amino terminus of the hormone, detect not only PTH(1-84) Accepted for publication December 13, 2002

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1 Kidney International, Vol. 63 (2003), pp CLINICAL NEPHROLOGY EPIDEMIOLOGY CLINICAL TRIALS Similar predictive value of bone turnover using first- and second-generation immunometric PTH assays in pediatric patients treated with peritoneal dialysis ISIDRO B. SALUSKY, WILLIAM G. GOODMAN, BEATRIZ D. KUIZON, JEFFREY R. LAVIGNE, RICHARD J. ZAHRANIK, BARBARA GALES, HE-JING WANG, ROBERT M. ELASHOFF, and HARALD JÜPPNER Division of Pediatric Nephrology, Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, California; Division of Nephrology, Department of Medicine and Department of Biomathematics, David Geffen School of Medicine at UCLA, Los Angeles, California; Immutopics, San Clemente, California; and Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts Similar predictive value of bone turnover using first- and second- The term renal osteodystrophy refers to a spectrum of generation immunometric PTH assays in pediatric patients treated bone diseases in patients with renal failure. The spectrum with peritoneal dialysis. Background. Accurate measurements of the concentration ranges from high-turnover lesions arising predominantly of parathyroid hormone (PTH) in serum or plasma are essential from excess of parathyroid hormone (PTH) secretion to for the proper assessment of renal osteodystrophy. The first- low-turnover lesions that are most often associated with generation immunometric PTH assay (1st PTH-IMA) not only normal or reduced serum PTH levels [1]. Determinations detects the intact hormone, but also additional PTH fragments truncated at the amino N-terminally truncated PTH-derived of PTH levels are used as surrogates for bone histology, fragments [ntpth(1-84)]. A second-generation immunometric which remains the most reliable method to establish the PTH assay (2nd PTH-IMA) recognizes only PTH(1-84) and diagnosis of renal osteodystrophy [2 4]. Accurate meapossibly PTH fragments that are truncated at the carboxylsurements of the concentration of PTH in serum or plasma terminus but not PTH(7-84). Whether estimates of the ratio between PTH(1-84) and ntpth(1-84) fragments are a better are therefore essential for the proper assessment of renal predictor of bone turnover remains controversial. osteodystrophy [5 9]. Methods. Thirty-three patients aged years treated The first clinically useful two-site immunometric PTH with continuous cycling peritoneal dialysis (CCPD) for 13 9 assay (PTH-IMA) employed two antibodies directed months underwent iliac crest bone biopsy. PTH levels were measured by two newly developed first-generation and secondtively, and was, therefore, predicted to measure only full- against amino- and carboxyl-terminal epitopes, respecgeneration PTH-IMA. The ntpth(1-84) fragments were calculated by subtracting PTH values determined using the 2nd length PTH(1-84) [10]. This first-generation immunomet- PTH-IMA from values obtained using 1st PTH-IMA that deric PTH assay (1st PTH-IMA) proved to be a reasonably tects both PTH(1-84) and relatively large ntpth(1-84). Results. Determinations of PTH levels by both assays were reliable predictor of the different subtypes of renal osteohighly correlated (r 0.89, P 0.001). The relationships be- dystrophy, and it performed well in assessing the theratween first-generation and second-generation PTH-IMA and peutic response to active vitamin D sterols in patients bone formation were similar (r 0.67, P and r 0.64, with renal failure [5, 8, 9]. However, recent observations P , respectively). When patients were grouped according to the presence or absence of secondary hyperparathyroidism, highlight important shortcomings of this and other PTH- the ratio PTH(1-84) to ntpth(1-84) did not differ between groups. IMAs [11, 12]. Conclusion. PTH concentrations determined by either the In particular, a series of studies by Brossard et al [11], first- or the second-generation PTH-IMA were found to be better predictors of bone formation than the PTH(1-84) to ntpth(1-84) Lepage et al [12], Brossard et al [13], and Brossard, Yamafragments ratio. moto, and D Amour [14], demonstrated that 1st PTH- IMAs detect not only the intact hormone, but also additional PTH fragments truncated at the amino terminus Key words: parathyroid hormone, immunometric PTH assay, peritoneal dialysis. [ntpth(1-84)]. Indeed, most of the detection antibodies, which are usually directed against epitopes within the Received for publication October 2, 2002 and in revised form November 8, 2002 amino terminus of the hormone, detect not only PTH(1-84) Accepted for publication December 13, 2002 but also one or several ntpth(1-84) fragments, some of 2003 by the International Society of Nephrology which coelute from reverse-phase high-performance liquid 1801

