239 Horm. Metab. Res. 9 (1977) 239-242

© Georg Thieme Verlag Stuttgart

Metabolism of Human PTH by the Kidney and tt'le liver J. Corvilain, T. Manderlier, J. Struyven, M. Fuss, A. Bergans, N. Nijs, H. Brauman Departments of Medicine, Radiology and Clinical Biology. Hopital Brugmann. Universite libre de Bruxelies et Fondation Reine Elisabeth, Bruxelles, Belgium

It is conc1uded that intaet PTH and possibly amino terminal fragments of the hormone are metabolized by the kidney and the liver. Considering the fact that a earboxyl terminal speeifie antiserum is also eapable of recognizing intaet hormone, the finding of a sm.all ~ositive arteri yenous difference for earboxyl termmal unmunoreaetJvlty does not permit us to exc1ude the possibility that the kidney and/or the Iiver are capable of generating carboxyl terminal fragments. Key-Words: Immunoreactive PTH - Kidney - Liver PTH Fragments

Introduction

..

.

dlstmct antJsera. We have investigated the role played by the kidney . the me t ab0 I'Ism 0 f th e tw 0 mam .. I t' f In CIICU a Ing ractions of PTH by measuring renal arterio venous ( ) 'f~ . PTH . r~nal A.V,. dIlerences for I. wlth two appropnate antJsera. We also measured the concentration of the intact . '

hormone and the carboxyl termmal fragment In the suprahepatic vein, considering the weH known ability of the liver to catabolize several polypeptide hormones and evidence that, in the rat, the liver plays a significant role in the metabolism of PTH (Fang and Tashijian 1972). Materials and Methods

There is some evidence that parathyroid hormone (PTH) is catabolized by the kidney. Bovine PTH labeled with p25 is rapidly destroyed by an enzyme in rat kidney (Fujita, Orimo, Ohata, Okano and Yoshikawa 1973, Martin, Melick and De Luise 1969) and nephrectomy prolongs the survival time of P 25 bovine PTH in rats, dogs (Martin, Melick and De Luise 1969) and human subjects (Meliek and Martin 1969). Little is known however concerning the mechanism and site of catabolism of human PTH in man. The resolution of this problem is complicated by the fact that plasma PTH in man consists of aleast two forms (Berson and Yalow 1968, Canterbury and Reiss 1972, Goldsmith, Furszy[er, Johnson, Fournier, Sizemore and Arnaud 1973, Habener, PoweIl, Murray, Mayer and Potts 1971, Segre, Habener, PoweIl, Tregear and Potts 1972, Silverman and Yalow 1973), the principal secretory product of the parathyroid gland is intact hormone (MW 95(0) (Habener et aL 1971, Martin, Greenberg and Melick 1972). Although a third immunoreactive form of PTH, an amino terminal fragment, has been found by Canterbury and Reiss in pooled peripheral hyperparathyroid serum (Canterbury and Reiss 1972, Canterbury, Levey and Reiss 1973) the major portion of the circulating Received: 30 Oet. 1976

PTH is a carboxyl terminal fragment of the molecule (MW ± 7(00) (Arnaud, Goldsmith, Bordier and Sizemore 1974, Segre, Niall, Habener and Potts 1974). The 9500 MW and 7000 MW forms of PTH are demonstrable by difference in their reactivity with two

Accepted: IDee. 1976

Subjects Blood was taken from the right renal vein (RRV) the suprahepatic vein (SHV) and the femoral artery (FA) in 10 hyperparathyroid patients during venous catheterization for preoperative localization of parathyroid adenoma. The position of the catheter in the renal or the suprahepatic vein was confirrned by the injection of Contrix 28 through it under fluoroscopic control. Arterial.and venous samplings were c10sely related in time in order to minimize any error in estimation of the A.V. difference due to spontaneous change in PTH secretion during the procedure. Radioimmunoassay of PTH In a11 ten patients plasma i PTH was measured by a modification (Arnaud, Tsao and Littledike 1971) of the procedure of Berson, Yalow, Aurbach and Potts (1963), using two antisera with different specificities (CH 14 M and GP 1 M) kindly provided by Dr. Cl. Arnaud. CH 14 M was developed against bovine PTH in a cockerel and was used at the final dilution of 1/3000. GP 1 M was developed against porcine PTH in a Guinea Pig and was used at a dilution of 1/100000. The specifieity charaeteristics of these two anitsera have been published (Aranud et al. 1974). CH 14 M antiserum reacts as we11 with the synthetie amino terminal human or bovine PTH fragment 1-34 as with the intaet human PTH 1-84 purified from parathyroid adenomas. In contrast, GP 1 M antiserum at the dilution used in this work does not recognize either the synthetie human or bovine amino terminal fragments. Analysis by gel chromatography of hyperparathyroid serum showed that CH 14 M antiserum reacted well with the i PTH that eluted in the same positions as 131 1 labeled bovine PTH 1-84 and with a species that eluted

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Summary Immunoreactive PTH was measured by amino terminal and carboxyl terminal specific assays in the femoral artery, the right renal vein and the suprahepatic vein of ten hyperparath~roid pat~ent~. A marked ~~erio venous diffe~ence for ammo terminal unmunoreactlVlty was observed m the kidney and the liver. In contrast, the arterio venous difference for carboxyl terminal immunoreactivity was small in the kidney and not significant in the liver.

