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Determination of bioactive rat parathyroid hormone (PTH) concentrations in vivo and in vitro by a 2-site homologous immunoradiometric assay
ELSEVIER
Bone and Mineral 27 (1994) 121-132
Determination of bioactive rat parathyroid hormone (PTH) concentrations in vivo and in vitro by a 2-site homologous immunoradiometric assay r Vijitha L. Schultz a, Sanford C. Garner c, Jeffrey R. Lavigne d, Svein U. Toverud *a'b aDental Research Center, University of North Carolina, Chapel Hill, NC 27599-7455, USA bDepartment of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA eDepartment of Surgery, Duke University Medical Center, Durham, NC 27710, USA dlmmutopics, Inc., San Clemente, CA 92673, USA Received 19 May 1994; revision received 5 August 1994; accepted 9 August 1994
Abstract A new homologous 2-site assay for rat parathyroid hormone (IRMA), developed by Immutopics, Inc., has been evaluated and compared with a bone cell cAMP bioassay. Circulating PTH for adult rats assayed with this IRMA are in the range 10-15 pg/ml, and of the same order of magnitude as published values for biologically active PTH. The standard curve for the IRMA was linear over the range 3.4-240 pg/ml of rPTH 1-34, and serum samples diluted in parallel with the standard curve. The within-assay and between-assay coefficients of variation ranged from 5.2% (n = 18) to 7.6% (n = 24) and 8.3% (n = 16) to 26.4% (n = 10), respectively. Serum PTH values (mean 4- S.E.) for parathyroidectomized rats were 3.5 4- 0.6 pg/ml (n = 18) versus 10.3 4- 1.4 pg/ml (n = 16) for intact non-mated rats. Calcium injections suppressed circulating PTH by 50%. Lactating rats had serum PTH levels 5-fold higher and vitamin D deficient rats 60-fold higher than non-mated controls. PTH secreted from parathyroid cells in vitro was in the range 60-490 pg/ml as determined by the IRMA. These values represented 86.0 4- 9.0% of the comparable bioassay values, indicating that the IRMA detects only bioactive PTH.
Keywords: Bone cAMP bioassay; Lactation; Serum calcium; Parathyroidectomy; Coefficient of variation r Presentedin part at the 15th Annual Meetingof the American Societyfor Boneand Mineral Research in Tampa, FL, 1993. * Corresponding author. 0169-6009/94/$07.00 9 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0169-6009(94)00748-6
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1. Introduction
Rat intact parathyroid hormone (PTH) is an 84-amino acid polypeptide secreted by the parathyroid gland with its biological activity residing in the N-terminal region of the peptide, specifically the 1-34 region. Although the first 34 amino acids in the rat PTH molecule differ at only five sites from human and bovine PTH Ill, determination of circulating levels of biologically active PTH in the rat has been difficult because of the lack of readily available antiserum specific for the active N-terminus of the polypeptide hormone. Previous assays used for rat PTH such as Nichols INSPTH (heterologous) [2] and recently published homologous assays [3,4] are time consuming, lacking in sensitivity, or are not commercially available. However, a new sensitive, homologous assay developed by Immutopics, Inc. (San Clemente, CA) is now available through the Nichols Institute. This assay is a two-site immunoradiometric assay (IRMA) which utilizes two different goat antibodies, purified by affinity chromatography, to the N-terminal region (1-34) of rat PTH. One of the antibodies is immobilized onto plastic beads to capture the PTH molecules and the other is radiolabeled for detection (Fig. l). In this study, we have tested the rat PTH (IRMA) kit for its sensitivity, accuracy, and its ability to detect changes in response to variations in serum Ca. In addition, we have compared this assay with a bioassay based on the cAMP response by rat osteosarcoma (ROS 17/2.8) cells [5] in the measurement of PTH secreted by freshly isolated rat parathyroid cells.
Immunoradiometric Assay (IRMA) for rat PTH
c)~ d) ~ . - ~
Fig. 1. Schematic of IRMA. The immunoradiometric assay (IRMA) for rat PTH consists of (a) a polystyrene bead coated with a goat antibody to the N-terminal (t-34) region of rat PTH and (b) an 1251-labeled antibody to the N-terminal (1-34) region of rat PTH. The standard curve is generated with (c) rat PTH (1-34) standards. Rat serum is believed to contain primarily (d) rat PTH (1-84) molecules; however, it is also possible that (c) PTH (I-34) is present in rat serum. The complete Bead/Anti-Rat PTH-Rat PTH- 125IAnti-Rat PTH sandwich complex is formed only when the biologically active forms of PTH, either rat PTH (1-34) or rat PTH (1-84) molecules, are present in the sample.
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123
Biological validation of the IRMA revealed that all manipulations of serum Ca yielded the expected changes in serum PTH. The removal of rat parathyroid glands was followed by a fall in serum PTH to levels at or close to the average level of detectability of the assay, which is estimated to be 2.9 pg/ml. 2. Methods and materials
2.1. Rats and diets Pregnant Holtzman rats were obtained at 12-18 days of gestation from Harlan Sprague Dawley (Indianapolis, IN) along with non-mated female controls of approximately the same age and weight. On arrival, the rats were fed a diet [6] consisting of 75% whole wheat flour, 13% casein, 5% cellulose (Alphacel), 4.4% fat (mostly corn oil), 2% salt mixture, 1.3% vitamin D-free vitamin mixture (ICN Biochemicals, Cleveland, OH), 5 IU cholecalciferol/g diet, and 0.8% CaCO3 (prepared as jumbo pellets by ICN Biochemicals). The final content of calcium was 0.4% or 0.1%, and the contents of phosphorus, and magnesium were 0.4% and 0.14%, respectively, by analysis. 2.2. Analytical procedures Total Ca was measured using either semiautomated fluorometric titration Calcette (Precision Systems, Inc., Sudbury, MA) or the Atomic Absorption Spectrophotometer (Model 5100, Perkin Elmer, South Pasadena, CA). Ionized Ca was measured with an ICA-I Ionized Calcium Analyzer (Radiometer America, Westlake, OH). Blood was drawn from the heart of ether-anesthetized rats and kept on ice approximately 20 rain after bleeding. The samples were spun at 2000 x g for 10 min after which serum was removed and stored at -70~ until time of assay. The IRMA has not been validated for rat plasma.
