CLINICAL CHEMISTRY
Endocrine Disorders (Parathyroid Hormone) Manjula K. Gupta' and Daphne Khoo Section of Immunopathology, Department of Clinical Pathology, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195-5131 INTRODUCTION
Manjula K. Gupta is Head of me E n d o d n e
Parathyroid hormone (PTH) and vitamin D govern the homeostatic mechanisms that regulateserum ionized calcium concentration. When these mechanisms fail to operate properly, hypo- or hypercalcemia develops,which in tUrn leads to bone and mineral disorders. These clinical disorders of calcium homeostasis have been recognized with increasing frequency primarily because of the routine measurement of serum calcium with use of multichannel automated instruments in the clinical lahoratory. Thus, low or high serum calcium levels are detected as an initial finding, which then requires measuring PTH for the differential diagnosis of the underlying cause. The precise measurement of PTH has been uncommonly difficultprimarily because of the heterogeneity of circulating PTH-related molecules. Itiswellestablished thatparathyroid hormone circulates in several chemically and immunologically distinct molecular forms, and dependin on antibody specificity, different molecular forms are fetected in a given radioimmunoassay. Various terminologies for PTH assays have also created much confusion in the literature regarding the accurate interpretation of PTH assays. The purpose of this article is to review the various PTH immunoassays developed to date (October 1992) and offer a perspective in their use for the differential diagnosis of calcium disorders.
Immunology Laboratory. In the Section of Immunopathology. Cepartment of Clinical
Pathology.TheClevelandClhicFoundation.
Cleveland, OH. and s h e has a secondary appointment in the Department of Endocrinology. ShealsoholdsafacultyposKin as ClinlCBIProfessor.DepartmentofClinical Chemistry, Cleveland State University, Cleveland. OH. Dr. Gupta received her Ph.D. from Agra University. India, and received her postgraduate training at the Cleveland Clinic Foundation. Her major area of research is in studying the role of hormones in the regulation of cancer growth.
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DaphneKhoograduated fromtheNational University of Singapore in 1983. She obtained her postgraduate internal medical qualificationsfromtheU n k d Kin@nnand Singapore. h.Khoo worked at the Cleveland Clinic Foundation as a Special Fellow in the Department of Endocrinology. She is currently a Senior Registrar in the Cepartment of Medicine I at Singapore General Hospital in Singapore.
BACKGROUND Parathyroid Physiology and Biochemistry. Parathyroid hormone is a linear polypeptideof Maminoacid residues and has a molecular mass of 9500 Da. It is secreted by the parathyroid glands a t a rate that is inversely proportional to calcium concentrations in serum. The primary action of arathyroid is toincreasecalciuminserum,whichisachieved y direct actions on the bone and kidney and by indirect actions via vitamin D in the gut. In bone, PTH causes resorption of calcium and phosphorus; in the kidney, it enhances calcium resorption from the glomerular filtrate, inhibits the resorption of phosphate, and promotes urinary phosphate excretion. More importantly, in the kidney, it stimulates the synthesisof 1,25-dihydroxyvitaminD, anactive metabolite of vitamin D, which in turn promotes the absorption of calcium from the gastrointestinal tract. Intact PTH is synthesized as a larger precursor of 115amino acid residues, the preproparatbyroid hormone (CI). Preproparathyroid hormone is converted intraglandularly to proparathyroid hormone, a polypeptide of 90 amino acid residues. The latter is further cleaved to intact PTH (CZ, CZ). The biologic activity of PTH resides in the first onethird or amino-terminal region (N-terminal) of polypeptide chain (C3). Thus, the intact hormone (1-84)and N-terminal fragments comprise the biologically active molecular species of PTH. Immunoreactive PTH in plasma includes active intact peptide and N-terminalfragment,as wellas inactive carboxylterminal fragments (C4). which contain the middle and carboxyl ends of the molecule. The principal form of PTH secreted by the parathyroid gland is intact PTH (C5). This molecule is degraded both within the parathyroid gland (C6) and a t peripheral sites ( C n to form biologically active aminoterminal fragments and inactive carboxyl-terminal fragments. Both intact PTH and its fragments are cleared by glomerular filtration. However, the intact molecule and aminoterminal fragments have much shorter half-lives because they are also metabolically degraded in the liver, possibly in the bone and at selective peritubular renal sites (C8). Because the carboxyl-terminal fragments are cleared only by glomerular filtration, their concentration in peripheral blood is markedly dependent on renal function. With normal renal function, the half-life of intact hormone is about 5 min, and the half-lifeof carboxyl-terminal fragments is about 30 min (C9). In patients with renal failure, the half-life of carboxylterminal fragments increases to 24-36 h (C9). This increase
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allows the carboxyl-terminal fragments in circulation to accumulate, leading to high levels of these fragments, which interferes in attempts to estimate the response of the parathyroid gland to physiologic stimuli. There is controversy in the literature regarding the relative concentrations of the different forms of PTH circulating in the peripheral blood. While some groups have detected circulatingamino-terminal fragments (CZO, CZZ),others have not (C4, C5). Overall, the consensus is that circulating aminoterminal fragments generally represent a minor component of the biological activity of PTH and that the intact hormone is the major component.
