Circulating intact parathyroid hormone measured by a two-site immunochemiluminometric assay.

A direct immunoassay for circulating intact human PTH (hPTH) is described. The method relies on the formation of an immune complex of labeled antiamino-terminal PTH antibody, intact hPTH, and solid phase antimidregion PTH antibody. A chemiluminescent aryl acridinium ester is used as label. Serum samples (100 microL) are incubated with labeled antibody, and subsequently the bound fraction is separated by the addition of solid phase antibody. The bound luminescence is quantitated in an automatic luminometer. Luminescence intensity is directly proportional to the amount of intact PTH present in the sample. Only intact PTH was found to react in this system; there was no significant interference from PTH fragments. The assay detection limit of 0.8 pmol/L hPTH-(1-84) allowed detection of intact PTH in the serum of all normal subjects tested. A clear distinction was found between hypercalcemic individuals subsequently proven to have primary hyperparathyroidism and those with malignancies. The assay offers several advantages over previously described PTH immunoassays with regard to specificity, rapidity, and reagent stability. It, thus, provides a valuable means of investigating parathyroid physiology and clinical disorders of extracellular calcium metabolism.

Comparison of poly- and monoclonal antibodies as labels in a two-site immunochemiluminometric assay for intact parathyroid hormone.

We have evaluated the potential of monoclonal antibodies in the development of a non-isotopic immunometric assay for intact human parathyroid hormone (PTH). The assay has been designed to utilise a chemiluminescent acridinium ester labelled anti-aminoterminal (anti-N) antibody and a solid-phase anti-carboxyterminal antibody in order to measure specifically the intact hormone. In this system the characteristics of the labelled antibody proved crucial to the performance of the assay. A low affinity monoclonal reagent yielded insufficient analytical sensitivity, while a higher affinity monoclonal reagent cross-reacted poorly with the intact molecule relative to the amino terminal PTH fragment to which it was raised. Neither antibody could therefore match the performance of an affinity-purified polyclonal anti-N PTH reagent. These results highlight the problems to be addressed in the selection of suitable reagents for immunometric assay development when specificity and sensitivity are crucial requirements.

Studies in patients with hyperparathyroidism using a new two-site immunochemiluminometric assay for circulating intact (1-84) parathyroid hormone.

A recently developed chemiluminescent immunoassay for 1-84 intact parathyroid hormone (PTH) demonstrated increased specificity by virtue of two-site antibody binding and increased sensitivity by use of a chemiluminescent technique. Basal PTH levels were measured in three groups of subjects: (1) normal (n = 82), (2) hyperparathyroidism (n = 31), and (3) patients with hypercalcemia of malignancy (n = 16). There was good discrimination between normal (1.2 to 9.4 pmol/L) and hyperparathyroid subjects (9.2 to 53.4 pmol/L). In persons with hypercalcemia of malignancy all PTH levels were within the normal range (0.8 to 5.2 pmol/L) or suppressed. PTH release was stimulated by the intramuscular injection of 100 IU salmon calcitonin in 6 normal controls, 10 patients with primary hyperparathyroidism due to adenoma, and 5 with four-gland hyperplasia. There was no significant rise in PTH concentration and out of the normal range in the control subjects, but the adenoma patients demonstrated a mean rise of 24.4%, 26%, and 33%, and hyperplasia patients, a mean rise of 37%, 47%, and 37% over basal levels at 120, 180, and 240 minutes. The mean absolute rise in PTH concentration was 13.4 +/- 7.7 pmol/gm of parathyroid tissue in the adenomas and 27.2 +/- 9.5 pmol/gm of parathyroid tissue in the hyperplastic glands; this difference was significant (p less than 0.05). Serial blood samples from a central vein were taken at surgery for hyperparathyroidism, and the rate of decay of the intact hormone was studied in 9 patients after removal of the parathyroid tissue. This decay was rapid with a half-life of 300 seconds. We conclude that this new specific and sensitive intact PTH assay will provide a valuable means of investigating dynamic aspects of parathyroid physiology.

Plasma pyruvate kinase and creatine kinase activity in Becker muscular dystrophy.

