Preliminary investigation of the effect of treating sheep during pregnancy with a vitamin A, D, E formulation on the incidence of vaginal prolapse.

Aim: To determine if vitamin D3 treatment reduced the incidence of vaginal prolapse in pregnant sheep on a North Canterbury sheep breeding property.Methods: Pregnant ewes from a single farm were allocated to three treatment groups in May 2018. At this time, the first group (EarlyVitADE; n = 512) received an I/M 1 mL dose of 500,000 IU/mL vitamin D3, 60,000 IU/mL vitamin A, and 25 mg/mL vitamin E. This was repeated in July 2018, when the second group (LateVitADE; n = 695) also received the same treatment. The third group (n = 737) were untreated controls. All cases of vaginal prolapse on the property were recorded from pregnancy diagnosis in June 2018 until ewes were set-stocked in August 2018. The planned start of lambing was 10 August 2018.Results: During the period of observation, vaginal prolapses were recorded in 3/699 (0.4%) 2-year-old ewes, and the odds of vaginal prolapse were not associated with treatment group in these ewes (p > 0.3). Amongst ewes aged ≥3 years, during the same period, there were 6/333 (1.8%), 6/443 (1.4%) and 25/469 (5.3%) cases in the EarlyVitADE, LateVitADE and control groups, respectively. Compared to control ewes, the odds of vaginal prolapse were reduced in both the EarlyVitADE (OR = 0.37; 95% CI = 0.15-0.92) and LateVitADE (OR = 0.25; 95% CI = 0.10-0.62) treatment groups.Conclusions and clinical relevance: In this preliminary study, administration of injectable vitamins A, D3, and E to pregnant ewes reduced the incidence of vaginal prolapse during the period from pregnancy diagnosis to set-stocking on one North Canterbury hill-country farm. Due to the restricted data collection period, this investigation should be replicated to better quantify the repeatability of the observed treatment effect over the complete lambing period.

Corticosteroid-binding globulin, cortisol, free cortisol, and sex hormone-binding globulin responses following oral glucose challenge in spinal cord-injured and able-bodied men.

Circulating cortisol, corticosteroid-binding globulin, and sex hormone-binding globulin were measured retrospectively in plasma samples following the oral glucose tolerance test in 20 spinal cord-injured men and 20 able-bodied controls. Plasma-free cortisol responses attenuated more rapidly in the able-bodied men, compared to spinal cord-injured subjects, due to significant rise in circulating corticosteroid-binding globulin whereas changes in total plasma cortisol were similar in both groups. The changes in plasma-free cortisol in both groups paralleled changes in insulin and glucose and show that spinal cord-injured men had heightened exposure to free cortisol during this dynamic test. This raises the possibility that the mechanism of abdominal obesity and the propensity towards insulin resistance in spinal cord-injured men could be subtly mediated by perturbations in free cortisol. There were no significant changes in plasma sex hormone-binding globulin in either group.

Comparison of indices of insulin resistance with metabolic syndrome classifications to predict the development of impaired fasting glucose in overweight and obese subjects: a 3-year prospective study.

