Kidney International, Vol. 63 (2003), pp. 1801–1808

CLINICAL NEPHROLOGY–EPIDEMIOLOGY–CLINICAL TRIALS

Similar predictive value of bone turnover using first- andsecond-generation immunometric PTH assays in pediatricpatients 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 JUPPNER

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 GeffenSchool 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 ofgeneration immunometric PTH assays in pediatric patients treated bone diseases in patients with renal failure. The spectrumwith peritoneal dialysis.

ranges from high-turnover lesions arising predominantlyBackground. Accurate measurements of the concentrationfrom excess of parathyroid hormone (PTH) secretion toof parathyroid hormone (PTH) in serum or plasma are essential

for the proper assessment of renal osteodystrophy. The first- low-turnover lesions that are most often associated withgeneration immunometric PTH assay (1st PTH-IMA) not only normal or reduced serum PTH levels [1]. Determinationsdetects the intact hormone, but also additional PTH fragments

of PTH levels are used as surrogates for bone histology,truncated at the amino N-terminally truncated PTH-derivedwhich remains the most reliable method to establish thefragments [ntPTH(1-84)]. A second-generation immunometric

PTH assay (2nd PTH-IMA) recognizes only PTH(1-84) and diagnosis of renal osteodystrophy [2–4]. Accurate mea-possibly PTH fragments that are truncated at the carboxyl- surements of the concentration of PTH in serum or plasmaterminus but not PTH(7-84). Whether estimates of the ratio

are therefore essential for the proper assessment of renalbetween PTH(1-84) and ntPTH(1-84) fragments are a betterosteodystrophy [5–9].predictor of bone turnover remains controversial.

Methods. Thirty-three patients aged 12.8 � 4.4 years treated The first clinically useful two-site immunometric PTHwith continuous cycling peritoneal dialysis (CCPD) for 13 � 9 assay (PTH-IMA) employed two antibodies directedmonths underwent iliac crest bone biopsy. PTH levels were

against amino- and carboxyl-terminal epitopes, respec-measured by two newly developed first-generation and second-tively, and was, therefore, predicted to measure only full-generation PTH-IMA. The ntPTH(1-84) fragments were calcu-

lated 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 de- ric PTH assay (1st PTH-IMA) proved to be a reasonablytects both PTH(1-84) and relatively large ntPTH(1-84).

reliable predictor of the different subtypes of renal osteo-Results. Determinations of PTH levels by both assays werehighly correlated (r � 0.89, P � 0.001). The relationships be- dystrophy, and it performed well in assessing the thera-tween first-generation and second-generation PTH-IMA and peutic response to active vitamin D sterols in patientsbone formation were similar (r � 0.67, P � 0.0001 and r � 0.64, with renal failure [5, 8, 9]. However, recent observationsP � 0.0001, respectively). When patients were grouped according

highlight important shortcomings of this and other PTH-to the presence or absence of secondary hyperparathyroidism,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 Lepage et al [12], Brossard et al [13], and Brossard, Yama-predictors of bone formation than the PTH(1-84) to ntPTH(1-84)

moto, and D’Amour [14], demonstrated that 1st PTH-fragments ratio.IMAs detect not only the intact hormone, but also addi-tional PTH fragments truncated at the amino terminusKey words: parathyroid hormone, immunometric PTH assay, perito-

neal dialysis. [ntPTH(1-84)]. Indeed, most of the detection antibodies,which are usually directed against epitopes within the

Received for publication October 2, 2002amino terminus of the hormone, detect not only PTH(1-84)and in revised form November 8, 2002

Accepted for publication December 13, 2002 but also one or several ntPTH(1-84) fragments, some ofwhich coelute from reverse-phase high-performance liquid 2003 by the International Society of Nephrology

1801

Salusky et al: First- and second-generation immunometric PTH assays and bone turnover1802

chromatography (HPLC) column with synthetic PTH(7-84) bone biopsy procedure; none of the patients receivedprior intermittent calcitriol therapy. The study was ap-[12]. In contrast, a more recently developed second-gen-

eration immunometric PTH assay (2nd PTH-IMA) using proved by the UCLA Human Subject Protection Com-mittee, and informed consent was obtained from all pa-a detection antibody raised against the first four amino

terminal amino acids of human PTH recognizes only tients and/or parents.Patients were admitted to the UCLA General ClinicalPTH(1-84) and possibly PTH fragments that are truncated

at the carboxyl terminus but not PTH(7-84) [15–17]. Research Center and bone biopsies were obtained fromthe anterior iliac crest using a modified Bordier trephineConsistent with this assessment human PTH(1-34), but

not human PTH(2-34) or other amino terminally truncated after double tetracycline as previously described [3]. Se-rum samples for measurements of calcium, phosphorus,fragments of human PTH(1-34) cross-reacted with the

detection antibody [15–17]. First-generation PTH-IMAs, alkaline phosphatase, and PTH levels were obtained atbone biopsy. Quantitative histomorphometry of bonethus, overestimate the true concentration of PTH(1-84)

in serum or plasma both in patients with ESRD and was done as previously described [3] and the terminologyestablished by the Nomenclature Committee of the Amer-those with normal renal function [14, 15, 17–19].