2 1802 Salusky et al: First- and second-generation immunometric PTH assays and bone turnover chromatography (HPLC) column with synthetic PTH(7-84) bone biopsy procedure; none of the patients received [12]. In contrast, a more recently developed second-generation prior intermittent calcitriol therapy. The study was apa immunometric PTH assay (2nd PTH-IMA) using proved by the UCLA Human Subject Protection Com- detection antibody raised against the first four amino mittee, and informed consent was obtained from all pa- terminal amino acids of human PTH recognizes only tients and/or parents. PTH(1-84) and possibly PTH fragments that are truncated Patients were admitted to the UCLA General Clinical at the carboxyl terminus but not PTH(7-84) [15 17]. Research Center and bone biopsies were obtained from Consistent with this assessment human PTH(1-34), but the anterior iliac crest using a modified Bordier trephine not human PTH(2-34) or other amino terminally truncated after double tetracycline as previously described [3]. Serum fragments of human PTH(1-34) cross-reacted with the samples for measurements of calcium, phosphorus, detection antibody [15 17]. First-generation PTH-IMAs, alkaline phosphatase, and PTH levels were obtained at thus, overestimate the true concentration of PTH(1-84) bone biopsy. Quantitative histomorphometry of bone in serum or plasma both in patients with ESRD and was done as previously described [3] and the terminology those with normal renal function [14, 15, 17 19]. established by the Nomenclature Committee of the Amer- Recent in vitro and in vivo experimental data also ican Society of Bone and Mineral Research was used for indicate that one or more amino terminally truncated all the results [22]. Reference values from subjects with PTH(1-84) fragments antagonize the calcemic actions of normal renal function were obtained from iliac crest PTH(1-84) and diminish bone cell activity, and may, bone biopsy done in 27 children between the ages of 2.5 thereby, modulate bone metabolism. These actions are and 17 years who had undergone elective orthopedic or possibly mediated through a receptor distinct from the urologic surgery procedures. Normal bone formation rate type I PTH/PTHrP receptor [16, 20]. Such findings sug- ranged between 97 and 613 m 2 /mm 2 /day and such range gest that ntpth(1-84) fragments may contribute to the was utilized for the classification of patients into high, nor- skeletal PTH resistance that occurs in patients with renal mal, or reduced rates of bone formation and turnover [3]. failure [16, 20]. It, therefore, appears plausible that an Serum calcium, phosphorus, and alkaline phosphatase independent assessment of PTH(1-84) and ntpth(1-84) levels were determined using a Technicon Autoanalyzer fragments may have a better value for diagnosis of the II. Serum PTH determinations were performed by two different subtypes of renal osteodystrophy than each pa- immunometric specific assays (Immutopics, San Clemente, rameter alone. CA, USA), one that specifically detects only the biologi- Indeed, it has been suggested that estimates of the cally active full-length PTH(1-84) (2nd PTH-IMA) and ratio between PTH(1-84) and ntpth(1-84) fragments another that detects with indistinguishable efficiency fullmore accurately predicts bone remodeling and turnover length PTH(1-84) and the PTH(7-84) fragment (1st PTH- in patients with ESRD [18]. On the other hand, a more IMA) as has been previously described with a similar assay recent report failed to confirm such findings [21]. To [15]. Both assays use goat polyclonal antibodies purified further examine the utility of 1st and 2nd PTH-IMAs as by affinity chromatography, which recognizes specific predictors of bone formation in ESRD, PTH measureused and distant epitopes along the molecule. Most reagents ments were performed with two newly developed assays in both assays are identical (i.e., the same capture in pediatric patients treated with peritoneal dialysis. antibody recognizing epitopes within the region of the PTH molecule, identical standards and controls, and METHODS identical buffer and