J. Corvilain, T. Manderlier, J. Struyven, M. Fuss, A. Bergans, N. Nijs, H. Brauman

240

immediately after 131 1 labeled bovine PTH 1-34. It reacted poorly with a component that eluted between these species (4000 to 8000 Daltons). In contrast GP 1 M antiserum re· cognized this intermediate component and also reacted with the PTH eluting in the region of the intaet moleeules. It appears therefore that CH 14 M antiserum is specific for the amino terminal region of the PTH molecule and recog· nizes in plasma mainly the intact form of the molecule but not the earboxyl terminal 7000 MW fragment. On the other hand, since GP 1 M antiserum did not reaet with the synthetie bovine or human PTH 1-34 fragments, it is by ex· c1usion carboxyl terminal specifie and recognizes both the carboxyl terminal 7000 MW fragment and the intact hor· mone.

was 0.44 ± 0.07 with eH 14 M antiserum and 0.09 ± 0.03 with GP 1 M antiserum. In the ease of eH 14 M antiserum there was a pos· itive eorrelation between individual renal (r = 0.89, p < 0.001) or hepatie (r = 0.73, p < 0.05) arterio venous differenees and eorresponding i PTH plasma levels in the FA Discussion

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As discussed in the methods careful immunologieal studies by ArTlllud et al. (1974) have shown that Microliter amounts of a partially purified extract of a para· eH 14 M antiserum reaets with the amino terminal thyroid adenoma were used as standards, eare being taken region of the PTH moleeule, and reeognizes intaet to use, for both antisera, oniy the portion of the standard hormone and amino terminal fragments of the mole· curve that was paralIel to the eurve obtained with multiple eule whereas GP 1 M antiserum reaets with the ear· dilutions of serum from a patient with end stage chronic renal failure. Results were expressed as microliter equivalents boxyl terminal region of the PTH moleeule and meas· of the diluted extract per ml of plasma (pI eq/ml). ures the 7000 MW cireulating fragment and intaet PTH. Results (Table 1) The renal arteriovenous differenee obtained in hyper· As it appears in the table, using eH 14 M antiserum parathyroid patients, for the moleeular form of PTH the mean plasma i PTH was signifieantly lower in recognized by eH 14 M antiserum, was proportional the R.R.V. than in the F .A .. The mean i PTH plasma to arterial blood eoneentration. The mean PTH level in the S.H.V. was also signifieantly lower than R.E.R. of 0.44 implies therefore that, within the in the F.A .. For every subjeet the plasma i PTH was limits of our studies, the renal clearanee of amino higher in the FA than in the R.R.V. or the S.H.V. terminal immunoreaetivity is of the order of 260 ml of plasma per minute, assuming a normal renal plasma With GP 1 M antiserum whieh measures mueh higher plasma i PTH levels, the difference between F .A. and flow of 650 ml. To our knowledge no similar stud· ies have been done in man. In the dog, the normal R.R.V. was smaller than with eH 14 M antiserum kidney extraets 20% of administered bovine PTH but still signifieant. In contrast with the results ob· and 37% of synthetie bovine PTH 1-34 fragment, served with eH 14 M antiserum, the plasma i PTH delivered to it (Hruska, Kopelman, Rutherford, Klahr was higher in the F.A. in seven eases and in the R.R.V. in one ease. There was no differenee between and Slatopolsky 1975). AIthough these data eannot be quantitatively compared to ours, they confirm the FA and the S.H.V. mean i PTH levels. the important role of the kidney in the metabolism The renal extraetion ratio for i PTH ( i PTH R.E.R.) of PTH in mammals. was ealeulated in each ease as the ratio between the Some i PTH is may be excreted by the kidney sinee renal A.V. differenee (differenee between the i PTH PTH like activity has been found in the urine by level in the renal artery and th i PTH in the R.R.V.) means of bioassays (Bethune and Turpin 1968, Fujita, and the i PTH in the F .A .. The mean PTH R.E.R.

