0013-7227/90/1264-l813$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 4 Printed in U.S.A.
Influence of Calcium on the Metabolism of Intact Parathyroid Hormone by Isolated Perfused Rat Kidney and Liver* HENRIK DAUGAARDf, MARTIN EGFJORD, AND KLAUS OLGAARD Medical Department P, Division of Nephrology, and Department of Experimental Pathology, Rigshospitalet, Copenhagen, Denmark
ABSTRACT. The metabolism of synthetic human PTH [PTH-(l-84)] 10~9 M was studied in isolated rat kidneys and livers, perfused at a calcium concentration of 1 mM or 4 mM. Clearances were measured by an assay specific for intact PTH, and by assays specific for NH2-terminal, mid-molecule, and COOH-terminal immunoreactive PTH (iPTH). Production of PTH fragments was analyzed by HPLC. The kidneys cleared PTH mainly by filtration. The glomerular filtration rate was not lower at 4 mM calcium than at 1 mM calcium, and no significant differences were found between the clearance of PTH at 4 mM and at 1 mM calcium. At 1 mM calcium the kidneys cleared intact PTH without release of detectable fragments. At 4 mM calcium there was significant (P
E
XTRACELLULAR calcium concentration is inversely related to the secretion of PTH by a direct feedback mechanism (1). However, circulating levels of intact PTH and biologically active PTH fragments, if any, are also determined by the peripheral metabolism of the hormone, mainly in the liver and the kidneys (2). Canterbury et al. (3) found an increased metabolism of intact bovine PTH [bPTH-(l-84)] at low perfusate calcium concentration ([Ca2+]), compared with high [Ca2+], in isolated rat livers, and Hruska et al. (4) found an increased metabolism of bPTH-(l-84) at low perfusate [Ca2+], compared with high [Ca2+], in isolated dog kidneys. These results were interpreted in accordance with the theory that intact PTH has to be "activated" (3, 5, 6) by metabolism to NH2-terminal fragments to exert its main biological effects. However, other studies have been unable to demonReceived September 26,1989. *The present investigation was kindly supported by grants from the Danish Medical Research Council, Novo Foundation, Foundation of 1870, Karla Marie Jensen of Kerteminde Foundation, Danish Foundation for the Advancement of Medical Science, Johann and Hanne Weimann born Seedorff s Foundation, Jacob and Olga Madsen Foundation, and Ruth I. E. Konig-Petersen Foundation. tAddress correspondence and reprint requests to: H. Daugaard, M.D., Medical Department P, Division of Nephrology 2131, Rigshospitalet, 9 Blegdamsvej, DK 2100 Copenhagen, Denmark.
< 0.05) accumulation of mid-molecule and COOH-terminal iPTH in the perfusate. Both at low and at high calcium the livers cleared NH2terminal iPTH at the same rate as intact PTH, whereas midmolecule and COOH-terminal iPTH was cleared significantly (P < 0.005) slower. In the livers, metabolic clearance of PTH was 60% faster at 4 mM calcium than at 1 mM calcium (P < 0.001). Assuming that the hepatic metabolism of PTH represents degradation of the biologically active hormone and hormone fragments, rather than activation of the hormone, the present results suggest a homeostatic control of PTH degradation in the liver to enhance inactivation of the hormone at high serum levels of calcium. (Endocrinology 126: 1813-1820,1990)
strate enhanced uptake or metabolism of PTH in the liver and the kidneys at low extracellular [Ca2+]. Neumann et al. (7) could not influence the uptake of [125I] bPTH-(l-84) by the rat liver in vivo by modifying serum calcium. D'Amour and Huet (8) found that serum calcium did not influence the extraction of [ 126 I]bPTH-(l84) by the dog liver in vivo. Fox et al. (6) could not influence total metabolic clearance rate of bPTH-(l-84) in thyroparathyroidectomized dogs by changes in plasma calcium. Oldham et al. (9) found no correlation between serum calcium and arteriovenous (AV) difference in NH2-terminal/intact immunoreactive PTH (iPTH) across the liver in nine hyperparathyroid patients. Furthermore, three studies have demonstrated decreased uptake or metabolism of PTH in the liver and the kidneys at low extracellular [Ca2+]. Hanano et al. (10) found decreased degradation of [125I]bPTH-(l-84) at lower perfusate [Ca2+] in isolated rat kidneys. Oldham et al. (9) found a positive correlation between serum calcium and AV difference in NH2-terminal/intact iPTH across the kidney in nine hyperparathyroid patients. A recent study by D'Amour and Huet (11) demonstrated decreased extraction of synthetic human PTH-(1-34) at low perfusate [Ca2+] in isolated rat livers. Thus, it is at present unclear, how extracellular [Ca2+] 1813
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Ca2+ AND PTH CLEARANCE BY RAT KIDNEY AND LIVER
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influences the metabolism of intact PTH in the liver and in the kidneys. The isolated, perfused organs are the models of choice to investigate this relationship unaffected by glandular secretion and metabolism in other organs. We, therefore, performed the present investigation employing pure, synthetic intact human PTH [synhPTH-(l-84)], which is biologically active in the rat (12), and an immunoradiometric assay (1-84 IRMA) specific for intact PTH (12). The formation of immunoreactive fragments in the two organs was investigated by HPLC and RIAs specific for NH2-terminal, midmolecule, and COOH-terminal iPTH. Materials and Methods Sources of PTH and PTH fragments. Synthetic human parathyroid hormone: synhPTH-(l-84)[asp76] (synthesized at the Peptide Institute, the Protein Research Foundation, Minoh, Osaka, Japan), synhPTH-(39-68), synhPTH-(39-84), synhPTH-(53-84), and synhPTH-(69-84) were obtained from Peninsula Laboratories. SynhPTH-(13-34), synhPTH-(l-34), synhPTH-(l-44), synhPTH-(28-48), synhPTH-(44-68), and synhPTH-(64-84) were obtained from Bachem. Immunoassay systems for intact PTH and fragments 1-34 Immunoradiometric assay (IRMA) (Allegro, Nichols Institute). A two-site IRMA using one goat antibody binding only PTH-(39-84) immobilized onto plastic beads and another radiolabeled goat antibody binding only PTH-(1-34). The standards were hPTH-(l-84). A distinct two-step procedure ensured that only molecules possessing both epitopes—i.e. exclusively intact PTH—was detected. 1-84 RIA (INS-PTH, Nichols Institute). This RIA used a chicken antibody raised against synhPTH-(l-34) and specific for the NH2-terminal part of the PTH molecule. 53-68 RIA (PTH OMEGA, Cambridge Medical Diagnostics). This RIA used a goat antiserum specific for the hPTH-(53-68) sequence. 53-84 RIA (MILAB, Malmo Immunlaboratorium AS, Malmo, Sweden). This assay used a chicken antibody highly specific for the COOH-terminal part of the PTH molecule. Performance of these assays has been validated in our laboratory (12), including dilution series to test parallelism with synhPTH-(l-84) in perfusion medium. The 1-84 RIA was found to be specific for intact PTH, the 1-34 RIA was specific for the NH2-terminal region, the 53-68 RIA was specific for the mid-molecule region, and the 53-84 RIA was specific for the COOH-terminal region of PTH. Media for organ perfusions All experiments were performed with modified Klebs-Henseleit bicarbonate buffer containing 5 mM glucose, 67 or 100 g/ liter BSA (fraction V [Pentex], Miles Laboratories; or charcoal treated [A7906], Sigma Chemical Company) and all 20 physiological L-amino acids. Electrolyte composition was (in mM)