OBJECTIVE: To compare the ability of biochemical indices of insulin resistance (IR) with metabolic syndrome (MetS) classifications to predict changes in blood glucose control over a 3-year period in overweight and obese subjects. DESIGN: This was a longitudinal, prospective study, with data collected at baseline, 18 and 36 months. SUBJECTS AND METHODS: A total of 175 overweight (body mass index (BMI)>25 kg m(-2)) and obese (BMI>30 kg m(-2)) subjects were enrolled in the study. The IR indices assessed included fasting insulin concentration, the insulin/glucose-derived indices, homeostasis assessment model of insulin resistance (HOMA-IR) and quantitative insulin sensitivity check index (QUICKI), the insulin/triglyceride-derived McAuley index, plasma adiponectin concentration and the triglyceride (trig) and high-density lipoprotein (HDL)-cholesterol ratio (trig:HDL). The two MetS classifications were assessed according to the definitions of the National Cholesterol Education Program-Third Adult Treatment Panel (NCEP-ATPIII) and the International Diabetes Federation (IDF). The potential of the IR indices and MetS classifications at baseline to predict the development of impaired fasting glucose (IFG) was examined using receiver-operator characteristic (ROC) curve analysis and analysis of variance. RESULTS: Complete data were collected on 158 subjects. In all, 51 (32%) subjects developed IFG during the study. The analysis of variance showed significant differences between the IFG and normoglycaemic group in the baseline values of the McAuley index, trig:HDL, plasma adiponectin concentration and prevalence of the MetS. The ROC curve analysis confirmed this result and showed that the strongest predictors of IFG were baseline trig:HDL and IDF MetS classification, followed in order by the McAuley index, plasma adiponectin concentration and NCEP-ATPIII MetS classification. In contrast, the baseline values of fasting insulin, HOMA-IR and QUICKI did not predict IFG. DISCUSSION: This study showed that the IR indices, derived, in part, from plasma triglyceride concentration, were sensitive predictors for the development of IFG in normoglycaemic overweight and obese subjects. Indices derived from glucose and insulin did not identify this at-risk group. The study also showed that the presence of MetS and its abnormalities of an increased trig:HDL ratio and low plasma adiponectin concentration were all sensitive predictors of IFG.

Plasma levels of sex hormone-binding globulin, corticosteroid-binding globulin and cortisol in overweight subjects who develop impaired fasting glucose: a 3-year prospective study.

Circulating sex hormone-binding globulin (SHBG), corticosteroid-binding globulin (CBG), and total and calculated free cortisol were measured in 206 overweight subjects to investigate whether or not they were markers of insulin resistance. Measurements were carried out on two occasions 36 months apart and subjects were grouped according to fasting plasma glucose. Fifty-one subjects, with a normal basal fasting glucose (<5.6 mmol/l) developed impaired fasting glucose 3 years later (> or = 5.6 mmol/l). Analysis either in toto or based on gender showed a highly significant increase in fasting insulin and insulin resistance, a modest increase in body mass index (BMI), but importantly no change in plasma SHBG, CBG, or cortisol concentrations. Subjects (n=101) with a normal fasting glucose both at baseline (<5.6 mmol/l) and at 36 months showed no significant change in fasting insulin, insulin resistance, SHBG, CBG, cortisol, or BMI. Cross-sectional analysis of the study population showed that plasma SHBG correlated negatively with insulin resistance both in men and women. Overall SHBG at baseline was not predictive of changes in fasting glucose. In females, plasma CBG correlated negatively with BMI. The major finding is that overweight subjects who developed impaired fasting glucose showed no significant change in plasma SHBG, CBG or cortisol, and therefore these indices are probably not early markers of insulin resistance in overweight subjects.

Allelotype of human bladder cancer.

To identify common regions of deletion in human bladder tumors, we have screened 83 cases of transitional cell carcinoma for loss of heterozygosity (LOH) on all autosomal chromosome arms. Seventy-two restriction fragment length polymorphism, variable number of tandem repeats, and minisatellite markers and 18 microsatellite markers were used to obtain a minimum of 50% informative results for each chromosome arm. A mean of 29.6 informative results per patient was obtained from 39 chromosome arms studied, representing information for 76% of chromosome arms. The most frequent losses were apparent monosomies of chromosome 9 (9p, 51%; 9q, 57%). Other frequent losses were on chromosomes 11p (32%), 17p (32%), 8p (23%), 4p (22%), and 13q (15%). LOH of 4p has not been reported previously in bladder carcinoma. The frequency of LOH on all other chromosome arms was < 12%. LOH on chromosome 8p showed a significant association with both high tumor grade and stage, and LOH on 13q showed a significant association with high tumor grade. Fractional allelic loss was calculated for all tumors and had a mean of 0.125 and a median of 0.110. A significant association was found between increased fractional allelic loss and higher tumor grade. An association was also found between LOH of chromosomes 8p and 9q and values for fractional allelic loss > or = the median value. No associations were found between LOH on different pairs of chromosome arms.