2.3. Parathyroid cell isolation and incubation Parathyroid glands from ether-anesthetized 18-20 day lactating and non-mated female rats were dissected free of thyroid tissue using fine-tipped forceps under a binocular dissecting microscope. Tissue was digested using the method of Brown et al. [7] with a slight modification. Parathyroid glands from 3 rats were incubated in 4.5 ml of Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS) with 2 mg/ml of collagenase D (Boehringer Mannheim, Indianapolis, IN), which has high collagenase activity and low ancillary protease and trypsin activities. The glands were placed in capped vials, gassed with 95% 02/5% CO2 and digested for 90-120 min in a 37~ water bath with continuous shaking at 90-100 cycles/rain. The parathyroid glands were also mechanically dispersed every 15-20 min with a glass pasteur pipet with the vials being gassed with 95% 02/5% CO2 each time. Dispersed parathyroid cells were then washed twice with 1 ml of DMEM/5% FBS and spun at 1000 x g for 5 rain. The pellet was resuspended in 500/~1 of DMEM and the cells counted on a hemacytometer. Approximately 95% of the cells excluded trypan blue, and the yield of cells was approximately 1.8-2.5 x 10 5 cells/ml per 3 pairs of parathyroid glands. The dispersed parathyroid cells were then
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incubated in minimum essential medium (MEM) containing either 0.5 or 2.5 mM CaCI2 for 4 h in a 37~ water bath with continuous shaking. Cells were spun down at 2000 x g for 5 min and the supernatant (secreted PTH) removed and stored at -70~ before assay. In addition, parathyroid cells from non-mated female rats, approximately 200 000 cells/ml (isolated as before using sterile techniques) were suspended in medium described by Sakaguchi et al. [8] and plated on 35 mm culture dishes precoated with laminin (Collaborative Biomedical Products, Bedford, MA). Ionized calcium of the medium was 1.2 mM. Cells were grown to confluence in 6-7 days.
2.4. Parathyroidectomy and Ca injections Parathyroid glands were removed from non-mated female ether-anesthetized rats as described above. The rats were fasted for approximately 24 h before they were bled. The serum was collected, as previously described, and assayed for PTH levels. Serum ionized calcium ranged from 0.8 to 1.11 mM compared with approximately 1.3 mM for intact rats. Another group of intact adult non-mated female rats were initially bled (time 0) and injected with 0.8 mg Ca/100 g body weight as CaCI2 and bled again 40 min later. Serum samples from both time points were then assayed for PTH levels. 2.5. Cell culture for the bioassay For the bioassay, rat osteosarcoma 17/2.8 cells, kindly provided by Dr Gideon Rodan (Merck Research Laboratories), were grown in DMEM with 5% FBS. Confluent cells were removed from culture flasks with 0.25% trypsin/EDTA and suspended in DMEM/5% FBS with 100 nM dexamethasone to enhance the response to PTH [5]. The cells were plated onto 24-well sterile plates (1.5 ml/well). Cells in the 24-well plates were allowed to grow to confluence in 3 days after which they were used in the bone cell cAMP bioassay. 2.6. cAMP bioassay Cells were incubated with 1 ml [3H]adenine solution (1 #Ci/ml) in DMEM for 2 h in a 37~ water bath. After the 2 h incubation, cells were washed with DMEM and 250 #1 of 0.2 mM 3-isobutyl-1-methyl-xanthine (IBMX), a phosphodiesterase inhibitor which prevents the breakdown of cAMP. Then 250 #1 of standards (rPTH 1-34), ranging from 10-s to 10 -11 M, or samples were added to wells in triplicate and incubated for 15 min in a 37~ water bath. Standards and samples were then aspirated and 1 ml of 0.5% TCA was added to each well and the plates were incubated at 4~ overnight. TCA extracts were eluted through Dowex and Alumina columns to collect ATP and cAMP counts, respectively, as described by Salomon et al. [9] and by Meeker and Harden [10]. 2. 7. Rat PTH immunoradiometric assay (IRMA) The rat PTH IRMA is a two site immunoradiometric assay for the measurement of PTH in rat serum, developed by Immutopics, Inc. (San Clemente, CA), and distributed by Nichols Institute Diagnostics (San Juan Capistrano, CA). A 200-/zl quanti-
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ty of standards, controls or samples was aliquoted into 12 x 75 mm polystyrene tubes in duplicate. The standards and Controls 1 and 2 contain a matrix (including proteins) to match that of normal rat serum. Then 100 gl of 125I-labeled rat PTH (1-34) goat antibody was added to each tube and vortexed. Another goat antibody to rat PTH (1-34), coated onto polystyrene beads, was added to each tube and incubated at room temperature for 18-24 h. The bead was washed three times with 2 ml of 0.01 M phosphate buffered saline containing 0.1% Triton X-100 and 0.05% sodium azide. The radioactivity bound to the bead was measured with a V-counter. Thus, results can be available within 24 h. 2.8. Recovery of P T H added to serum Recovery of PTH added to rat serum was tested by adding rat PTH 1-84 to two serum samples containing different amounts of PTH. PTH 1-84 was chosen because it is assumed that this is the major circulating form of PTH, and because preliminary tests indicated that recovery of rat PTH 1-34 (aliquots of the assay standards) was incomplete (a range of 55-90% for 4 samples, and a mean of 70.1%). Rat PTH 1-34 and rat PTH 1-84 are synthetic peptides obtained from Peninsula Laboratories, Inc. (Belmont, CA). 2.9. Statistics Values are means • standard error of the mean. Asterisks represent significant differences at the P < 0.01 level as determined by Student's t-test. 3. Results 3.1. Rat PTH IRMA characteristics The standard curve for the rat PTH IRMA is shown in Fig. 2. Three dilutions of the lowest standard provided in the kit (27 pg/ml) were also plotted on the standard 5" :! a. r
O All DataPoints 0__ P Linear ortion
4"
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I .,,j'---'~'O2"=O2400 ~700
13.~7 73
3"
0
2"
240
f 6.75 ~ " 3.375
0 ..J y
1 0
1
= 1.57 + 1.02x
R^2
2
3
= 0.095
4
Log Standards Fig. 2. Rat PTH IRMA standard curve. A representative curve for the rat laTH IRMA is drawn on a log-log scale. The open symbols represent all the points of the standard curve, and the closed symbols represent the linear region. The numbers next to the symbols represent standards in pg/mi.
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Table 1 Rat PTH IRMA characteristics: coefficients of variation (CV) and assay sensitivity Samples
PTH mean (pg/ml)
Intra-assay Serum NM Serum L lnterassay Control 1a Control 2 a Serum pool (high) Serum pool (NM)
Coefficient of variation (%)
No. samples
No. assays
13 56
7.62 5.21
24 18
6 6
48 485 36 7.5
1i.72 8.26 5.1 26.4
16 16 4 10
16 16 4 5
Sensitivity of the IRMA was 2.94 • 1.9 (mean 9 S.D.) as determined by 2 standard deviations of the zero dose response based on 15 duplicates, each from a separate assay over a period of 1 year. Abbreviations: NM, non-mated; L, lactating. aControl samples provided in the IRMA kit.
curve. Six out of the 8 standards fell on a straight line (3.3-240 pg/ml) with a correlation coefficient of 0.995. Values below 240 pg/ml are reliable since they fall on the linear portion of the curve. However, values above 240 pg/ml are usually less reliable since the standard curve begins to plateau around the 700-pg/ml standard. The interassay CV was determined in (a) 16 assays by 16 duplicates of Controls 1 and 2,
Dilutions ~1)
50~ 1~0(200
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.
.
.
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.
.
.
.
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.
.
.
.
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2 3 Log Standards
.
.
.
.