IMMUNOASSAYS While bioassays have been used to measure PTH in the past, most laboratories currently use immunoassays. The first radioimmunoassay for measuring PTH was described in 1963(CZ2). Earlystudiesshowedwidevariationintheability of the immunoassay to differentiate between normal and hyperparathyroid sera (CZ3-Cl5). This variation was later explained in 1968 by the finding that PTH in human plasma is immunochemically heterogenous (CZ6). Parathyroid hormone immunoassays are divided into four categories according to the major epitopes on the molecule against which the antisera are directed (1) The amino-terminal assays use antisera directed to the N-terminal(1-34) regionof PTH. Theseassays recognize intact hormone as well as N-terminal fragments. (2) In carboxyl-terminal assays,the antiseraaredirected to the C-terminal region (53-84). These assays detect intact hormone as well as carboxyl fragments. (3)Midmolecule assays recognize intact PTH as well as fragments of hormone containing the midregion (44+8) sequence of the molecule. (4) Intact P T H assays use either a single antiserum directed against the region where PTH is cleaved (2848) or two antisera directed to the N-terminal and C-terminal regions. Because these assays detect different molecular species of PTH in circulation, the actual amount measured in different clinical situations varies greatly from one assay to another. This variation, in addition to the natural variation in
CLINICAL CHEMISTRY
specificities of the polyclonal antibodies used, makes assay comparisons even more difficult and requires careful characterization of each individual assay.
HYPERPARATHYROIDISM Among the disorders of calcium homeostasis, hypercalcemia is far more common than reviously realized, occurring in 1-2% of the general popuyation. Also, the most common causes of h ercalcemia in ambulatory patients is primary hyperparatgoidism, which results from increased and inap ropriate production of PTH from the parathyroid glan&. The disease is caused by a sin le adenoma in 80% of patients and b hyperplasia in t i e majority of the remainder. Multi re adenomas or arathyroid carcinomas are rare causes. 8ccasionally the &+easemay be familial, usually occurring as part of the syndromes of multiple endocrine neoplasia. The elevated parathyroid hormone levels act on bone and kidney, resulting in hypercalcemia, the biochemical hallmark of the disease. Diagnosis of Hyperparathyroidism. Primary hyperparathyroidism is usually diagnosed by demonstrating persistent hypercalcemia in the presence of elevated PTH concentrations. Because of the widespread use of multiphasic screening for hypercalcemia, the frequency of dia osis of hyperparathyroidism has risen in recent years. WEle normocalcemic hyperparathyroidism has been well documented (C17, CM),its true incidence is unknown. Ionized calcium is more sensitive to alterations in PTH concentrations and may be elevated in mild caws of hyperparathyroidismin which total calcium levels may be within the normal reference range (C19).