Recent in vitro and in vivo experimental data also ican Society of Bone and Mineral Research was used forall the results [22]. Reference values from subjects withindicate that one or more amino terminally truncated

PTH(1-84) fragments antagonize the calcemic actions of normal renal function were obtained from iliac crestbone biopsy done in 27 children between the ages of 2.5PTH(1-84) and diminish bone cell activity, and may,

thereby, modulate bone metabolism. These actions are and 17 years who had undergone elective orthopedic orurologic surgery procedures. Normal bone formation ratepossibly mediated through a receptor distinct from the

type I PTH/PTHrP receptor [16, 20]. Such findings sug- ranged between 97 and 613 �m2/mm2/day and such rangewas utilized for the classification of patients into high, nor-gest that ntPTH(1-84) fragments may contribute to the

skeletal PTH resistance that occurs in patients with renal mal, or reduced rates of bone formation and turnover [3].Serum calcium, phosphorus, and alkaline phosphatasefailure [16, 20]. It, therefore, appears plausible that an

levels were determined using a Technicon Autoanalyzerindependent assessment of PTH(1-84) and ntPTH(1-84)II. Serum PTH determinations were performed by twofragments may have a better value for diagnosis of theimmunometric specific assays (Immutopics, San Clemente,different subtypes of renal osteodystrophy than each pa-CA, USA), one that specifically detects only the biologi-rameter alone.cally active full-length PTH(1-84) (2nd PTH-IMA) andIndeed, it has been suggested that estimates of theanother that detects with indistinguishable efficiency full-ratio between PTH(1-84) and ntPTH(1-84) fragmentslength PTH(1-84) and the PTH(7-84) fragment (1st PTH-more accurately predicts bone remodeling and turnoverIMA) as has been previously described with a similar assayin patients with ESRD [18]. On the other hand, a more[15]. Both assays use goat polyclonal antibodies purifiedrecent report failed to confirm such findings [21]. Toby affinity chromatography, which recognizes specificfurther examine the utility of 1st and 2nd PTH-IMAs asand distant epitopes along the molecule. Most reagentspredictors of bone formation in ESRD, PTH measure-used in both assays are identical (i.e., the same capturements were performed with two newly developed assaysantibody recognizing epitopes within the 39-84 region ofin pediatric patients treated with peritoneal dialysis.the PTH molecule, identical standards and controls, andidentical buffer and incubation conditions). The differ-

METHODS ences between both assays are two distinct detectionA 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 aminobiopsy was 12.8 � 4.4 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 thewith peritoneal dialysis (Daniel, Baxter, Deerfield, IL, 13 to 34 region of the molecule. Thus, the assays measureUSA) with a dialysate calcium concentration of 2.5 different PTH fragment concentrations without influencemEq/L and glucose concentration that was adjusted ac- by matrix, standard, or incubation procedures. Values forcording to the needs for ultrafiltration. All patients were subjects with normal renal function range from 10 to 65receiving calcium carbonate as phosphate-binding agent. pg/mL for the 1st PTH-IMA and 2 to 32 pg/mL for theNone of the patients were treated with steroids or cyclo- 2nd PTH-IMA.sporine for at least 12 months prior to the biopsy and only

Evaluation of PTH fragment specificitytwo patients were previously treated with recombinanthuman growth hormone, and therapy was discontinued 5 The specificity of the two immunometric PTH assaysand 11 months before the performance of the procedure. was determined by assessing cross-reactivity with increas-

ing concentrations of human (h) hPTH(7-84) (Bachem Cal-Therapy with calcitriol was held for 4 weeks before the

Salusky et al: First- and second-generation immunometric PTH assays and bone turnover 1803

RESULTS

Characterization of the two PTH assays

To assess the specificity of both immunometric assays,each was characterized through in vitro competition ex-periments using several synthetic PTH peptides. The 1stPTH-IMA demonstrated equal cross-reactivity for bothhPTH(1-84) and hPTH(7-84), while the 2nd PTH-IMAfailed to detect hPTH(7-84) at concentrations as high as20,000 pg/mL; (data not shown) these findings are consis-

Fig. 1. A description of the technical aspects of the first- and second-tent with previous reports using a similar assay [15–17].generation immunometric parathyroid hormone assay (1st generation

and 2nd generation PTH-IMAs). Both assays employ capture antibodies The specificity of the two distinct detection antibodies,that recognize epitopes within the 39 to 84 region of the PTH molecule. which are directed for both assays against an epitopeThe differences between the assays are two distinct antibodies. In the

within the amino terminal portion of hPTH(1-84), was1st PTH-IMA, the detection antibody recognizes an epitope withinthe13 to 34 region of the molecule. In contrast, for the 2nd PTH-IMA, further examined by incubating a constant concentrationthe antibody recognizes an initial epitope within the amino terminus of hPTH(1-84) (545 pg/mL) with increasing amounts of(residues 1 to 3).