Endo • 1990 Voll26«No 4
141 Na, 5 K, 0.7 Mg, 0.7 SO4, 25 HCO3> 122 Cl, 3 PO4, 4 lactate, and 1, 3, or 4 Ca. The resulting ionized calcium concentrations, [Ca2+], are given under Results. [Ca2+] was measured with a calcium ion electrode (ICA 2, Radiometer, Copenhagen, Denmark). Kidney perfusion technique Experiments were performed on male Wistar rats allowed water ad libitum and fed with Altromin pellets with 0.9% (wt/ wt) calcium and 0.7% phosphorus. Normal plasma [Ca2+] for the Wistar rats in our laboratory free fed on this diet is 1.28 ± 0.04 mM (12). The animals were anesthetized with 2.5% thiopental sodium 0.5 ml per 100 g rat body weight, given ip. The cannulation of the right kidney with a 19-gauge blunted needle via the superior mesenteric artery without interruption of flow, and its isolation has been described earlier (13). The kidneys were not perfused along with the perfusion of the liver. Filtering kidneys were perfused with BSA 67 g/liter at 96 mm Hg pressure. Nonfiltering kidneys were perfused with BSA 100 g/liter, 75 mm Hg pressure, and ligature of the ureter. Liver perfusion technique Female Wistar rats were fasted 24 h before the experiments but allowed free access to water. Pentobarbital sodium anesthesia, 6 mg per 100 g rat body weight, was given ip. The bile duct was cannulated with PE-50 tubing for bile collection. The portal vein and the inferior caval vein were cannulated with 14-gauge catheters. The liver was isolated, and the perfusion was conducted as described before (13). The livers were not perfused along with the kidneys. The animal studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Danish Ethical Committees. HPLC Reverse-phase HPLC was performed using a water/acetonitrile/trifluoroacetic acid (TFA) solvent system, and Waters Ci8 juBondapak 2-mm X 30-cm steel columns, operated at ambient temperature. Solvent system. The two limit solvents, 0.1% TFA in water and 50% acetonitrile in 0.1% TFA, were mixed by two pumps in proportions determined by a Waters 721 System Controller. The gradient used in all separations is shown in Fig. 3. Flow rate was 1 ml/min. The elution positions of various PTH fragments was established by injecting 5-200-jug quantities of the different fragments dissolved in 200 n\ 0.1% TFA into the system and measuring ultraviolet absorption of column eluates at 215 nm. Perfusate samples (6 ml each) were extracted on three C18 Sep-Paks before HPLC. Priming and rinsing procedures for the Sep-Paks were as described by Bennett et al. (14). Lyophilized Sep-Pak eluates were redissolved in 300 ^10.1% TFA, 200 /A of which were injected on the HPLC column. Column eluates were collected in 1-ml fractions, lyophilized, redissolved in 1 ml of pentobarbital sodium buffer with EDTA and human serum proteins, and analyzed in the PTH assays. Recovery of
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Ca2+ AND PTH CLEARANCE BY RAT KIDNEY AND LIVER 100-1000 pmol/liter of different NH2- and COOH-terminal synhPTH fragments in perfusate was also tested in this system.
TABLE 1. Function of perfused rat kidney
Assay
Experimental design Organ perfusions. The initial stabilizing and control period was 40-50 min for both kidneys and livers. At the start of the experimental period synhPTH-(l-84) was added to the perfusates of the kidneys and livers at an initial concentration of 1000 pmol/liter. Circulating volume at the start of the experimental period was chosen to allow diuresis, sampling for all assays, and HPLC separations. Six filtering kidneys were perfused at 1 mM Ca, and six filtering kidneys were perfused at 4 mM Ca with a circulating volume of 191 ml. Three nonfiltering kidneys were perfused at 1 mM Ca, and three at 4 mM Ca with a circulating volume of 176 ml. Four nonfiltering kidneys were perfused at 3 mM Ca with a circulating volume of 149 ml. The small circulating volume in this series was chosen to permit optimal determination of peritubular clearance of PTH at normal calcium, but did not permit sampling for HPLC analyses. The BSA used in this series was from Sigma, because BSA from Miles ensuring acceptable function of the kidneys was not available at the time of these experiments. All other perfusions used BSA from the same batch of Miles Pentex. Six livers were perfused at 1 mM Ca, and six at 4 mM Ca with a circulating volume of 175 ml. Two experiments were carried out at 1 mM Ca, and two at 4 mM Ca without any organs in the perfusion circuit. Perfusate was sampled 5, 15, 30, 60, and 90 min after the addition of PTH and analyzed in four PTH assays. Results were corrected for sampling and plotted us. time on a semilogarithmic scale. Clearances were calculated from the regression lines for each experimental perfusion. For Figures 1 and 2 linear scales were chosen, because they are easier to read and can have a dimension; therefore these curves are not quite linear. HPLC studies. Perfusate samples from two kidneys perfused at 1 mM Ca, two kidneys perfused at 4 mM Ca, two livers perfused at 1 mM Ca, and two livers perfused at 4 mM Ca were analyzed by HPLC. The samples were drawn 90 min after the addition of synhPTH-(l-84) at 1000 pmol/liter, fractionated by HPLC, and analyzed in the 1-34 RIA and the 53-68 RIA. Every third HPLC separation was a control, drawn from the perfusions without organs and synhPTN-(l-84) added. Data and statistical analysis. The data are given as means ± SD where nothing else is stated. Student's t test and parametric linear regression were used. All significance limits are twosided.
