Next generation physiologically based kinetic (NG-PBK) models in support of regulatory decision making.

The fields of toxicology and chemical risk assessment seek to reduce, and eventually replace, the use of animals for the prediction of toxicity in humans. In this context, physiologically based kinetic (PBK) modelling based on in vitro and in silico kinetic data has the potential to a play significant role in reducing animal testing, by providing a methodology capable of incorporating in vitro human data to facilitate the development of in vitro to in vivo extrapolation of hazard information. In the present article, we discuss the challenges in: 1) applying PBK modelling to support regulatory decision making under the toxicology and risk-assessment paradigm shift towards animal replacement; 2) constructing PBK models without in vivo animal kinetic data, while relying solely on in vitro or in silico methods for model parameterization; and 3) assessing the validity and credibility of PBK models built largely using non-animal data. The strengths, uncertainties, and limitations of PBK models developed using in vitro or in silico data are discussed in an effort to establish a higher degree of confidence in the application of such models in a regulatory context. The article summarises the outcome of an expert workshop hosted by the European Commission Joint Research Centre (EC-JRC) - European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), on "Physiologically-Based Kinetic modelling in risk assessment - reaching a whole new level in regulatory decision-making" held in Ispra, Italy, in November 2016, along with results from an international survey conducted in 2017 and recently reported activities occurring within the PBK modelling field. The discussions presented herein highlight the potential applications of next generation (NG)-PBK modelling, based on new data streams.

Radioimmunoassay of cortisone in serum, urine, and saliva to assess the status of the cortisol-cortisone shuttle.

We have developed a new assay for cortisone (E) in serum, saliva, and urine involving Celite chromatography followed by RIA with 125I-labeled E and scintillation proximity assay. The chromatography step separates cortisol (F) from E, and in combination with their RIAs, permits assessment of the status of the F-E shuttle. We report the results of basal, postcorticotropin (ACTH), and postdexamethasone E and F concentrations and their circadian fluctuations in the serum, saliva, and urine of healthy volunteers. The serum and urine F/E ratios were increased in patients with ectopic ACTH secretion, whereas in adrenal adenoma and Cushing disease only the urinary ratio was increased. In chronic renal insufficiency this ratio was increased in serum (23.5 +/- 3.9) but diminished in saliva (0.38 +/- 0.11), and in apparent mineralocorticoid excess the ratios were high in serum (44.3 +/- 9.3) and urine (5.35 +/- 0.85) compared with those of healthy subjects (serum 9.8 +/- 3.5, urine 0.52 +/- 0.29, saliva 0.52 +/- 0.29).

Direct colorimetric monoclonal antibody enzyme immunoassay for estradiol-17 beta in saliva.

We developed a direct microtiter plate enzyme immunoassay to measure estradiol-17 beta in saliva. The assay has a commercially available monoclonal antibody, raised against estradiol-17 beta-6-carboxymethyloximebovine serum albumin, and a homologous horseradish peroxidase conjugate measured colorimetrically. The detection limit (equivalent to B0-3 SD) is 365 amol/well or 7.3 pmol/L when 50-microL samples are assayed. Cross-reactivity with estrone and estriol, testosterone, or progesterone is < 0.2%. Estradiol-17 beta was measured in daily samples over five natural menstrual cycles and eight cycles stimulated as a preliminary to in vitro fertilization, and the concentrations and fluctuations found agreed with previously published data. This method gives results in approximately 3 h and may be useful for fertility monitoring and management.

The dependence of radioimmunoassay detection limits on antibody affinity.

Competitive radioimmunoassay standard curves for progesterone were established with tritiated progesterone and six different anti-progesterone monoclonal antibodies ranging in affinity from 1.23 x 10(8) to 2.82 x 10(10) l/mol. The detection limits for these curves ranged from 133 to 6,670 pmol/l final assay concentration (16.7 to 839.1 pg/tube). Separately, a mass-action mathematical model was used to predict lower detection limits for assays run under equivalent conditions with the six antibodies. When compared to the experimental data the predictions were reliable for the higher affinity antibodies but became less accurate as the affinity decreased.

Hirsutism and acne in women: coordinated radioimmunoassays for eight relevant plasma steroids.

We developed and validated a coordinated set of RIAs for the following eight steroids in single small aliquots (< or = 1 mL) of plasma: androstenedione, dehydroepiandrosterone, 11-deoxycortisol, 21-deoxycortisol (21-DF), 11 beta-hydroxyandrostenedione, 17 alpha-hydroxypregnenolone (17-Hpreg), 17 alpha-hydroxyprogesterone, and testosterone. Samples were extracted and then chromatographed on celite microcolumns. Radioiodinated tracers were used for two of the assays (17-Hpreg and 21-DF). Tritiated tracers and scintillation proximity assay counting were used to give separation-free procedures for the other six assays, which considerably improved their practicability and reproducibility. The basal and postadrenocorticotropic hormone plasma values for these steroids in normal women sampled in the follicular phase are presented. Finally, the measurement of the eight steroids as a diagnostic method is evaluated with reference to data from 203 patients with hirsutism and (or) acne.