incubation conditions). The differences between both assays are two distinct detection A total of 33 patients, 13 females and 20 males, were antibodies (Fig. 1). For the 2nd PTH-IMA, the detection entered into the study; the mean age at the time of bone antibody recognizes an initial epitope within the amino biopsy was years and they have been treated terminus (residues 1 to 3), and for the 1st PTH-IMA, with CCPD for 13 9 months. All patients were treated the detection antibody recognizes an epitope within the with peritoneal dialysis (Daniel, Baxter, Deerfield, IL, 13 to 34 region of the molecule. Thus, the assays measure USA) with a dialysate calcium concentration of 2.5 different PTH fragment concentrations without influence meq/l and glucose concentration that was adjusted ac- by matrix, standard, or incubation procedures. Values for cording to the needs for ultrafiltration. All patients were subjects with normal renal function range from 10 to 65 receiving calcium carbonate as phosphate-binding agent. pg/ml for the 1st PTH-IMA and 2 to 32 pg/ml for the None of the patients were treated with steroids or cyclo- 2nd PTH-IMA. sporine for at least 12 months prior to the biopsy and only two patients were previously treated with recombinant Evaluation of PTH fragment specificity human growth hormone, and therapy was discontinued 5 The specificity of the two immunometric PTH assays and 11 months before the performance of the procedure. was determined by assessing cross-reactivity with increasing Therapy with calcitriol was held for 4 weeks before the concentrations of human (h) hpth(7-84) (Bachem Cal-

3 Salusky et al: First- and second-generation immunometric PTH assays and bone turnover 1803 Fig. 1. A description of the technical aspects of the first- and second- generation immunometric parathyroid hormone assay (1st generation and 2nd generation PTH-IMAs). Both assays employ capture antibodies that recognize epitopes within the 39 to 84 region of the PTH molecule. The differences between the assays are two distinct antibodies. In the 1st PTH-IMA, the detection antibody recognizes an epitope within the13 to 34 region of the molecule. In contrast, for the 2nd PTH-IMA, the antibody recognizes an initial epitope within the amino terminus (residues 1 to 3). ifornia, Inc., Torrance, CA, USA). Further characterization of both assays was obtained by coincubating hpth(1-84) (545 pg/ml) with increasing concentrations of [Tyr34] hpth(1-34)amide (hpth(1-34)), [Tyr34]hPTH(2-34)am- ide (hpth(2-34)), [Tyr-34]hPTH(3-34)amide (hpth(3-34)), [Tyr-34]hPTH(4-34) amide (hpth(4-34)), and [Tyr34]hPTH (5-34) amide (hpth(5-34)) as described [15 17]. All pep- tides were synthesized and quantified by the Massachu- setts General Hospital Polymer Core Facility. RESULTS Characterization of the two PTH assays To assess the specificity of both immunometric assays, each was characterized through in vitro competition experiments using several synthetic PTH peptides. The 1st PTH-IMA demonstrated equal cross-reactivity for both hpth(1-84) and hpth(7-84), while the 2nd PTH-IMA failed to detect hpth(7-84) at concentrations as high as 20,000 pg/ml; (data not shown) these findings are consistent with previous reports using a similar assay [15 17]. The specificity of the two distinct detection antibodies, which are directed for both assays against an epitope within the amino terminal portion of hpth(1-84), was further examined by incubating a constant concentration of hpth(1-84) (545 pg/ml) with increasing amounts of several different PTH fragments. In the 1st PTH-IMA, specific binding of the detection antibody to hpth(1-84) was progressively and with similar efficacy reduced in the presence of increasing concentrations of hpth(1-34), hpth(2-34), hpth(3-34), hpth(4-34), and hpth(5-34) (data not shown). In contrast, in the 2nd PTH-IMA, hpth(1-34) revealed the highest efficacy to reduce binding of the detection antibody, while hpth(2-34) and hpth(3-34) showed 50% and 4%, respectively, of the potency of hpth(1-34); hpth(4-34) had a potency of 0.1%, and hpth(5-34) completely failed to displace the detection antibody. Statistical analysis Comparison of 1st and 2nd PTH-IMAs in patients treated with dialysis All results are presented as the mean one standard deviation (SD). Patients were classified in two groups Among the 33 bone biopsies with concurrent determi- nations of serum PTH levels by both assays, there was according to the upper value for bone formation rate evidence