4
Fig. 3. Rat PTH IRMA dilutions. Serum dilutions plotted with the rat PTH IRMA standard curve. Rat serum from non-mated and lactating rats were diluted to 50% and 25~ with 0 standard provided with the IRMA kit and plotted against the standard curve, which is the same as in Fig. 1. Serum numbers I-3 were from 19-21-day lactating rats on a 0.4% Ca diet, and serum 4 was from an adult male on a 0. !% Ca diet.
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127
(b) 4 assays by 4 duplicates of a high PTH serum pool, and (c) in 5 assays by 10 samples of a non-mated serum pool (Table 1). The interassay CVs were 11.7%, 8.3%, 15.1%, and 26.4%, respectively, for Control 1, Control 2, high PTH serum pool, and the non-mated serum pool. The intra-assay CV was determined by 24 duplicates of non-mated rat serum samples and 18 duplicates of lactating rat serum samples over 6 different assays. The percent CV for non-mated and lactating serum samples were 7.6% and 5.2%, respectively. The limit of detection based on two standard deviations of the zero dose response is 2.94 pg/ml (n = 15 duplicates each from a separate assay over a period of 1 year). Overall, the serum dilutions paralleled the standard curve (Fig. 3), and there was no consistent trend in the directionality of the dilution curves based on an analysis of a total of 11 samples. 3.2. Recovery of PTH added to serum When 104 pg/ml of rat PTH 1-84 was added to serum samples containing 8 or 101 pg/ml PTH, the observed concentrations represented 95% of the expected value for both samples. Furthermore, when equal volumes of two serum samples containing 9 and 112 pg/ml, respectively, were combined, the observed concentration was 99% of the expected value. 3.3. Assay validation To validate the IRMA against the bone cell bioassay, medium collected after incubation of freshly isolated or cultured parathyroid cells in 0.5, 1.2, or 2.5 mM CaCl2 were tested in both assays simultaneously. The results show that the values from the IRMA represent 86.0 • 9.0% (n = 13) of the comparable values as determined by the bioassay (Fig. 4). The difference in the result between the two methods is due primarily to the greater error in the bioassay. 800 r= 0.671 600-
~
r2=0.450
400-
200-
0 200
4 0
600
800
rPTH, pg/ml (Bioassay) Fig. 4. Regression of rPTH by IRMA on PTH bioactivity. PTH was determined in 13 samples of medium from freshly isolated cells and cultured cells from rat parathyroid glands. The slope of the regression line is significantly different from zero at P= 0.012. Based on these observations, the IRMA detects 86.0 • 9.0% of bioactivity, indicating that the IRMA detects only bioactive rPTH.
128
V.L. Schultz et al./ Bone Miner. 27 (1994) 121-132 12-
_~
8
E 6
z
4
m
2
PTX
Intact Controls
N=18
N=16
Serum Ca (mg/dl)
9.78• 0.11
a
7.46•
Fig. 5. Decreased Serum PTH in parathyroidectomized (PTX) rats. Parathyroid glands were removed from adult non-mated rats (PTX) and serum was collected 24 h after surgery. Rats were fasted overnight. aOnly a small subset of samples (n = 5) was available to determine serum Ca values for the control group.
Several biological validations were also performed with the rat P T H I R M A . Fig. 5 shows P T H levels in pg/ml in both intact control rats and parathyroidectomized (PTX) rats. P T H values for the P T X rats were 3.5 • 0.6 pg/ml (n = 18) versus 10.2 • 1.4 pg/ml (n = 16) for the intact control rats. The P T X value is not significantly different from the limit o f detection as given above. A small subset included in this PTX mean had P T H values overlapping with the controls, even though they were clearly hypocalcemic, and therefore, apparently parathyroidectomized.
20. 15
I
E o~ Q.
10
.
-r
Control
+Calcium
Serum Ca
10.7 + 0.2
11.4 + 0.4
(mg/dl)
N=5
N-5
Fig. 6. Suppression of serum PTH after calcium injection. Adult non-mated female rats were initially bled for baseline values, then injected i.p. with CaCI2, 0.8 mg Ca/100 g body wt., and bled 40 min later. Serum was collected and assayed in the IRMA.
V.L. Schultz et aL / Bone Miner. 27 (1994) 121-132
129
7O
,ot 50-
E o~
Iz
40~
3o~ 2o~ lO2
o2 Nonmated Lactating Serum Ca (mg/dl)
10.3:1:0.1 N=5
8.7 + 0.2 NI6
Weaned for 2 Days 10.7:1:O.Z N=8
Fig. 7. Elevated serum PTH in lactating rats. Serum was collected from lactating rats on 19-22 days of lactation, and from a separate group of lactating rats 2 days after they were weaned on 18-20 days of lactation. *P < 0.01 vs. lactating group.
The reasons for these discrepant values are not clear. The control and the PTX rats had serum calcium levels of 9.78 • 0.1 and 7.46.4- 0.2 mg/dl, respectively. Only a small subset (n = 5) of serum Ca values were available for the control group. When adult female non-mated rats were injected i.p. with Ca, 0.8 rag/100 g body weight, and bled 40 rain later, PTH levels decreased almost 50% compared with the zero time values (Fig. 6), but the PTH values were still higher than the PTH values in the PTX group shown in Fig. 5. The serum Ca values did not show a significant rise, presumably because after a Ca injection, serum Ca peaks before 40 min [11] and because of the low dosage of Ca. Elevated serum PTH concentrations were obtained by giving rats a vitamin-D free (-D) diet. Young rats (56 days old) had been kept on a - D diet as had also their mothers since beginning of pregnancy and through lactation. PTH values were 699 4- 147 pg/ml (n = 5). The serum calcium level was 5.5 • 0.3 mg/dl for the - D group. Fig. 7 shows elevated serum PTH concentrations in 19-22-day lactating rats, which represent a 5-6-fold increase in serum PTH compared with non-mated controls. However, 2 days after the rats were weaned, they showed a significant decrease in serum PTH relative to the lactating group. Serum Ca levels in the lactating and the weaned rats were 8.7 4. 0.2 and 10.7 4. 0.2 mg/dl, respectively. 4. Discussion
The results of these studies represent the performance of a new specific and sensitive homologous amino-terminal immunoradiometric assay (IRMA) to measure circulating rat PTH as well as PTH secreted from rat parathyroid cells in vitro. On a log-log scale, the standard curve for the IRMA is linear from 3.4 pg/ml to