Development and validation of a monoclonal antibody enzyme immunoassay for measuring progesterone in saliva.

A nonextraction, competitive, solid-phase enzyme immunoassay with a monoclonal antibody was developed and validated for measuring progesterone in saliva. The antibody was raised against 11 alpha-hydroxyprogesterone hemisuccinate-bovine serum albumin conjugate and was indirectly immobilized to the walls of microtiter wells. The labeled analyte (progesterone-horseradish peroxidase conjugate) was homologous with the immunogen. The lower detection limit (concentration equivalent to B0-3 SD) was 38 pmol/L of saliva sample. We validated the assay with studies to establish the independence of the concentration determined from the volume of saliva assayed, quantitative recovery of progesterone added to saliva, interference from possible cross-reactants, and agreement with a similar assay that incorporated an extraction step. In addition, we determined luteal-phase concentrations of salivary progesterone in normal women and compared the results with those of an older assay involving a polyclonal antibody.

Standardization of hapten immunoprocedures: total cortisol.

The broad objectives of this report are to enhance the clinical utility of the results of hapten measurement immunoprocedures by encouraging standardization of the procedures. It was decided to restrict the subject of the report to one analyte, and cortisol was chosen because of its diagnostic importance, the need for improved comparability with routine procedures, and its relevance to many other analytes. Therefore, the immediate objectives are to improve the clinical utility of total serum cortisol concentrations measured on different occasions and in different laboratories, to make diagnostic reference intervals more reliable and widely applicable, and to improve diagnostic accuracy by: Improving the agreement between results from reference measurement procedures and the results obtained with normal routine immunoprocedures for the measurement of total serum cortisol, and hence the comparability of routine results; and Reducing the susceptibility of the immunoprocedures to crossreactivity and interference. It is recognized that good comparability of results depends on procedures which are specific, properly calibrated and validated, and that the most important factors are resistance to crossreactants and interferants. Analytical goals for cortisol immunoprocedures are zero bias for 'all' samples with respect to a reference procedures, with the total analytical standard deviation equal to, or less than, half the within-individual standard deviation for cortisol, or 7.6% according to a recent estimate. To these ends the group proposes the following measures: 1. An international network of reference laboratories for cortisol measurement should be established, and progressively developed to include other hapten analytes. The service of the participating laboratories would be necessary to make feasible other recommendations listed below. 2. Procedures should be comprehensively validated, including a thorough crossreactivity study with the results presented to indicate the possible significance of each crossreactivity, a direct comparison of results with those obtained by a reference procedure for an adequate number of varied fresh/frozen patient samples, and suitable clinical validation studies. 3. External quality assessment schemes should assess participating laboratory performance against reference procedure values, and not consensus values related to values determined by any group of participating procedures. They should also assess the ability to determine added standard and to resist common interferants. 4. Master calibrators for use in the production of calibrators included in commercial kits, and formulated identically to these, should be certified with at least one reference measurement procedure, so that the concentrations of analyte in calibrators can be traced back to concentrations determined by a reference procedure.(ABSTRACT TRUNCATED AT 400 WORDS)

The frequency of salivary progesterone sampling and the diagnosis of luteal phase insufficiency.

A profile of salivary progesterone concentrations, based on daily samples taken over a full menstrual cycle, provides a detailed picture of changes in luteal function, at the expense of analyzing a large number of samples. Strain can be placed on analytical services by assaying daily samples instead of one or a few serum (or saliva) samples. This study sought to determine the minimum number of salivary progesterone determinations which adequately describe luteal function. Daily salivary progesterone levels from 215 cycles, of which 29 cycles had progesterone profiles indicative of luteal phase insufficiency, were analyzed to ascertain the efficiencies of various sampling patterns of reduced frequency. A single mid-luteal salivary progesterone estimation or the mid-luteal Lenton progesterone index (n = 4) satisfactorily reflected the normal luteal phase, but a frequency of one sample every 3 days over the luteal phase (n = 5-6) was necessary to allow recognition of a short luteal phase or poor progesterone surge.

Standardization of immunoassays for hapten analytes.

The pace of the development and commercialization of immunoassay methodology has been so great, that the parallel development of ways to ensure comparability between the results of different immunoassays for the same substance has fallen behind. If, as is the case with many hapten analytes, the analyte is chemically defined, homogeneous and available at very high purity, the rigorous standardization of different immunoassays developed to determine the same would appear to be feasible. However, many published details of external quality assessment schemes and of studies on the comparability of commercial assays indicate that the results of immunoassays for hapten analytes are not generally and reliably comparable. Lack of comparability is most marked for assays detecting a variety of analyte subforms or metabolites. This problem is surveyed with respect to the variability of hapten immunoassays, factors related to details of immunoassays. Finally, the assessment of assay comparability and some potential strategies for improving it are discussed.

A decade of development in immunoassay methodology.