of increased bone formation rate, above the upobtained in subjects with normal renal function after per limit of normal, in 23 patients, lesion consistent with double tetracycline labeling [3]. PTH determinations obsecondary hyperparathyroidism; indices of bone formatained with 1st and 2nd PTH-IMA as well as the tion were within the normal range in seven patients, and ntpth(1-84) fragments and the ratio PTH(1-84) to values were below the lower limit of normal in three ntpth(1-84) fragments were compared between the two patients, lesion consistent with adynamic osteodystrophy groups using Wilcoxon rank sum test. Under each group, as previously shown [3]. Patients were classified into two the receiver operating curves (ROC) were generated for groups: (1) high bone turnover group, which includes all the different tests (2nd PTH-IMA, 1st PTH-IMA, and the patients (N 23) with bone formation rates above the ratio). The ntpth(1-84) fragments were calculated by upper limit of normal from our laboratory [24]; and (2) subtracting PTH values determined using the 2nd PTH- patients with normal/reduced bone turnover group (N IMA, which detects PTH(1-84) exclusively, from values 10), because these patients do not require vitamin D therobtained using 1st PTH-IMA that detects both PTH(1- apy, and due to the small sample size of patients with 84) and ntpth(1-84). The PTH(1-84)/ ntpth(1-84) frag- adynamic bone. The mean age of the patients were 13.3 ments ratio was calculated as previously described [18]. 3.6 years and years in the high and normal/ The areas under the ROC curves and their standard low turnover groups, respectively (NS), (Table 1) and errors were obtained and comparisons were carried out the mean duration of treatment with peritoneal dialysis as previously described [23]. For given cut off points, did not differ between the two groups. The mean values sensitivity, specificity, number of true positive, true nega- for calcium, phosphorus, alkaline phosphatase activity, tive, false positive, and false negative were calculated. and PTH determinations measured by 1st and 2nd PTH- All statistical analyses were performed using software IMA in each of the two groups are shown in Table 1. package SAS and two-sided significance level of Serum calcium and phosphorus levels were similar in both

4 1804 Salusky et al: First- and second-generation immunometric PTH assays and bone turnover Table 1. Patient characteristics and serum biochemical determinations according to indices of bone turnover High bone Normal/low turnover bone turnover (N 23) (N 10) Bone formation rate a lm 2 /mm 2 /day Age years Dialysis months Serum calcium mg/dl Serum phosphorus mg/dl Serum total alkaline phosphatase IU/L b Serum 1st PTH-IMA pg/ml c Serum 2nd PTH-IMA pg/ml c NtPTH(1-84) fragments pg/ml c Ratio PTH(1-84)/ntPTH(1-84) fragments Values are presented as mean SD. a Range of normal bone formation, 98 to 613 m 2 /mm 2 /day; b P 0.02; c P refer to differences between both groups, respectively. Fig. 3. Relationship between bone formation rate and parathyroid hormone (PTH) levels determined by first-generation immunometric parathyroid hormone assay (1st PTH-IMA) (A ) and second generation immunometric parathyroid hormone assay (2nd PTH-IMA) (B ). Fig. 2. Relationship between serum parathyroid hormone (PTH) levels determined by the first- and second-generation immunometric para- thyroid hormone assay (1st PTH-IMA and 2nd PTH-IMA). bone formation rate are shown in Figure 3. The coefficients of correlation were 0.67 and 0.64, P and P , respectively, for these two assays. A lower degree of correlation was found between bone groups. In contrast, as expected, serum total alkaline phostivity formation rate and serum total alkaline phosphatase acphatase activity was higher in patients with elevated bone (r 0.38, P 0.03). On the other hand, the coeffi- turnover. Similarly serum PTH concentrations determined cient of correlation between PTH(1-84)/ntPTH(1-84) by 1st and 2nd PTH-IMA were higher in patients with fragment ratio and bone formation was 0.30 (not signifihigh turnover osteodystrophy, pg/ml and 455 cant) (Fig. 4). Serum calcium levels and phosphorus lev- 256 pg/ml, respectively, versus those with normal/ady- els did not correlate with bone formation rate. namic lesion, pg/ml and pg/ml, To determine how accurately values of PTH obtained respectively (Table 1). with either assay can differentiate between high and nor- Overall, PTH levels obtained with 1st PTH-IMA were