Results Function of isolated kidneys perfused at low and high [Ca2+] Results from six filtering kidneys perfused at low [Ca2+] and six filtering kidneys perfused at high [Ca2+] are given in Table 1. Mean perfusion pressure and perfusate flow was identical in the two groups. Glomerular
1815
Perfusate [Ca2+], mmol/ liter 0.49 ± 0.02
Number of perfusions, n Kidney weight, g Perfusion pressure, mm Hg Perfusate flow, ml • min"1 g"1 Urine production, ^1-min"1 g"1 Inulin clearance, /J-min"1 g"1 Filtration fraction, % Na excretion, ^molmin" 1 g"1 Fractional reabsorption of Na, % K excretion, /imol-min"1 g"1 Fractional excretion of K, % Ca excretion, nmolmin" 1 g"1 Fractional excretion of Ca, % Pi excretion, nmol-min"1 g"1 Fractional excretion of Pi, %
1.79 ± 0.06
6
6
1.45 ± 0.15 96 ± 2 31 ± 3 55 ±32 465 ± 144 1.6 ± 0.5 4.3 ± 2.7 93.6 ± 3.7 1.7 ± 1.3 79 ± 4 3 19 ± 8** 8.1 ± 3.1 285 ± 121* 28.5 ± 13.4"
1.65 ± 0.18 96 ± 3 31 ± 3 50 ± 17 541 ± 140 1.7 ± 0.4 3.9 ± 1.4 94.9 ± 1.9 1.6 ± 0.6 70 ±19 67 ± 2 8 6.3 ± 3.6 140 ± 72 9.18 ± 3.3
Values are means ± SD based on averages for each kidney of results obtained during the whole experimental period with synhPTH-(l-84) added at an initial concentration of 10"9 M. The experimental period was 90 min, starting 47 min after cannulation of the kidneys in both groups. Significant differences between the two groups: * P < 0.05; ** P < 0.01.
filtration rate (GFR) determined as inulin clearance was unchanged by high perfusate [Ca2+]. Total Ca excretion was significantly higher (P < 0.01) at high perfusate [Ca2+], but fractional Ca excretion was not significantly different. Ultrafilterable Ca was assumed to be 56% of total Ca in the calculations (15). Inorganic phosphate (Pi) was assumed to be 100% ultrafilterable. Both total and fractional Pi excretion was significantly lower (P < 0.05) at high perfusate [Ca2+]. Compared with the initial control period, fractional Pi excretion was significantly higher after addition of synhPTH(l-84) at 10~9 M, both at low [Ca2+] (P < 0.05), and at high [Ca2+] (P < 0.01). Clearance of PTH-(l-84) in perfused kidneys The effect of perfusate [Ca2+] on the disappearance of iPTH from the perfusates of filtering and nonfiltering isolated kidneys is shown in Fig. 1. The disappearance of synhPTH-(l-84) added at 10"9 M at time "0" was measured in four different assays. At low [Ca2+], both NH2-terminal, mid-molecule, and COOH-terminal iPTH disappeared parallel with the intact PTH (1-84). At high [Ca2+], mid-molecule and COOH-terminal iPTH disappeared significantly slower (P < 0.05) than intact PTH from the perfusates of the filtering kidneys. Due to great variation, the disappearance of NH2-terminal iPTH was neither significantly different from the disappearance of intact PTH nor from the disappearance of mid-molecule and COOH-terminal iPTH. The disappearance of intact PTH at low [Ca2+] was not significantly different from the disappearance at high [Ca2+].
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Ca2+ AND PTH CLEARANCE BY RAT KIDNEY AND LIVER
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TABLE 2. Effect of perfusate Ca2+ concentration on clearance of synhPTH-(l-84) 10"9 M in isolated rat kidney
Kidney = 0.49mmol/l
100
Assay 90
"o I
80
el's
100
= 1.79mmol/l
9080
[Ca2+] = 0.49 mmol/liter n=6 Clearance, ^lmin" 1
[Ca2+] = 1.79 mmol/liter n=6 Clearance, julmin"1
P
1-84 IRMA 1-34 RIA 53-68 RIA 53-84 RIA
677 ± 602 ± 528 ± 551 ±
219 217 204 149
878 ± 247 744 ± 257 586 ± 65* 543 ± 70*
NS NS NS
Inulin
667 ± 171
901 ± 274
NS
NS
Values are means ± SD. Clearance of the added intact PTH was measured in four assay systems as indicated. P values in right column pertain to differences between clearances at low and high Ca2+ concentration measured by the same assay. Significant differences from clearance measured by the 1-84 IRMA at same Ca2+ concentration: * P < 0.05. TABLE 3. Function of perfused rat liver Perfusate [Ca2+ ], mmol/liter
• 5 3 - 8 4 RIA
Assay
-o 5 3 - 6 8 RIA 70-
Endo • 1990 Vol 126-No 4
•*
1-34 RIA
•°
1-84 IRMA
D Non-filtering 60 15
30
60
90
Minutes 2+
FIG. 1. Effect of perfusate Ca concentration on the disappearance of iPTH from the perfusates of isolated kidneys. Six filtering kidneys and three nonfiltering kidneys were perfused with low [Ca2+]. Six filtering and three nonfiltering kidneys were perfused with high [Ca2+]. synhPTH-(l-84) was added at 1000 pmol/liter at time 0. iPTH was measured in four assays. Measured values corrected for sampling are expressed as percent of estimated initial values. Results are means ± SE. Significant differences from iPTH (percent of initial reactivity) measured in the 1-84 IRMA at same time and same [Ca2+]: *P < 0.05; **P < 0.01. Nonfiltering kidneys: results with the four assays did not differ significantly, and only disappearance measured in the 1-84 IRMA is shown.
