l) (Jlg/L) CJlkat/L) n=23 n=23 n=23 Basal 20 5 (21'1) (286) 1 4 (1'1) 15 days 13 4 (9'9) 37 6 (42.8) 1 33 (0'77) 30 days 12 6 (12'1) 32 1 (30'5) 1-45 (0'67) 90 days 8 2 (7'1) 50 3 (34'4) 2 03 (1'26) 180 days 8.2 (6'2) 75 9 (42) 2 65 (2) Results are mean (SD). Reference ranges for PTH are 1 I--4 6pmol/L, for osteocalcin Jlg/L, and for alkaline phosphatase Jlkat/L

4 654 Bonnin et al. and 6 month follow-up, probably due to the persistence of glandular hypertrophy. Calcium and phosphate concentrations remain in the normal range after transplantation, so these analytes are unlikely to be causing the high PTH concentrations. Alkaline phosphatase levels were within the normal range before transplantation, with a subsequent increase between 3 to 6 months after transplantation. In this study, serum osteocalcin concentrations were found to be high before kidney transplantation, reflecting both the high bone turnover and decreased clearance associated with renal insufficiency. The osteocalcin concentrations decreased between 15 and 30 days after transplantation. These decreases were probably due to a combination of the corticosteroid therapy and the improvement in renal function. The rise in serum osteocalcin levels observed in our patients 3 and 6 months after renal transplantation is not in accordance with previous reports." This difference might be attributed to the higher doses of steroids used by these authors in comparison with lower doses administered in our patients. It is known that total alkaline phosphatase is a less sensitive marker of osteoblastic activity than osteocalcin because it is also synthesized in other tissues.p In our patients the highly significant correlations obtained between osteocalcin and alkaline phosphatase after kidney transplantation indicate that both parameters show a similar behaviour with respect to the osteoblastic status. However, osteocalcin measurements should be used if liver disease is present. In contrast to other reports." no correlation was found between PTH and osteocalcin concentrations at any time after kidney transplant, suggesting that increased osteocalcin levels reported in this study are not due to an increased bone turnover provoked by the persistence of some degree of secondary hyperparathyroidism. In conclusion, we have presented evidence that osteocalcin concentrations can remain above the reference range, in spite of significant decreases in creatinine and PTH levels, within the first 6 months after kidney transplantation. Furthermore, the significant correlation with total alkaline phosphatase shows that both markers are reflecting an increase in the osteoblastic activity. The low doses of corticosteroids used in our transplant schedule do not seem to interfere in serum osteocalcin production as much as previously reported. REFERENCES Garnero P, Grimaux M, Seguin P, Delmas PD. Characterization of immunoreactive forms of human osteocalcin generated in vivo and in vitro. J Bone Mineral Res 1994; 9: 255--M 2 Worsfold M, Sharp CA, Davie MW1. Serum osteocalcin and other indices of bone formation: an 8-decade population study in healthy men and women. Clin Chim Acta 1988; 178: Malluche HH, Faugere MC, Fanti P, Price PA. Plasma levels of bone gla-protein reflect bone formation in patients on chronic maintenance dialysis. Kidney Int 1984; 26: Gundberg CM, Weinstein RS. Muliple immunoreactive forms of osteocalcin in uremic serum. J Clin Invest 1986; 77: Epstein S, Traberg H, Raja R, Poser 1. Serum and dialysate osteocalcin levels in hemodialysis and peritoneal dialysis patients and after renal transplantation. J Clin Endocrinol Metab 1985; 60: I 253--{j 6 Gonzalez MT, Gonzalez C, Grifio 1M, Castelao AM, Marifioso ML, Serrano S, et al. Long-term evolution of renal osteodystrophy after kidney transplantation: comparative study between intact PTH levels and bone biopsy. Transplant Proc 1990; 22: Taylor AK, Lueken SA, Libanati C, Baylink 01. Biochemical markers of bone turnover for the clinical assessment of bone metabolism. Clin N Am 1994; 20: 589--{j06 8 Boiskin I, Epstein S, Ismail F, Thomas SB, Raja R. Serum osteocalcin and bone mineral metabolism following successful renal transplantation. Clin Nephrol1989; 31: Bonnin MR, Gonzalez MT, Huguet 1, Guillen E, Navarro MA. Serum osteocalcin levels in patients after kidney transplantation. Clin Nephrol 1990; 3: Gundberg CM, Wilson PS, Gallop PM, Parfitt AM. Determination of osteocalcin in human serum: results with two kits compared with those by a well characterized assay. Clin Chern 1985; 34: 172Q--3 II Lukertand BP, Raisz LG. Glucocorticoid-induced osteoporosis: pathogenesis and management. Ann Int Med 1990; 112: 352--M 12 Kaspersen H, Charles P, Mosekilde L. The effect of single oral doses of predisone on the circadian rhythm of serum osteocalcin in normal subjects. J Clin Endocrinol Metab 1988; 67: Grifio 1M, Alsina 1, Sabater R, Caste1ao AM, Gil-Vernet S, Andres E, et al. Antilymphoblast globulin, cyclosporine, and steroids in cadaveric renal transplantation. Transplantation 1990; 49: Grifio 1M, Castelao AM, Seron 0, Gonzalez C, Galceran 1M, Gil-Vernet S, et al. Antilymphocyte Globulin versus OKT3 induction therapy in cadaveric kidney transplantation: A prospective randomized study. Am J Kidney Dis 1992; 20: Ann C/in Biochem 1997: 34

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