Radioimmunoassay of Human Proparathyroid Hormone: Analysis of Hormone Content in Tissue Extracts and in Plasma

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Abstract
A radioimmunoassay for human proparathyroid hormone (hProPTH) has been developed and applied to an evaluation of prohormone content in parathyroid tissues and in plasma. Antisera were produced in rabbits by immunization with a synthetic octadecapeptide fragment of bovine ProPTH (bProPTH). A synthetic analogue of the prohormone peptide fragment containing tyrosine was used as the radioiodinated tracer, and a synthetic prohormone peptide consisting of the first 40 amino acids of hProPTH was used as the assay standard. The immunological activity of synthetic hProPTH peptide was unstable in plasma and buffers containing plasma or serum, but degradation was prevented by using heat-inactivated serum and EDTA in the assay solutions. The sensitivity of the assay for detection of hProPTH peptides (0.2 ng) was 5,000-times greater than for the detection of cross-reacting parathyroid hormone (PTH) peptides (1 μg), and the prohormone-specific hexapeptide did not react in the assay. The amounts of ProPTH in extracts of freshly-collected human parathyroid adenomas and in extracts of bovine parathyroid glands were 0.35 ± 0.13 and 0.38 ± 0.13 μg/mg protein, respectively, whereas the amounts of ProPTH in extracts of adenomas stored for up to a year before extraction and in extracts of human parathyroid glands obtained at autopsy were approximately ten times less than in freshly collected and extracted tissues. PTH in parathyroid adenomas (0.32 ± 0.14 μg/mg tissue protein) was much less than it was in bovine parathyroid glands (4.96 ± 1.34); in parathyroid adenomas, ProPTH contributed 50% of total immunoreactive glandular hormone compared with 7% in bovine parathyroids. No prohormone was detected in parathyroid venous effluent blood (>2.0 ng/ml) under conditions in which ProPTH was shown to remain immunologically stable. PTH levels in effluents were estimated separately and found to be as high as 190 ng/ml. Thus, ProPTH, if released, can only comprise less than 1-2% of the secreted immunoreactive hormone. Although studies of prohormone content in parathyroid tissue under various circumstances may provide important information concerning biosynthetic mechanisms, it does not appear likely that assays for proparathyroid hormone will be helpful as markers of parathyroid-gland secretory activity.

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