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 inthe presence of increasing concentrations of hPTH(1-34),ifornia, Inc., Torrance, CA, USA). Further characterizationhPTH(2-34), hPTH(3-34), hPTH(4-34), and hPTH(5-34)of both assays was obtained by coincubating hPTH(1-84)(data not shown). In contrast, in the 2nd PTH-IMA,(545 pg/mL) with increasing concentrations of [Tyr34]hPTH(1-34) revealed the highest efficacy to reduce bind-hPTH(1-34)amide (hPTH(1-34)), [Tyr34]hPTH(2-34)am-ing of the detection antibody, while hPTH(2-34) andide (hPTH(2-34)), [Tyr-34]hPTH(3-34)amide (hPTH(3-34)),hPTH(3-34) showed �50% and �4%, respectively, of[Tyr-34]hPTH(4-34) amide (hPTH(4-34)), and [Tyr34]hPTHthe potency of hPTH(1-34); hPTH(4-34) had a potency(5-34) amide (hPTH(5-34)) as described [15–17]. All pep-of �0.1%, and hPTH(5-34) completely failed to displacetides were synthesized and quantified by the Massachu-the detection antibody.setts General Hospital Polymer Core Facility.

Comparison of 1st and 2nd PTH-IMAs in patientsStatistical analysistreated with dialysis

All results are presented as the mean � one standardAmong the 33 bone biopsies with concurrent determi-deviation (SD). Patients were classified in two groups

nations of serum PTH levels by both assays, there wasaccording to the upper value for bone formation rateevidence of increased bone formation rate, above the up-obtained in subjects with normal renal function afterper limit of normal, in 23 patients, lesion consistent withdouble tetracycline labeling [3]. PTH determinations ob-secondary hyperparathyroidism; indices of bone forma-

tained with 1st and 2nd PTH-IMA as well as thetion 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 threentPTH(1-84) fragments were compared between the two patients, lesion consistent with adynamic osteodystrophygroups using Wilcoxon rank sum test. Under each group, as previously shown [3]. Patients were classified into twothe receiver operating curves (ROC) were generated for groups: (1) high bone turnover group, which includes allthe different tests (2nd PTH-IMA, 1st PTH-IMA, and the patients (N � 23) with bone formation rates above theratio). 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 ther-obtained using 1st PTH-IMA that detects both PTH(1- apy, and due to the small sample size of patients with84) 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 11.7 � 5.8 years in the high and normal/The areas under the ROC curves and their standard low turnover groups, respectively (NS), (Table 1) anderrors were obtained and comparisons were carried out the mean duration of treatment with peritoneal dialysisas previously described [23]. For given cut off points, did not differ between the two groups. The mean valuessensitivity, 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.

Serum calcium and phosphorus levels were similar in bothpackage SAS and two-sided significance level of 0.05.

Salusky et al: First- and second-generation immunometric PTH assays and bone turnover1804

Table 1. Patient characteristics and serum biochemicaldeterminations according to indices of bone turnover

High bone Normal/lowturnover bone turnover(N � 23) (N � 10)

Bone formation ratea lm2/mm2/day 1314�622 243�208Age years 13.3�3.6 11.7�5.8Dialysis months 13�10 12�9Serum calcium mg/dL 8.6�0.9 8.4�1.0Serum phosphorus mg/dL 6.7�1.5 6.1�0.9Serum total alkaline phosphatase IU/L 469�278 264�137b

Serum 1st PTH-IMA pg/mL 893�412 350�325c

Serum 2nd PTH-IMA pg/mL 455�256 150�147c

NtPTH(1-84) fragments pg/mL 438�212 200�218c

Ratio PTH(1-84)/ntPTH(1-84)fragments 1.11�0.57 1.07�1.27

Values are presented as mean � SD. aRange of normal bone formation, 98 to613 �m2/mm2/day; bP � 0.02; cP � 0.002 refer to differences between both groups,respectively.

Fig. 3. Relationship between bone formation rate and parathyroid hor-mone (PTH) levels determined by first-generation immunometric para-thyroid hormone assay (1st PTH-IMA) (A ) and second generationimmunometric parathyroid hormone assay (2nd PTH-IMA) (B ).

