Comparison of steroid and thyroid hormone concentrations in blood serum and plasma of captive tigers.

Analysis of steroid and thyroid hormones is often performed in blood serum. Occasionally though, plasma samples are submitted in lieu of serum for exotic species such as tigers. However, blood tube anticoagulants may affect hormone values. We compared serum and heparin plasma results for 7 hormones in tigers. Serum and plasma samples were collected from 25 tigers and analyzed for progesterone, 17-hydroxyprogesterone, cortisol, androstenedione, testosterone, estradiol, and thyroxine. Using Lin concordance correlation, serum and heparin plasma measures agreed for all hormones except cortisol. However, Passing-Bablok regression only found agreement between serum and heparin plasma measures for androstenedione, testosterone, and estradiol. Median values between the 2 sample types were significantly (p < 0.05) different for progesterone, 17-hydroxyprogesterone, cortisol, and thyroxine. Our results suggest that, for the aforementioned hormones, serum and heparin plasma values may not always be comparable.

Analytic performance evaluation of a veterinary-specific ELISA for measurement of serum cortisol concentrations of dogs.

OBJECTIVE To investigate the precision of an ELISA for measurement of serum cortisol concentration (SCC) in dogs, assess agreement between this ELISA and 2 validated chemiluminescence assays (CLAs), and evaluate the clinical implications of any bias associated with this ELISA when measuring SCC in dogs. DESIGN Evaluation study. SAMPLE 75 stored, frozen serum samples from client-owned dogs. PROCEDURES Enzyme-linked immunosorbent assay precision was evaluated by measuring SCC of pooled serum samples. Agreement with standard methods was evaluated with Spearman rank correlation, Passing-Bablok regression, and Bland-Altman analysis to compare SCCs obtained with the ELISA and the 2 CLAs. An error grid was used to evaluate identified bias. RESULTS Within-laboratory coefficients of variation for pooled serum samples with low, medium, and high SCCs were 21.4%, 28.9%, and 13.0%, respectively. There was a high correlation between ELISA results (for all samples combined) and results of the 2 CLAs (CLA 1, r = 0.96; CLA 2, r = 0.97), but constant and proportional biases between the ELISA and CLAs were present at all concentrations. Clinically important disagreement between ELISA results and CLA results occurred in 16 of 63 (25%) samples, particularly with low and high SCCs. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that the rate of clinical disagreement between the ELISA and CLAs was sufficiently high to recommend that equivocal results obtained with the ELISA be confirmed by a reference laboratory. Further evaluation of analytic performance of the ELISA should focus on samples with very high and very low SCCs.

Early life triclocarban exposure during lactation affects neonate rat survival.

Triclocarban (3,4,4'-trichlorocarbanilide; TCC), an antimicrobial used in bar soaps, affects endocrine function in vitro and in vivo. This study investigates whether TCC exposure during early life affects the trajectory of fetal and/or neonatal development. Sprague Dawley rats were provided control, 0.2% weight/weight (w/w), or 0.5% w/w TCC-supplemented chow through a series of 3 experiments that limited exposure to critical growth periods: gestation, gestation and lactation, or lactation only (cross-fostering) to determine the susceptible windows of exposure for developmental consequences. Reduced offspring survival occurred when offspring were exposed to TCC at concentrations of 0.2% w/w and 0.5% w/w during lactation, in which only 13% of offspring raised by 0.2% w/w TCC dams survived beyond weaning and no offspring raised by 0.5% w/w TCC dams survived to this period. In utero exposure status had no effect on survival, as all pups nursed by control dams survived regardless of their in utero exposure status. Microscopic evaluation of dam mammary tissue revealed involution to be a secondary outcome of TCC exposure rather than a primary effect of compound administration. The average concentration of TCC in the milk was almost 4 times that of the corresponding maternal serum levels. The results demonstrate that gestational TCC exposure does not affect the ability of dams to carry offspring to term but TCC exposure during lactation has adverse consequences on the survival of offspring although the mechanism of reduced survival is currently unknown. This information highlights the importance of evaluating the safety of TCC application in personal care products and the impacts during early life exposure.

Clinical features of progressive vacuolar hepatopathy in Scottish Terriers with and without hepatocellular carcinoma: 114 cases (1980-2013).

