Primary hyperparathyroidism: review and recommendations on evaluation, diagnosis, and management. A Canadian and international consensus.

The purpose of this review is to assess the most recent evidence in the management of primary hyperparathyroidism (PHPT) and provide updated recommendations for its evaluation, diagnosis and treatment. A Medline search of "Hyperparathyroidism. Primary" was conducted and the literature with the highest levels of evidence were reviewed and used to formulate recommendations. PHPT is a common endocrine disorder usually discovered by routine biochemical screening. PHPT is defined as hypercalcemia with increased or inappropriately normal plasma parathyroid hormone (PTH). It is most commonly seen after the age of 50 years, with women predominating by three to fourfold. In countries with routine multichannel screening, PHPT is identified earlier and may be asymptomatic. Where biochemical testing is not routine, PHPT is more likely to present with skeletal complications, or nephrolithiasis. Parathyroidectomy (PTx) is indicated for those with symptomatic disease. For asymptomatic patients, recent guidelines have recommended criteria for surgery, however PTx can also be considered in those who do not meet criteria, and prefer surgery. Non-surgical therapies are available when surgery is not appropriate. This review presents the current state of the art in the diagnosis and management of PHPT and updates the Canadian Position paper on PHPT. An overview of the impact of PHPT on the skeleton and other target organs is presented with international consensus. Differences in the international presentation of this condition are also summarized.

Early events in the cellular formation of proparathyroid hormone.

Early events in the cellular synthesis and subsequent transfer into membrane-limited compartments of pre-proparathyroid hormone (pre-proPTH) and proparathyroid hormone (proPTH) were investigated by electrophoretic analyses of newly synthesized proteins in subcellular fractions of parthyroid gland slices pulse-labeled for 0.5-5 min with [(35)S] methionine. During these short times of incubation, both pre-proPTH and proPTH were confined to the microsomal fraction. Labeled pre-proPTH and proPTH were detected in a 30-s interval between 0.5 and 1.0 min of incubation. The radioactivity in proPTH became relatively constant between 3 and 5 min, whereas the radioactivity in ProPTH increased markedly over this period. When corrected for the known content of methionine in the prohormone and the prohormone, we found four times as much radiolabeled prohormone as prehormone between 0.5 and 1.0 min of synthesis. Sequestration of labeled prohomrone into endoplasmic reticulum compartments was shown by treatment of the microsomal fraction with chymotrypsin and trypsin, which resulted in the degradation of the prehormone but not of the prohormones. Approximately 50 percent of pre-prohormone and 25 percent of prohormone were released from the microsomes by their extraction with 1.0 M KCl, whereas 80-90 percent of both was released by treatment with Triton X-100. These results in intact cells support the signal hypothesis proposed by Blobel and his co-workers in studies utilizing cell-free systems, inasmuch as the results indicate transfer of prohormone into the cisternal space of the rough endoplasmic reticulum concomitant with the growth of the nascent polypeptide chain. Appearance of membrane-sequestered proPTH takes place without entry of pre-proPTH into the cisternal space, suggesting that proteolytic removal of the leader peptide occurs during transfer of the polypeptide through the lipid bilayer. Further evidence in support of this process is that pre-proPTH is only partly extracted from the microsomes by treatment with 1.0 M KCl, suggesting that a substantial fraction of the nascent pre-proPTH is integrally inserted into the membranes before it is cleaved to form proPTH.

Parathyroid secretion: discovery of a major calcium-dependent protein.

Bovine parathyroid tissue incubated in vitro secretes a protein that is distinct from both parathyroid hormone and proparathyroid hormone and comprises about 50 percent of the total secreted protein. This protein appears to be an aggregate consisting of two or more subunits of molecular weight 70,000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Although the function of this protein is unknown, the secretion rates of both the protein and parathyroid hormone respond in parallel to changes in the concentration of calcium in the medium.

A parathyroid hormone inhibitor in vivo: design and biological evaluation of a hormone analog.