Fig. 2. Relationship between serum parathyroid hormone (PTH) lev- bone formation rate are shown in Figure 3. The coeffi-els determined by the first- and second-generation immunometric para-

cients of correlation were 0.67 and 0.64, P � 0.00001thyroid hormone assay (1st PTH-IMA and 2nd PTH-IMA).and P � 0.00001, respectively, for these two assays. Alower degree of correlation was found between boneformation rate and serum total alkaline phosphatase ac-groups. In contrast, as expected, serum total alkaline phos-tivity (r � 0.38, P � 0.03). On the other hand, the coeffi-phatase activity was higher in patients with elevated bonecient of correlation between PTH(1-84)/ntPTH(1-84)turnover. Similarly serum PTH concentrations determinedfragment ratio and bone formation was 0.30 (not signifi-by 1st and 2nd PTH-IMA were higher in patients withcant) (Fig. 4). Serum calcium levels and phosphorus lev-high turnover osteodystrophy, 893 � 412 pg/mL and 455 �els did not correlate with bone formation rate.256 pg/mL, respectively, versus those with normal/ady-

To determine how accurately values of PTH obtainednamic lesion, 350 � 325 pg/mL and 150 � 147 pg/mL,with either assay can differentiate between high and nor-respectively (Table 1).mal/low osteodystrophy, receiver operating curves (ROCs)Overall, PTH levels obtained with 1st PTH-IMA werewere constructed. For the detection of high bone forma-on average 40% to 50% higher than those from the 2ndtion a value greater than 613 �mm2/mm2/day was utilizedPTH-IMA, which is similar to previously reported databecause it corresponds to the upper limit of normal of[15, 16, 18, 19, 21]. Determinations of PTH levels by 1sttetracycline-based measurements of bone formation rateand 2nd PTH-IMA were highly correlated (r � 0.89,in individuals with normal renal function in our labora-P � 0.0001) (Fig. 2). Furthermore, the concentrationtory (Fig. 5) [3]. The areas under the curve for the 1stof ntPTH(1-84) fragments was higher in patients withand 2nd PTH-IMA were 0.866 and 0.870, respectivelyelevated bone formation rates than in those with normal(not significant). In contrast, the area under the curveor reduced indices of bone turnover (Table 1), and the

ratio of PTH(1-84) over ntPTH(1-84) fragments did not for the PTH(1-84)/ntPTH(1-84) ratio was 0.694 and suchvalue is significantly lower than that obtained with 2nddiffer between the two groups (Table 1).

The relationships between 1st and 2nd PTH-IMA and PTH-IMA, P � 0.01 and 1st PTH-IMA, P � 0.05 (Fig. 6).

Salusky et al: First- and second-generation immunometric PTH assays and bone turnover 1805

Fig. 4. Relationship between bone formation rate and the ratio ofparathyroid 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 hormoneassay (1st PTH-IMA and 2nd PTH-IMA) and the ratio PTH(1-84)/[Nterminally truncated (ntPTH(1-84)] fragments. *P � 0.05 and P � 0.01,refers to 1st PTH-IMA and 2nd PTH-IMA, respectively, versus theratio PTH(1-84)/ntPTH(1-84) fragments.

tions. These findings are consistent with previous obser-vations and indicate that 1st PTH-IMA may overestimateparathyroid gland function [15–19, 21]. On the other hand,PTH concentrations were highly correlated and valueswith both assays showed indistinguishable correlation withindices of bone formation, as previously reported withdifferent 1st and 2nd PTH-IMAs [18, 21]. When patientswere grouped according to the presence or absence ofbone biopsy findings consistent with secondary hyper-parathyroidism, serum PTH concentrations differed be-tween both groups, as expected. PTH levels were consis-Fig. 5. Receiving operating curves (ROC) for the detection of bonetently higher in the group with high bone turnover lesionsturnover with the first- and second-generation immunometric parathy-

roid hormone assay [1st PTH-IMA (dotted line) and 2nd PTH-IMA than in those with normal/adynamic osteodystrophy as(broken line)] and the ratio PTH(1-84)/[N terminally truncated determined by both assays [18, 21]. Similar to PTH(1-84),(ntPTH(1-84)] fragments (solid line).

the ntPTH(1-84) fragments concentrations were higherin patients with high rates of bone turnover than in thosewith normal or reduced rates of bone formation as pre-

DISCUSSION viously shown [18, 21]. However, in contrast to a previousstudy [18], the ratio PTH(1-84)/ntPTH(1-84) fragmentsThe results indicate that the 2nd PTH-IMA used indid not differ between both groups. Furthermore, PTHthe current study detects only PTH(1-84), while the 1stconcentrations determined by either the 1st or the 2ndPTH-IMA cross-reacts equivalently with hPTH(7-84) andPTH-IMA were found to be better predictors of bonehPTH(1-84) as has been previously shown for similar assaysformation than the ratio PTH(1-84)/ntPTH(1-84) frag-[15–17]. Furthermore, high concentrations of syntheticments, consistent with data recently reported by CoenhPTH(1-34) and to a lesser extent hPTH(2-34), hPTH(3-34),et al [21] in adult patients treated with hemodialysis.hPTH(4-34), and hPTH(5-34) interfered with hPTH(1-