Deletion of two regions on chromosome 4 in bladder carcinoma: definition of a critical 750kB region at 4p16.3.

Loss of heterozygosity (LOH) of chromosome 4 has been identified in several human tumours including carcinomas of colorectum, ovary, liver and head and neck. LOH at loci on chromosome 4p has previously been identified in 22% of primary bladder tumours. We have assessed LOH in 178 bladder tumours using a panel of six microsatellite markers. Thirty-four tumours (19%) showed LOH at one or more loci. Twenty-three deletions involved restricted regions of 4p and could be used to define two common regions of deletion. A very small common region between D4S43 and D4S127 (estimated to be approximately 750 kB) was involved in 14/23 (61%) of 4p deletions. A second common region centromeric to D4S174 was deleted in 7/23 (30%) of tumours with deletions. Two tumours (9%) had deletions involving both regions independently. Previous functional studies have demonstrated both a senescence function and a suppressor of tumorigenicity on human chromosome 4. Localisation of the common regions of deletion in bladder tumours provides a starting point for positional cloning of the gene(s) concerned and for more precise comparative functional studies.

Detailed deletion mapping of chromosome 9q in bladder cancer: evidence for two tumour suppressor loci.

Loss of heterozygosity (LOH) at loci on chromosome 9p and/or 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder. However, localisation of the tumour suppressor locus or loci on 9q has been hampered by the relative infrequency of tumours with subchromosomal deletions. We have used 24 microsatellite markers to examine LOH in 70 new cases of TCC of the bladder and upper urinary tract. Forty tumours (57%) showed LOH at one or more loci on 9q and partial deletions were detected in five tumours (7%). Combined data from the five cases with partial deletions place one tumour suppressor locus at 9q34 between D9S61 and D9S66 (an estimated distance of 13-14 cM). This region is frequently deleted in other sporadic tumours and encompasses one of the loci for tuberous sclerosis (TSC1). One tumour contained a distinct deletion between D9S153 and D9S109 (9q13-q31), which encompasses the locus for the familial nevoid basal cell carcinoma syndrome (Gorlin syndrome). This may indicate the presence of another tumour suppressor locus on 9q for TCC. Our findings significantly reduce the regions of 9q within which suppressor genes for TCC may reside. The possible involvement of two deletion targets on 9q in addition to the locus at 9p21 implicated in TCC may explain why LOH at all loci on chromosome 9 is frequent in TCC.

Circulating levels of isoflavones and markers of 5alpha-reductase activity are higher in Japanese compared with New Zealand males: what is the role of circulating steroids in prostate disease?

Epidemiological evidence implicates dietary isoflavone intake as protective against prostate disease. A putative mechanism is attenuated circulating androgen levels in male populations consuming an isoflavone rich diet. We investigated this hypothesis by collecting plasma from 60 Japanese and 60 New Zealand males aged between 21 and 31 years each consuming their traditional diets. We measured plasma testosterone, dihydrotestosterone (DHT), androstenedione, dehydroepiandrosterone sulfate (DHEAS), the combined levels of androsterone sulfate and epiandrosterone sulfate (AoS/epiAoS), sex hormone-binding globulin, and cortisol and corticosteroid-binding globulin as well as the isoflavones genistein and equol. Plasma genistein and equol levels were several times higher in Japanese males as would be expected from an isoflavone rich diet. However, androstenedione, DHEAS, calculated free testosterone and paradoxically markers of 5alpha-reductase, DHT and AoS/epiAoS were all also significantly higher in Japanese rather than the New Zealand male counterparts. All other comparisons were not significant. Plasma DHT and DHEAS correlated positively with plasma equol and plasma AoS/epiAoS correlated positively with genistein levels. Taken together the results suggest that, rather than reduced levels of steroidogenesis, Japanese males may have increased 5alpha-reductase activity and possibly altered 17beta OH steroid dehydrogenase activity. Significantly the positive association between isoflavones levels and 5alpha-steroids is counter-intuitive to isoflavone intake offering prostate protection, unless this is postulated to occur through other mechanisms.

