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.

Plasma variation of corticosteroid-binding globulin and sex hormone-binding globulin.

Sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) circulate in plasma and bind their cognate ligands with high affinity, offering a steroid delivery system to target tissues by a variety of mechanisms. Analysis of these steroid-binding proteins is gaining importance in the clinical setting, although more information is warranted on their diurnal and biological variation. This study shows that plasma SHBG (in normal subjects) exhibits little diurnal or biological variation over the 30 day period studied, in contrast to CBG, where plasma levels peak in the early afternoon. This leads to attenuation of the diurnal free cortisol level rhythm compared to total cortisol. We also show that plasma CBG is significantly lower in male subjects with the metabolic syndrome compared to age-matched lean counterparts, and may therefore act as a surrogate marker of insulin resistance. The consequence of lower levels of CBG in these obese male subjects is reflected by higher levels of circulating free cortisol, potentially offering a more favourable environment for adipogenesis.

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.

Plasma sex hormone-binding globulin rather than corticosteroid-binding globulin is a marker of insulin resistance in obese adult males.

AIM: Plasma levels of corticosteroid-binding globulin (CBG) and sex hormone-binding globulin (SHBG) may be regulated by insulin. The aim of this study was to test the hypothesis that these steroid-binding proteins are markers of insulin resistance and obesity in adult patients with the metabolic syndrome. METHODS: Fasting blood samples were obtained from 108 male and 88 female overweight adult patients who had varying degrees of dyslipidaemia, adiposity and insulin resistance. We measured plasma levels of SHBG and CBG and investigated their correlation with insulin resistance [homeostasis model assessment (HOMA) % sensitivity] and anthropometric markers of adiposity. RESULTS: In male patients, plasma SHBG correlated positively with HOMA (% sensitivity) and negatively with anthropometric measurements, including body mass index, waist circumference (cm) and percentage body fat. There was no correlation with CBG and any other parameter in the male patients. The female patients were treated as two groups, those not using oral contraceptives or hormone replacement therapy (n = 67) and those taking steroid medications (n = 21). Female patients using steroid medications had significantly higher SHBG levels but neither group showed any correlation between SHBG, insulin resistance and adiposity. Correlation studies of CBG with other parameters in the female subgroups did not reach statistical significance. CONCLUSIONS: We conclude that plasma SHBG is another surrogate marker for insulin resistance in obese males but not in obese females. It also appears that plasma CBG is not a useful marker of insulin resistance in patients with the metabolic syndrome.

Plasma adiponectin in overweight, nondiabetic individuals with or without insulin resistance.

AIM: Adiponectin is a protein produced exclusively by adipocytes with putative insulin-sensitizing and anti-atherogenic properties. This cross-sectional study investigated the relationship between plasma adiponectin and a range of anthropometric, glycaemic, lipid and inflammatory parameters in overweight and obese subjects expressing characteristics of the metabolic syndrome. METHODS: Subjects were selected for the study from a clinical database, if they were non-diabetic, overweight [body mass index (BMI) > 25] and had features of the metabolic syndrome. The subjects were grouped according to BMI (25-30, 31-35 and >35 kg/m2) and then stratified for insulin resistance [homeostasis model assessment (HOMA) %S]. One hundred and ninety-seven patients (109 males and 88 females) were selected for the study by taking an equal number with the highest and lowest HOMA indices from each of the three BMI groups. Plasma adiponectin concentration was measured in duplicate by radioimmunoassay, and the relationship between these levels and the other parameters was investigated using correlation and multiple linear regression analyses. RESULTS: Plasma adiponectin concentration was higher in females than males (median 10.3 vs. 7.1 micro g/ml, p < 0.001) despite being matched for BMI. In both genders, adiponectin levels were inversely related to BMI, waist circumference, percentage body fat, insulin resistance and the fasting plasma concentration of leptin. A direct correlation in both sexes was found between adiponectin levels and high-density lipoprotein (HDL)-cholesterol, apolipoprotein A1 and age. Multiple linear regression analyses showed that the independent determinants of low plasma adiponectin concentrations were gender, age, BMI, insulin resistance and HDL-cholesterol. An association between reduced adiponectin and increased high-sensitivity plasma C-reactive protein concentration was observed only in female subjects and was independent of anthropometric variables. Our observation that adiponectin levels increase with age differs from the majority of other studies and may simply reflect the demographics of the population studied. CONCLUSIONS: This study shows that adiponectin is an important molecular link between obesity, insulin resistance and atherogenic lipoproteins. It is possible that plasma adiponectin concentration may be a convenient marker for identifying subjects with the metabolic syndrome who may progress to impaired glucose tolerance. Longitudinal studies are required in order to verify this clinical application of adiponectin.

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.

Partial allelotype of schistosomiasis-associated bladder cancer.