The ratio PTH(1-84)/ntPTH(1-84) fragments was pre-84) binding to the labeled detection antibody in the 2ndviously reported to be a superior predictor of bone turn-PTH-IMA. In contrast, hPTH(3-34), hPTH(4-34), orover when compared to PTH values determined by twohPTH(5-34) interacted poorly with this detection anti-different IRMA assays and other biochemical markersbody suggesting that the first and second amino terminalin adult patients treated with maintenance hemodialysisresidue of full-length PTH(1-84) are particularly impor-[18]. These earlier results, therefore, differ not only fromtant for antibody recognition. Serum PTH determina-those observed in the current study; but also from an-tions with the 1st PTH-IMA have been consistentlyother study carried out in adult patients treated withhigher when compared to those obtained with the 2nd

PTH-IMA over a wide range of serum PTH concentra- hemodialysis [21]. Although different PTH assays were

Salusky et al: First- and second-generation immunometric PTH assays and bone turnover1806

utilized in the current study, in vitro characterization of tween 65 to 450 pg/mL, determined with 1st PTH-IMAwere poor predictors of bone turnover in patients under-these new assays did not differ significantly from those

previously described [15–17]. In fact, the use of different going maintenance dialysis. These differences comparedto those obtained in previous studies may be related toassays leading to the same conclusions makes it unlikely

that differences between PTH assays are responsible for selection bias, geographic differences, inclusion of pa-tients treated with different dialytic modalities, and pre-the discrepant findings observed in the study by Monier-

Faugere et al [18]. Moreover, the criteria utilized for the vious vitamin D treatment, as well as the use of differenttypes of phosphate binders.grouping of patients according to indices of bone for-

mation differed between both studies. Indeed, Monier- On the other hand, discrepancies between serum PTHlevels as assessed by 1st PTH-IMA and indices of boneFaugere et al [18] included patients with normal bone

formation rate within the “high turnover” group, while formation have been described during intermittent calci-triol therapy in pediatric patients treated with peritonealsuch patients were grouped in the current study with

those patients with adynamic osteodystrophy. Such crite- dialysis [25, 32]. Similar findings have been reported byAndress et al [33], after treatment with intravenous cal-rion was used because of the different need for therapy

with vitamin D in these subtypes of renal bone diseases citriol in adult patients undergoing hemodialysis. The dis-parity between the histologic and biochemical findings[24, 25]. Yet, even when ROC analysis was performed

according to the criterion established by Monier-Faugere highlights the potential limitation of single PTH determi-nations to predict the skeletal manifestations of renal os-et al [18], the conclusions remained the same despite the

small number of patients with adynamic bone and are teodystrophy. Diurnal variations in serum ionized calcium,as well as short-term variations in serum PTH levels duefurthermore consistent with the observations reported

by Coen et al [21]. to pulsatile PTH secretion could account for these findings[34]. Another potential factor may be a direct inhibitoryDifferences in bone remodeling between adult and

pediatric patients may have played a role, but values of effect of calcitriol on differentiated osteoblastic functionnot mediated through PTH [25, 32]. An alternative expla-bone formation rates were quite similar between prepu-

bertal, adolescents, and adult subjects with normal renal nation may be due to the overestimation of PTH levelsby 1st PTH-IMAs because of cross-reactivity with ntPTHand parathyroid gland function in our laboratory [25].

Moreover, the threshold of PTH levels as predictors (1-84) fragments [14–16, 18]. Although the precise struc-ture of these putative large carboxyl-terminal PTH frag-of bone turnover was comparable between adult and

pediatric patients treated with dialysis using the 1st PTH- ments has yet to be identified, some of these fragmentsexhibit chromatographic similarities to those of PTH(7-84)IMA [5–9]. In addition, diabetes was present in 40% of

the patients with low bone turnover in the study by [12] and may contribute to the discrepancy between se-rum PTH levels and indices of bone formation duringMonier-Faugere et al [18], and it has been shown that

those patients have less biochemical evidence of second- intermittent calcitriol therapy [25, 32, 33, 35].Indeed, recent studies have provided evidence thatary hyperparathyroidism than patients with other etiolo-

gies of ESRD [27, 28]. Insulin deficiency or tissue resis- such circulating fragments of truncated PTH may havephysiologic effects on bone metabolism. For example,tance to the actions of insulin may contribute to

reductions in osteoblastic activity [29]. It is interesting synthetic PTH(7-84) has been shown to have hypocalce-mic properties when tested in vivo in parathyroidecto-to note, however, that diabetes was present only in 14%

