J. Stuart Woodhead

EFFECT OF DICHLOROMETHYLENE DIPHOSPHONATE IN PAGET'S DISEASE OF BONE AND IN HYPERCALCÆMIA DUE TO PRIMARY HYPERPARATHYROIDISM OR MALIGNANT DISEASE

30 patients with disorders of calcium metabolism were treated with dichloromethylene diphosphonate (C1(2)MDP, or clodronate disodium), an inhibitor of bone resorption. 13 patients with Pagets disease of bone were given C1(2)MDP by mouth (1.6 g/day). Serum-alkaline-phosphatase and urinary hydroxyproline fell to normal or near-normal within 3-7 months, and there was a clinical improvement in all but 1 patient. C1(2)MDP (0.8-3.2 g/day) also reduced plasma-calcium and urinary calcium in 17 patients with hypercalcaemia due to primary hyperparathyroidism or secondary to malignant disease. C1(2)MDP seems to be an effective oral drug for inhibiting excessive bone resorption in man.

[31] Immunoassays using acridinium esters

Publisher Summary This chapter describes recent studies using chemiluminescent acridinium esters as labels. Apart from providing the advantages of nonisotopic compounds such as stability, these molecules can provide superior immunoassay performance in the form of increased sensitivity when compared with radioisotopes. Certain acridinium species are among the most highly chemiluminescent synthetic molecules currently known. The light emitting reaction is rather simpler than the luminol and isoluminol systems. First, it requires none of the many and varied catalysts of these phthalhydrazide molecules, and second, it does not proceed with as many active oxygen species as do these systems. The sensitive measurement of visible photons emitted during the acridinium reaction requires the use of a photomultiplier tube (PMT) detector and is analogous to the measurement of radioactivity. The measurement of acridinium chemiluminescence is more straightforward since the emission wavelength (∼430 nm) is easily accessible to high efficiency photomultiplier tubes. The chapter describes the chemiluminescence immunoassays are: (1) polypeptide immunoassays utilizing acridinium esters; (2) preparation of solid phase antibody; (3) immunochemiluminometric assay of α 1 -fetoprotein; (4) immunochemiluminometric assay of thyrotropin; and (5) competitive immunochemiluminometric assay for free thyroxine.

Prolactin response to thyrotropin‐releasing hormone stimulation and dopaminergic inhibition in benign breast disease

Pituitary function was tested in predefined clinical groups of benign breast disease under strictly controlled clinical and laboratory conditions. Two different tests of prolactin storage and control mechanisms, direct stimulation by thyrotropin‐releasing hormone (TRH) and inhibition of dopaminergic control by dom‐peridone, indicate a significant abnormality in patients with severe cyclical mastalgia and nodular breast disease (P < 0.05 and P < 0.002), but not in those with noncyclical mastalgia. No abnormalities of thyroid function were found. Cancer 53:1311‐1315, 1984.

The relationship between adenoma weight and intact (1-84) parathyroid hormone level in primary hyperparathyroidism.

The relationship between preoperative serum levels of intact parathyroid hormone (PTH), serum calcium, and the weight of parathyroid adenoma has been investigated in 44 patients undergoing surgery for primary hyperparathyroidism due to single gland disease. There was no significant correlation between preoperative serum calcium and either intact PTH concentration or adenoma weight (r = 0.465 and 0.381, respectively). Although there was a significant correlation between PTH concentration and adenoma weight (r = 0.850, p less than 0.0005), this correlation was lost when two unusually heavy adenomas weighing 10.98 and 15.23 g were removed from the analysis. Clearly, a preoperative direct prediction of gland weight determined from PTH level was not possible. Patients with adenomata heavier than 750 mg had a significantly lower circulating PTH level per mg of adenoma than patients with glands lighter than 750 mg. PTH secretion in vitro in low calcium medium by adenoma cells from glands weighing less than 1 g was higher than secretion by cells from adenomas heavier than 1 g. Larger parathyroid adenomata appear to secrete less PTH per unit weight in vivo and per unit cell in vitro under conditions of maximal stimulation.

Comparison of the performance and clinical utility of a carboxy-terminal assay and an intact assay for parathyroid hormone

A comparison of the performance of a two-site immunochemiluminometric assay for intact parathyroid hormone with that of an in-house radioimmunoassay for carboxy terminal parathyroid hormone has been performed on samples from unselected patients being investigated for hypercalcaemia. The intact parathyroid hormone assay was found to be a simple and robust technique with a broad working assay range (CV less than 10% between 1.8-212 pmol/l) and a detection limit of 0.2 pmol/l. Clinically it is superior to the carboxy terminal assay in its ability to distinguish between patients with hyperparathyroidism from those with other causes of hypercalcaemia especially in the presence of impaired renal function.

