Parathyroid Hormone Assays

Assaying for PTH in vivo has been notoriously difficult because of its picomolar concentrations in the circulation as well as its molecular heterogeneity. This heterogeneity is due to parathyroid gland secretion of various carboxyterminal PTH fragments (especially when PTH release is suppressed), hepatic proteolysis, and accumulation of midregional and carboxyterminal fragments caused by slow renal clearance in kidney disease. In principle, PTH may be assayed for bioac-tivity as well as by radioimmunologic and immunometric methods. Although the former two are currently of limited clinical interest, their mechanisms deserve mention.

PTH bioassays use either directly analyzed cAMP concentrations in urine or cAMP production of cell systems to assess biologic PTH activity of patients' sera.124 125 Although such assays may be sensitive in the range of picograms of PTH (per milliliter), they are limited in routine use. These techniques are laborious. They are hampered by the concomitant activation of the PTH-PTHrP receptor by both ligands despite their very limited sequence homology. PTHrP is the most common cause for humoral hypercalcemia of malignancy. PTH bioassays do not distinguish between these two hormones. Moreover, cAMP responses of the PTH-PTHrP receptor might mirror its activation only partially because of the involvement of multiple second messenger systems, whereby bioassays may underestimate activity of circulating PTH.

Radioimmunoassays were previously the most widely used means of PTH determination. This technique primarily uses a polyclonal, high-affinity antibody to which binding of radioiodinated PTH is allowed to compete with PTH to be measured in serum. By generating standard curves for binding of the radioactive ligand as a function of its concentration, the amount of iodinated PTH not displaced from the antibody can be used to approximate the concentration of PTH in the serum sample. Naturally, a host of factors require characterization and optimization to secure performance of such analyses. Some of these factors are antibody affinity, radiolabeling of PTH without interference with its immunoreactivity, and stability of the tracer peptide to be displaced.126 Particular problems have been caused by the use of bovine PTH as a tracer. Bovine PTH was selected because of its availability and the presence of tyrosine residues suitable for iodination. To reduce other difficulties, results have been reported in equivalents of pooled human sera. Moreover, analysis with characterized human PTH fragments has demonstrated multiple immunoreactivity of available displacement assays despite their characterization as "aminoterminal," "midregional," or "carboxyterminal." In view of the substantially lower circulating concentration of intact PTH in comparison with fragments encompassing various portions distal to the aminoterminal region, radioimmunoassays have been dependent on satisfactory renal function for reliable use in vivo and are variably limited in the recognition of mild to moderate primary HPT as well as the discrimination of HPT from other causes of hypercalcemia.127 •128

Displacement assays separate patients with primary HPT from overtly euparathyroid control subjects, provided that renal function is normal.129 This separation, however, naturally depends on the biochemical decision levels by which HPT is considered to prevail and current limits for the application of operative treatment verifying existence of the disease. Even with these assays, however, some 20% of individuals with malignancy-associated hypercalcemia demonstrate false-positive PTH elevations. This phenomenon is possibly related to circumstances other than limited specificity toward PTHrP.129 130 Hypothetically, nonspecific interaction of serum in these patients cannot be excluded as a cause of this phenomenon. It might also be related to disproportionally increased parathyroid gland secretion of midregional and carboxyterminal PTH fragments recognized by these assays. Moreover, the total weight of abnormal parathyroid tissue is generally strongly correlated with serum PTH measured by displacement assay, which indeed also applies to total serum calcium.63 Because the variation among patients is considerable, however, measurements from many patients are required to establish these relationships. Neither biochemical variable, therefore, should be expected to be applicable to predict the degree of glandular enlargement.

Immunometric Assays

Routine diagnosis of primary HPT and evaluation of the extent of secondary HPT have been greatly facilitated by the development of immunometric "sandwich" assays. Briefly, these assays use a pair of antibodies that recognize different regions of PTH.131"134 One of these antibodies, preferentially monoclonal, is immobilized, whereas a polyclonal antiserum with greater affinity is labeled with radioiodine (immunoradiometric assay) or chemoluminescence. Because of cooperation of the antibodies, such a sandwich assay is more sensitive than either antibody alone in displacement radioimmunoassays. With careful selection of antibodies, the immunometric assays are specific and sensitive for intact PTH, which allows identification of insufficient PTH secretion of hypoparathyroidism as well as a wide range of PTH levels without sample dilution. Moreover, it is technically favorable to tag antibodies rather than peptides. A vast excess of PTH fragments might hinder a small fraction of intact PTH from binding to the immobilized antibody and from reacting in the assay, a circumstance that has been suggested to occur in analyses for PTH of rare parathyroid gland aspirates.135 The immunometric analyzing process is faster than with radioimmunoassays. By reducing incubation times, the analysis can be done in 15 to 30 minutes and, consequently, can be used intraoperative ly.136_ 138 Such speed of processing, however, is accompanied by a reduction in assay sensitivity.

Clinical analysis with immunometric PTH assays usually separates hypercalcemic patients with HPT from patients with other causes of hypercalcemia. This is particularly evident with respect to malignancies of nonparathyroid origin, although some 5% to 10% of these patients demonstrate intact serum PTH in the lower portion of the normal range. Nonparathyroid tumors that produce intact PTH are exceptionally rare. Examples of such tumors include ovarian and small cell carcinomas as well as thymomas.139"142 The immunometric assays demonstrate that clinically overt derangements in renal function are accompanied by very early elevation in intact serum PTH values. This circumstance reflects the onset of parathyroid gland hyperactivity rather than accumulation from impaired clearance, although indirect evidence suggests that a prolonged half-life of intact serum PTH may exist, at least occasionally, in uremia.143

The distribution of intact serum PTH values and recognition of primary HPT among otherwise healthy individuals largely depend on how patients are recruited. In a health screening study of 5200 menopausal women, biochemical levels for the recognition of primary HPT were deliberately set low to include those with mild disease (unpublished data). Briefly, individuals demonstrating hypercalcemia (albumin-corrected total serum calcium > 2.60 mmol/L) and intact serum PTH greater than or equal to 25 ng/L (reference range, 12 to 55 ng/L), serum calcium 2.50 to 2.60 mmol/L and serum PTH greater than or equal to 35 ng/L, or serum PTH greater than or equal to 55 ng/L in combination with a previous serum calcium concentration exceeding 2.55 mmol/L were presumed to have primary HPT. Among the identified individuals, about two thirds showed normal total and ionized serum calcium values, and a similar proportion (69%) showed intact serum PTH within the reference range. Under a stratified case-control treatment program, more than half the patients have been subjected to parathyroid surgery. These individuals were representative of the entire group with respect to total serum calcium values, and operation verified the presence of parathyroid abnormalities in all of them. These findings are described not to provoke arguments about the indications for parathyroid exploration but to demonstrate that even these limits appear to underestimate the prevalence of HPT, and that intact serum PTH measured by immunometric assay may be normal in many patients with mild HPT. The proportion of such patients is assumed to be 5% to 20%.

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