More than 907% of the cases of hypercalcemia are caused by primary hyperparathyroidism or malignancy. While hypercalcemia associated with malignancy is the most common cause of hypercalcemia in hospitalized patients (C20), the great ma’ority of hypercalcemic patients detected by routine health surveillance in the community will have hyperparathyroidism ((221). In selectin a PTH assa for the screening of hyperparathyroidism, tfie assay shouYd be able to distinguish hyperparathyroid from normal subsets and to identify patients with non-parathyroid causes of hypercalcemia. Because biological activity of PTH resides in the amino terminal of the molecule (C22),amino-terminal assa s would be expected to be hi hly sensitiveand specificin the Ltection of hyperparath oifism. While this has been the case in some reports (C23, g 4 ) , most of these assa s have shown poor discrimination (C25427). Possible expLations of this poor discrimination include the low concentrations of the aminoterminal fr ents, their short half-lives, the episodic secretion of P H, and the use of low-affinity antisera (C28). While amino-terminal assays may be of limited use in the dia osis of h erparathyroidism, they may be useful in stuges of PTpmetabolism (C24, C25, C29). A reverse immunoextraction assay for quantitating amino-terminal ents has been recently described (C29). While intact assays have proven effective in discriminating between various states of calcium metabolism, they do not measure amino-terminal activity and may, therefore, underestimate biological activity. Development of improved amino-terminal assays will help to answer the question of whether circulating amino fragments are present and will contribute greatly to a better understanding of PTH physiology and metabolism. C-terminal assays for PTH are based on antisera that react against the 53-84 sequence of PTH. These assays are generally superior to amino-terminal assays in the diagnosis of hyperparath oidism (C25, C27, C28). While excellent discrimination Etween hyperparathyroid and normal sera has been reported (C30-C32), commercialassays usually show a considerable overlap (C23, C27). These assays are also greatly affected by renal function because renal failure markedly reduces clearance of carboxyl fragments (C33). Of the re ion-specific assays, the midmolecule (44-68) assays are t e most sensitive for detecting hyperparathyroidism. Good midregion assays were able to detect elevated PTH levels in 90-100% of atients with hyperparath oidism ((227,C30,C34-C36). In aidition to detecting intact Xformone and fragments containing the middle region, these assays may also measure small midregion fragments that contain
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the midregion but not the C-terminal region of PTH. These fragments have been detected in patients with hyperparathyroidism but not in normal sera (C37). This insensitivity to the C-terminal region may account for the superior erformance of the midmolecule assays compared to the 6-terminal assays in these patients. However, other authors have not been able to demonstrate the existence of these small fragments (C38).The d i s a d v a n y of midregion assays is that they are of limited use in the di ferential diagnosis of hypercalcemia. Parathyroid hormone levels are often normal or even elevated in malignancy-associated hypercalcemia when measured by these assays (39). Furthermore, like C-terminal assays, levels are affected by renal function. Earlier assays for intact PTH involved the use of antisera against the proteolytic region (24-48)of intact PTH (C40)or immunoextraction of N-terminal fragments and intact PTH by use of solid-phaseN-terminal-specificantibodies followed by midregion radioimmunoassay (C41). The former lacked sensitivity whereas the latter differentiated normal from hyperparathyroid sera but was technically difficult, which precluded ita routine use. Modifications of the two-step immunochemical method (C41) have further enhanced the analytical and diagnostic sensitivity of this assay (C42, C43). However, this assay lacks simplicity and precision as a result of the immunoextraction step that precedes the radioimmunoassay. The recent development of immunoradiometric assays (IRMA) for intact PTH is a definite advancement in technology and provides the desired simplicity and sensitivity in PTH measurement (C44). The immunoradiometric (C45,C46) assays for intact PTH were first described in 1987. These earlier studies detected increased PTH levels in greater than 94% of patients with hyperparathyroidismand differentiated all hyperparathyroid patients from those with hypercalcemia of malignancy (C45, C47). While some authors have reported higher diagnostic sensitivity of immunometric assays compared to regionspecific assays (C38,C39) in separatin hyper arathyroid from normal sera, others have reported %at the t e s t regions ecific assays have equal or even superior sensitivity (C30, &6). However, immunometric assays show excellent discrimination between hyperparathyroid hormone and hypercalcemia of malignancy, whereas carboxyl-terminal and midregion assays do not (C36, C45,C47449). For this reason, and because the intact PTH assay is not affected by renal function, the immunometric assays for intact PTH are currently the PTH assay of choice in the diagnosis of hyperparathyroidism.
HYPOPARATHYROIDISM Hypoparathyroidism, characterized by low serum calcium, high serum phosphate, and low levels of PTH, arises from a number of causes. Surgical hypoparathyroidism primarily from thyroid or parathyroid sur ery accounts for 80-9057, of cases, and idiopathic hypoparat yroidism is the next largest Idiopathic hypoparathyroidism may be sporadic or ami ‘al. It is thought to be autoimmune in nature and may be associated with the failure of other endocrine glands (type I autoimmune polyglandular syndromes). Rarely, it may occur as a feature of congenital syndromes,such as DiGeorge’s or Keams-Sayne syndrome. Gland destruction resulting from Wilson’s disease, hemochromatosis, tumor infiltration or metastasis, or radioactive iodine for Graves’disease have been reported. Chronic magnesium deficiency is another unusual cause. Diagnosis of Hypoparathyroidism. As with hyperparathyroidism,the immunometricassays are superior to regionspecific assays in confirming hypoparathyroidism, primarily because of their hi h analytical sensitivity. C-terminal assays discriminate poox!y between hypoparathyroid and normal subjects (C31, C32), whereas sensitivity of the midregion assays for this discrimination ranged from poor ((239) to excellent (C35,C36) when highly sensitive assays were used. Results with the immunometricassays have been consistent1 good, with several studies reporting undetectable levels in cases of hypoparathyroidism (C44,C47, C50) and others reporting levels below normal in 92-1005% of hypoparathyroid patients (C31, C36, C39).