A synthetic analog of bovine parathyroid hormone (bPTH), [tyrosine-34] bPTH-(7-34)NH2, was found to inhibit parathyroid hormone action in vivo. When the analog and parathyroid hormone were infused simultaneously to rats at a molar ratio of 200 to 1, the analog inhibited the excretion of urinary phosphate and adenosine 3',5'-monophosphate. When infused alone at the same dose rate, the analog was devoid of agonist activity. The compound was prepared by following design principles developed for inhibitors of parathyroid hormone, and is believed to be the first antagonist of parathyroid hormone that is effective in vivo.

Proparathyroid hormone: biosynthesis by human parathyroid adenomas.

Biosynthesis of a precursor (proparathyroid hormone) to human parathyroid hormone was demonstrated during incubation of tissue from parathyroid adenomas. The proparathyroid hormone is labeled more rapidly than parathyroid hormone during incubation with amino acids labeled with carbon-14 and is progressively converted to the hormone. Apparent differences in the relative rate of conversion of precursor to hormone found in different tumors suggest that proparathyroid hormone may accumulate in some of the tumors and be secreted into the circulation.

Parathyroid hormone production in vitro by human parathyroid cells transformed by Simian virus 40.

Cells froma human parathyroid adenoma were infected with simian virus 40 and maintained through 13 subcultures in monolayer tissue culture. For more than 9 months, these "transformed" cells contimed to produce parathyroid hormone which was identified by radioimmunoassay and density-gradient ultra centrifugation.

Photosynthesis of previtamin D3 in human skin and the physiologic consequences.

Photosynthesis of previtamin D3 can occur throughout the epidermis in the dermis when hypopigmented Caucasian skin is exposed to solar ultraviolet radiation. Once previtamin D3 is formed in the skin, it undergoes a temperature-dependent thermal isomerization that takes at least 3 days to complete. The vitamin D-binding protein preferentially translocates the thermal product, vitamin D3, into the circulation. These processes suggest a unique mechanism for the synthesis, storage, and slow, steady release of vitamin D3 from the skin into the circulation.

Calcitonin messenger RNA encodes multiple polypeptides in a single precursor.

Recombinant DNA techniques were used to analyze the structure of the messenger RNA encoding a precursor of calcitonin, a small calcium-regulating hormone of 32 amino acids. Analyses of the nucleotide sequences of cloned complementary DNA's comprising the entire coding sequence of the messenger RNA revealed that calcitonin is flanked at both its amino and carboxyl termini by peptide extensions linked to the hormone by short sequences of basic amino acids. The location of glycine next to the carboxyl terminal prolinamide of calcitonin is consistent with indications that glycine is required for the enzymatic amidation of proline to the prolinamide. During cellular biosynthesis, calcitonin arises from a large precursor protein by cleavages at both amino and carboxyl terminal residues of the hormone. These findings raise questions concerning the regulation of these cleavages and the potential biological functions of the precursor extensions derived from these cleavages.

A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide.

The complementary DNA encoding a 585-amino acid parathyroid hormone-parathyroid hormone-related peptide (PTH-PTHrP) receptor with seven potential membrane-spanning domains was cloned by COS-7 expression using an opossum kidney cell complementary DNA (cDNA) library. The expressed receptor binds PTH and PTHrP with equal affinity, and both ligands equivalently stimulate adenylate cyclase. Striking homology with the calcitonin receptor and lack of homology with other G protein-linked receptors indicate that receptors for these calcium-regulating hormones are related and represent a new family.

Circulating bovine lymphocytes contain receptors for parathyroid hormone.

No cell type practicably obtainable in vivo, such as blood cells, is known to contain parathyroid hormone (PTH) receptors; this deficiency has hampered investigation of receptor regulation. Second, PTH in vivo is among the potent stimulators of osteoclastic activity, although no direct hormonal effects on these cells have been identified. Several lines of evidence suggest that cells of the immune system may mediate PTH effects on osteoclasts. We, therefore, studied bovine blood cells for the presence of PTH receptors and PTH-stimulated adenylate cyclase. Using an analogue of bovine PTH, (125)I-labeled [Nle(8),Nle(18),Tyr(34)]bPTH-(1--34)amide, we found PTH-specific binding to intact, nonadherent mononuclear cells (lymphocytes) and PTH-stimulated adenylate cyclase in plasma membranes prepared from these cells, and not with cells or membranes from other blood cells. Lymphocytes may serve to study the effects of physiologic and pathologic perturbations on PTH-receptor function in vivo. Exploration of PTH-related lymphocyte responses may help define the relation between cells of the immune system and osteoclastic bone resorption.