An enzyme linked immunosorbent assay (ELISA) for plasma medroxyprogesterone acetate (MPA).

A single extraction fixed antigen enzyme-linked immunosorbent assay (ELISA) that can be completed in less than 24 h is described for the measurement of medroxyprogesterone acetate (MPA) in plasma. MPA is covalently coupled to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to a standard 96-well microtitre plate where it competes with MPA in the extracted plasma sample for goat anti-MPA. Antibody binding to the solid phase is determined via binding of a horse-radish peroxidase second antibody which reacts colorimetrically with its substrate. The reaction is stopped by addition of 1.25 M H2SO4 and absorbance read at 492 nm. All steps except for sample addition and extraction can be performed on an automatic ELISA processing machine. The assay is sensitive, specific and precise, with intra- and inter-assay coefficients of variation of less than 10 and 15%, respectively. Assay sensitivity is 0.08 ng/ml. The assay follows established methodology for other assays in this laboratory which assists standardization, cost structure and sample throughput and thus is a useful alternative to radioimmunoassays for the determination of MPA in plasma.

Plasma 11-deoxycortisol and cortisol following dexamethasone in psychiatric patients.

As it has been suggested that calculating the ratio of cortisol to its biosynthetic precursor, 11-deoxycortisol, may enhance the sensitivity of the dexamethasone suppression test (DST) for depression, cortisol and 11-deoxycortisol were measured in 90 subjects undergoing this test. Among these subjects, post-dexamethasone cortisol and 11-deoxycortisol levels were significantly correlated (r = 0.65, P less than 0.001) and evaluating the ratio of cortisol to 11-deoxycortisol decreased rather than enhanced sensitivity of the DST.

An enzyme-linked immunosorbent assay (ELISA) for plasma progesterone: immobilised antigen approach.

A single extraction ELISA for plasma progesterone is described using the fixed antigen approach. Progesterone is covalently coupled to bovine thyroglobulin and adsorbed onto a 96-well microtitre plate in guanidine hydrochloride. The assay, performed on an automatic ELISA processor, follows an established methodology used for other steroid hormones analysed in this laboratory with concomitant advantages in assay standardisation, cost structure and result throughput. A comparison with an established RIA shows the assay to be rapid, of similar specificity and accuracy with a sensitivity of less than 0.5 nmol/l and is suitable for use in a routine endocrine laboratory for determination of luteal function.

Individual differences in plasma cortisol changes during mania and depression.

Daytime plasma cortisol levels in four rapid cycling bipolar affective disorder patients were measured longitudinally over multiple affective episodes, and changes in levels with mood state assessed for each individual patient. While three of the four patients had, as expected, increased cortisol levels during depression, higher cortisol levels were also found in the days immediately preceding depressive episodes. Daytime cortisol levels in mania were more variable but were lower in mania for two of the patients. It is hypothesized that in the early stages of mania decreased cortisol levels reflect early neurochemical changes, but that, as manic episodes become dysphoric and/or severe, elevated cortisol levels occur.

The unipolar-bipolar depressive dichotomy and the relationship between afternoon prolactin and cortisol levels.

Afternoon prolactin and cortisol levels were measured in 29 patients suffering from a current major depressive episode. Among the 15 unipolar depressed patients the afternoon prolactin and cortisol levels were positively correlated, but 14 bipolar depressed patients did not show a similar relationship, and had prolactin levels lower than the unipolar patients. This finding adds to the growing list of ways in which the neurobiology of bipolar and unipolar depression may differ.