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HUMORAL HYPERCALCEMIA OF MALIGNANCY
PSEUDOHYPOPARATHYROIDISM The term seudohypoparathyroidism describes a heterogenous syngome characterized by biochemical hypoparathyroidism (i.e., hypocalcemia and hyperphosphatemia), elevated immunoreactive PTH levels, and peripheral unresponsivenessto the biologic actions of PTH. It is an inherited disorder. Diagnosisis confiied by a subnormalphosphaturic response to exogenous PTH. Patients with pseudohypoparathyroidism are further categorized by the urinary CAMP response to exogenous PTH, showing blunted (type I) or normal (t e 11) responses. Recent studies demonstrated that altereyactivity of the guanyl nucleotide-bindingprotein of the adenylated cyclase system may be the cause of PTH resistance in type I syndrome of pseudohypoparathyroidism (C51).
Dissociation between immunoreactive and bioactive PTH levels in type I PHP has been reported (C52). In these cases, immunoreactive PTH levels measured by a C-terminal assay after HPLC fractionation were elevated, whereas bioactive PTH levels were low or low normal. Intact PTH levels have been reported to be elevated in these patients (C39, C53),but a recent paper reported a wide variation of PTH levels in type I patients (C54). Two subgroups were identified. The fiit ou showed a similar relationship between serum intact PTI-?an calcium levels as in normal subjects, and the second group had high PTH levels with normocalcemia.
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SECONDARY HYPERPARATHYROIDISM I N RENAL FAILURE Renal failure si nificantly alters calcium and phos horus metabolism, whic% in turn is reflected in increase PTH secretion. Secondary hyperparathyroidism occurs early in the course of chronic renal insufficiency and results from hypocalcemia. It is possible that phosphate retention and altered vitamin D metabolism may cause hypocalcemia in renal failure (C55,C56). Consistent hyperparathyroidism in renal failure results in substantial alteration in bone histology. In this circumstance, in addition to causing increased bone remodeling, PTH promotes formation of marrow fibrosis. Renal Osteodystrophy. “Renal osteodystrophy” describesmetabolicbone disorders that accompany chronicrenal failure. On the basis of bone histomorphomet , patients can usually be divided into five groups: osteitis fi rosa, mild hyperparathyroidism, osteomalacia, adynamic bone disease, and mixed bone disease (osteitis fibrosis and osteomalacia). Renal osteodystrophy occurs as a result of hyperparathyroidism, which is principally caused by the retention of phosphorus and the altered metabolism of vitamin D (C56). One of the main aims in managing patients with renal impairment is preventing secondary h erparathyroidism or the suppression of existing hyperparat yroidism. Accurate PTH measurements are therefore important to assess the degree of hyperparathyroidism as well as to monitor the effects of treatment. Because C-terminal fragments accumulate in renal failure (C33),PTH levels are markedly elevated when C-terminal or midregion assays are used. Because these fragments are biologically inactive, raised PTH levels measured by these assays do not necessarily reflect secretionrates of PTH and imply hyperparathyroidism. Studies using a sensitive amino-terminal assay showed that the assay was superior to a midregion assay in predictin osteitis fibrosa (C57). A bioactive assay has also been d e s c r i b (C58),which like the amino-terminalassay (C57)accuratelyidentifies those patients with osteomalacia or low bone turnover disease, as well asthose with severe hyperparathyroidism. A recent study comparing intact, midregion and C-terminal PTH assays in the prediction of histolo ical type of bone disease in hemodialyzed patients showe! that the IRMA assay was clearly more sensitive to adynamic bone disease and hyperparathyroidism. However, as in the earlier studies (C57, C58),PTH levels could not discriminate between patients with osteomalacia or adynamic bone disease and those with mild hyperparathyroidism. Even with the IRMA, five out of eight atients with mild hyperparathyroidism as determined by one histomorphometry had normal PTH levels (59).