The calculated clearances of PTH in the filtering kidneys are given in Table 2. No significant differences were found between the clearance at low and at high perfusate [Ca2+] of the added synhPTH-( 1-84) measured in any of the assays. The delayed disappearance of midmolecule and COOH-terminal iPTH at high [Ca2+] was reflected in clearances that were significantly lower (P < 0.05) than the clearance of intact PTH. Clearance of intact PTH was 106 ± 105 ^1 min"1 in the three nonfiltering kidneys perfused at low [Ca2+], and 185 ± 107 iA min"1 in the three nonfiltering kidneys perfused at high [Ca2+]. No significant differences were found between the clearances of intact PTH, NH2-terminal, mid-molecule, or COOH-terminal iPTH at low and at high perfusate [Ca2+] in the non-filtering kidneys. The clearances were not significantly different from zero
Number of perfusions, n Rat weight, g Liver wet weight, g Portal vein pressure, cmH 2 0 Bile flow, ^1-min"1 Potassium release, K+ jimol • min"1 Oxygen consumption O2 jumol • min"1 O2 jurnol-min"1 g"1 Perfusate flow (fixed), ml-min" 1
0.47 ± 0.01
1.79 ± 0.03
6
6
189 ± 12 6.57 ± 0.98 9±1
197 ± 16 6.49 ± 0.65 9±2
2.4 ± 0.9 1.3 ± 0.4
3.1 ± 1.2 1.1 ± 0.3
18.13 ± 2.29 2.82 ± 0.60
17.60 ± 2.21 2.72 ± 0.21
35
35
Values are means ± SD based on averages for each liver of results obtained during the whole experimental period with synhPTH-(l-84) added at an initial concentration of 10"9 M. The experimental period was 90 min, starting 41 min after cannulation of the livers in both groups. None of the differences between the two groups was significant.
either. Four nonfiltering kidneys perfused with a smaller circulating volume, perfusate [Ca2+] = 1.21 ± 0.03 mmol/ liter, and a different batch of BSA, cleared intact PTH at a rate of 293 ± 34 iA min"1, significantly different from zero (r = 0.93; P < 0.0001). As a different source of albumin was used for these experiments at normal [Ca2+] they were not directly comparable with those performed at low or high [Ca2+]. All other experiments were conducted with albumin from one batch of Miles' Pentex, as function of the perfused kidney (but not the liver) varies from batch to batch. Function of isolated livers perfused at low and high [Ca2+]
Results from six livers perfused at low [Ca2+] and six livers perfused at high [Ca2+] are given in Table 3. No significant differences were found between the functional
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Ca2+ AND PTH CLEARANCE BY RAT KIDNEY AND LIVER
parameters of livers perfused with low [Ca2+] and high [Ca 2+1 Clearance of PTH-(1-84) in perfused livers The effect of perfusate [Ca2+] on the disappearance of iPTH from the perfusates of isolated livers is shown in Fig. 2. The disappearance of synhPTH-(l-84) added at 10~9 M at time "0" was measured in four different assays. Both at low and at high [Ca2+] NH2-terminal PTH immunoreactivity disappeared parallel with intact PTH (1-84), whereas mid-molecule and COOH-terminal iPTH disappeared significantly slower (P < 0.001), and mutually parallel. The disappearance of iPTH measured in all four assays was faster at high [Ca2+] than at low [Ca2+]. These differences were significant (P < 0.01) (not indicated by symbols in Fig. 2) after 30 minutes and throughout the rest of the experimental period, except in the 1-34 RIA,
Liver = 0.47mmol/l
100-
1817
that exhibited large variation. The metabolic clearances calculated from the disappearance curves are given in Table 4. The delayed disappearance of mid-molecule and COOH-terminal iPTH at both low and high perfusate [Ca2+] resulted in clearances that were significantly lower (P < 0.001) than clearance of intact PTH (1-84) at the same Ca concentration. At low [Ca2+] the regression calculations yielded a clearance of NH2-terminal iPTH that was significantly higher (P < 0.05) than the clearance of intact PTH, although no measuring points along these two disappearance curves (Fig. 2) were significantly different from each other. Clearance of intact PTH in the isolated livers was 60% faster at perfusate [Ca2+] = 1.79 mmol/liter than at perfusate [Ca2+] = 0.47 mmol/liter. This difference was highly significant (P < 0.0005). Clearance of mid-molecule iPTH was also significantly faster at high [Ca2+] (P < 0.005), as was the clearance of COOH-terminal iPTH ( P < 0.0001). Clearances of iPTH measured in all assays were not significantly different from zero in the control experiments without any organs in the perfusion circuit, and synhPTH-(l-84) added at 10~9 M from start, neither at low nor at high perfusate [Ca2+]. HPLC studies
X
70
Q_
2
60-
E 50 100
[ C a + + ] = 1.79mmol/l
90 80 7060-
Livers. Samples from two livers perfused at low [Ca2+], and two livers perfused at high [Ca2+], drawn at the end of the perfusions, were analyzed by HPLC (Fig. 3). Every third HPLC separation was a control, drawn from the control experiments with zero iPTH clearance. In the control separations, only one slim peak was detected in all assays, representing the added synhPTH-(l-84). This peak was also found in all liver perfusates as expected, as 50-70% of the initially added intact PTH was still present in the liver perfusates at the end of the experiTABLE 4. Effect of perfusate Ca2+ concentration on the clearance of synhPTH-(l-84) 10~9 M in isolated rat liver
— 53-84 RIA o—o 53-68 RIA * — A 1-34 RIA o—a 1-84 IRMA
Assay
50 15
60
30
90
Minutes 2+
FIG. 2. Effect of perfusate Ca concentration on the disappearance of iPTH from the perfusates of isolated livers. Six livers were perfused with low [Ca2+], and six livers were perfused with high [Ca2+]. synhPTH-(l-84) was added at 1000 pmol/liter at time 0. iPTH was measured in four assays. Measured values corrected for sampling are expressed as percent of estimated initial values. Results are means ± SE. Significant differences from iPTH (percent of initial reactivity) measured in the 1-84 IRMA at same time and same [Ca2+]: *P < 0.05; **P< 0.01; ***P< 0.001.
1-84 IRMA 1-34 RIA 53-68 RIA 53-84 RIA
[Ca2+] = 0.47 mmol/liter n=6 Clearance, /ul-min"1
[Ca2+] = 1.79 mmol/liter n=6 Clearance,
765 ± 1010 ± 378 ± 304 ±
1223 ± 1381 ± 609 ± 687 ±
162 201* 106** 71***
P
jul-min"1
151 432 76*** 91***
Vol. 126, No. 4 Printed in U.S.A.