Immunoassays are now very widely used in the clinical laboratory, either because no other type of assay system is feasible or because they are often the most effective and suitable of the possible analytical methods. The last decade has seen the development and refinement of many new immunoassay reagents and systems. The major trend has been away from liquid-phase assays involving radioisotopic labels, towards fast homogeneous or solid-phase assays capable of operation anywhere; and towards precise and reliable nonisotopic, automated or semi-automated laboratory assays, often with detection limits measured in pico- or attomoles. The use of monoclonal antibodies is now widespread, and the methodologies of labels and of solid-phase components are much more sophisticated. New assay formulations, novel homogeneous systems, immunosensors, free-analyte assays, the importance of thorough validation and of interfering substances, and future trends are discussed.

Follicular growth and corpus luteum function in women with unexplained infertility, monitored by ultrasonography and measurement of daily salivary progesterone.

Ovarian function was evaluated over a minimum of 3 consecutive menstrual cycles from each of 41 women with unexplained infertility. Follicular development and ovulation were monitored using real time ultrasonography and luteal function was evaluated by daily salivary progesterone measurement. In 129 spontaneous cycles, normal single ovulations were detected in 121 (93.8%). Luteal phase insufficiency was identified in 21 (17.4%) of these 121 cycles and this was a recurrent phenomenon in the cycles of 5 of the 41 women (12.2%). A successful pregnancy was seen only in association with consistently normal salivary progesterone profiles or where the empirical use of clomiphene citrate therapy had corrected previously diagnosed luteal phase insufficiency. Basal body temperature records or mid-luteal serum progesterone measurements were less satisfactory indices of luteal function than a salivary progesterone profile.

Direct solid-phase enzymoimmunoassay of testosterone in saliva.

This competitive, solid-phase enzymoimmunoassay for testosterone in saliva is carried out on microtiter plates and involves no chromatographic or extraction steps. With an overnight incubation the detection limit of the assay is 230 fg per well (16.1 pmol/L). There was a good correlation (correlation coefficient 0.95) between testosterone concentrations measured with and without prior extraction of the saliva samples. Repeated assay of three control saliva samples containing a range of testosterone concentrations (200-1000 pmol/L) gave within- and between-assay coefficients of variation of 5.5-13.2%. The analytical procedure is simple and closely resembles already published procedures for the determination of progesterone and estrone (with extraction) in saliva. One person can assay 200 samples in 24 h and the assay is suitable for reproductive and sports medical applications, particularly for projects involving serial sampling and yielding large numbers of samples.

Radioimmunoassay of osteocalcin with polyclonal and monoclonal antibodies.

We have developed and thoroughly validated radioimmunoassays (RIAs) for osteocalcin in plasma and serum, demonstrating independence of volume and determining the recovery of added standard and within- and between-assay precision. Antibodies were raised in rabbits and mice by immunization with either osteocalcin adsorbed to polyvinylpyrrolidone or osteocalcin conjugated to bovine serum albumin. In both species, use of the conjugated osteocalcin was more efficacious. Correlation of the results obtained when groups of plasma or serum samples were analyzed by RIAs with different antibodies (two polyclonal and one monoclonal) indicated that the normal range observed for osteocalcin concentrations in serum depends on the antibody used. These findings may account for the wide range of "normal" osteocalcin values reported by different groups.

Solid-phase enzymoimmunoassay of estrone in saliva.

In this solid-phase enzymoimmunoassay for estrone in saliva, microtiter plates are used after extraction of the sample with diethyl ether. No chromatographic step is involved. The detection limit of the assay is 450 fg per well (33 pmol/L). Intra- and interassay coefficients of variation for the assay of low, medium, and high concentrations of estrone in saliva were respectively 4.2, 12.7; 5.2, 8.7; and 2.7, 5.8%. Using this assay, we found a highly significant correlation (P less than 0.001) between estrone concentrations in time-matched serum and saliva samples. The analytical procedure is rapid and relatively simple. One person can assay 50-60 saliva samples during a normal working day. We conclude that the assay is very suitable, even in small laboratories, for saliva estrone measurements, which, in facilitating serial sampling, enables dynamic observations of estrone concentrations and ovarian activity to be more easily made.

Normal salivary progesterone levels throughout the ovarian cycle as determined by a direct enzyme immunoassay.

This study, which is concerned with establishing clinically relevant corridors defining normal progesterone (P) levels in saliva, has addressed some of the shortcomings associated with previous equivalent studies (e.g., limited data, inadequately monitored ovulations, inadequately defined subjects). A sensitive, direct, microtiter plate, enzymeimmunoassay (EIA) for progesterone developed in the authors' unit, was used to determine daily salivary progesterone concentrations over 41 menstrual cycles in 41 women. Each woman was without associated factors known to affect ovarian function and each cycle included was judged to be (single) ovulatory by serial pelvic ultrasound examinations. Daily salivary progesterone levels from these cycles were statistically analyzed and corridors of progesterone concentrations associated with normal luteal function were established.