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The association between hypercalcemia and m a l i p n c y was recognized in the 19208,but it was Fuller Albright in 1941 who first ostulated that a humoral substance could account for the Eypercalcemiain those patients with little or no metastatic lesions. This substance was initially thought to be PTH, and the finding of elevated PTH concentrations in plasma and tissue extracts of patients with humoral hypercalcemia (C60,C61)lent support to this theory of ectopic hyperparathyroidism. However, others reported low or suppressed PTH levels in the hypercalcemia of malignancy (C23, C26). With the development of the immunometric assays, it has been shown conclusively that PTH levels are usually suppressed in most patients (C36,C43, C45,C47,C50) unless there is associatedhype arathyroidism. Furthermore, PTH mRNA is not present in x e tumor tissue of the m ‘ority of these patients (C62). However, true cases of 9 T H producing tumors have been described (C63) with PTH mRNA in tumor extracts. Carboxyl-terminaland midregion assays are less reliable in these situations because they measure inactive carboxyl fragments. In high-calcium states, parathyroid glands preferentially secrete C-terminal fragments (C64, C65) whereas intact PTH secretion is markedly suppressed (17.24,C W . Furthermore,the glomerular fitration rate may be compromised in hypercalcemia, resulting in further elevation of carbox 1 fr ent levels. More recently a humoral factor called P J H - r Z d peptide (PTHRP) has been isolated from solid tumors (C67). It is immunolo ‘cally distinct from PTH and is considered responsible for &percalcemia of malignancy in most patients. In 1987,the structure of PTHRP was first re orted (C67). The molecule was composed of 141 amino aci&, of which 8 of the f i s t 13 amino acids were homologous with PTH. No homology with PTH was found in the rest of the molecule. Parathyroid hormone-relatedpeptide has most of the biol ic effects of PTH ((268).The peptide also binds to the PTH receptor (C69). It does not demonstrate any cross-reactivity with PTH antisera, includingthose specific for the N-terminus (C36), and its concentrations are not increased in hyperparathyroidism (C70). On the other hand, amajority of patients with cancer-associated hypercalcemia show increased concentrations of PTHRP as detected by immunoradiometric assay (C71, C72). These results suggest PTHRP may be a causative factor for humoral hypercalcemia of malignancy.
SUMMARY In summary,precise PTH measurements have been difficult and complex as a result of low circulating concentrations of biolo ically active hormone and the immunoheterogeneityof clrc&ing PTH molecules. The differencesin secretion ratee and metabolism of various PTH fragmentsin different clinical disorders further complicated the interpretation of various radioimmunoassable PTH levels. In recent years, the development of two-site or intact immunometric assays represents significant advancements in PTH measurement. These assays, for the first time, provide the lon awaited sensitivity and precision of PTH measurement. &e IRMA assay has certainly overcome the difficulties in accurately osing parathyroid disorders and the difficulties in the diagnosis of hypercalcemia. Above all, these assays are more convenientand more specific in patienta with compromised renal function. LITERATURE CITED (C1) Habener, J. F.; Po&, J. T., Jr. Mw€ngl. J. kkd. 1978, 288, 580-585. (C2) HabemK,J. F.; Potts, J. T., Jr. M w € q # . J. A M . 1978, 288, 835-844. (C3) Potts, J. T., Jr.; Muray, T. M.; Pee&, M.; NlaU, H. D.; T-r, Q. W.; Keutmann, H. T.; Powell, D.;Deftm, L. J. Am. J. W .1971,50,839-649. (C4) w e . Q. V.; NlaU, H.D.;Habenor, J. F.; Potts, J. T., Jr. Am. J. kkd. 1974, 56, 774-784. (C5) w e , G. V.; Habenw J. F.; Powell, D.; Tregear, 0. W.; Potts, J. T., Jr. J. CUn. Invest. 1972, 51, 3183-3172. (C8) Flueck, J. A.; DI Bella, F. P.; Edb. A. J.; Kehrwald. J. M.; Arnaud, C. D. J. M. Invest. 1977, 60, 1367-1375. (C7) Habenor, J. F.; Mayef, Q. P.; Dee, P. C.; Potts, J. T., Jr. Msteb. CUn. Efp. 1976, 25, 385-395. (C8) Martin, K. J.; liruska, K. A.; Freltag, J. J.; Klahr, S.; SlatopoQky, E. New E@. J. W .1879. 301, 1092-1098.
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