Influence of Calcium on the Metabolism of Intact Parathyroid Hormone by Isolated Perfused Rat Kidney and Liver* HENRIK DAUGAARDf, MARTIN EGFJORD, AND KLAUS OLGAARD Medical Department P, Division of Nephrology, and Department of Experimental Pathology, Rigshospitalet, Copenhagen, Denmark
ABSTRACT. The metabolism of synthetic human PTH [PTH-(l-84)] 10~9 M was studied in isolated rat kidneys and livers, perfused at a calcium concentration of 1 mM or 4 mM. Clearances were measured by an assay specific for intact PTH, and by assays specific for NH2-terminal, mid-molecule, and COOH-terminal immunoreactive PTH (iPTH). Production of PTH fragments was analyzed by HPLC. The kidneys cleared PTH mainly by filtration. The glomerular filtration rate was not lower at 4 mM calcium than at 1 mM calcium, and no significant differences were found between the clearance of PTH at 4 mM and at 1 mM calcium. At 1 mM calcium the kidneys cleared intact PTH without release of detectable fragments. At 4 mM calcium there was significant (P
E
XTRACELLULAR calcium concentration is inversely related to the secretion of PTH by a direct feedback mechanism (1). However, circulating levels of intact PTH and biologically active PTH fragments, if any, are also determined by the peripheral metabolism of the hormone, mainly in the liver and the kidneys (2). Canterbury et al. (3) found an increased metabolism of intact bovine PTH [bPTH-(l-84)] at low perfusate calcium concentration ([Ca2+]), compared with high [Ca2+], in isolated rat livers, and Hruska et al. (4) found an increased metabolism of bPTH-(l-84) at low perfusate [Ca2+], compared with high [Ca2+], in isolated dog kidneys. These results were interpreted in accordance with the theory that intact PTH has to be "activated" (3, 5, 6) by metabolism to NH2-terminal fragments to exert its main biological effects. However, other studies have been unable to demonReceived September 26,1989. *The present investigation was kindly supported by grants from the Danish Medical Research Council, Novo Foundation, Foundation of 1870, Karla Marie Jensen of Kerteminde Foundation, Danish Foundation for the Advancement of Medical Science, Johann and Hanne Weimann born Seedorff s Foundation, Jacob and Olga Madsen Foundation, and Ruth I. E. Konig-Petersen Foundation. tAddress correspondence and reprint requests to: H. Daugaard, M.D., Medical Department P, Division of Nephrology 2131, Rigshospitalet, 9 Blegdamsvej, DK 2100 Copenhagen, Denmark.
< 0.05) accumulation of mid-molecule and COOH-terminal iPTH in the perfusate. Both at low and at high calcium the livers cleared NH2terminal iPTH at the same rate as intact PTH, whereas midmolecule and COOH-terminal iPTH was cleared significantly (P < 0.005) slower. In the livers, metabolic clearance of PTH was 60% faster at 4 mM calcium than at 1 mM calcium (P < 0.001). Assuming that the hepatic metabolism of PTH represents degradation of the biologically active hormone and hormone fragments, rather than activation of the hormone, the present results suggest a homeostatic control of PTH degradation in the liver to enhance inactivation of the hormone at high serum levels of calcium. (Endocrinology 126: 1813-1820,1990)
strate enhanced uptake or metabolism of PTH in the liver and the kidneys at low extracellular [Ca2+]. Neumann et al. (7) could not influence the uptake of [125I] bPTH-(l-84) by the rat liver in vivo by modifying serum calcium. D'Amour and Huet (8) found that serum calcium did not influence the extraction of [ 126 I]bPTH-(l84) by the dog liver in vivo. Fox et al. (6) could not influence total metabolic clearance rate of bPTH-(l-84) in thyroparathyroidectomized dogs by changes in plasma calcium. Oldham et al. (9) found no correlation between serum calcium and arteriovenous (AV) difference in NH2-terminal/intact immunoreactive PTH (iPTH) across the liver in nine hyperparathyroid patients. Furthermore, three studies have demonstrated decreased uptake or metabolism of PTH in the liver and the kidneys at low extracellular [Ca2+]. Hanano et al. (10) found decreased degradation of [125I]bPTH-(l-84) at lower perfusate [Ca2+] in isolated rat kidneys. Oldham et al. (9) found a positive correlation between serum calcium and AV difference in NH2-terminal/intact iPTH across the kidney in nine hyperparathyroid patients. A recent study by D'Amour and Huet (11) demonstrated decreased extraction of synthetic human PTH-(1-34) at low perfusate [Ca2+] in isolated rat livers. Thus, it is at present unclear, how extracellular [Ca2+] 1813
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Ca2+ AND PTH CLEARANCE BY RAT KIDNEY AND LIVER
1814
influences the metabolism of intact PTH in the liver and in the kidneys. The isolated, perfused organs are the models of choice to investigate this relationship unaffected by glandular secretion and metabolism in other organs. We, therefore, performed the present investigation employing pure, synthetic intact human PTH [synhPTH-(l-84)], which is biologically active in the rat (12), and an immunoradiometric assay (1-84 IRMA) specific for intact PTH (12). The formation of immunoreactive fragments in the two organs was investigated by HPLC and RIAs specific for NH2-terminal, midmolecule, and COOH-terminal iPTH. Materials and Methods Sources of PTH and PTH fragments. Synthetic human parathyroid hormone: synhPTH-(l-84)[asp76] (synthesized at the Peptide Institute, the Protein Research Foundation, Minoh, Osaka, Japan), synhPTH-(39-68), synhPTH-(39-84), synhPTH-(53-84), and synhPTH-(69-84) were obtained from Peninsula Laboratories. SynhPTH-(13-34), synhPTH-(l-34), synhPTH-(l-44), synhPTH-(28-48), synhPTH-(44-68), and synhPTH-(64-84) were obtained from Bachem. Immunoassay systems for intact PTH and fragments 1-34 Immunoradiometric assay (IRMA) (Allegro, Nichols Institute). A two-site IRMA using one goat antibody binding only PTH-(39-84) immobilized onto plastic beads and another radiolabeled goat antibody binding only PTH-(1-34). The standards were hPTH-(l-84). A distinct two-step procedure ensured that only molecules possessing both epitopes—i.e. exclusively intact PTH—was detected. 1-84 RIA (INS-PTH, Nichols Institute). This RIA used a chicken antibody raised against synhPTH-(l-34) and specific for the NH2-terminal part of the PTH molecule. 53-68 RIA (PTH OMEGA, Cambridge Medical Diagnostics). This RIA used a goat antiserum specific for the hPTH-(53-68) sequence. 53-84 RIA (MILAB, Malmo Immunlaboratorium AS, Malmo, Sweden). This assay used a chicken antibody highly specific for the COOH-terminal part of the PTH molecule. Performance of these assays has been validated in our laboratory (12), including dilution series to test parallelism with synhPTH-(l-84) in perfusion medium. The 1-84 RIA was found to be specific for intact PTH, the 1-34 RIA was specific for the NH2-terminal region, the 53-68 RIA was specific for the mid-molecule region, and the 53-84 RIA was specific for the COOH-terminal region of PTH. Media for organ perfusions All experiments were performed with modified Klebs-Henseleit bicarbonate buffer containing 5 mM glucose, 67 or 100 g/ liter BSA (fraction V [Pentex], Miles Laboratories; or charcoal treated [A7906], Sigma Chemical Company) and all 20 physiological L-amino acids. Electrolyte composition was (in mM)