In Egypt and other regions of the Middle East where the trematode Schistosoma haematobium is endemic, bladder cancer is the most common adult cancer. Unlike bladder cancers in Western countries, which are predominantly transitional-cell carcinoma (TCC), these schistosomiasis-associated bladder cancers are predominantly squamous-cell carcinoma (SCC). Our aim was to assess a large series of schistosomiasis-associated bladder tumours for genetic alterations commonly found in TCC in the United Kingdom and the United States. We have carried out a partial allelotype of 70 tumours from patients with schistosomiasis. LOH was found on all chromosome arms studied (3p, 4p, 4q, 8p, 9p, 9q, 11p, 11q, 13q, 14q, 17p, 18q). The most frequent regions of LOH were 9p (65%), 17p (58%), 3p (40%), 9q (39%) and 8p (37%). LOH on 17p, where the TP53 gene is located, was more common in Egyptian TCC than in SCC. Similarly, 8p LOH was more common in TCC than SCC. The most striking difference between this group of tumours and TCCs from the United Kingdom and the United States was the high frequency of 9p LOH in the region of the CDKN2 gene (65%) and the relatively low frequency of 9q LOH (39%); 15 of 43 tumours with LOH of at least one marker on chromosome 9 showed LOH of 9p only. This suggests that a 9p gene, possibly CDKN2, may contribute to the development of the majority of schistosomiasis-associated bladder tumours but that genes on 9q play a much less important role.

Structure and methylation-based silencing of a gene (DBCCR1) within a candidate bladder cancer tumor suppressor region at 9q32-q33.

Loss of heterozygosity (LOH) on chromosome 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder, indicating the presence of one or more relevant tumor suppressor genes. We previously mapped one of these putative tumor suppressor loci to 9q32-q33 and localized the candidate region within a single YAC 840 kb in size. This locus has been designated DBC1 (for deleted in bladder cancer gene 1). We have identified a novel gene, DBCCR1, in this candidate region by searching for expressed sequence tags (ESTs) that map to YACs spanning the region. Database searching using the entire DBCCR1 cDNA sequence identified several human ESTs and a few homologous mouse. ESTs. However, the predicted 761-amino-acid sequence had no significant homology to known protein sequences. Mutation analysis of the coding region and Southern blot analysis detected neither somatic mutations nor gross genetic alterations in primary TCCs. Although DBCCR1 was expressed in multiple normal human tissues including urothelium, mRNA expression was absent in 5 of 10 (50%) bladder cancer cell lines. Methylation analysis of the CpG island at the 5' region of the gene and the induction of de novo expression by a demethylating agent indicated that this island might be a frequent target for hypermethylation and that hypermethylation-based silencing of the gene occurs in TCC. These findings make DBCCR1 a good candidate for DBC1.

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.

A monoclonal antibody recognizing the chlorohydrin derivatives of oleic acid for probing hypochlorous acid involvement in tissue injury.

A monoclonal antibody against hypochlorous acid-modified oleic acid has been raised to investigate involvement of HOCI in tissue injury. Mice were immunized with an isomeric mixture of chlorohydrin derivatives of oleic acid (18:0-chlorohydrin) conjugated to keyhole limpet haemocyanin (CH-KLH). The chlorohydrin was formed by the treatment of oleic acid with hypochlorous acid. Monoclonal antibodies were raised and the fusion was screened with 18:0-chlorohydrin-bovine serum albumin (CH-BSA) conjugate. A number of antibody-secreting clones were identified and the supernatants were characterized by binding studies and dose-response curves. In ELISA, mAb CH-1 had an equivalent titre when either the chlorohydrin or bromohydrin derivative of oleic acid, complexed to bovine serum albumin, was used as screening antigen. The mAb CH-1 recognition of CH-BSA was competed with chlorohydrin and bromohydrin conjugates of BSA and KLH. Similarly, free 18:0-chlorohydrin and the 18:0-chlorohydrin-phosphatidyl choline treated with hypochlorous acid competed with mAb CH-1 binding. The mAb CH-1 also recognised the chlorohydrin derivative of linoleic acid and chlorohydrin formed from palmitoyl, oleyl phosphatidyl choline but with a decreased avidity. Weak cross-reactivity was observed with hydroxy-linoleic acid and linoleic acid hdroperoxide, either as free fatty acid or in phosphatidyl choline. There was minimal competitive binding of mAb CH-1 to free oleic acid, 16:0/18:1 phosphatidylcholine, cholesterol, or cholesterol chlorohydrin. The mAb CH-1 described here may be a useful probe for assessing the involvement of hypochlorous acid in tissue injury.

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.

High frequency of chromosome 9 deletion in ovarian cancer: evidence for three tumour-suppressor loci.

We have screened 33 ovarian tumours of various grades and stages for the loss of heterozygosity (LOH) of markers on chromosome 9. LOH was detected in 26 cases (79%). Eleven tumours (33%) showed LOH of all informative markers. The remaining 15 cases had partial deletions. Of these, six (18%) had losses on 9p only, three (9%) had LOH confined to 9q and six (18%) had losses on both chromosome arms, four of which had a retention of hetereozygosity in between. There was no association between tumour grade stage or histopathology and any losses. High-density deletion mapping was carried out in 12 selected cases that had partial deletions of 9p and/or 9q. The deleted region on 9p included the cyclin-dependent kinase inhibitor 2 (CDKN2) locus and one tumour was found to have a homozygous deletion of CDKN2. LOH on 9q extended over a larger region. We found evidence for two regions of deletion on 9q, one at 9q34 and the other encompassing the nevoid basal cell carcinoma (Gorlin) syndrome locus on proximal 9q.

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.