Short-term effects of synthetic human parathyroid hormone-(1--34) administration on bone mineral metabolism in osteoporotic patients.

Since studies in animals and humans have shown that parathyroid hormone can stimulate bone formation and increase trabecular bone, and patients with primary and secondary hyperparathyroidism may exhibit osteosclerosis, we evaluated the effect of short-term administration of human parathyroid hormone, hPTH-(1--34), in patients with osteoporosis. Six patients with osteoporosis underwent detailed studies including blood and urinary measurements of calcium, phosphate, and magnesium; 47Ca kinetic studies; and 18-d balance studies before and during the short-term administration (3--4 wk) of a daily subcutaneous injection of hPTH fragment 1--34 given as 450 or 750 U/dose. The mean fasting plasma calcium values rose slightly after hPTH-(1--34) administration, primarily in the high-dose group. There was no difference in the mean fasting plasma inorganic phosphate levels. The mean daily urinary excretion of calcium and phosphate was significantly increased in patients given the higher dose. In patients given 750 U, net intestinal calcium absorption increased, phosphate absorption increased, calcium balance improved, and phosphate balance improved. In patients given 450 U, calcium balance and phosphate balance worsened. 47Ca kinetic studies showed a minimal increase in bone accretion rate, a decrease in the mean transit time of calcium in the exchangeable pools, and a decrease in the exchangeable-pool size. In all six patients there was an increased renal clearance of 47Ca as a result of hPTH-(1--34) administration. These studies indicate that low doses of parathyroid hormone may promote bone formation, whereas higher doses clearly have an adverse effect on the skeleton.

Parathyroid hormone secretion in vivo. Demonstration of a calcium-independent nonsuppressible component of secretion.

The response of normal bovine parathyroid glands to hypercalcemia was assessed in vivo by radioimmunoassay of immunoreactive parathyroid hormone concentrations in parathyroid effluent blood obtained by surgical cannulation of both anesthetized and nonanesthetized calves. Hypercalcemia was induced for periods of 0.3-35 h by intravenous infusion of a solution of calcium chloride. Assessment of immunoreactivity in effluent and peripheral blood included measurements of selected samples by use of a radioimmunoassay specific for a site residing in the biologically active portion of the hormone molecule. In all instances, the concentration of immunoreactive parathyroid hormone in hypercalcemic venous effluent from a superior parathyroid gland exceeded that of the peripheral blood. Failure of hypercalcemia to suppress completely secretion by normal parathyroids indicates that a portion of parathyroid hormone secretion occurs independent of blood calcium concentration. Consequently, continued parathyroid hormone secretion despite hypercalcemia can no longer be regarded as a unique feature of parathyroid neoplasia.

Analysis of parathyroid hormone and its fragments in rat tissues: chemical identification and microscopical localization.

