Kao, Pei-Chun

Abstract This new bioassay for parathyrin (PTH) in plasma (bio-PTH) combines immunoextraction on affinity columns [goat anti-hPTH (1-44) conjugated to Sepharose 4B] and a receptor assay involving an osteosarcoma cell line. The mean extraction efficacy ranges from 87% (as determined with immunopurified 125I-labeled PTH) to 62% for hPTH bioactivity. The assay is standardized with synthetic hPTH (1-84) and can detect as little as 0.9 pmol/L of PTH in 2 mL of plasma. In 100 healthy adults, the 95% reference interval for bio-PTH was less than 0.9 to 6.1 pmol/L (median, 2.0 pmol/L). In 185 patients with surgically confirmed hyperparathyroidism, bio-PTH concentrations ranged from 1.0 to greater than 120 pmol/L (median, 12.9 pmol/L); 80% of values were greater than 6.1 pmol/L. In 50 patients with both preoperative and postoperative determinations, the mean (+/--SD) concentrations of calcium in serum were 113 +/- 10 and 89 +/- 6 mg/L, respectively; the median bio-PTH concentrations were 13.6 and 2.0 pmol/L, respectively. In 22 patients with nonparathyroid-mediated hypercalcemia, the concentration of bio-PTH ranged from less than 0.9 to 5.3 pmol/L (median, 1.8 pmol/L). This bio-PTH assay is slightly less sensitive than our GP235 immunoreactive PTH (iPTH) immunoassay for detecting hyperparathyroidism (Clin Chem 1982;28:69-74); however, the bioassay is more specific and detected some cases missed by the iPTH assay. Overall, 95% of the hyperparathyroid patients had an increased test result for either the bio-PTH or the iPTH assay.

Abstract We developed a competitive chemiluminoimmunoassay of osteocalcin that is similar to radioimmunoassay but uses acridinium-ester-labeled antigen instead of 125I-labeled osteocalcin; a second antibody immobilized on plastic beads is used to separate free and bound fractions. There was good correlation of the new chemiluminoimmunoassay (y) with a polyclonal antiserum (R102) radioimmunoassay (x) used in many previous clinical studies (r = 0.96, y = 0.968x + 2.69, Sy/x = 0.029, n = 86). The new assay recognized both the intact and the small fragment of osteocalcin in plasma and detected decreases of them (total, approximately 47%) after a 24-h infusion of parathyroid hormone. Patients with primary hyperparathyroidism had increased concentrations of intact osteocalcin. Children had higher concentrations of osteocalcin than adults did. Healthy women had greater osteocalcin concentrations at ages 50-70 years than earlier. Inverse correlations of bone mineral density and osteocalcin were found in healthy women and in women with osteoporosis.

Abstract The immunochemiluminometric assay described uses immobilized anti-human parathyrin (parathyroid hormone, hPTH)(1-44) and anti-hPTH(44-68) antisera and acridinium ester-labeled anti-hPTH(1-34) to simultaneously measure both intact hPTH and its amino-terminal fragments. Results by the assay correlate well with those by a cAMP-based bioassay and the Nichols Allegro immunoradiometric assay. The minimal detection limit is 0.08 pmol/L. The normal range is 1.0-5.0 pmol/L, and values are higher in older women. About 90% of study patients with surgically proven parathyroid adenomas had above-normal preoperative PTH concentrations, whereas patients with hypercalcemia of malignancy had normal or suppressed values. This assay was designed to detect both intact PTH and amino-terminal PTH fragments; however, chromatographic fractionation of pools of primary and secondary hyperparathyroid plasma showed virtually no amino-terminal fragment activity. Nonetheless, the design is important because the absence of carboxyl-terminal binding sites prevents interference by carboxyl-terminal fragments and because bioactive amino-terminal fragments will react in the assay if they are present in the patients' sera or if they are produced by in vitro proteolysis of intact PTH.