Endo • 1990 Voll26«No 4
141 Na, 5 K, 0.7 Mg, 0.7 SO4, 25 HCO3> 122 Cl, 3 PO4, 4 lactate, and 1, 3, or 4 Ca. The resulting ionized calcium concentrations, [Ca2+], are given under Results. [Ca2+] was measured with a calcium ion electrode (ICA 2, Radiometer, Copenhagen, Denmark). Kidney perfusion technique Experiments were performed on male Wistar rats allowed water ad libitum and fed with Altromin pellets with 0.9% (wt/ wt) calcium and 0.7% phosphorus. Normal plasma [Ca2+] for the Wistar rats in our laboratory free fed on this diet is 1.28 ± 0.04 mM (12). The animals were anesthetized with 2.5% thiopental sodium 0.5 ml per 100 g rat body weight, given ip. The cannulation of the right kidney with a 19-gauge blunted needle via the superior mesenteric artery without interruption of flow, and its isolation has been described earlier (13). The kidneys were not perfused along with the perfusion of the liver. Filtering kidneys were perfused with BSA 67 g/liter at 96 mm Hg pressure. Nonfiltering kidneys were perfused with BSA 100 g/liter, 75 mm Hg pressure, and ligature of the ureter. Liver perfusion technique Female Wistar rats were fasted 24 h before the experiments but allowed free access to water. Pentobarbital sodium anesthesia, 6 mg per 100 g rat body weight, was given ip. The bile duct was cannulated with PE-50 tubing for bile collection. The portal vein and the inferior caval vein were cannulated with 14-gauge catheters. The liver was isolated, and the perfusion was conducted as described before (13). The livers were not perfused along with the kidneys. The animal studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Danish Ethical Committees. HPLC Reverse-phase HPLC was performed using a water/acetonitrile/trifluoroacetic acid (TFA) solvent system, and Waters Ci8 juBondapak 2-mm X 30-cm steel columns, operated at ambient temperature. Solvent system. The two limit solvents, 0.1% TFA in water and 50% acetonitrile in 0.1% TFA, were mixed by two pumps in proportions determined by a Waters 721 System Controller. The gradient used in all separations is shown in Fig. 3. Flow rate was 1 ml/min. The elution positions of various PTH fragments was established by injecting 5-200-jug quantities of the different fragments dissolved in 200 n\ 0.1% TFA into the system and measuring ultraviolet absorption of column eluates at 215 nm. Perfusate samples (6 ml each) were extracted on three C18 Sep-Paks before HPLC. Priming and rinsing procedures for the Sep-Paks were as described by Bennett et al. (14). Lyophilized Sep-Pak eluates were redissolved in 300 ^10.1% TFA, 200 /A of which were injected on the HPLC column. Column eluates were collected in 1-ml fractions, lyophilized, redissolved in 1 ml of pentobarbital sodium buffer with EDTA and human serum proteins, and analyzed in the PTH assays. Recovery of
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Ca2+ AND PTH CLEARANCE BY RAT KIDNEY AND LIVER 100-1000 pmol/liter of different NH2- and COOH-terminal synhPTH fragments in perfusate was also tested in this system.
TABLE 1. Function of perfused rat kidney
Assay
Experimental design Organ perfusions. The initial stabilizing and control period was 40-50 min for both kidneys and livers. At the start of the experimental period synhPTH-(l-84) was added to the perfusates of the kidneys and livers at an initial concentration of 1000 pmol/liter. Circulating volume at the start of the experimental period was chosen to allow diuresis, sampling for all assays, and HPLC separations. Six filtering kidneys were perfused at 1 mM Ca, and six filtering kidneys were perfused at 4 mM Ca with a circulating volume of 191 ml. Three nonfiltering kidneys were perfused at 1 mM Ca, and three at 4 mM Ca with a circulating volume of 176 ml. Four nonfiltering kidneys were perfused at 3 mM Ca with a circulating volume of 149 ml. The small circulating volume in this series was chosen to permit optimal determination of peritubular clearance of PTH at normal calcium, but did not permit sampling for HPLC analyses. The BSA used in this series was from Sigma, because BSA from Miles ensuring acceptable function of the kidneys was not available at the time of these experiments. All other perfusions used BSA from the same batch of Miles Pentex. Six livers were perfused at 1 mM Ca, and six at 4 mM Ca with a circulating volume of 175 ml. Two experiments were carried out at 1 mM Ca, and two at 4 mM Ca without any organs in the perfusion circuit. Perfusate was sampled 5, 15, 30, 60, and 90 min after the addition of PTH and analyzed in four PTH assays. Results were corrected for sampling and plotted us. time on a semilogarithmic scale. Clearances were calculated from the regression lines for each experimental perfusion. For Figures 1 and 2 linear scales were chosen, because they are easier to read and can have a dimension; therefore these curves are not quite linear. HPLC studies. Perfusate samples from two kidneys perfused at 1 mM Ca, two kidneys perfused at 4 mM Ca, two livers perfused at 1 mM Ca, and two livers perfused at 4 mM Ca were analyzed by HPLC. The samples were drawn 90 min after the addition of synhPTH-(l-84) at 1000 pmol/liter, fractionated by HPLC, and analyzed in the 1-34 RIA and the 53-68 RIA. Every third HPLC separation was a control, drawn from the perfusions without organs and synhPTN-(l-84) added. Data and statistical analysis. The data are given as means ± SD where nothing else is stated. Student's t test and parametric linear regression were used. All significance limits are twosided.