Plasma creatine kinase (CK) and pyruvate kinase (PK) were measured in 31 obligate carriers of Becker muscular dystrophy (BMD), 36 BMD patients and appropriate controls. Mean plasma CK was 108 U/l in obligate carriers and 62 U/l in 43 age- and sex-matched controls (P less than 0.001 carriers vs controls). Control CK reference range was 31-125 U/l (mean +/- 2 SD of log transformed values). Mean plasma PK was 40 U/l in obligate carriers and 34 U/l in 56 controls (P less than 0.02 carriers vs controls). Control PK reference range was 18-61 U/l. Values of CK above the reference range upper limit were found in 13 of 31 BMD obligate carriers but only 2 showed elevated PK values. The sensitivity of CK in determining BMD carrier status, although only 42%, was markedly better than PK at 6.5%. Mean plasma CK in BMD patients was 2366 U/l, a 19-fold increase over the control value of 127 U/l (P less than 0.001 patients vs controls). Control CK reference range was 40-316 U/l. In contrast, mean plasma PK in BMD patients was 353 U/l, only 7-fold higher than the mean control value of 57 U/l (P less than 0.001 patients vs controls). Control PK reference range was 22-126 U/l. Clearly, the estimation of plasma PK as a means of determining BMD carrier status is markedly inferior to CK. Previous reports of increased sensitivity of PK compared with CK may have been due to artefactually elevated PK levels produced during sample preparation.

Effect of exercise on plasma pyruvate kinase and creatine kinase activity.

Plasma pyruvate kinase (PK) and creatine kinase (CK) were measured in healthy subjects engaging in (a) mild exercise, 30 min on an exercise cycle maintaining a pulse rate of 150/min, (b) moderate exercise, squeezing a ball until exhaustion with a sphygmomanometer cuff inflated above systolic pressure around the arm (max. 2 min) and (c) severe exercise, completing a marathon race. Mild exercise resulted in no change in enzyme levels over 24 h. Moderate exercise produced a small increase in PK but no change in CK. PK activity rose from 35.3 +/- 10 U/l pre-exercise to 41.3 +/- 13 U/l 15 min post-exercise (n = 8, p less than 0.025). Severe exercise (completing a marathon race) resulted in a 3-fold increase in PK from 26 (4-87) U/l pre-race to 69 (21-156) U/l immediately post-race, and also, as expected, an increase in CK from 60 (15-164) U/l to 257 (72-1535) U/l (results are means and ranges, n = 69, p less than 0.001 for both enzymes). Runners showed parallel increases in PK and CK (p less than 0.05 by Spearman rank correlation). The mean post-race activity of CK-MB was less than 5% of total CK but 18 runners had values greater than 6% (mean 4.8, range 1-18). We conclude that PK, like CK, is increased following exercise due to liberation of muscle enzyme. However, only severe exercise is likely to lead to a substantial increase in plasma PK activity and therefore prejudice its clinical usefulness as a diagnostic test.

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.

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 immunoaffinity chromatography enrichment and nuclease P1 procedures for 32P-postlabelling analysis of PAH-DNA adducts.

32P-postlabelling analysis for detecting DNA adducts formed by polycyclic aromatic compounds is one of the most widely used techniques for assessing genotoxicity associated with these compounds. In cases where the formation of adducts is extremely low, a crucial step in the analysis is an enrichment procedure for adducts prior to the radiolabelling step. The nuclease P1 enhancement procedure is the most established and frequently used of these methods. An immunoaffinity procedure developed for class specific recognition for polycyclic aromatic hydrocarbon (PAH)-DNA adducts has therefore been compared with the nuclease P1 method for a range of DNA adducts formed by PAHs. The evaluation was carried out with skin DNA from mice treated topically with benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 5-methylchrysene or chrysene. The immobilised antibody had the highest affinity for adducts structurally similar to the BPDE-I-deoxyguanosine adduct ([+/-]-N2-(7r,8t,9r-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-1 0t-yl)-2'-deoxyguanosine) against which the antibody had been raised. Of the PAH-modified DNAs evaluated, the maximum adduct recovery was obtained for DNA containing the BPDE I-deoxyguanosine adduct. With DMBA-modified DNA, the profiles of adducts recovered from the column were similar when the column material was treated either with a digest of DMBA-modified DNA or with 32P-labelled DMBA adducts. I-compounds (endogenous adducts in tissue DNA of unexposed animals), which had similar chromatographic properties to PAH-DNA adducts, were not enriched by the immunoaffinity procedure. Compared to the simple nuclease P1 enhancement procedure, the immunoaffinity methods were lengthier and more labour intensive. Advantages of the immunoaffinity procedure include: specificity, allowing the selective detection of a certain class of adducts: efficient adduct enrichment, providing a viable alternative to other enrichment procedures; adequate sensitivity for model studies and the potential to purify adducts for further characterisation. However, as a general screen for detecting the formation of DNA adducts, the nuclease P1 procedure was viewed as the initial method of choice since it was capable of detecting a wider range of PAH-DNA adducts.

Construction of a database of benzene biological monitoring.