After intravenous injection of [(125)I]-iodo-parathyroid hormone in the rat, uptake of the hormone was greatest in the liver and kidneys. Uptake was rapid, reaching a maximal concentration by 4 and 8 min, respectively. Extracts, prepared from both these organs at intervals soon after the injection of intact hormone, showed three main radioactive peaks when samples were subjected to gel filtration under protein-denaturing conditions. The first peak coeluted with intact hormone. The second eluted at a position corresponding to the carboxy-terminal fragments previously described in plasma, and the last eluted at the salt volume of the column. Microsequence analysis of the radioiodinated fragments, a method that has proved valuable for chemically defining the circulating fragments resulting from metabolism of injected hormone, showed that extracts of liver and kidney, prepared at 4 and 8 min after injection of the intact hormone, contained different fragments. The radioiodinated fragments in liver extracts were identical to those previously reported in the plasma of rats and dogs, fragments resulting principally from proteolysis between positions 33 and 34, and 36 and 37 of the intact hormone. Although the same fragments were also present in the kidneys, they constituted less than 15% of the amount present in the liver. More than 50% of the labeled renal fragments consisted of a peptide whose amino-terminal amino acid was position 39 of the intact hormone, a fragment not present in plasma. The rate of appearance of radioiodinated fragments that were chemically identical to those in plasma was more rapid in the liver than in plasma. Correlation of these chemical analyses with studies of the localization of (125)I by autoradiography showed that at the times when the intact hormone and the carboxy-terminal fragments comprised nearly all of the (125)I-labeled moieties in the tissues, the proximal convoluted tubules of the kidney and sinusoidal lining cells of the liver, which probably are Kupffer cells, contained the highest concentration of (125)I. Preferential localization of immunoreactive parathyroid hormone to these tissue sites also was shown by immunoperoxidase staining in studies with unlabeled hormone. Our results suggest that, unless multiple renal mechanisms are present for release of hormonal fragments, one of which releases the circulating fragments preferentially, the liver, rather than the kidney, is principally responsible for generating the carboxy-terminal fragments in plasma after injection of intact hormone, and the Kupffer cells may contain the enzymes that hydrolyze parathyroid hormone.

Pre - proparathyroid hormone identified by cell - free translation of messenger RNA from hyperplastic human parathyroid tissue.

An 8-15S fraction of RNA isolated from hyperplastic human parathyroid tissue (primary chief-cell hyperplasia) and translated in a cell-free extract of wheat germ directs the synthesis of a protein that shares antigenic determinants and tryptic peptides with parathyroid hormone and its previously recognized immediated precursor, proparathyroid hormone. In addition, the protein contains tryptic peptides not found in proparathyroid hormone and migrates more slowly than does proparathyroid hormone on both urea-acid and urea-sodium dodecyl sulfate polyacrylamide gels, indicating that it is more acidic and larger than proparathyroid hormone. Sequential Edman degradation of the cell-free protein, radiolabeled with [35S]methionine, for 25 cycles released [35S]methionine at cycles 1, 7, 11, and 14, indicating that the NH2-terminal peptide sequence of the protein differs from that of both proparathyroid hormone and parathyroid hormone. We propose that this protein is an early biosynthetic precursor of human parathyroid hormone, pre-proparathyroid hormone, analogous to that identified recently by in vitro translation of bovine parathyroid mRNA.

Effects of hepatectomy, nephrectomy, and nephrectomy/uremia on the metabolism of parathyroid hormone in the rat.

Reports from several laboratories, showing extensive hepatic extraction of circulating parathyroid hormone, led us to examine the effect of near-total hepatectomy on the metabolism of the hormone to circulating fragments, and on its clearance from plasma. The rate of disappearance of (125)I-labeled and unlabeled bovine parathyroid hormone from plasma, and the appearance, disappearance, and chemical and immunochemical characteristics of circulating fragments were examined by gel filtration and either sequence-specific radioimmunoassays or sequence analysis using the Edman reaction. Results from awake rats subjected to near-total hepatectomy were compared with those found in sham-treated, nephrectomized, and short-term uremic rats (studied 2 d after nephrectomy). When compared with the sham-treated group, all other groups clear (125)I-labeled hormone more slowly; after hepatectomy, however, the clearance rate is most strikingly decreased. After injection of intact hormone, the concentration of carboxy-terminal fragments in the circulation of hepatectomized rats is greatly reduced at all time intervals when compared with that in sham-treated rats. Sequence analysis of plasma samples, collected from rats into which (125)I-labeled hormone had been injected, shows that carboxy-terminal fragments having positions 34 and 37 of the intact hormone sequence as their amino-terminal amino acids are abundant in sham-treated, nephrectomized, and nephrectomized/uremic rats, but are undetectable in hepatectomized rats. The data suggest that inasmuch as the liver in vivo generates most of the carboxy-terminal fragments resulting from the metabolism of injected hormone, specific cell types within the liver must be the principal locus of the responsible enzyme(s); thus, studies of the enzymic properties of isolated hepatic cells in vitro most likely will yield information of physiologic relevance to the metabolism of the hormone in the intact animal.