Results Function of isolated kidneys perfused at low and high [Ca2+] Results from six filtering kidneys perfused at low [Ca2+] and six filtering kidneys perfused at high [Ca2+] are given in Table 1. Mean perfusion pressure and perfusate flow was identical in the two groups. Glomerular
1815
Perfusate [Ca2+], mmol/ liter 0.49 ± 0.02
Number of perfusions, n Kidney weight, g Perfusion pressure, mm Hg Perfusate flow, ml • min"1 g"1 Urine production, ^1-min"1 g"1 Inulin clearance, /J-min"1 g"1 Filtration fraction, % Na excretion, ^molmin" 1 g"1 Fractional reabsorption of Na, % K excretion, /imol-min"1 g"1 Fractional excretion of K, % Ca excretion, nmolmin" 1 g"1 Fractional excretion of Ca, % Pi excretion, nmol-min"1 g"1 Fractional excretion of Pi, %
1.79 ± 0.06
6
6
1.45 ± 0.15 96 ± 2 31 ± 3 55 ±32 465 ± 144 1.6 ± 0.5 4.3 ± 2.7 93.6 ± 3.7 1.7 ± 1.3 79 ± 4 3 19 ± 8** 8.1 ± 3.1 285 ± 121* 28.5 ± 13.4"
1.65 ± 0.18 96 ± 3 31 ± 3 50 ± 17 541 ± 140 1.7 ± 0.4 3.9 ± 1.4 94.9 ± 1.9 1.6 ± 0.6 70 ±19 67 ± 2 8 6.3 ± 3.6 140 ± 72 9.18 ± 3.3
Values are means ± SD based on averages for each kidney of results obtained during the whole experimental period with synhPTH-(l-84) added at an initial concentration of 10"9 M. The experimental period was 90 min, starting 47 min after cannulation of the kidneys in both groups. Significant differences between the two groups: * P < 0.05; ** P < 0.01.
filtration rate (GFR) determined as inulin clearance was unchanged by high perfusate [Ca2+]. Total Ca excretion was significantly higher (P < 0.01) at high perfusate [Ca2+], but fractional Ca excretion was not significantly different. Ultrafilterable Ca was assumed to be 56% of total Ca in the calculations (15). Inorganic phosphate (Pi) was assumed to be 100% ultrafilterable. Both total and fractional Pi excretion was significantly lower (P < 0.05) at high perfusate [Ca2+]. Compared with the initial control period, fractional Pi excretion was significantly higher after addition of synhPTH(l-84) at 10~9 M, both at low [Ca2+] (P < 0.05), and at high [Ca2+] (P < 0.01). Clearance of PTH-(l-84) in perfused kidneys The effect of perfusate [Ca2+] on the disappearance of iPTH from the perfusates of filtering and nonfiltering isolated kidneys is shown in Fig. 1. The disappearance of synhPTH-(l-84) added at 10"9 M at time "0" was measured in four different assays. At low [Ca2+], both NH2-terminal, mid-molecule, and COOH-terminal iPTH disappeared parallel with the intact PTH (1-84). At high [Ca2+], mid-molecule and COOH-terminal iPTH disappeared significantly slower (P < 0.05) than intact PTH from the perfusates of the filtering kidneys. Due to great variation, the disappearance of NH2-terminal iPTH was neither significantly different from the disappearance of intact PTH nor from the disappearance of mid-molecule and COOH-terminal iPTH. The disappearance of intact PTH at low [Ca2+] was not significantly different from the disappearance at high [Ca2+].
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Ca2+ AND PTH CLEARANCE BY RAT KIDNEY AND LIVER
1816
TABLE 2. Effect of perfusate Ca2+ concentration on clearance of synhPTH-(l-84) 10"9 M in isolated rat kidney
Kidney = 0.49mmol/l
100
Assay 90
"o I
80
el's
100
= 1.79mmol/l
9080
[Ca2+] = 0.49 mmol/liter n=6 Clearance, ^lmin" 1
[Ca2+] = 1.79 mmol/liter n=6 Clearance, julmin"1
P
1-84 IRMA 1-34 RIA 53-68 RIA 53-84 RIA
677 ± 602 ± 528 ± 551 ±
219 217 204 149
878 ± 247 744 ± 257 586 ± 65* 543 ± 70*
NS NS NS
Inulin
667 ± 171
901 ± 274
NS
NS
Values are means ± SD. Clearance of the added intact PTH was measured in four assay systems as indicated. P values in right column pertain to differences between clearances at low and high Ca2+ concentration measured by the same assay. Significant differences from clearance measured by the 1-84 IRMA at same Ca2+ concentration: * P < 0.05. TABLE 3. Function of perfused rat liver Perfusate [Ca2+ ], mmol/liter
• 5 3 - 8 4 RIA
Assay
-o 5 3 - 6 8 RIA 70-
Endo • 1990 Vol 126-No 4
•*
1-34 RIA
•°
1-84 IRMA
D Non-filtering 60 15
30
60
90
Minutes 2+
FIG. 1. Effect of perfusate Ca concentration on the disappearance of iPTH from the perfusates of isolated kidneys. Six filtering kidneys and three nonfiltering kidneys were perfused with low [Ca2+]. Six filtering and three nonfiltering kidneys were perfused with high [Ca2+]. synhPTH-(l-84) was added at 1000 pmol/liter at time 0. iPTH was measured in four assays. Measured values corrected for sampling are expressed as percent of estimated initial values. Results are means ± SE. Significant differences from iPTH (percent of initial reactivity) measured in the 1-84 IRMA at same time and same [Ca2+]: *P < 0.05; **P < 0.01. Nonfiltering kidneys: results with the four assays did not differ significantly, and only disappearance measured in the 1-84 IRMA is shown.