Biological monitoring of occupational exposure to benzene has been conducted in the petroleum, steel and chemical industries. The urinary benzene-specific biomarker, S-phenylmercapturic acid (PMA), was quantified in post-shift samples using a sensitive enzyme-linked immunosorbent assay (ELISA) and expressed as a function of urinary creatinine concentration. The assay, based on a PMA-specific antiserum, is sufficiently sensitive to measure PMA levels in non-occupationally exposed control subjects. The assay delivers batch results in a timely manner which may be as short as 3 h. Samples were analysed from groups of workers engaged in coke oven combustion processes, petroleum refining and decontamination of a benzene land spill. The construction of a database of results provides an index of benzene uptake as a consequence of the respective work processes and tasks and readily enables benchmarking exercises aimed at comparing degrees of exposure across segments of industry.

Secretion of intact PTH by dispersed human hyperparathyroid cells.

The rate of secretion of intact PTH by cells dispersed from adenomatous and hyperplastic human parathyroid tissue was measured using a two-site immunochemiluminometric assay specific for the intact peptide. Secretion rates were monitored following incubation in high and low-calcium media. The mean rate of secretion from hyperplastic cells was significantly greater (P less than 0.05) at low calcium and significantly more suppressible at high calcium (P less than 0.01) compared with adenomatous cells. These studies have demonstrated: (1) the calcium-dependent secretion of intact PTH, (2) that intact hormone release contributes to the non-suppressible component of secretion, and (3) differences in the secretory characteristics of adenomatous and hyperplastic parathyroid cells.

Water immiscible solvent based immunoassay.

A competitive monoclonal antibody-based immunoassay which quantifies a hydrophobic hapten (Rx) in water immiscible solvents, obviating the need of a pre-extraction step, has been developed. Approximately linear dose response profiles of analyte, over the range 1-20 ugml-1 in the hydrophobic solvents, hexane, toluene and xylene were obtained. UV spectrophotometric analyses of Rx dosed hexane confirm the phenomenon of antibody-mediated transfer of analyte from the organic to the aqueous milieu. Preliminary data on the effect of water immiscible solvents on the immunoreactivity of a monoclonal antibody in free solution are presented. The potential industrial applications of water immiscible solvent based immunoassays are discussed.

Development and validation of a competitive immunoassay for urinary S-phenylmercapturic acid and its application in benzene biological monitoring.

An immunoassay that quantifies urinary S-phenylmercapturic acid (PMA), a benzenespecific biomarker, has been developed and its potential usefulness as a screening tool for monitoring occupational exposure to benzene has been demonstrated. Analytical reliability has been confirmed by correlation of results with gas chromatography-mass spectrometry (GC/MS) data (R = 0.92). The assay has been configured as a competitive enzyme-linked immunosorbent assay (ELISA) to facilitate rapid throughput of samples. The ELISA has a working range of 40-1200 nmoll-1 urinary PMA and appears to be unaffected by the presence of structurally related urinary metabolites. Background levels of 0-1.9 mumol PMA/mol creatinine (mean 0.9 mumol mol-1, n = 32) were measured in nonsmoking control subjects. Recent exposures to benzene (8 h time-weighted averages-TWA), during diverse industrial processes, over the range 0-4.8 ppm were identified by application of the assay in biological monitoring programmes.

Measurement of circulating parathyroid hormone.

The measurement of parathyroid hormone (PTH) is important in the investigation of disorders of mineral metabolism. Though the first immunoassay for PTH was described more than 25 years ago, it is only recently that methods have been developed which provide the required sensitivity and specificity to detect small changes in hormone secretion in normal subjects. These new methods take advantage of modern labelled antibody technology and have already been shown capable of yielding improvements in the diagnosis and monitoring of disorders which involve changes in PTH secretion. In addition, they are now providing fresh insight into the basic physiology of PTH secretion.

Detection of 1,N6-etheno-2'-deoxyadenosine and 3,N4-etheno-2'-deoxycytidine occurring endogenously in DNA.