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240 pg/ml, after which it begins to plateau (Fig. 1). This suggests that PTH values above 240 pg/ml are not as accurate as values below that concentration. As can be seen from Table 1, the lower the PTH concentration of the sample, the higher the percentage CV. At the lower end of the standard curve, as illustrated by the serum pool from non-mated rats, the percentage CV is the highest of the three concentrations. Overall, rat serum dilutions paralleled the standard curve (Fig. 3) indicating that the binding of the tracer is indeed due to rat PTH. The improved recovery of rat PTH 1-84 added to rat serum samples compared with the recovery of rat PTH 1-34 may be due to greater vulnerability of PTH 1-34 to serum proteases. The IRMA was validated with a bioassay based on the cAMP response by ROS 17/2.8 cells. In this experiment we tested PTH secreted from freshly isolated and cultured parathyroid cells (Fig. 4). The results indicate that values for PTH secreted in vitro are within 14% on the average of the values determined by a bioassay, suggesting that the IRMA detects only bioactive PTH. Validation of the IRMA could not be carried out with rat serum samples, because the level of detectability of the bioassay is seldom lower than 150 pg/ml, and all serum samples, except for the - D rat samples, would be undetectable in the bioassay. The bioassay in which the secreted PTH samples were included happened to have an exceptionally low level of detectability of 40 pg/ml. We also assessed the capability of the assay to detect changes in PTH in a variety of in vivo conditions known to alter PTH secretion. Surgically parathyroidectomized rats had a mean PTH value of 3.5 • 0.6 pg/ml, which was not significantly different from the limit of detection calculated as 2 S.D. from the zero standard (Table 1). The PTX value is much lower than that reported for the previously published rat PTH immunoassay [4], but is similar to the value obtained with the INS assay [2]. The highly elevated serum PTH values for the hypocalcemic vitamin D-deficient rats are similar to the values reported for such rats with the INS assay [2]. Furthermore, PTH values in lactating rats were 5-6-fold higher than the non-mated controls, a much greater rise than that obtained with the previous INS assay [11]. Thus, the IRMA for rat PTH is similar in sensitivity to the earlier commercial assay, but the dynamic undiluted range is much greater, 3.4-240 pg/ml for the IRMA versus 4-35 pg/ml for the INS assay. The mean serum PTH value for adult female rats of 10.3 4- 1.4 is close to the value of 17 pg/ml reported by Seshadri et al. [12] for the mean serum bioactivity of adult Wistar rats based on a renal membrane cyclase method. The cytochemical bioassay, on the other hand, gave a lower value, approximately 3 pg/ml, for serum PTH in adult Fischer rats [13]. Although the serum PTH values obtained with the present assay are similar to those obtained with the INS assay for several conditions (PTX, intact adult rats and vitamin D-deficient rats), they may be specific for the particular rat strain, diet, method of bleeding, anesthesia, and handling of the serum, because these variables were the same as reported with the INS assay [21. The effect of any of these variables has not been determined, except that preliminary data indicate that some anesthetics, including methoxyflurane and sodium pentobarbital, cause elevation of serum PTH [141. The rat PTH IRMA is the first commercially available homologous assay, and it
KL. Schultz et al./ Bone Miner. 27 (1994) 121-132
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is more sensitive than previously published h o m o l o g o u s r a d i o i m m u n o a s s a y s [3,4]. The I R M A is simple to use, a n d results can be o b t a i n e d in less than 24 h. Previously published i m m u n o a s s a y s [2-4] were time consuming a n d required 5 - 6 days for completion. The rat P T H I R M A has also been found to give valid measurements o f P T H in mice [15]. In conclusion, the h o m o l o g o u s I R M A for rat P T H is a reliable, convenient and commercially available assay that a p p e a r s to measure only bioactive PTH. The I R M A allows d e t e r m i n a t i o n o f rat P T H in serum a n d m e d i u m o f incubated p a r a t h y r o i d cells over a 100-fold range, a n d induced changes in serum Ca and p a r a t h y r o i d e c t o m y p r o d u c e the expected alterations in serum PTH.
Acknowledgments This w o r k was s u p p o r t e d in p a r t by grant HD-12496 to S.U. T o v e r u d from the N a t i o n a l Institute o f Child Health and H u m a n Development.
References [I] Keutmann HT, Griscom AW, Nussbaum SR, Reiner BF, Goud AN, Potts JT, Rosenblatt M. Rat parathyroid hormone-(1-34) fragment: renal adenylate cyclase activity and receptor binding properties in vitro. Endocrinology 1985;117:1230-1234. [2] Toverud SU, Boass A, Garner SC, Endres DB. Circulating parathyroid hormone concentrations in normal and vitamin D-deprived rat pups determined with an N-terminal-specific radioimmunoassay. Bone Miner 1986;1:145-155. [3] Calvo MS, Gundberg CM, Heath 1II H, Fox J. Homologous amino-terminal radioimmunoassay for rat parathyroid hormone. Am J Physiol (Endocrinol Metab 24) 1991;261:E262-E268. [4] Finch JL, Rapp N, Martin KJ, Slatopolsky E. A new sensitive homologous radioimmunoassay for amino-terminal parathyroid hormone in the rat. J Bone Miner Res 1992;7:229-233. [5] Rodan SB, Fischer MK, Egan J J, Epstein PM, Rodan GA. The effect of dexamethasone on parathyroid hormone stimulation of adenylate cyclase in ROS 17/2.8 cells. Endocrinology 1984;115:951-958. 16l Boass A, Lovdal JA, Toverud SU. Pregnancy- and lactation-induced changes in active intestinal calcium transport in rats. Am J Physiol (Gastrointest Liver Physiol 26) 1992;263:GI27-G134. [71 Brown EM, Hurv,itz S, Aurbach GD. Preparation of viable isolated bovine parathyroid cells. Endocrinology 1976;99:1582-1588. [81 Sakaguchi K, Santora A, Zimering M, Curcio F, Aurbach GD, Brandi ML. Functional epithelial cell line cloned from rat parathyroid glands. Proc Natl Acad Sci USA 1987;84:3269-3273. 191 Salomon Y, Londos D, Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem 1974;58:541-548. [lOl Meeker RB, Harden TK. Muscarinic cholinergic receptor-mediated activation of phosphodiesterase. Mol Pharmacol 1982;22:310-319. Jill Garner SC, Boass A, Toverud SU. Hypercalcemia fails to suppress elevated serum parathyroid hormone concentrations during lactation in rats. J Bone Miner Res 1989;4:577-583. [121 Seshadri MS, Frankel TL, Lissner D, Mason RS, Posen S. Bioactive parathyroid hormone m the rat: effects of calcium and calcitriol. Endocrinology 1985;117:2417-2423. [131 Peng T, Garner SC, Hirsch PF, Posillico JT. Cytochemical bioassay of circulating concentrations of rat parathyroid hormone: application to a study of age and sex. J Bone Miner Res 1986;1: 351-357.
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[14] Schultz VL, Boass A, Toverud SU, Garner SC. Elevated serum parathyroid hormone concentrations in rats under pentobarbital or methoxyflurane but not under ether or ketamine anesthesia [abstract]. In; The 16th Annual Meeting of The American Society for Bone and Mineral Research, J Bone Miner Res 1994;9,Suppl. 1:$408. [15] Meyer RA, Jr., Garges PL, Meyer MH. Measurement of parathyroid hormone in the mouse: secondary hyperparathroidism in the x-linked hypophosphatemic (GYRO, Gy) mouse [abstract]. FASEB J 1994;8:A836.