mal/low osteodystrophy, receiver operating curves (ROCs) on average 40% to 50% higher than those from the 2nd were constructed. For the detection of high bone forma- PTH-IMA, which is similar to previously reported data tion a value greater than 613 mm 2 /mm 2 /day was utilized [15, 16, 18, 19, 21]. Determinations of PTH levels by 1st because it corresponds to the upper limit of normal of and 2nd PTH-IMA were highly correlated (r 0.89, tetracycline-based measurements of bone formation rate P ) (Fig. 2). Furthermore, the concentration in individuals with normal renal function in our laboraof ntpth(1-84) fragments was higher in patients with tory (Fig. 5) [3]. The areas under the curve for the 1st elevated bone formation rates than in those with normal and 2nd PTH-IMA were and 0.870, respectively or reduced indices of bone turnover (Table 1), and the (not significant). In contrast, the area under the curve ratio of PTH(1-84) over ntpth(1-84) fragments did not for the PTH(1-84)/ntPTH(1-84) ratio was and such differ between the two groups (Table 1). value is significantly lower than that obtained with 2nd The relationships between 1st and 2nd PTH-IMA and PTH-IMA, P 0.01 and 1st PTH-IMA, P 0.05 (Fig. 6).

5 Salusky et al: First- and second-generation immunometric PTH assays and bone turnover 1805 Fig. 4. Relationship between bone formation rate and the ratio of parathyroid hormone [PTH(1-84)]/[N terminally truncated (ntpth(1-84)] fragments. Fig. 6. Areas under the curve of the receiving operating curves (ROC) for first- and second-generation immunometric parathyroid hormone assay (1st PTH-IMA and 2nd PTH-IMA) and the ratio PTH(1-84)/[N terminally truncated (ntpth(1-84)] fragments. *P 0.05 and P 0.01, refers to 1st PTH-IMA and 2nd PTH-IMA, respectively, versus the ratio PTH(1-84)/ntPTH(1-84) fragments. Fig. 5. Receiving operating curves (ROC) for the detection of bone turnover with the first- and second-generation immunometric parathyroid hormone assay [1st PTH-IMA (dotted line) and 2nd PTH-IMA (broken line)] and the ratio PTH(1-84)/[N terminally truncated (ntpth(1-84)] fragments (solid line). DISCUSSION The results indicate that the 2nd PTH-IMA used in the current study detects only PTH(1-84), while the 1st PTH-IMA cross-reacts equivalently with hpth(7-84) and hpth(1-84) as has been previously shown for similar assays [15 17]. Furthermore, high concentrations of synthetic hpth(1-34) and to a lesser extent hpth(2-34), hpth(3-34), hpth(4-34), and hpth(5-34) interfered with hpth(1-84) binding to the labeled detection antibody in the 2nd PTH-IMA. In contrast, hpth(3-34), hpth(4-34), or hpth(5-34) interacted poorly with this detection anti- body suggesting that the first and second amino terminal residue of full-length PTH(1-84) are particularly important for antibody recognition. Serum PTH determinations with the 1st PTH-IMA have been consistently higher when compared to those obtained with the 2nd PTH-IMA over a wide range of serum PTH concentra- tions. These findings are consistent with previous observations and indicate that 1st PTH-IMA may overestimate parathyroid gland function [15 19, 21]. On the other hand, PTH concentrations were highly correlated and values with both assays showed indistinguishable correlation with indices of bone formation, as previously reported with different 1st and 2nd PTH-IMAs [18, 21]. When patients were grouped according to the presence or absence of bone biopsy findings consistent with secondary hyperparathyroidism, serum PTH concentrations differed between both groups, as expected. PTH levels were consistently higher in the group with high bone turnover lesions than in those with normal/adynamic osteodystrophy as determined by both assays [18, 21]. Similar to PTH(1-84), the ntpth(1-84) fragments concentrations were higher in patients with high rates of bone turnover than in those with normal or reduced rates of bone formation as previously shown [18, 21]. However, in contrast to a previous study [18], the ratio PTH(1-84)/ntPTH(1-84) fragments did not differ between both groups. Furthermore, PTH concentrations determined by either the 1st or the 2nd PTH-IMA were found to be better predictors of bone formation than the ratio PTH(1-84)/ntPTH(1-84) fragments, consistent with data recently reported by Coen et al [21] in adult patients treated with hemodialysis. The ratio PTH(1-84)/ntPTH(1-84) fragments was previously reported to be a superior predictor of bone turnover when compared to PTH values determined by two different IRMA assays and other biochemical markers in adult patients treated with maintenance hemodialysis [18]. These earlier results, therefore, differ not only from those observed in the current study; but also from an- other study carried out in adult patients treated with hemodialysis [21]. Although different PTH assays were