of the patients reported by Coen et al [21] and in none mized rats fed a low calcium diet [16, 20]. In this bioassaysystem, removal of the parathyroid glands leads to aof the patients in the current study. Finally, the tech-

niques for determination of bone turnover were different rapid decrease in serum calcium levels unless PTH(1-34)or PTH(1-84) are given parenterally, and PTH(7-84) an-between studies. Ballant et al, however, demonstrated

that bone remodeling rates were comparable with both tagonized these calcemic activities of PTH moleculeswith an intact amino terminus. Furthermore, whentechniques in the context of renal osteodystrophy and the

superiority of activation frequency was more conceptual PTH(1-84) was given simultaneously with PTH(7-84),the PTH fragment decreased the phosphaturic effect ofthan practical [30].

Over the last decade, the 1st PTH-IMA was shown to the intact hormone by 50% [16]. Antagonistic effects ofPTH(7-84), thus, occurred not only at the level of bonebe a good predictor of the different subtypes of renal

bone diseases in children and adults treated with dialysis but also the kidney [16]. In addition, Nguyen-Yamamotoet al [20, 35] have shown that PTH(7-84) with or without[5–9]. In such cross-sectional studies, patients at the time

of bone biopsy were treated with either daily calcitriol a mixture of other carboxyl terminal PTH fragments inhib-ited the calcemic effect of PTH(1-34), indicating that thesetherapy or were not given vitamin D metabolites at all,

and calcium containing phosphate-binding agents were actions are not mediated through the PTH/PTHrP recep-tor, but instead involve a receptor that interacts only withthe main phosphate-binding agents [5, 6, 8, 9]. Only the

study by Qi et al [31], documented that PTH levels be- the carboxyl terminal portion of PTH. Furthermore, Di-

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 providean advantage over 1st PTH-IMAs for the diagnosis oftrations that are likely to occur in renal failure, exerts a

direct antiresorptive effect on bone that it is not medi- the different subtypes of renal bone diseases [18]. Insummary, further studies are needed to establish theated through the PTH/PTHrP receptor, but probably

through a receptor specific for carboxyl-terminal frag- value of the new generation of PTH assays for assessingments of PTH, as mentioned above. Serum calcium levels bone disease in renal failure, during therapy with vitaminmay also act as regulators of the relative amounts of circu- D, and to address the need for higher PTH levels deter-lating PTH(1-84) and ntPTH(1-84) fragments [37, 38]. mined by either assay for preventing the developmentIndeed, it has been demonstrated that serum calcium of adynamic osteodystrophy and its consequences [26].concentrations correlated with the percentage of thesePTH-derived fragments [38]. Thus, there is a growing ACKNOWLEDGMENTSbody of evidence that at least some of these different This study was supported in part, by USPHS grants DK-35423, RR-carboxyl terminal fragments of PTH are biologically ac- 00865, DK-52905, and DK-60107 and by funds from the Casey Lee

Ball Foundation. The results of the study were presented in part attive and their secretion by the parathyroid glands maythe 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 Ne-

these 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 951752, Los Angeles, CA 90095.the upper limit of normal, as determined by 1st PTH- E-mail: isalusky@mednet.ucla.eduIMA, to maintain normal rates of bone formation in pa-tients treated with dialysis has been recommended by REFERENCESdifferent investigators [6, 7]. Such findings have been con- 1. Goodman WG, Coburn JW, Slatopolsky E, Salusky IB: Renalsistent with our previous observation and they are further osteodystrophy in adults and children, in Primer on the Metabolic

Bone Diseases and Disorders of Mineral Metabolism, 4th ed, editedsupported by the current findings [8, 9]. Indeed, PTHby Favus MJ, Philadelphia, Lippincott, Williams and Wilkins, 1999,levels in patients with bone formation rates within thepp 347–363

normal range were 196 � 180 pg/mL and 126 � 165 2. Sherrard DJ, Baylink DJ, Wergedal JE, Maloney NA: Quanti-tative histological studies on the pathogenesis of uremic bone dis-pg/mL, when determined by 1st and 2nd PTH-IMAs,ease. J Clin Endocrinol Metab 39:119–135, 1974respectively. Thus, with both PTH assays, values should

3. Salusky IB, Coburn JW, Brill J, et al: Bone disease in pediatricbe maintained within three to four times the upper limit patients undergoing dialysis with CAPD or CCPD. Kidney Int