Bone and Mineral 27 (1994) 121-132
Determination of bioactive rat parathyroid hormone (PTH) concentrations in vivo and in vitro by a 2-site homologous immunoradiometric assay r Vijitha L. Schultz a, Sanford C. Garner c, Jeffrey R. Lavigne d, Svein U. Toverud *a'b aDental Research Center, University of North Carolina, Chapel Hill, NC 27599-7455, USA bDepartment of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA eDepartment of Surgery, Duke University Medical Center, Durham, NC 27710, USA dlmmutopics, Inc., San Clemente, CA 92673, USA Received 19 May 1994; revision received 5 August 1994; accepted 9 August 1994
Abstract A new homologous 2-site assay for rat parathyroid hormone (IRMA), developed by Immutopics, Inc., has been evaluated and compared with a bone cell cAMP bioassay. Circulating PTH for adult rats assayed with this IRMA are in the range 10-15 pg/ml, and of the same order of magnitude as published values for biologically active PTH. The standard curve for the IRMA was linear over the range 3.4-240 pg/ml of rPTH 1-34, and serum samples diluted in parallel with the standard curve. The within-assay and between-assay coefficients of variation ranged from 5.2% (n = 18) to 7.6% (n = 24) and 8.3% (n = 16) to 26.4% (n = 10), respectively. Serum PTH values (mean 4- S.E.) for parathyroidectomized rats were 3.5 4- 0.6 pg/ml (n = 18) versus 10.3 4- 1.4 pg/ml (n = 16) for intact non-mated rats. Calcium injections suppressed circulating PTH by 50%. Lactating rats had serum PTH levels 5-fold higher and vitamin D deficient rats 60-fold higher than non-mated controls. PTH secreted from parathyroid cells in vitro was in the range 60-490 pg/ml as determined by the IRMA. These values represented 86.0 4- 9.0% of the comparable bioassay values, indicating that the IRMA detects only bioactive PTH.
Keywords: Bone cAMP bioassay; Lactation; Serum calcium; Parathyroidectomy; Coefficient of variation r Presentedin part at the 15th Annual Meetingof the American Societyfor Boneand Mineral Research in Tampa, FL, 1993. * Corresponding author. 0169-6009/94/$07.00 9 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0169-6009(94)00748-6
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1. Introduction
Rat intact parathyroid hormone (PTH) is an 84-amino acid polypeptide secreted by the parathyroid gland with its biological activity residing in the N-terminal region of the peptide, specifically the 1-34 region. Although the first 34 amino acids in the rat PTH molecule differ at only five sites from human and bovine PTH Ill, determination of circulating levels of biologically active PTH in the rat has been difficult because of the lack of readily available antiserum specific for the active N-terminus of the polypeptide hormone. Previous assays used for rat PTH such as Nichols INSPTH (heterologous) [2] and recently published homologous assays [3,4] are time consuming, lacking in sensitivity, or are not commercially available. However, a new sensitive, homologous assay developed by Immutopics, Inc. (San Clemente, CA) is now available through the Nichols Institute. This assay is a two-site immunoradiometric assay (IRMA) which utilizes two different goat antibodies, purified by affinity chromatography, to the N-terminal region (1-34) of rat PTH. One of the antibodies is immobilized onto plastic beads to capture the PTH molecules and the other is radiolabeled for detection (Fig. l). In this study, we have tested the rat PTH (IRMA) kit for its sensitivity, accuracy, and its ability to detect changes in response to variations in serum Ca. In addition, we have compared this assay with a bioassay based on the cAMP response by rat osteosarcoma (ROS 17/2.8) cells [5] in the measurement of PTH secreted by freshly isolated rat parathyroid cells.
Immunoradiometric Assay (IRMA) for rat PTH
c)~ d) ~ . - ~
Fig. 1. Schematic of IRMA. The immunoradiometric assay (IRMA) for rat PTH consists of (a) a polystyrene bead coated with a goat antibody to the N-terminal (t-34) region of rat PTH and (b) an 1251-labeled antibody to the N-terminal (1-34) region of rat PTH. The standard curve is generated with (c) rat PTH (1-34) standards. Rat serum is believed to contain primarily (d) rat PTH (1-84) molecules; however, it is also possible that (c) PTH (I-34) is present in rat serum. The complete Bead/Anti-Rat PTH-Rat PTH- 125IAnti-Rat PTH sandwich complex is formed only when the biologically active forms of PTH, either rat PTH (1-34) or rat PTH (1-84) molecules, are present in the sample.
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123
Biological validation of the IRMA revealed that all manipulations of serum Ca yielded the expected changes in serum PTH. The removal of rat parathyroid glands was followed by a fall in serum PTH to levels at or close to the average level of detectability of the assay, which is estimated to be 2.9 pg/ml. 2. Methods and materials
2.1. Rats and diets Pregnant Holtzman rats were obtained at 12-18 days of gestation from Harlan Sprague Dawley (Indianapolis, IN) along with non-mated female controls of approximately the same age and weight. On arrival, the rats were fed a diet [6] consisting of 75% whole wheat flour, 13% casein, 5% cellulose (Alphacel), 4.4% fat (mostly corn oil), 2% salt mixture, 1.3% vitamin D-free vitamin mixture (ICN Biochemicals, Cleveland, OH), 5 IU cholecalciferol/g diet, and 0.8% CaCO3 (prepared as jumbo pellets by ICN Biochemicals). The final content of calcium was 0.4% or 0.1%, and the contents of phosphorus, and magnesium were 0.4% and 0.14%, respectively, by analysis. 2.2. Analytical procedures Total Ca was measured using either semiautomated fluorometric titration Calcette (Precision Systems, Inc., Sudbury, MA) or the Atomic Absorption Spectrophotometer (Model 5100, Perkin Elmer, South Pasadena, CA). Ionized Ca was measured with an ICA-I Ionized Calcium Analyzer (Radiometer America, Westlake, OH). Blood was drawn from the heart of ether-anesthetized rats and kept on ice approximately 20 rain after bleeding. The samples were spun at 2000 x g for 10 min after which serum was removed and stored at -70~ until time of assay. The IRMA has not been validated for rat plasma.