Synthesis and properties of novel chemiluminescent biological probes: substituted 4-(2-succinimidyloxycarbonylethyl)phenyl 10-methylacridinium-9-carboxylate trifluoromethanesulphonate

Substituted aryl acridinium esters, 2,6-dimethyl-4-(2-succinimidyloxycarbonylethyl)phenyl 10-methylacridinium-9-carboxylate trifluoromethanesulphonate (3), 2,6-dimethoxy-4-(2-succinimidyloxycarbonylethyl)phenyl 10-methylacridinium-9-carboxylate trifluoromethanesulphonate (4), 2-methoxy-4-(2-succinimidyloxycarbonylethyl)phenyl 10-methylacridinium-9-carboxylate trifluoromethanesulphonate (5) and 2,6-dibromo-4-(2-succinimidyloxycarbonylethyl)phenyl 10-methylacridinium-9-carboxylate trifluoromethanesulphonate (6), have been synthesised and coupled to immunoglobulin G (IgG). The chemiluminescent properties and hydrolytic stabilities of the conjugates have also been examined. The different substituents on the phenoxy ring affected the chemiluminescent efficiency and kinetics. All the human IgG conjugates of these substituted acridinium esters demonstrated much higher stability than the conjugate of the corresponding unsubstituted acridinium ester.

A new and rapid immunochemiluminometric assay for the measurement of Tamm-Horsfall glycoprotein

Tamm-Horsfall glycoprotein was purified to apparent homogeneity from human urine by repeated precipitation with 0.58 mol/l NaCl and gel permeation chromatography under dissociating conditions on Bio-Gel A1.5M. The protein was found to consist of a single polypeptide chain of Mr 100,000 under non-reducing conditions and Mr 75,000 under reducing conditions. Antibodies to Tamm-Horsfall glycoprotein were raised in rabbits and subsequently purified by affinity chromatography using the glycoprotein linked to Sepharose 4B. The specificity of these antibodies was confirmed by Western blotting and by indirect immunofluorescence staining of human kidney tissue. The purified antibodies were labelled with 4-(2-succinimidyloxycarbonylethyl)phenyl-10-methyl-9-acridinium carboxylate fluorosulphonate, an acridinium ester, to a specific activity of 6 X 10(5) photon counts/ng of protein, and used to establish a two-site immunochemiluminometric assay for the measurement of Tamm-Horsfall glycoprotein in serum and urine. The bound and the free fractions were separated by a second antibody to Tamm-Horsfall glycoprotein linked to paramagnetic particles. The bound antibodies were quantified by chemiluminescence. The assay had a sensitivity of detection of 2 ng/ml and a working range, as determined by inter-assay precision profiles, of 30-500 ng/ml. The range in serum samples from volunteers with normal renal function (n = 92) was 74-520 ng/ml and the mean 24-h excretion rate in healthy subjects (n = 32) was 70 +/- 26 mg.

The use of antiserum with specific reactivity toward fat-cell surface antigen(s) to follow the progression of 3T3-L1 preadipocyte differentiation in vitro.

Using an i n d i r e c t , labelled-second-antibody cellular immunoassay technique, an adipocyte-specific antiserum haS been investigated. Components of the antiserum were shown to bind to differentiated 3T3-L1 cells; the cellular capacity for binding increased progressively during the induced differentiation of these ceils in vitro.

A rapid and sensitive method for estimating low concentrations of albumin in human urine

A chemiluminescence immunoassay has been developed for the measurement of albumin concentrations in human urine, as an indicator of diabetic nephropathy. The assay involved competition between analyte albumin and an acridinium ester labelled albumin tracer for binding to a rabbit (anti-human albumin) antibody. Immune complexes were separated using sheep (anti-rabbit immunoglobulin G) antibodies coupled to paramagnetic particles. The total incubation time was ninety minutes at room temperature followed by sedimentation and washing of the solid-phase using a magnetic rack. Chemiluminescence emission was quantified rapidly (2 s) using a commercially available luminometer. The assay was sufficiently sensitive (10 ng/ml) for the detection of microalbuminuria with the advantages of rapidity and use of stable reagents. The assay correlated well with both RIA and rate nephelometry.

Synthesis and chemiluminescent evaluation of a series of phenyl N-alkylacridinium 9-carboxylates

The nitrogen atom of phenyl acridine-9-carboxylate was alkylated with several alkylating agents using thermal, photochemical and sonochemical techniques. Thermal reactions were only suitable for methylation and ethylation, but irradiation with UV light for ultrasound allowed the synthesis of n-propyl iso-propyl and benzyl derivatives. Use of ultrasound produced cleaner product mixtures than UV irradiation. The quantum yields and kinetics of the chemiluminescent reactions of the products with alkaline hydrogen peroxide were found to be independent of the nature of the N-alkyl group.