33:975–982, 1988of the respective normal range in order to prevent the4. Malluche HH, Monier-Faugere MC: The role of bone biopsydevelopment of adynamic bone. When serum PTH levels

in the management of patients with renal osteodystrophy. J Amwere within two times the upper limit of either the 1st Soc Nephrol 4:1631–1642, 1994or the 2nd PTH-IMAs, 117 � 56 pg/mL and 44 � 44 5. Sherrard DJ, Hercz G, Pei Y, et al: The spectrum of bone disease

in end-stage renal failure—An evolving disorder. Kidney Intpg/mL, respectively, the bone turnover rate was below43:436–442, 1993the lower limit of normal and, therefore, consistent with 6. Cohen-Solal ME, Sebert JL, Boudailliez B, et al: Comparison

adynamic osteodystrophy [8]. Skeletal resistance to the of intact, midregion, and carboxy-terminal assays of parathyroidhormone for the diagnosis of bone disease in hemodialyzed pa-actions of PTH has been described in patients with ad-tients. J Clin Endocrinol Metab 73:516–524, 1991vanced renal insufficiency [39]; therefore, increments in 7. Quarles LD, Lobaugh B, Murphy G: Intact parathyroid hormone

serum PTH levels are necessary to maintain normal indi- overestimates the presence and severity of parathyroid-mediatedosseous abnormalities in uremia. J Clin Endocrinol Metab 75:145–ces of bone remodeling. Multiple factors have been im-150, 1992plicated in the pathogenesis of this abnormality in renal 8. Salusky IB, Ramirez JA, Oppenheim WL, et al: Biochemical mark-

failure, including a decrease in PTH/PTHrP receptor ers of renal osteodystrophy in pediatric patients undergoingCAPD/CCPD. Kidney Int 45:253–258, 1994expression observed in kidney and growth plate of rats

9. Mathias RS, Salusky IB, Harmon WH, et al: Renal bone diseasewith renal insufficiency [40–42]. Furthermore, the studies in pediatric patients and young adults treated by hemodialysis inoutlined above make it plausible that ntPTH(1-84) frag- a children’s hospital. J Am Soc Nephrol 12:1938–1946, 1993

10. Nussbaum SR, Zahradnik RJ, Lavigne JR, et al: Highly sensitivements may have direct inhibitory actions [16, 20, 35, 36].two-site immunoradiometric assay of parathyrin, and its clinicalTaking into consideration the crucial role of serum PTHutility in evaluating patients with hypercalcemia. Clin Chem

concentrations in the diagnosis and treatment of renal os- 33:1364–1367, 198711. Brossard JH, Cloutier M, Roy L, et al: Accumulation of a non-teodystrophy, 2nd PTH-IMA may provide important new

(1–84) molecular form of parathyroid hormone (PTH) detectedinsights into the physiology of parathyroid gland function.by intact PTH assay in renal failure: Importance in the interpreta-

However, our findings demonstrated that measurements tion of PTH values. J Clin Endocrinol Metab 81:3923–3929, 199612. Lepage R, Roy L, Brossard JH, et al: A non-(1–84) circulatingof PTH using either 1st or 2nd PTH-IMAs provided

parathyroid hormone (PTH) fragment interferes significantly withsimilar accuracy for predicting bone turnover in pediatricintact PTH commercial assay measurements in uremic samples.

patients undergoing maintenance peritoneal dialysis. Clin Chem 44:805–809, 199813. Brossard JH, Lepage R, Cardinal H, et al: Influence of glomerularThus, the current data, as well those from others [21],

Salusky et al: First- and second-generation immunometric PTH assays and bone turnover1808

filtration rate on non-(1–84) parathyroid hormone (PTH) detected 28. Pei Y, Hercz G, Greenwood C, et al: Renal osteodystrophy indiabetic patients. Kidney Int 44:159–164, 1993by intact PTH assays. Clin Chem 46:697–703, 2000

29. Shires R, Teitelbaum SL, Bergfeld MA, et al: The effect of14. Brossard JH, Yamamoto LN, D’Amour P: Parathyroid hormonestreptozotocin-induced chronic diabetes mellitus on bone and min-metabolites in renal failure: Bioactivity and clinical implications.eral homeostasis in the rat. J Lab Clin Med 97:231–240, 1981Semin Dial 15:196–201, 2002

30. Ballanti P, Coen G, Mazzaferro S, et al: Histomorphometric15. John MR, Goodman WG, Gao P, et al: A novel immunoradiome-assessment of bone turnover in uraemic patients: Comparison be-tric assay detects full-length human PTH but not amino-terminallytween activation frequency and bone formation rate. Histopathol-truncated fragments: Implications for PTH measurements in renalogy 38:571–583, 2001failure. J Clin Endocrinol Metab 84:4287–4290, 1999