2.3. Parathyroid cell isolation and incubation Parathyroid glands from ether-anesthetized 18-20 day lactating and non-mated female rats were dissected free of thyroid tissue using fine-tipped forceps under a binocular dissecting microscope. Tissue was digested using the method of Brown et al. [7] with a slight modification. Parathyroid glands from 3 rats were incubated in 4.5 ml of Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS) with 2 mg/ml of collagenase D (Boehringer Mannheim, Indianapolis, IN), which has high collagenase activity and low ancillary protease and trypsin activities. The glands were placed in capped vials, gassed with 95% 02/5% CO2 and digested for 90-120 min in a 37~ water bath with continuous shaking at 90-100 cycles/rain. The parathyroid glands were also mechanically dispersed every 15-20 min with a glass pasteur pipet with the vials being gassed with 95% 02/5% CO2 each time. Dispersed parathyroid cells were then washed twice with 1 ml of DMEM/5% FBS and spun at 1000 x g for 5 rain. The pellet was resuspended in 500/~1 of DMEM and the cells counted on a hemacytometer. Approximately 95% of the cells excluded trypan blue, and the yield of cells was approximately 1.8-2.5 x 10 5 cells/ml per 3 pairs of parathyroid glands. The dispersed parathyroid cells were then
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incubated in minimum essential medium (MEM) containing either 0.5 or 2.5 mM CaCI2 for 4 h in a 37~ water bath with continuous shaking. Cells were spun down at 2000 x g for 5 min and the supernatant (secreted PTH) removed and stored at -70~ before assay. In addition, parathyroid cells from non-mated female rats, approximately 200 000 cells/ml (isolated as before using sterile techniques) were suspended in medium described by Sakaguchi et al. [8] and plated on 35 mm culture dishes precoated with laminin (Collaborative Biomedical Products, Bedford, MA). Ionized calcium of the medium was 1.2 mM. Cells were grown to confluence in 6-7 days.
2.4. Parathyroidectomy and Ca injections Parathyroid glands were removed from non-mated female ether-anesthetized rats as described above. The rats were fasted for approximately 24 h before they were bled. The serum was collected, as previously described, and assayed for PTH levels. Serum ionized calcium ranged from 0.8 to 1.11 mM compared with approximately 1.3 mM for intact rats. Another group of intact adult non-mated female rats were initially bled (time 0) and injected with 0.8 mg Ca/100 g body weight as CaCI2 and bled again 40 min later. Serum samples from both time points were then assayed for PTH levels. 2.5. Cell culture for the bioassay For the bioassay, rat osteosarcoma 17/2.8 cells, kindly provided by Dr Gideon Rodan (Merck Research Laboratories), were grown in DMEM with 5% FBS. Confluent cells were removed from culture flasks with 0.25% trypsin/EDTA and suspended in DMEM/5% FBS with 100 nM dexamethasone to enhance the response to PTH [5]. The cells were plated onto 24-well sterile plates (1.5 ml/well). Cells in the 24-well plates were allowed to grow to confluence in 3 days after which they were used in the bone cell cAMP bioassay. 2.6. cAMP bioassay Cells were incubated with 1 ml [3H]adenine solution (1 #Ci/ml) in DMEM for 2 h in a 37~ water bath. After the 2 h incubation, cells were washed with DMEM and 250 #1 of 0.2 mM 3-isobutyl-1-methyl-xanthine (IBMX), a phosphodiesterase inhibitor which prevents the breakdown of cAMP. Then 250 #1 of standards (rPTH 1-34), ranging from 10-s to 10 -11 M, or samples were added to wells in triplicate and incubated for 15 min in a 37~ water bath. Standards and samples were then aspirated and 1 ml of 0.5% TCA was added to each well and the plates were incubated at 4~ overnight. TCA extracts were eluted through Dowex and Alumina columns to collect ATP and cAMP counts, respectively, as described by Salomon et al. [9] and by Meeker and Harden [10]. 2. 7. Rat PTH immunoradiometric assay (IRMA) The rat PTH IRMA is a two site immunoradiometric assay for the measurement of PTH in rat serum, developed by Immutopics, Inc. (San Clemente, CA), and distributed by Nichols Institute Diagnostics (San Juan Capistrano, CA). A 200-/zl quanti-
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ty of standards, controls or samples was aliquoted into 12 x 75 mm polystyrene tubes in duplicate. The standards and Controls 1 and 2 contain a matrix (including proteins) to match that of normal rat serum. Then 100 gl of 125I-labeled rat PTH (1-34) goat antibody was added to each tube and vortexed. Another goat antibody to rat PTH (1-34), coated onto polystyrene beads, was added to each tube and incubated at room temperature for 18-24 h. The bead was washed three times with 2 ml of 0.01 M phosphate buffered saline containing 0.1% Triton X-100 and 0.05% sodium azide. The radioactivity bound to the bead was measured with a V-counter. Thus, results can be available within 24 h. 2.8. Recovery of P T H added to serum Recovery of PTH added to rat serum was tested by adding rat PTH 1-84 to two serum samples containing different amounts of PTH. PTH 1-84 was chosen because it is assumed that this is the major circulating form of PTH, and because preliminary tests indicated that recovery of rat PTH 1-34 (aliquots of the assay standards) was incomplete (a range of 55-90% for 4 samples, and a mean of 70.1%). Rat PTH 1-34 and rat PTH 1-84 are synthetic peptides obtained from Peninsula Laboratories, Inc. (Belmont, CA). 2.9. Statistics Values are means • standard error of the mean. Asterisks represent significant differences at the P < 0.01 level as determined by Student's t-test. 3. Results 3.1. Rat PTH IRMA characteristics The standard curve for the rat PTH IRMA is shown in Fig. 2. Three dilutions of the lowest standard provided in the kit (27 pg/ml) were also plotted on the standard 5" :! a. r
O All DataPoints 0__ P Linear ortion
4"
~
'1o r,)
P k.
I .,,j'---'~'O2"=O2400 ~700
13.~7 73
3"
0
2"
240
f 6.75 ~ " 3.375
0 ..J y
1 0
1
= 1.57 + 1.02x
R^2
2
3
= 0.095
4
Log Standards Fig. 2. Rat PTH IRMA standard curve. A representative curve for the rat laTH IRMA is drawn on a log-log scale. The open symbols represent all the points of the standard curve, and the closed symbols represent the linear region. The numbers next to the symbols represent standards in pg/mi.
V.L. Schultz et al. / Bone Miner. 27 (1994) 121-132
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Table 1 Rat PTH IRMA characteristics: coefficients of variation (CV) and assay sensitivity Samples
PTH mean (pg/ml)
Intra-assay Serum NM Serum L lnterassay Control 1a Control 2 a Serum pool (high) Serum pool (NM)
Coefficient of variation (%)
No. samples
No. assays
13 56
7.62 5.21
24 18
6 6
48 485 36 7.5
1i.72 8.26 5.1 26.4
16 16 4 10
16 16 4 5
Sensitivity of the IRMA was 2.94 • 1.9 (mean 9 S.D.) as determined by 2 standard deviations of the zero dose response based on 15 duplicates, each from a separate assay over a period of 1 year. Abbreviations: NM, non-mated; L, lactating. aControl samples provided in the IRMA kit.
curve. Six out of the 8 standards fell on a straight line (3.3-240 pg/ml) with a correlation coefficient of 0.995. Values below 240 pg/ml are reliable since they fall on the linear portion of the curve. However, values above 240 pg/ml are usually less reliable since the standard curve begins to plateau around the 700-pg/ml standard. The interassay CV was determined in (a) 16 assays by 16 duplicates of Controls 1 and 2,
Dilutions ~1)
50~ 1~0(200
f
"5 O.
"(g 0
1,=
o ~J
o~ o .J .
0
.
.
.
!
1
.
.
.
.
)
.
.
.
.
|
2 3 Log Standards
.