6 1806 Salusky et al: First- and second-generation immunometric PTH assays and bone turnover utilized in the current study, in vitro characterization of tween 65 to 450 pg/ml, determined with 1st PTH-IMA these new assays did not differ significantly from those were poor predictors of bone turnover in patients under- previously described [15 17]. In fact, the use of different going maintenance dialysis. These differences compared assays leading to the same conclusions makes it unlikely to those obtained in previous studies may be related to that differences between PTH assays are responsible for selection bias, geographic differences, inclusion of patients the discrepant findings observed in the study by Monier- treated with different dialytic modalities, and prethe Faugere et al [18]. Moreover, the criteria utilized for the vious vitamin D treatment, as well as the use of different grouping of patients according to indices of bone formation types of phosphate binders. differed between both studies. Indeed, Monier- On the other hand, discrepancies between serum PTH Faugere et al [18] included patients with normal bone levels as assessed by 1st PTH-IMA and indices of bone formation rate within the high turnover group, while formation have been described during intermittent calcitriol such patients were grouped in the current study with therapy in pediatric patients treated with peritoneal those patients with adynamic osteodystrophy. Such crite- dialysis [25, 32]. Similar findings have been reported by rion was used because of the different need for therapy Andress et al [33], after treatment with intravenous cal- with vitamin D in these subtypes of renal bone diseases citriol in adult patients undergoing hemodialysis. The disparity [24, 25]. Yet, even when ROC analysis was performed between the histologic and biochemical findings according to the criterion established by Monier-Faugere highlights the potential limitation of single PTH determinations et al [18], the conclusions remained the same despite the to predict the skeletal manifestations of renal oset small number of patients with adynamic bone and are teodystrophy. Diurnal variations in serum ionized calcium, furthermore consistent with the observations reported as well as short-term variations in serum PTH levels due by Coen et al [21]. to pulsatile PTH secretion could account for these findings Differences in bone remodeling between adult and [34]. Another potential factor may be a direct inhibitory pediatric patients may have played a role, but values of effect of calcitriol on differentiated osteoblastic function bone formation rates were quite similar between prepubertal, not mediated through PTH [25, 32]. An alternative expla- adolescents, and adult subjects with normal renal nation may be due to the overestimation of PTH levels and parathyroid gland function in our laboratory [25]. by 1st PTH-IMAs because of cross-reactivity with ntpth Moreover, the threshold of PTH levels as predictors (1-84) fragments [14 16, 18]. Although the precise structure of bone turnover was comparable between adult and of these putative large carboxyl-terminal PTH fragof pediatric patients treated with dialysis using the 1st PTH- ments has yet to be identified, some of these fragments IMA [5 9]. In addition, diabetes was present in 40% of exhibit chromatographic similarities to those of PTH(7-84) the patients with low bone turnover in the study by [12] and may contribute to the discrepancy between serum Monier-Faugere et al [18], and it has been shown that PTH levels and indices of bone formation during those patients have less biochemical evidence of second- intermittent calcitriol therapy [25, 32, 33, 35]. ary hyperparathyroidism than patients with other etiologies Indeed, recent studies have provided evidence that of ESRD [27, 28]. Insulin deficiency or tissue resis- such circulating fragments of truncated PTH may have tance to the actions of insulin may contribute to physiologic effects on bone metabolism. For example, reductions in osteoblastic activity [29]. It is interesting synthetic PTH(7-84) has been shown to have hypocalcemic to note, however, that