31. Qi Q, Monier-Faugere MC, Geng Z, Malluche HH: Predictive16. Slatopolsky E, Finch J, Clay P, et al: A novel mechanism for value of serum parathyroid hormone levels for bone turnover inskeletal resistance in uremia. Kidney Int 58:753–761, 2000 patients on chronic maintenance dialysis. Am J Kidney Dis 26:622–17. Gao P, Scheibel S, D’Amour P, et al: Development of a novel 631, 1995

immunoradiometric assay exclusively for biologically active whole 32. Salusky IB, Kuizon BD, Belin T, et al: Intermittent calcitriolparathyroid hormone 1–84: Implications for improvement of accu- therapy in secondary hyperparathyroidism: A comparison betweenrate assessment of parathyroid function. J Bone Miner Res 16:605– oral and intraperitoneal administration. Kidney Int 54:907–914,614, 2001 1998

18. Monier-Faugere MC, Geng Z, Mawad H, et al: Improved assess- 33. Andress DL, Norris KC, Coburn JW, et al: Intravenous calcitriolin the treatment of refractory osteitis fibrosa of chronic renal fail-ment of bone turnover by the PTH-(1–84)/large C-PTH fragmentsure. N Engl J Med 321:274–279, 1989ratio in ESRD patients. Kidney Int 60:1460–1468, 2001

34. Schmitt CP, Huber D, Mehls O, et al: Altered instantaneous and19. Salomon R, Charbit M, Gagnadoux MF, et al: High serum levelscalcium-modulated oscillatory PTH secretion patterns in patientsof a non-(1–84) parathyroid hormone (PTH) fragment in pediatricwith secondary hyperparathyroidism. J Am Soc Nephrol 9:1832–haemodialysis patients. Pediatr Nephrol 16:1011–1014, 20011844, 199820. Nguyen-Yamamoto L, Rousseau L, Brossard JH, et al: Synthetic

35. Nguyen-Yamamoto L, Rousseau L, Brossard JH, et al: Origincarboxyl-terminal fragments of parathyroid hormone (PTH) de-of parathyroid hormone (PTH) fragments detected by intact-PTHcrease ionized calcium concentration in rats by acting on a receptor assays. Eur J Endocrinol 147:123–131, 2002

different from the PTH/PTH-related peptide receptor. Endocrinol- 36. Divieti P, John MR, Juppner H, Bringhurst FR: Human PTH-ogy 142:1386–1392, 2001 (7–84) inhibits bone resorption in vitro via actions independent of

21. Coen G, Bonucci E, Ballanti P, et al: PTH 1–84 and PTH “7–84” the type 1 PTH/PTHrP receptor. Endocrinology 143:171–176, 2002in the noninvasive diagnosis of renal bone disease. Am J Kidney 37. Mayer GP, Keaton JA, Hurst JC, Habener JF: Effects of plasmaDis 40:348–354, 2002 calcium concentration on the relative proportion of hormone and

22. Parfitt AM, Drezner MK, Glorieux FH, et al: Bone histomor- carboxyl fragments in parathyroid venous blood. Endocrinologyphometry: Standardization of nomenclature, symbols, and units. J 104:1778–1784, 1979

38. D’Amour P, Rousseau L, Rocheleau B, et al: Influence of Ca2�Bone Miner Res 2:595–610, 1987concentration on the clearance and circulating levels of intact and23. Hanley JA, MeNeil BJ: The meaning and use of the area undercarboxy-terminal iPTH in pentobarbital-anesthetized dogs. J Bonea receiving operating charactersitic (ROC) curve. Radiology 143:Miner Res 11:1075–1085, 199629–36, 1982

39. Massry SG, Coburn JW, Lee DBN, et al: Skeletal resistance to24. Sanchez CP, Salusky IB, Kuizon BD, et al: Bone disease inparathyroid hormone in renal failure. Ann Intern Med 78:357–364,children and adolescents undergoing successful renal transplanta-1973tion. Kidney Int 58:1358–1364, 1998

40. Urena P, Mannstadt M, Hruby M, et al: Parathyroidectomy does25. Goodman WG, Ramirez JA, Belin TR, et al: Development of not prevent the renal PTH/PTHrP receptor down-regulation inadynamic bone in patients with secondary hyperparathyroidism uremic rats. Kidney Int 47:1797–1805, 1995after intermittent calcitriol therapy. Kidney Int 46:1160–1166, 1994 41. Urena P, Ferreira A, Morieux C, et al: PTH/PTHrP receptor

26. Salusky IB, Goodman WG: Adynamic renal osteodystrophy: Is mRNA is downregulated in epiphyseal cartilage growth plate ofthere a problem? J Am Soc Nephrol 12:1978–1985, 2001 uraemic rats. Nephrol Dial Transplant 11:2008–2016, 1996

27. Andress DL, Hercz G, Kopp JB, et al: Bone histomorphometry 42. Sanchez CP, Salusky IB, Kuizon BD, et al: Growth of long bonesof renal osteodystrophy in diabetic patients. J Bone Miner Res in renal failure: roles of hyperparathyroidism, growth hormone

and calcitriol. Kidney Int 54:1879–1887, 19982:525–531, 1987