.
.
.
4
Fig. 3. Rat PTH IRMA dilutions. Serum dilutions plotted with the rat PTH IRMA standard curve. Rat serum from non-mated and lactating rats were diluted to 50% and 25~ with 0 standard provided with the IRMA kit and plotted against the standard curve, which is the same as in Fig. 1. Serum numbers I-3 were from 19-21-day lactating rats on a 0.4% Ca diet, and serum 4 was from an adult male on a 0. !% Ca diet.
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127
(b) 4 assays by 4 duplicates of a high PTH serum pool, and (c) in 5 assays by 10 samples of a non-mated serum pool (Table 1). The interassay CVs were 11.7%, 8.3%, 15.1%, and 26.4%, respectively, for Control 1, Control 2, high PTH serum pool, and the non-mated serum pool. The intra-assay CV was determined by 24 duplicates of non-mated rat serum samples and 18 duplicates of lactating rat serum samples over 6 different assays. The percent CV for non-mated and lactating serum samples were 7.6% and 5.2%, respectively. The limit of detection based on two standard deviations of the zero dose response is 2.94 pg/ml (n = 15 duplicates each from a separate assay over a period of 1 year). Overall, the serum dilutions paralleled the standard curve (Fig. 3), and there was no consistent trend in the directionality of the dilution curves based on an analysis of a total of 11 samples. 3.2. Recovery of PTH added to serum When 104 pg/ml of rat PTH 1-84 was added to serum samples containing 8 or 101 pg/ml PTH, the observed concentrations represented 95% of the expected value for both samples. Furthermore, when equal volumes of two serum samples containing 9 and 112 pg/ml, respectively, were combined, the observed concentration was 99% of the expected value. 3.3. Assay validation To validate the IRMA against the bone cell bioassay, medium collected after incubation of freshly isolated or cultured parathyroid cells in 0.5, 1.2, or 2.5 mM CaCl2 were tested in both assays simultaneously. The results show that the values from the IRMA represent 86.0 • 9.0% (n = 13) of the comparable values as determined by the bioassay (Fig. 4). The difference in the result between the two methods is due primarily to the greater error in the bioassay. 800 r= 0.671 600-
~
r2=0.450
400-
200-
0 200
4 0
600
800
rPTH, pg/ml (Bioassay) Fig. 4. Regression of rPTH by IRMA on PTH bioactivity. PTH was determined in 13 samples of medium from freshly isolated cells and cultured cells from rat parathyroid glands. The slope of the regression line is significantly different from zero at P= 0.012. Based on these observations, the IRMA detects 86.0 • 9.0% of bioactivity, indicating that the IRMA detects only bioactive rPTH.
128
V.L. Schultz et al./ Bone Miner. 27 (1994) 121-132 12-
_~
8
E 6
z
4
m
2
PTX
Intact Controls
N=18
N=16
Serum Ca (mg/dl)
9.78• 0.11
a
7.46•
Fig. 5. Decreased Serum PTH in parathyroidectomized (PTX) rats. Parathyroid glands were removed from adult non-mated rats (PTX) and serum was collected 24 h after surgery. Rats were fasted overnight. aOnly a small subset of samples (n = 5) was available to determine serum Ca values for the control group.
Several biological validations were also performed with the rat P T H I R M A . Fig. 5 shows P T H levels in pg/ml in both intact control rats and parathyroidectomized (PTX) rats. P T H values for the P T X rats were 3.5 • 0.6 pg/ml (n = 18) versus 10.2 • 1.4 pg/ml (n = 16) for the intact control rats. The P T X value is not significantly different from the limit o f detection as given above. A small subset included in this PTX mean had P T H values overlapping with the controls, even though they were clearly hypocalcemic, and therefore, apparently parathyroidectomized.
20. 15
I
E o~ Q.
10
.
-r
Control
+Calcium
Serum Ca
10.7 + 0.2
11.4 + 0.4
(mg/dl)
N=5
N-5
Fig. 6. Suppression of serum PTH after calcium injection. Adult non-mated female rats were initially bled for baseline values, then injected i.p. with CaCI2, 0.8 mg Ca/100 g body wt., and bled 40 min later. Serum was collected and assayed in the IRMA.
V.L. Schultz et aL / Bone Miner. 27 (1994) 121-132
129
7O
,ot 50-
E o~
Iz
40~
3o~ 2o~ lO2
o2 Nonmated Lactating Serum Ca (mg/dl)
10.3:1:0.1 N=5
8.7 + 0.2 NI6
Weaned for 2 Days 10.7:1:O.Z N=8
Fig. 7. Elevated serum PTH in lactating rats. Serum was collected from lactating rats on 19-22 days of lactation, and from a separate group of lactating rats 2 days after they were weaned on 18-20 days of lactation. *P < 0.01 vs. lactating group.
The reasons for these discrepant values are not clear. The control and the PTX rats had serum calcium levels of 9.78 • 0.1 and 7.46.4- 0.2 mg/dl, respectively. Only a small subset (n = 5) of serum Ca values were available for the control group. When adult female non-mated rats were injected i.p. with Ca, 0.8 rag/100 g body weight, and bled 40 rain later, PTH levels decreased almost 50% compared with the zero time values (Fig. 6), but the PTH values were still higher than the PTH values in the PTX group shown in Fig. 5. The serum Ca values did not show a significant rise, presumably because after a Ca injection, serum Ca peaks before 40 min [11] and because of the low dosage of Ca. Elevated serum PTH concentrations were obtained by giving rats a vitamin-D free (-D) diet. Young rats (56 days old) had been kept on a - D diet as had also their mothers since beginning of pregnancy and through lactation. PTH values were 699 4- 147 pg/ml (n = 5). The serum calcium level was 5.5 • 0.3 mg/dl for the - D group. Fig. 7 shows elevated serum PTH concentrations in 19-22-day lactating rats, which represent a 5-6-fold increase in serum PTH compared with non-mated controls. However, 2 days after the rats were weaned, they showed a significant decrease in serum PTH relative to the lactating group. Serum Ca levels in the lactating and the weaned rats were 8.7 4. 0.2 and 10.7 4. 0.2 mg/dl, respectively. 4. Discussion
The results of these studies represent the performance of a new specific and sensitive homologous amino-terminal immunoradiometric assay (IRMA) to measure circulating rat PTH as well as PTH secreted from rat parathyroid cells in vitro. On a log-log scale, the standard curve for the IRMA is linear from 3.4 pg/ml to