The calculated clearances of PTH in the filtering kidneys are given in Table 2. No significant differences were found between the clearance at low and at high perfusate [Ca2+] of the added synhPTH-( 1-84) measured in any of the assays. The delayed disappearance of midmolecule and COOH-terminal iPTH at high [Ca2+] was reflected in clearances that were significantly lower (P < 0.05) than the clearance of intact PTH. Clearance of intact PTH was 106 ± 105 ^1 min"1 in the three nonfiltering kidneys perfused at low [Ca2+], and 185 ± 107 iA min"1 in the three nonfiltering kidneys perfused at high [Ca2+]. No significant differences were found between the clearances of intact PTH, NH2-terminal, mid-molecule, or COOH-terminal iPTH at low and at high perfusate [Ca2+] in the non-filtering kidneys. The clearances were not significantly different from zero
Number of perfusions, n Rat weight, g Liver wet weight, g Portal vein pressure, cmH 2 0 Bile flow, ^1-min"1 Potassium release, K+ jimol • min"1 Oxygen consumption O2 jumol • min"1 O2 jurnol-min"1 g"1 Perfusate flow (fixed), ml-min" 1
0.47 ± 0.01
1.79 ± 0.03
6
6
189 ± 12 6.57 ± 0.98 9±1
197 ± 16 6.49 ± 0.65 9±2
2.4 ± 0.9 1.3 ± 0.4
3.1 ± 1.2 1.1 ± 0.3
18.13 ± 2.29 2.82 ± 0.60
17.60 ± 2.21 2.72 ± 0.21
35
35
Values are means ± SD based on averages for each liver of results obtained during the whole experimental period with synhPTH-(l-84) added at an initial concentration of 10"9 M. The experimental period was 90 min, starting 41 min after cannulation of the livers in both groups. None of the differences between the two groups was significant.
either. Four nonfiltering kidneys perfused with a smaller circulating volume, perfusate [Ca2+] = 1.21 ± 0.03 mmol/ liter, and a different batch of BSA, cleared intact PTH at a rate of 293 ± 34 iA min"1, significantly different from zero (r = 0.93; P < 0.0001). As a different source of albumin was used for these experiments at normal [Ca2+] they were not directly comparable with those performed at low or high [Ca2+]. All other experiments were conducted with albumin from one batch of Miles' Pentex, as function of the perfused kidney (but not the liver) varies from batch to batch. Function of isolated livers perfused at low and high [Ca2+]
Results from six livers perfused at low [Ca2+] and six livers perfused at high [Ca2+] are given in Table 3. No significant differences were found between the functional
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Ca2+ AND PTH CLEARANCE BY RAT KIDNEY AND LIVER
parameters of livers perfused with low [Ca2+] and high [Ca 2+1 Clearance of PTH-(1-84) in perfused livers The effect of perfusate [Ca2+] on the disappearance of iPTH from the perfusates of isolated livers is shown in Fig. 2. The disappearance of synhPTH-(l-84) added at 10~9 M at time "0" was measured in four different assays. Both at low and at high [Ca2+] NH2-terminal PTH immunoreactivity disappeared parallel with intact PTH (1-84), whereas mid-molecule and COOH-terminal iPTH disappeared significantly slower (P < 0.001), and mutually parallel. The disappearance of iPTH measured in all four assays was faster at high [Ca2+] than at low [Ca2+]. These differences were significant (P < 0.01) (not indicated by symbols in Fig. 2) after 30 minutes and throughout the rest of the experimental period, except in the 1-34 RIA,
Liver = 0.47mmol/l
100-
1817
that exhibited large variation. The metabolic clearances calculated from the disappearance curves are given in Table 4. The delayed disappearance of mid-molecule and COOH-terminal iPTH at both low and high perfusate [Ca2+] resulted in clearances that were significantly lower (P < 0.001) than clearance of intact PTH (1-84) at the same Ca concentration. At low [Ca2+] the regression calculations yielded a clearance of NH2-terminal iPTH that was significantly higher (P < 0.05) than the clearance of intact PTH, although no measuring points along these two disappearance curves (Fig. 2) were significantly different from each other. Clearance of intact PTH in the isolated livers was 60% faster at perfusate [Ca2+] = 1.79 mmol/liter than at perfusate [Ca2+] = 0.47 mmol/liter. This difference was highly significant (P < 0.0005). Clearance of mid-molecule iPTH was also significantly faster at high [Ca2+] (P < 0.005), as was the clearance of COOH-terminal iPTH ( P < 0.0001). Clearances of iPTH measured in all assays were not significantly different from zero in the control experiments without any organs in the perfusion circuit, and synhPTH-(l-84) added at 10~9 M from start, neither at low nor at high perfusate [Ca2+]. HPLC studies
X
70
Q_
2
60-
E 50 100
[ C a + + ] = 1.79mmol/l
90 80 7060-
Livers. Samples from two livers perfused at low [Ca2+], and two livers perfused at high [Ca2+], drawn at the end of the perfusions, were analyzed by HPLC (Fig. 3). Every third HPLC separation was a control, drawn from the control experiments with zero iPTH clearance. In the control separations, only one slim peak was detected in all assays, representing the added synhPTH-(l-84). This peak was also found in all liver perfusates as expected, as 50-70% of the initially added intact PTH was still present in the liver perfusates at the end of the experiTABLE 4. Effect of perfusate Ca2+ concentration on the clearance of synhPTH-(l-84) 10~9 M in isolated rat liver
— 53-84 RIA o—o 53-68 RIA * — A 1-34 RIA o—a 1-84 IRMA
Assay
50 15
60
30
90
Minutes 2+
FIG. 2. Effect of perfusate Ca concentration on the disappearance of iPTH from the perfusates of isolated livers. Six livers were perfused with low [Ca2+], and six livers were perfused with high [Ca2+]. synhPTH-(l-84) was added at 1000 pmol/liter at time 0. iPTH was measured in four assays. Measured values corrected for sampling are expressed as percent of estimated initial values. Results are means ± SE. Significant differences from iPTH (percent of initial reactivity) measured in the 1-84 IRMA at same time and same [Ca2+]: *P < 0.05; **P< 0.01; ***P< 0.001.
1-84 IRMA 1-34 RIA 53-68 RIA 53-84 RIA
[Ca2+] = 0.47 mmol/liter n=6 Clearance, /ul-min"1
[Ca2+] = 1.79 mmol/liter n=6 Clearance,
765 ± 1010 ± 378 ± 304 ±
1223 ± 1381 ± 609 ± 687 ±
162 201* 106** 71***
P
jul-min"1
151 432 76*** 91***