OBJECTIVE: To characterize signalment, clinical features, clinicopathologic variables, hepatic ultrasonographic characteristics, endocrinologic profiles, treatment response, and age at death of Scottish Terriers with progressive vacuolar hepatopathy (VH) with or without hepatocellular carcinoma (HCC). DESIGN: Retrospective case series. ANIMALS: 114 Scottish Terriers with progressive VH. PROCEDURES: Electronic databases from 1980 to 2013 were searched for adult (age > 1 year) Scottish Terriers with histopathologic diagnoses of diffuse glycogen-like VH. Available sections of liver specimens were histologically reevaluated to confirm diffuse VH with or without HCC; 8 dogs with HCC only had neoplastic tissue available. Physical examination, clinicopathologic, treatment, and survival data were obtained. RESULTS: 39 of 114 (34%) dogs with VH had HCC detected at surgery or necropsy or by abdominal ultrasonography. Histologic findings indicated that HCC was seemingly preceded by dysplastic hepatocellular foci. No significant differences were found in clinicopathologic variables or age at death between VH-affected dogs with or without HCC. Fifteen of 26 (58%) dogs with high hepatic copper concentrations had histologic features consistent with copper-associated hepatopathy. Although signs consistent with hyperadrenocorticism were observed in 40% (46/114) of dogs, definitive diagnosis was inconsistently confirmed. Assessment of adrenal sex hormone concentrations before and after ACTH administration identified high progesterone and androstenedione concentrations in 88% (22/25) and 80% (20/25) of tested dogs, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that VH in Scottish Terriers may be linked to adrenal steroidogenesis and a predisposition to HCC. In dogs with VH, frequent serum biochemical analysis and ultrasonographic surveillance for early tumor detection are recommended.

Effect of combined lignan phytoestrogen and melatonin treatment on secretion of steroid hormones by adrenal carcinoma cells.

OBJECTIVE: To investigate the in vitro effect of the combination of lignan enterolactone (ENL) or lignan enterodiol (END) with melatonin on steroid hormone secretion and cellular aromatase content in human adrenal carcinoma cells. SAMPLE: Human adrenocortical carcinoma cells. PROCEDURES: Melatonin plus ENL or END was added to cell culture medium along with cAMP (100µM); control cells received cAMP alone. Medium and cell lysates were collected after 24 and 48 hours of cultivation. Samples of medium were analyzed for progesterone, 17-hydroxyprogesterone, androstenedione, aldosterone, estradiol, and cortisol concentration by use of radioimmunoassays. Cell lysates were used for western blot analysis of aromatase content. RESULTS: The addition of ENL or END with melatonin to cAMP-stimulated cells (treated cells) resulted in significant decreases in estradiol, androstenedione, and cortisol concentrations at 24 and 48 hours, compared with concentrations in cells stimulated with cAMP alone (cAMP control cells). The addition of these compounds to cAMP-stimulated cells also resulted in higher progesterone and 17-hydroxyprogesterone concentrations than in cAMP control cells; aldosterone concentration was not affected by treatments. Compared with the content in cAMP control cells, aromatase content in treated cells was significantly lower. CONCLUSIONS AND CLINICAL RELEVANCE: The combination of lignan and melatonin affected steroid hormone secretion by acting directly on adrenal tumor cells. Results supported the concept that this combination may yield similar effects on steroid hormone secretion by the adrenal glands in dogs with typical and atypical hyperadrenocorticism.

Blood steroid concentrations in domestic Mongolian horses.

Traditionally, analysis of blood cortisol alone has been used to evaluate adrenal function. Currently, multisteroid analyses are considered more informative than analysis of a single hormone to assess adrenal function. The objective of the present research was to create a database for steroid reference values for domestic Mongolian horses. Seven adrenal steroid levels were determined in the blood of 18 colts, 34 stallions, 25 geldings, 17 fillies, and 29 mares. Results were as follows (lowest and highest group median, in nanograms per milliliter): progesterone: <0.030 (fillies), 4.30 (mares), and 0.070 (all horses); 17-OH-progesterone: 0.070 (colts), 0.520 (mares), and 0.110 (all horses); androstenedione: 0.101 (colts), 0.256 (stallions), and 0.181 (all horses); testosterone: <0.040 (mares, stallions, and fillies), 0.040 (geldings and colts), and <0.40 (all horses); estradiol: 0.066 (stallions), 0.093 (fillies), and 0.085 (all horses); cortisol: 23.040 (colts), 70.210 (geldings), and 50.770 (all horses); and aldosterone: 0.018 (colts), 0.297 (geldings), and 0.191 (all horses). Overall medians indicate that cortisol (98.70%) is the predominant steroid, followed by aldosterone (0.37%), androstenedione (0.35%), 17-OH-progesterone (0.21%), estradiol (0.17%), progesterone (0.14%), and testosterone (0.06%). This information provides adrenal and gonadal steroid reference concentrations to assist in physiological characterization and diagnosis of endocrine disorders in domestic Mongolian horses.

