Evolution of PTH assays

Abstract

PTH metabolism is complex and the circulating forms include the intact 1-84 molecule as well as several carboxyl-terminal fragments. The first generation of PTH assays included several types of competitive assays, with specificities that spanned carboxyl, mid-region and amino-terminal portions of the molecule. The limitations of these assays and the methodological evolution led to the description of 2 nd generation non-competitive immunometric assays for PTH in the late 80's, based on the recognition of the PTH molecule by two different antibodies, one directed against de amino-terminal and other against the carboxyl-terminal segments. The observation that in some circumstances "long" carboxyl-terminal segments were also measured by 2 nd generation assays led to the development of 3 rd generation assays based on amino-terminal specific antibodies that are specific for the first amino acids, measuring only the molecular forms that activate PTH1R. The practical and cost-benefit advantages of these assays are still debatable. The recent observation that carboxyl-terminal fragments of PTH have biological activity via a distinct receptor than PTH1R, points to the future need of more than one assay in order to evaluate parathyroid hormone function. RESUMO Evolução dos Ensaios para Paratormônio.O metabolismo do PTH e complexo e as formas circulantes incluem o PTH 1-84, assim como fragmentos C-terminal. A primeira geração de ensaios para o PTH incluía vários ensaios competitivos com especificidades para as regiões carboxi, meio da molécula e amino-terminal. A limitação destes ensaios e a evolução metodológica, levaram ao desenvolvimento dos ensaios não competitivos de 2ª. geração no final dos anos 80, baseados no reconhecimento por dois anticorpos diferentes, contra a porção amino e carboxi-terminal respectivamente. A observação que em algumas circunstâncias segmentos carboxiterminais longos também eram detectados, levou ao desenvolvimento dos ensaios de 3ª. geração, baseados em anticorpos específicos para a porção aminoterminal com maior especificidade para os primeiros aminoácidos, e assim mensurando apenas a forma molecular que ativa o PTH1R. As vantagens práticas e o custo-benefício deste ensaio ainda e motivo de debate. A observação recente de que fragmentos carboxiterminais têm atividade biológica via receptor distinto, aponta para a necessidade futura de mais de um ensaio para avaliar a função do paratormônio.

Extraction and purification of recombinant intact human Parathyroid Hormone (hPTH) from bacterial cell

“…[27,28] Also the HPLC chromatogram for both the standard and produced hPTH showed the same main peaks at nearly the same retention time as shown in Figure 9, and the hPTH peaks in both samples are mostly appeared at a retention time of about 45 minutes, the peaks type mainly similar in both samples and they are also resemble to the human PTH (1-84) peaks which appeared in HPLC chromatogram for human serum sample analyzed by RP-HPLC using elution solution contain a gradient range of Acetonitril (CAN) which was reached to the 33% of the total elution solution components and it's near the Acetonitrile percent (35%) that was used in HPLC analysis of this study. [29,30] According to the ability of using the produced hPTH as a drug for treatment of severe bone osteoporosis for human that open the door for doing multiple test to checking its safety for human, one of the more important test is the studying of its effect on the human RBC. By doing the haemolysis assay for produced hormone which show the ability of the hormone to hydrolyze the human RBC at a high concentration while in a low concentration there is no haemolysis effect on the RBC that's because the hPTH regard as a major factor influencing red blood cell osmotic fragility.…”

Experience with a third-generation parathyroid hormone assay (BIO-PTH) in the diagnosis of primary hyperparathyroidism in a Brazilian population

“…These latter forms are quickly metabolized in the liver and have a halflife of fewer than 4 minutes. The carboxyl-terminal fragments are eliminated by glomerular filtration and have a longer half-life in the circulation, although their biological effects are not completely known (4,5). These fragments have an activity potentially independent from their binding to the PTHR1 receptor which, according to some studies, can result in hypocalcemia, hypophosphatemia, and increase in urinary phosphorus (6).…”

“…These fragments have an activity potentially independent from their binding to the PTHR1 receptor which, according to some studies, can result in hypocalcemia, hypophosphatemia, and increase in urinary phosphorus (6). Thus, circulating forms of PTH are quite heterogeneous and, considering that immunoassays depend on sequences recognized by antibodies, measurement of the so-called intact molecule (PTH 1-84) becomes a challenge (4).…”

“…in 1963 and, since then, several different assays have emerged (3,7). The first RIA used polyclonal antibodies generated against PTH from bovine or porcine parathyroid glands, with specificity mainly directed against the carboxyl-terminal region (4,8). They were also called first-generation assays, and their major limitation was interference from PTH carboxyl fragments, especially in patients with impaired renal function (8).…”

Comparison of iPTH values in serum and plasma samples depending on the time and temperature in patients with chronic kidney disease

“…Classical biological activities of PTH are mediated by PTH1R receptors, which are present in some tissues, while the amino-terminal region of the molecule contains the sequence necessary to active PTH1R. PTH present in circulation is very heterogeneous, and this is a consequence of a complex metabolism (1). PTH is a linear peptide of 84 amino acids, produced in the parathyroid glands, encoded in humans by a gene located on the short arm of chromosome 11.…”