diabetes was present only in 14% properties when tested in vivo in parathyroidectoto of the patients reported by Coen et al [21] and in none mized rats fed a low calcium diet [16, 20]. In this bioassay of the patients in the current study. Finally, the techniques system, removal of the parathyroid glands leads to a for determination of bone turnover were different rapid decrease in serum calcium levels unless PTH(1-34) between studies. Ballant et al, however, demonstrated or PTH(1-84) are given parenterally, and PTH(7-84) an- that bone remodeling rates were comparable with both tagonized these calcemic activities of PTH molecules techniques in the context of renal osteodystrophy and the with an intact amino terminus. Furthermore, when superiority of activation frequency was more conceptual PTH(1-84) was given simultaneously with PTH(7-84), than practical [30]. the PTH fragment decreased the phosphaturic effect of Over the last decade, the 1st PTH-IMA was shown to the intact hormone by 50% [16]. Antagonistic effects of be a good predictor of the different subtypes of renal PTH(7-84), thus, occurred not only at the level of bone bone diseases in children and adults treated with dialysis but also the kidney [16]. In addition, Nguyen-Yamamoto [5 9]. In such cross-sectional studies, patients at the time et al [20, 35] have shown that PTH(7-84) with or without of bone biopsy were treated with either daily calcitriol a mixture of other carboxyl terminal PTH fragments inhibited therapy or were not given vitamin D metabolites at all, the calcemic effect of PTH(1-34), indicating that these and calcium containing phosphate-binding agents were actions are not mediated through the PTH/PTHrP receptor, the main phosphate-binding agents [5, 6, 8, 9]. Only the but instead involve a receptor that interacts only with study by Qi et al [31], documented that PTH levels be- the carboxyl terminal portion of PTH. Furthermore, Di-

7 Salusky et al: First- and second-generation immunometric PTH assays and bone turnover 1807 vieti et al [36] demonstrated that PTH(7-84), at concen- do not yet support the claim that 2nd PTH-IMAs provide trations that are likely to occur in renal failure, exerts a an advantage over 1st PTH-IMAs for the diagnosis of direct antiresorptive effect on bone that it is not medi- the different subtypes of renal bone diseases [18]. In ated through the PTH/PTHrP receptor, but probably summary, further studies are needed to establish the through a receptor specific for carboxyl-terminal frag- value of the new generation of PTH assays for assessing ments of PTH, as mentioned above. Serum calcium levels bone disease in renal failure, during therapy with vitamin may also act as regulators of the relative amounts of circu- D, and to address the need for higher PTH levels deterlating PTH(1-84) and ntpth(1-84) fragments [37, 38]. mined by either assay for preventing the development Indeed, it has been demonstrated that serum calcium of adynamic osteodystrophy and its consequences [26]. concentrations correlated with the percentage of these PTH-derived fragments [38]. Thus, there is a growing ACKNOWLEDGMENTS body of evidence that at least some of these different This study was supported in part, by USPHS grants DK-35423, RRcarboxyl terminal fragments of PTH are biologically ac , DK-52905, and DK and by funds from the Casey Lee tive and their secretion by the parathyroid glands may Ball Foundation. The results of the study were presented in part at the 2001 American Society of Nephrology Meeting in San Francisco. be regulated by calcium. However, the precise role of The current data was presented at the 2002 American Society of Nethese fragments in the context of renal osteodystrophy phrology Meeting in Philadelphia. remains to be elucidated. Reprint requests to Isidro B. Salusky, M.D., UCLA Medical Center, The need for PTH levels to be one to three times above Division of Pediatric Nephrology, Box , Los Angeles, CA the upper limit of normal, as determined by 1st PTH- isalusky@mednet.ucla.edu IMA, to maintain normal rates of bone formation in patients treated with dialysis has been recommended by REFERENCES different investigators [6, 7]. Such findings have been con- 1. Goodman WG, Coburn JW, Slatopolsky E, Salusky IB: Renal sistent with our previous observation and they are further osteodystrophy in adults and children, in Primer on the Metabolic supported by the current findings [8, 9]. 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