Monoclonal antibodies to human sex hormone-binding globulin (SHBG): characterization and use in a simple enzyme-linked immunosorbent assay (ELISA) of SHBG in plasma.

Four monoclonal antibodies to human sex hormone-binding globulin were raised and characterized. Three of the four antibodies recognised different antigenic determinants on SHBG. Two of the distinct antibodies were useful for Western blotting and recognized a major 48 kDa band in human plasma as well as a 46 kDa minor component. Carbohydrate residues do not form part of the antigenic determinants of these two antibodies, although one of these showed increased signal following removal of N-linked oligosaccharides. Some of the antibodies were selected to form a basis of a same-day, non-competitive, enzyme-linked immunosorbent assay (ELISA) for SHBG in plasma. The assay employs a purified IgG2a SHBG monoclonal antibody adsorbed to the wells of a microtitre plate. After blocking any further adsorption to the plate, standards or diluted patient samples were added for a 5-h incubation at room temperature, after which the plate was washed and antibody-bound SHBG was detected with an anti-SHBG IgG1 monoclonal antibody followed by peroxidase-labeled antimouse-IgG1 and o-phenylenediamine substrate. The assay correlated well with an existing 2-day ELISA for SHBG in plasma using polyclonal antibodies and also correlated with a dihydrosterone (DHT) ligand-binding assay. The monoclonal antibody-based ELISA shows excellent performance characteristics and is unaffected by added testosterone or estradiol.

A competitive enzyme-linked immunosorbent assay for plasma 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione).

An enzyme-linked immunosorbent assay (ELISA) for 11-deoxycortisol is described for the first time. 11-Deoxycortisol-thyroglobulin conjugate is adsorbed onto the wells of a 96-well ELISA plate and competes with 11-deoxycortisol in the standards or plasma extract for antibody binding sites. After washing, immobilized primary antibody is probed with peroxidase-labeled goat anti-rabbit IgG. The ELISA plate is further washed and o-phenylenediamine added, color developed and the absorbance read at 492 nm. The ELISA shows good agreement with our existing 11-deoxycortisol radioimmunoassay (RIA) and has similar specificity and performance which allow its use in the routine steroid laboratory for assessing pituitary adrenal function by the metyrapone test.

Monoclonal antibodies to pregnanediol-3-glucuronide: application to a direct enzyme-linked immunosorbent assay of urine.

Monoclonal antibodies to pregnanediol-3-glucuronide were produced and characterized. One of three clones investigated provided antibody suitable for a direct urinary enzyme-linked immunosorbent assay (ELISA). The ELISA uses a pregnanediol-thyroglobulin conjugate adsorbed onto the wells of a standard 96-well microtiter plate. Pregnanediol-3-glucuronide in standards or diluted urine competes with the immobilized steroid for antibody-binding sites. After washing, mouse monoclonal antibody bound to the plate is probed with antimouse immunoglobulin peroxidase. After further washing, o-phenylenediamine substrate is added and, finally, the absorbance is read at 492 nm. The ELISA shows excellent performance and agreement with the previous gas chromatographic method. The ELISA is ideal for aiding the assessment of ovarian function in the routine laboratory.

Plasma androsterone/epiandrosterone sulfates as markers of 5 alpha-reductase activity: effect of finasteride in normal men.

Plasma androsterone/epiandrosterone sulfates, dehydroepiandrosterone sulfate, dihydrotestosterone, testosterone, androstenedione, and cortisol were measured in three normal adult men before and following finasteride administration (5 mg/day). Plasma androsterone/epiandrosterone sulfates and dihydrotestosterone declined in parallel to 50% of basal levels with little change in either dehydroepiandrosterone sulfate, cortisol, or androstenedione. The results suggest that the direct measurement of plasma androsterone/epiandrosterone sulfates by enzyme-linked immunosorbent assay provide similar information to plasma dihydrotestosterone and therefore provide a simple alternative for the assessment of 5 alpha-reductase activity.