Metabolism of parathyroid hormone by isolated rat Kupffer cells and hepatocytes.

Data from several laboratories indicate that hepatic mechanisms may have a distinctive role in the metabolism of intact hormone after secretion, a process that accounts, at least partly, for the heterogeneity of circulating parathyroid hormone. Accordingly, we studied the proteolysis of intact hormone by isolated rat Kupffer cells and hepatocytes. Kupffer cells (10(6) cells/ml) and hepatocytes (10(7) cells/ml) were incubated with unlabeled and (125)I-labeled bovine parathyroid hormone at 37 degrees C for periods ranging up to 2 h. When incubated with Kupffer cells, intact hormone disappeared with a t((1/2)) of 12+/-4 min. Radio-immunoassays using sequence-specific antisera showed that the dominant hormonal fragments recovered in the medium have an apparent molecular weight of approximately 6,000, lack amino-terminal antigenic determinants, and react in assays that specifically recognize determinants in the carboxy-terminal portion of the intact hormone. Amino-terminal fragments also were detected in high concentrations, particularly after short incubation periods. Radioiodinated fragments resulting from incubation of (125)I-labeled bovine parathyroid hormone with Kupffer cells had the same apparent size as fragments derived from the metabolism of unlabeled, intact hormone; when analyzed by Edman degradation, positions 34 and 37 of the intact hormone sequence were the amino-terminal amino acids of these dominant carboxy-terminal fragments. Hepatocytes did not hydrolyze the hormone. Thus, metabolism of parathyroid hormone by Kupffer cells results in the appearance of fragments in the media that are immunochemically indistinguishable from, and chemically identical with, those found in plasma when intact hormone is injected intravenously. This indicates that the proteolysis observed in vitro accurately reflects the metabolism of the hormone in vivo. The detection of amino-terminal fragments in concentrations nearly equal to those of carboxy-terminal fragments indicates that cleavage of intact hormone is, initially, by an endopeptidase(s). Kupffer cells may be a source from which specific protease(s) that hydrolyze parathyroid hormone can be characterized, particularly in terms of enzymic specificity and requirements for inhibition. Detailed analysis of the cellular and molecular events during incubation of parathyroid hormone with these cells may help to clarify the biologic significance of the peripheral metabolism of the hormone.

Secretion of calcitonin in hypocalcemic states in man.

The control of calcitonin secretion in humans has been studied extensively only in patients with medullary thyroid carcinoma since the peripheral concentration of the hormone in normal subjects is too low for accurate measurement by existing assay procedures. However, we have recently found that the concentrations of calcitonin in the peripheral plasma of hypocalcemic subjects during provocative tests of hormone secretion were high enough to be measured by radioimmunoassay. Accordingly, the effect of calcium and pentagastrin infusions on plasma calcitonin was studied in nine patients with pseudohypoparathyroidism, seven patients with idiopathic hypoparathyroidism, and six patients with hypocalcemia not due to parathyroid disease. The infusion of calcium in these hypocalcemic subjects resulted in increases in plasma calcitonin to levels that could be readily detected by our radioimmunoassay. Pentagastrin infusion also caused an increase of plasma calcitonin in some subjects, but calcium was approximately 10 times more effective than gastrin in its stimulatory effect on hormone secretion. These results demonstrate that in humans as well as other mammals the secretion of calcitonin by parafollicular cells that are not involved by medullary thyroid carcinoma is directly related to plasma calcium and that gastrin can also stimulate hormone secretion. The results are consistent with the thesis that the secretion of calcitonin by normal human subjects does occur but at peripheral concentrations of the hormone below the detection limits of most existing immunoassays; hypocalcemia leads to increased stores of hormone that can be related by the appropriate stimuli.

Metabolism and biological activity of parathyroid hormone in renal cortical membranes.