Table 1 i PTH concentrations (pI eq/ml) determined with eH 14 M and GP 1 M antiserum, in the femoral artery (FA), the right renal vein (RRV) and the suprahepatic vein (SHV) in 10 hyperparathyroid patients. FA

RRV GP I M

SHV eH 14 M CP I M

Patient

Sex

Age

eH 14 M

CP I M

CH 14 M

1 2 3 4 5 6 7 8 9 10

M F M F F F F F F F

74 75 56 62 56 52 62 48 63 49

36 68 30

90 102 100 130 88 88 75 105

16 24 20 33 25 20 15 20 16 12

80 102 100 115 80 77 59 100 80

5 29 20 29 20 16 13 22 5 5

85 108 98 115 88 90 74 125 68 68

20.1 2.3 < 0.005

84.3 6.5. < 0.05

16.4 2.9 < 0.001

91.9 6.2 NS

Mean

± SEM p

SO 37 25 35 29 25 44 37.9 4.2

71

76 92.5 5.6

so

Morii,Ibayashz: Takahashi and Okinaka 1961, NijsDewolf, De Nutte, Brauman and Corvilain 1975). Until now the moleeule responsible far the PTH like aetivity in the urine has not been charaeterized, so that its nature, its size and the magnitude of its urinary clearanee are not known. Urinary extraction of bovine immunoreaetive PTH aecounted for less than 1% of the renal extraction in the dog (Hruska et al. 1975). Even if the urinary clearanee of amino terminal immunoreactivity was equal to that of inulin (± 120 ml per min) in man, it would aecount for less than 50% of its renal clearanee. It is therefore necessary to postulate either an improbable tubular secretion of intaet PTH or mueh more likely a metabolie clearing of that intact PTH. If the second alternative is true, at least half the intaet PTH must be taken up by the tubular eells directly from the blood since the renal extraction of intact PTH is more than twiee the glomerular filtration rate. Study by fluorescent microscopy and light and electron autoradiography of the loealization in the rat kidney of infused labeled PTH suggest that the PTH destruetion might take plaee in proximal tubular eells (Nordquist and Palmieri 1974). A true liver extraction ratio for i PTH eannot be ealeulated from our data. Plasma i PTH in the F .A. is not really representative of the i PTH eoneentration in the blood entering the liver, since only about 30% of the hepatic blood flow is of arte rial origin. However the size of the differenee in PTH concentration between the F .A. and the S.H.V., using CH 14 M antiserum, is such as to suggest a metabolie References Arnaud. C.D.• H.S. Tsao. T. Littledike: Radioimmunoassay of human parathyroid hormone in serum. J. Clin Invest 50: 21-34 (1971) Arnaud. C.D.• R.S. Goldsmith. P.J. Bordier. G. W. Sizemore: lnfluence of immunoheterogeneity of circulating parathyroid hormone on results of radioimmunoassays of serum in man. Am J Med 56: 785-793 (1974) Benon. S.A .• R.S. Yalow. G.D. Aurbach. J. T. Potts Jr.: lmmunoassay of bovine and human parathyroid hormone. Proc Natl Acad Sci USA 49: 613-617 (1963) Benon. S.A .• R.S. Yalow: Immunochemical heterogeneity of parathyroid hormone in plasma. J Clin Endocrinol Metab 28: 1037-1047 (1968) Bethune J.E.. R.A. Turpin: A study of urinary excretion of parathyroid hormone in man. J Clin lnvest 47: 15831589 (1968) Canterbury. IM. E. Reiss: Multiple irnmunoreactive molecular forms of parathyroid hormone in human serum. Proc Soc Exp Biol Med 140: 1393-1398 (1972) Canterbury J.M.. G.S. Levey. E. Reiss: Activation of renal cortical adenylate cyclase by circulating immunoreactive parathyroid hormone fragments. J Clin Invest 52: 524527 (1973) Canterbury. J.M.• L.A. Bricker. G.S. Levey. P.L. Koz10 vskis. E. Ruiz. J.E. Zull. E. Reiss: Metabolisrn of bovine parathyroid hormone. Immunological and biological characteristics of fragments genera ted by liver perfusion. J Clin Invest 55: 1245-1253 (1975)

241

clearing of intact PTH by the liver similar to that demonstrated for the kidney. The high degree of that clearing is eonsistent with the short half life of intaet PTH (Goldsmith et al. 1973, Silverman and Yalow 1973). Our data do not indicate whether the intaet PTH is eompletely eatabolized or transformed into a shorter fragment by loss of the amino terminal part of the molecule. In contrast the data obtained with GP 1 M antiserum shows that litde metabolie consumption of 7000 MW PTH takes plaee in the kidney and the liver, in accordanee with the long half life generally attributed to that moleeule (Goldsmith et al. 1973, Silverman and Yalow 1973). On the other hand the slight positive mean renal A.Y. differenee observed might be an artefact since GP 1 M antiserum ean also reaet with intaet hormone. Under these conditions, a marked positive renal A.V. difference for the intaet hormone might mask a small negative renal A.V. difference for the 7000 MW form although the latter is 5 to 20 times mare abundant than the 9500 MW moleeule in the peripheral blood (Amaud et aL 1974, Segre et al. 1974). Therefore our data do not allow us to exclude that the 7000 MW fragment is produced in the kidney and/or the liver from the intaet hormone. Such a possibility has indeed reeeived supporting evidence from the work of Canterbury, Bricker, Levey, Kozlovskis, Ruiz, Zull and Reiss (1975), who have demonstrated that carboxyl terminal immunoreaetivity ean be generated in the rat liver perfused with intaet bovine PTH.