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240 pg/ml, after which it begins to plateau (Fig. 1). This suggests that PTH values above 240 pg/ml are not as accurate as values below that concentration. As can be seen from Table 1, the lower the PTH concentration of the sample, the higher the percentage CV. At the lower end of the standard curve, as illustrated by the serum pool from non-mated rats, the percentage CV is the highest of the three concentrations. Overall, rat serum dilutions paralleled the standard curve (Fig. 3) indicating that the binding of the tracer is indeed due to rat PTH. The improved recovery of rat PTH 1-84 added to rat serum samples compared with the recovery of rat PTH 1-34 may be due to greater vulnerability of PTH 1-34 to serum proteases. The IRMA was validated with a bioassay based on the cAMP response by ROS 17/2.8 cells. In this experiment we tested PTH secreted from freshly isolated and cultured parathyroid cells (Fig. 4). The results indicate that values for PTH secreted in vitro are within 14% on the average of the values determined by a bioassay, suggesting that the IRMA detects only bioactive PTH. Validation of the IRMA could not be carried out with rat serum samples, because the level of detectability of the bioassay is seldom lower than 150 pg/ml, and all serum samples, except for the - D rat samples, would be undetectable in the bioassay. The bioassay in which the secreted PTH samples were included happened to have an exceptionally low level of detectability of 40 pg/ml. We also assessed the capability of the assay to detect changes in PTH in a variety of in vivo conditions known to alter PTH secretion. Surgically parathyroidectomized rats had a mean PTH value of 3.5 • 0.6 pg/ml, which was not significantly different from the limit of detection calculated as 2 S.D. from the zero standard (Table 1). The PTX value is much lower than that reported for the previously published rat PTH immunoassay [4], but is similar to the value obtained with the INS assay [2]. The highly elevated serum PTH values for the hypocalcemic vitamin D-deficient rats are similar to the values reported for such rats with the INS assay [2]. Furthermore, PTH values in lactating rats were 5-6-fold higher than the non-mated controls, a much greater rise than that obtained with the previous INS assay [11]. Thus, the IRMA for rat PTH is similar in sensitivity to the earlier commercial assay, but the dynamic undiluted range is much greater, 3.4-240 pg/ml for the IRMA versus 4-35 pg/ml for the INS assay. The mean serum PTH value for adult female rats of 10.3 4- 1.4 is close to the value of 17 pg/ml reported by Seshadri et al. [12] for the mean serum bioactivity of adult Wistar rats based on a renal membrane cyclase method. The cytochemical bioassay, on the other hand, gave a lower value, approximately 3 pg/ml, for serum PTH in adult Fischer rats [13]. Although the serum PTH values obtained with the present assay are similar to those obtained with the INS assay for several conditions (PTX, intact adult rats and vitamin D-deficient rats), they may be specific for the particular rat strain, diet, method of bleeding, anesthesia, and handling of the serum, because these variables were the same as reported with the INS assay [21. The effect of any of these variables has not been determined, except that preliminary data indicate that some anesthetics, including methoxyflurane and sodium pentobarbital, cause elevation of serum PTH [141. The rat PTH IRMA is the first commercially available homologous assay, and it
KL. Schultz et al./ Bone Miner. 27 (1994) 121-132
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is more sensitive than previously published h o m o l o g o u s r a d i o i m m u n o a s s a y s [3,4]. The I R M A is simple to use, a n d results can be o b t a i n e d in less than 24 h. Previously published i m m u n o a s s a y s [2-4] were time consuming a n d required 5 - 6 days for completion. The rat P T H I R M A has also been found to give valid measurements o f P T H in mice [15]. In conclusion, the h o m o l o g o u s I R M A for rat P T H is a reliable, convenient and commercially available assay that a p p e a r s to measure only bioactive PTH. The I R M A allows d e t e r m i n a t i o n o f rat P T H in serum a n d m e d i u m o f incubated p a r a t h y r o i d cells over a 100-fold range, a n d induced changes in serum Ca and p a r a t h y r o i d e c t o m y p r o d u c e the expected alterations in serum PTH.
Acknowledgments This w o r k was s u p p o r t e d in p a r t by grant HD-12496 to S.U. T o v e r u d from the N a t i o n a l Institute o f Child Health and H u m a n Development.
References [I] Keutmann HT, Griscom AW, Nussbaum SR, Reiner BF, Goud AN, Potts JT, Rosenblatt M. Rat parathyroid hormone-(1-34) fragment: renal adenylate cyclase activity and receptor binding properties in vitro. Endocrinology 1985;117:1230-1234. [2] Toverud SU, Boass A, Garner SC, Endres DB. Circulating parathyroid hormone concentrations in normal and vitamin D-deprived rat pups determined with an N-terminal-specific radioimmunoassay. Bone Miner 1986;1:145-155. [3] Calvo MS, Gundberg CM, Heath 1II H, Fox J. Homologous amino-terminal radioimmunoassay for rat parathyroid hormone. Am J Physiol (Endocrinol Metab 24) 1991;261:E262-E268. [4] Finch JL, Rapp N, Martin KJ, Slatopolsky E. A new sensitive homologous radioimmunoassay for amino-terminal parathyroid hormone in the rat. J Bone Miner Res 1992;7:229-233. [5] Rodan SB, Fischer MK, Egan J J, Epstein PM, Rodan GA. The effect of dexamethasone on parathyroid hormone stimulation of adenylate cyclase in ROS 17/2.8 cells. Endocrinology 1984;115:951-958. 16l Boass A, Lovdal JA, Toverud SU. Pregnancy- and lactation-induced changes in active intestinal calcium transport in rats. Am J Physiol (Gastrointest Liver Physiol 26) 1992;263:GI27-G134. [71 Brown EM, Hurv,itz S, Aurbach GD. Preparation of viable isolated bovine parathyroid cells. Endocrinology 1976;99:1582-1588. [81 Sakaguchi K, Santora A, Zimering M, Curcio F, Aurbach GD, Brandi ML. Functional epithelial cell line cloned from rat parathyroid glands. Proc Natl Acad Sci USA 1987;84:3269-3273. 191 Salomon Y, Londos D, Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem 1974;58:541-548. [lOl Meeker RB, Harden TK. Muscarinic cholinergic receptor-mediated activation of phosphodiesterase. Mol Pharmacol 1982;22:310-319. Jill Garner SC, Boass A, Toverud SU. Hypercalcemia fails to suppress elevated serum parathyroid hormone concentrations during lactation in rats. J Bone Miner Res 1989;4:577-583. [121 Seshadri MS, Frankel TL, Lissner D, Mason RS, Posen S. Bioactive parathyroid hormone m the rat: effects of calcium and calcitriol. Endocrinology 1985;117:2417-2423. [131 Peng T, Garner SC, Hirsch PF, Posillico JT. Cytochemical bioassay of circulating concentrations of rat parathyroid hormone: application to a study of age and sex. J Bone Miner Res 1986;1: 351-357.
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[14] Schultz VL, Boass A, Toverud SU, Garner SC. Elevated serum parathyroid hormone concentrations in rats under pentobarbital or methoxyflurane but not under ether or ketamine anesthesia [abstract]. In; The 16th Annual Meeting of The American Society for Bone and Mineral Research, J Bone Miner Res 1994;9,Suppl. 1:$408. [15] Meyer RA, Jr., Garges PL, Meyer MH. Measurement of parathyroid hormone in the mouse: secondary hyperparathroidism in the x-linked hypophosphatemic (GYRO, Gy) mouse [abstract]. FASEB J 1994;8:A836.