Production and characterization of monoclonal antibodies to dehydroepiandrosterone sulfate: application to direct enzyme-linked immunosorbent assays of dehydroepiandrosterone sulfate and androsterone/epiandrosterone sulfates in plasma.

Mice were immunized with 5-androstene-3 beta-ol-7,17-dione-7-CMO:bovine serum albumin (DHEA-7-O-CMO-BSA) or 5-androstene-3 beta-ol-17-one hemisuccinate-bovine serum albumin (DHEA-3HS-BSA) conjugates and monoclonal antibodies were produced, characterized, and selected for maximum DHEAS binding. Of these hybridomas, four clones from DHEA-3HS-BSA-immunized mice had acceptable criteria for the development of a competitive enzyme-linked immunosorbent assay (ELISA) for DHEAS in plasma. One hybridoma supernatant from DHEA-7-O-CMO-BSA-immunized mice showed 360% cross-reactivity to both androsterone sulfate and epiandrosterone sulfate. This allows the possibility of the direct determination of androsterone sulfate and epiandrosterone sulfate in plasma after correction for the DHEAS contribution. Both ELISAs employ a DHEA-3HS-thyroglobulin conjugate adsorbed to the wells of a standard 96-well microtiter plate. DHEAS in the standards or diluted plasma sample competes with immobilized DHEA-3HS-thyroglobulin for antibody-binding sites. Antibody is detected with anti-mouse-lg peroxidase by further washing, adding o-phenylenediamine substrate, and reading the absorbance at 492 nm. The ELISAs are simple, reproducible, and reliable and, to our knowledge, they are the first tests employing monoclonal antibodies to DHEAS.

Re-examining steroid hormone metabolites as ovulation markers using monoclonal antibodies.

Plasma progesterone and plasma estradiol levels are commonly used to monitor ovulation in women although for the adequate documentation of ovulation the expense and discomfort of multiple venipuncture sampling may be required. Accurate and definitive information on ovulation in women can be obtained by the simple measurement of metabolites of progesterone and estradiol in early morning urine samples. These analyses have been made possible by the use of our own highly specific monoclonal antibodies to both pregnanediol-3-glucuronide (P-3-G) and estrone-3-glucuronide (E-3-G) and the development of simple, direct, automated enzyme-linked immunosorbent assays. Sequential sampling and the generation of ovulation profiles allows detection of ovulation and identification of the infertile/fertile phases of the cycle for either planned pregnancies or natural family planning. Aberrations in ovulation are easily detected as is documentation of the transition to menopause. The use of sequential, spaced, early morning urine samples for P-3-G and E-3-G allows accurate assessment of ovulatory function rather than relying on the usual single plasma sampling. The data presented also show that the direct determination of plasma pregnanediol-3-glucuronide in plasma is as informative as plasma progesterone measurement.

Production of a monoclonal antibody to cortisol: application to a direct enzyme-linked immunosorbent assay of plasma.

Cortisol mouse monoclonal antibodies were produced and characterized. Of the four clones studied, supernatant from one clone (A2), compared with other cortisol monoclonal antibodies, showed minimal cross-reactivity to other C21 steroids and was suitable for the direct determination of cortisol in plasma by enzyme-linked immunosorbent assay using a standard 96-well microtiter plate. The enzyme-linked immunosorbent assay uses the immobilized antigen approach, in which cortisol in plasma samples or standards competes with immobilized steroid for antibody-binding sites. After washing, the cortisol antibody bound to the wells of the microtiter plate is detected with antimouse immunoglobulin conjugated to horseradish peroxidase. Following further washing, o-phenylenediamine substrate is added. The enzyme-linked immunosorbent assay is robust and semiautomated. The mean +/- SD recovery from plasma was 97% +/- 6%. Precision studies on three different plasma pools showed mean coefficients of variation of 7.6% and 8.6% for within- and between-assay variation, respectively. The satisfactory performance criteria allow its use in the routine laboratory.