Fang. V.S.• A.H. Tashjian: Studies on the role of the liver in the metabolism of parathyroid hormone I Effects of partial hepatectomy and incubation of the hormone with tissue homogenates. Endocrinology 90: 1177-1184 (1972) Fujita, T., H. Morii, H. lbayashi, Y. Takahashi, S. Okinaka: Assay of parathyroid hormone in human urine using 32p excretion in parathyroidectomized rat. Acta Endocrinol (Kbh) 38: 321-329 (1961) Fujita, T.• H. Orimo. M Ohata, K. Okano. M Yoshikawa: Loss of immunoreactivity of human serum parathyroid hormone by the action of rat kidney enzyme preferentially hydrolysing parathyroid hormone. Horm Res 4: 213-218 (1973) Goldsmith, R.S., J. Furszyfer, W.J. Johnson, A.E. Fournier: G. W. Sizemore. C.D. Arnaud: Etiology of hyperparathyroidisrn and bone disease during chronic hemodialysis 111 Evaluation of parathyroid suppressibility. lehn Invest 52: 173-180 (1973) Habener J.F., D. Po weil. T.M. Mu"ay, G. Mayer, J. T. Potts Jr.: Parathyroid hormone: secretion and metabolisrn in vitro. Proc Natl Acad Sei USA 68: 2986-2991 (1971) Hruska, K.A .• R. Kopelman, W.E. Rutherford. S. Klahr, E. Slatopolsky: Metabolism of irnmunoreactive parathyroid hormone in the dog. The role of the kidney and the effects of chronic renal disease. I Clin Invest 56: 39-48 (1975) Martin, T.J., R.A. Melick, M. De Luise: Metabolisrn of parathyroid hormone. Degradation of 125 1 labelIed hormone by a kidney enzyme. Biochm I 111: 509-514 (1969)

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PTH Metabohsm in Kidney and Liver

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1. Corvilain, T. Manderlier, 1. Struyven, M. Fuss, A. Bergans, N. Nijs, H. Brauman

Martin, T.J., R.A. Melick, M. De Luise: The effect of nephrectomy on the metabolism of labelJed parathyroid hormone. Clin Sei 37: 137-142 (1969) Martin, T.J., P.B. Breenberg, R.A. Melick: Nature of human parathyroid hormone secreted by monolayer celJ cultures. 1 Clin Endocrinol Metab 34: 437-440 (1972) Melick, R.A., T.I. Martin: Parathyroid hormone metabolism in man: effect of nephrectomy. Clin Sei 37: 667-674 (1969) Nijs-Dewolf, N., N. De Nutte, H. Brauman, I. Corvilain: PTH like substance in human urine. In: Vitamine D and Problems related to Uremic bone disease, p. 467-473. A.W. Norman, K. Schaefer, H.G. Grigoleit, D. Von Herrath, and E. Ritz (eds), W. de Gruyter, Berlin, New York

Nordquist. R.E., G.M.A. Palmieri: IntracelJular localization of parathyroid hormone in the kidney. Endocrinology 95: 229-237 (1974) Sgre. G. V.• J.F. Habener, D. PoweIl, G. W. Tregear. J. T. Potts Ir.: Parathyroid hormone in human plasma. Immunochemical characterization and biological implications. 1 Clin Invest 51: 3163-3172 (1972) Segre. G. V.. H.D. Niall. I.F. Habener. I. T. Potts Ir.: Metabolism of parathyroid hormone. Am 1 Med 56: 774-784 (1974) Silvennan. R .• R.S. Yalow: Heterogeneity of parathyroid hormone. Clinical and physiologie implications. 1 Clin Invest 52: 1958-1971 (1973)

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Requests for reprints should be addressed to: I Corvilain, Hopital Universitaire Brugmann, 4, Place Van Gehuchten, 1020 BruxelJes (Belgium)

Metabolism of human PTH by the kidney and the liver.

239 Horm. Metab. Res. 9 (1977) 239-242 © Georg Thieme Verlag Stuttgart Metabolism of Human PTH by the Kidney and tt'le liver J. Corvilain, T. Mander...
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