BACKGROUND beta-Atrial natriuretic factor (beta-ANF) is an antiparallel dimer of alpha-ANF (alpha-ANF) with diminished cyclic GMP generation in vitro. To date, the presence of beta-ANF in the circulation of humans with severe congestive heart failure (CHF) remains controversial. The current study was designed to determine the presence and magnitude of circulating beta-ANF in severe CHF, to correlate plasma beta-ANF with the degree of ventricular dysfunction, and to investigate the role of human plasma and atrial tissue in the degradation of beta-ANF. METHODS AND RESULTS Venous plasma samples were obtained from patients (n = 12) with severe CHF and normal volunteers (n = 8). Total plasma ANF was measured by radioimmunoassay. alpha-ANF and beta-ANF in nonextracted plasma were separated by gel filtration chromatography using a P-6 column. Right atrial tissue samples (n = 5) were collected from a different group of patients at the time of open-heart surgery. 125I beta-ANF and I125 ANF were incubated with atrial tissue or plasma. The corresponding peak areas of beta-ANF were determined by Tamaya Digital Planimeter. beta-ANF represented 61% of total plasma ANF in CHF patients and was not detected in normal human plasma. The elevation of beta-ANF correlated with the severity of ventricular dysfunction. Thirty percent of beta-ANF and 100% alpha-ANF were converted to smaller peptide fragments in atrial tissue no conversion in plasma. CONCLUSIONS beta-ANF is the principal form of circulating ANF in patients with severe CHF and correlates with the degree of left ventricular dysfunction. beta-ANF is not generated from alpha-ANF and may be degraded rapidly in atrial tissue to smaller peptide fragments that do not occur in plasma. As beta-ANF is reported to have reduced biological action, the current studies may support the conclusion that the ANF system in CHF has reduced functional activity despite increases in circulation concentrations.

BACKGROUND Although recent investigations report the elevation of plasma endothelin (ET) in congestive heart failure (CHF), it remains unclear if this elevation is that of the biologically active peptide ET-1 or of its precursor big-ET. Furthermore, it is unclear if such elevation is associated with increased myocardial ET and if the molecular form from cardiac tissue is altered ET. Last, it remains to be established whether circulating ET is increased at the earliest stage of CHF in patients with asymptomatic left ventricular dysfunction and correlates with the magnitude of ventricular dysfunction. METHODS AND RESULTS The present study was designed to investigate concentrations and molecular forms of ET in plasma and cardiac tissue in healthy subjects and CHF patients with New York Heart Association (NYHA) class I through IV using cardiac radionuclide angiogram, cardiac myocardial biopsy, radioimmunoassay, gel permeation chromatography (GPC), and immunohistochemical staining (IHCS). Plasma ET was increased only in patients with moderate (NYHA class III) or severe (NYHA class IV) CHF compared with healthy subjects and individuals with asymptomatic (NYHA class I) or mild (NYHA class II) CHF. The elevation of circulating ET in CHF showed a negative correlation with left ventricular ejection fraction and cardiac index and a positive correlation with functional class and left ventricular end-diastolic volume index. GPC demonstrated that immunoreactive plasma ET was ET-1 in healthy subjects and both mature ET-1 and its precursor big-ET in severe CHF patients, with big-ET the predominant molecular form. Cardiac tissue concentrations and IHCS revealed ET presence in healthy atrial and ventricular tissue, which were not different in severe CHF. GPC revealed that the molecular form of cardiac ET was ET-1 in both healthy and CHF hearts. CONCLUSIONS The present study establishes for the first time that the elevation of plasma ET in severe human CHF represents principally elevation of big-ET. Second, ET is present in healthy and failing myocardia, and its activity by both immunohistochemistry and radioimmunoassay is not changed in CHF. Furthermore, the elevated plasma ET is characteristic of severe CHF and not asymptomatic or mild CHF. In addition, the degree of plasma elevation of ET correlates with the magnitude of alterations in cardiac hemodynamics and functional class. The present study confirms and extends previous investigations of ET in human CHF and establishes the evolution of circulating and local cardiac ET in the spectrum of human CHF.