1,N6-Etheno-2'-deoxyadenosine (epsilon dA) and 3,N4-etheno-2'-deoxycytidine (epsilon dC) are DNA adducts formed by a number of genotoxic chemicals, including vinyl chloride. They are also formed endogenously in tissue DNA, probably from a reactive metabolite of lipid peroxidation. Both the qualitative and quantitative detection of endogenous adducts is important in order to place adduct formation by chemicals such as vinyl chloride in the context of this natural background level. Methods with sufficient sensitivity are therefore being developed to measure the natural background of epsilon dA and epsilon dC adducts. We have developed a high-performance liquid chromatography (HPLC)-32P-postlabelling method to measure epsilon dA and epsilon dC at alkylation frequencies of 1 adduct in 10(7)-10(8) nucleotides in 10-microgram samples of DNA. In HPLC-32P-postlabelling analysis of liver DNA from control Wistar rats, epsilon dA and epsilon dC were determined at levels of 1 adduct in 8.1 x 10(7) and 1 adduct in 1.8 x 10(7) nucleotides, respectively. The levels of epsilon dA and epsilon dC measured in liver DNA of animals exposed orally to five daily doses of 50 mg/kg body weight vinyl chloride were found by this method to be 1 adduct in 2.9 x 10(7) and 1 adduct in 1.4 x 10(7) nucleotides, respectively. In contrast, in a direct labelling study, radiolabelled epsilon dA and epsilon dC were not detected in liver DNA of rats exposed for 6 h by nose-only inhalation to [1,2-14C]vinyl chloride at up to 45 ppm v/v. Immunochemical procedures are also being developed for recognizing etheno adducts. Thus, a monoclonal antibody raised to protein conjugates of epsilon dC showed high selectivity in the recognition of this DNA adduct. When the antibody was immobilized on a solid support and used in an immunoenrichment procedure to purify epsilon dC from a large excess of normal nucleotides, one epsilon dC adduct from about 10(8) normal nucleotides could be resolved. Coupling the immunoaffinity enrichment procedure with capillary zone electrophoresis permitted the detection of approximately one epsilon dC adduct in 3 x 10(6) nucleotides.

Laboratory comparison of diagnostic specificity of intact hormone and region-specific immunoassays of parathyroid hormone.

A 2-site immunochemiluminometric assay (ICMA) for intact parathyroid hormone (PTH) has been developed to overcome the problems of limited sensitivity and specificity associated with conventional PTH immunoassays. The present investigation assesses the relative merits of the 2-site intact PTH (ICMA) and a midmolecule PTH radioimmunoassay (RIA). Immunoassay profiles of dispersed hyperparathyroid cell supernatants and hyperparathyroid sera obtained during parathyroidectomy and from renal failure patients, fractionated by gel filtration chromatography, facilitated a direct comparison of the specificity of PTH immunoassays. Using gel filtration matrices which permitted the separation of intact hormone, large C-terminal fragment(s), and a small midmolecule fragment in adenomatous cell supernatant and peripheral sera, the intact PTH (ICMA) assay consistently yielded a single peak of immunoreactivity. In contrast, the midmolecule RIA yielded 2 and 3 broad peaks of immunoreactivity after fractionation of serum and adenomatous cell supernatant, respectively. There was no evidence for the secretion of a small midregion fragment in vitro or its peripheral derivation in vivo. These studies demonstrate the superior specificity of the 2-site intact PTH assay compared with a midmolecule RIA and, thus, confirm the potential diagnostic superiority of the ICMA.

Class-specific immunoadsorption purification for polycyclic aromatic hydrocarbon-DNA adducts.

An immunoenrichment procedure has been developed for applications in the detection and identification of a broad range of polycyclic aromatic hydrocarbon (PAH)-DNA adducts at very low abundance. The procedures are based on a monoclonal antibody raised to r-7,trans-8,trans-9-trihydroxy-cis-10-(N2-deoxyguanosyl-5'-phospha te)- 7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE-N2-dG) which has been tested for cross-reactivity towards DNA and proteins (bovine serum albumin, chicken gamma globulin and human globin) covalently modified with a range of PAH diol-epoxides. The antibody recognised DNA adducted with the diol-epoxides of benzo[a]pyrene, benz[a]anthracene, chrysene, dibenz[a,h]anthracene or picene. The antibody also cross-reacted with the 7,8,9,10-tetraol derived from benzo[a]pyrene and the 1,2,3,4-tetraols of benz[a]anthracene and chrysene. The degree of cross-reactivity was greatest for PAH adducts with structural features similar to anti-BPDE-N2-dG proximate to the base attachment. The antibody also recognised a range of PAHs adducted to human globin; these included adducts of benzo[a]pyrene, benz[a]anthracene and chrysene diol-epoxides. This wide range of recognition provides good evidence for the class-specific recognition of PAH adducts by the antibody. When immobilized on Sepharose 4B and used in the immunoadsorption purification of adducted nucleotides, the antibody selectively enriched adducts of benzo[a]pyrene, benz[a]anthracene and chrysene from normal nucleotides. Quantitative measurements with [14C]benzo[a]pyrene-DNA adducts showed that the immobilized antibody was able to enrich benzo[a]pyrene adducts from a DNA hydrolysate containing adducts at a level of 1 adduct/10(10) normal nucleotides. In addition, this immunoadsorption technique was effective in enriching a mixture of DNA adducts formed in the skin of CF1 mice treated cutaneously with a mixture of [3H]benzo[a]pyrene and [3H]chrysene. Class-specific immunoenrichment procedures for DNA adducts are important in assisting the identification of genotoxic components in complex mixtures. The performance characteristics of this immobilized antibody suggest that it may be suitable for application in the detection, identification and monitoring of human exposures to low levels of PAHs.