Recent studies from several laboratories have documented the presence of fragments of parathyroid hormone in blood or peripheral tissues or in both. Inasmuch as amino-terminal fragments are known to be biologically active, it has been suggested that fragments, rather than the intact polypeptide of 84 amino acids, might be the active molecular species in tissue fluids. Accordingly, the metabolism of native bovine parathyroid hormone, bPTH-(1-84), was studied in purified renal cortical membranes from several species and correlated with hormonal stimulation of adenylyl cyclase in these membranes in vitro. Analysis of whole incubation mixtures or membrane-bound hormone by gel electrophoresis and gel chromatography after incubation of [3H]bPTH-(1-84) or 125-I-labeled bPTH-(1-84) or unlabeled biologically active bPTH-(1-84) with purified canine renal cortical membranes revealed no evidence of proteolysis, and yet the uncleaved hormone readily stimulated adenylyl cyclase. Kinetic studies of hormone-stimulated adenylyl cyclase activity revealed no difference in rate of onset of activity between bPTH-(1-84) And the active synthetic amino-terminal tetratriacontapeptide bPTH-(1-34), and hence there was no evidence of precursor-product relationship between the native hormone and an active amino-terminal fragment. The results suggest, insofar as the activity detected in these membranes reflects the biological response of the hormone in vivo, that the native hormone is indeed biologically active at the receptor level directly without the requirement for cleavage into active fragments.

An evaluation of antibodies and clinical resistance to salmon calcitonin.

21 patients with Paget's disease of bone and one with osteoporosis were studied to detect development of antibodies to salmon calcitonin during chronic therapy. Antibody titers ranged from 1:40 to 1:30,000 in plasma obtained after treatment of 11 patients. Radio-immunoelectrophoresis revealed that the antibodies were restricted to the gammaG class. One patient, W. O., with Paget's disease initially responded to treatment with a decrease in bone turnover, but later became resistant to the hormone in association with the appearance of a very high titer (1:30,000) of antibody against salmon calcitonin. A 1:10 dilution of his plasma was shown to completely inactivate 20 mMRC units/ml of salmon calcitonin as detected by bioassay in rats; slight inactivation was detected at a 1:200 dilution. All other patients continued to respond to salmon calcitonin despite the development of antibody to the hormone in ten cases. No evidence of systemic allergic reactions or other toxicity was found in any patient. The data suggest that although antibody formation may occur in as many as 50% of patients treated with salmon calcitonin, this antibody response is unlikely to be of clinical significance in most patients. However, in an occasional patient, a marked antibody response may occur which interferes with the therapeutic use of the hormone.

Parathyroid Hormone in Human Plasma: IMMUNOCHEMICAL CHARACTERIZATION AND BIOLOGICAL IMPLICATIONS.

Antigenic recognition of four anti-bovine parathyroid hormone antisera was characterized by their reactivity with bovine hormonal fragments (1-34, 1-13, 14-34, 19-34, 53-84) and human hormone extracted from parathyroid adenomas. All antisera were found to have antibody populations which recognized more than one antigenic determinant and all antisera differed in their specificity and reactivity for the fragments of bovine hormone. By modification of two antisera, GP-1 and GP-133, by preincubation with excess concentrations of 1-34 or 53-84 fragments, antigenic recognition was restricted to defined regions of the hormonal sequence.When assays using these modified antisera were applied to the study of hormones extracted from glands, greater immunochemical similarities were seen between bovine and human parathyroid hormone using assays that were specific for the measurement of amino-terminal portions of the hormones than of the carboxy-terminal portions.When assays using these antisera were applied to the study of endogenous parathyroid hormone in human plasma, the immunoreactive hormone in the general circulation was shown to substantially lack an amino-terminal portion of the sequence of the intact hormone, including an antigenic determinant requiring all or some of the 14-19 region. This deletion accounts, at least in part, for the immunochemical heterogeneity of plasma parathyroid hormone in man. Radioimmunoassay of fractions of peripheral plasma subjected to gel filtration confirms that the dominant form of the immunoreactive hormone in the general circulation of man is a hormonal fragment that is totally devoid of amino-terminal reactivity. Because of this deletion, it can be concluded that most of the immunoreactive parathyroid hormone in the general circulation of man must be biologically inactive.