Comparison of testis structure, function and thyroid hormone levels in control C57BL/6 mice and anti-mullerian hormone over expressing mice.

Anti-Mullerian hormone (AMH) is considered as a negative regulator of postnatal Leydig cell (LC) differentiation, because AMH over expressing mice (Mt-hAMH mice) testes are deficient in LC. Therefore, in the present study Mt-hAMH mice was used as a model to examine the process of postnatal LC differentiation. Testis structure-function studies were performed in age-matching Mt-hAMH and C57BL/6 (controls) mice; testicular components were quantified and circulating testosterone and thyroid hormone levels (thyroxine/T4 and triiodothyronine/T3; necessary for postnatal LC differentiation) were determined. Results revealed that Mt-hAMH mice were heavier and their testis weights were smaller compared to controls. Mast cells were present in Mt-AMH testis interstitium, but absent in controls. The absolute volumes of seminiferous tubules (ST), testis interstitium, LC and blood vessels per testis were lower and lymphatic space was higher in Mt-hAMH mice than in controls (p<0.05). The average cell LC volume and their number per testis, ST length, plasma testosterone, luteinizing hormone-stimulated testosterone secretion per testis and per LC in vitro, plasma T4 and T3 were significantly lower in Mt-hAMH mice compared to controls (p<0.05). Increased body weight in Mt-hAMH mice could be attributed to the reduced T4 and T3. Reduced testis weight in Mt-AMH mice is explained by the reduced ST volume in them. Reduced plasma testosterone, testicular and LC testosterone secretion in vitro in Mt-hAMH mice can be explained by the reduced number, size and steroidogenic potential of LC in Mt-hAMH mice. Study revealed several structure-function deficiencies in Mt-AMH mouse compared to controls, which were not documented in previous investigations. As hypothyroidism causes arrest in postnatal LC differentiation, it is suggested that the reduced LC number in Mt-hAMH testes could be at least in part due to their reduced thyroid hormone levels. However, latter concept needs to be further tested in future investigations.

Physiologic assessment of blood glucose homeostasis via combined intravenous glucose and insulin testing in horses.

OBJECTIVE: To characterize the physiologic response to i.v. bolus injection of glucose and insulin for development of a combined glucose-insulin test (CGIT) in horses. ANIMALS: 6 healthy mares and 1 mare each with pituitary adenoma and urolithiasis. PROCEDURE: Horses were given a CGIT (glucose, 150 mg/kg; insulin, 0.1 U/kg); results were compared with a singular i.v. glucose tolerance test (GTT; 150 mg/kg) and a singular i.v. insulin sensitivity test (IST; 0.1 U/kg). Healthy horses were also given a CGIT after receiving xylazine and undergoing stress. RESULTS: Physiologically, the CGIT resulted in a 2-phase curve with positive (hyperglycemic) and negative (hypoglycemic) portions; the positive phase came first (250% of baseline at 1 minute). The descending segment declined linearly to baseline by approximately 30 minutes and to a nadir at 58% of baseline by 75 minutes. After a 35-minute valley, a linear ascent to baseline began. Addition of insulin in the CGIT increased glucose utilization by approximately 4.5 times during the positive phase but not during the negative phase. The diseases' effects and experimental inhibition of insulin secretion with xylazine and stress were detectable by use of the 2 phases of the CGIT. Only a single positive phase resulted from the GTT and a single negative phase from the IST CONCLUSIONS AND CLINICAL RELEVANCE: The CGIT resulted in a consistent, well-defined glycemia profile, which can be disrupted experimentally or by a disease process. The CGIT has clinical potential because it provides integrated information and more information than either the singular GTT or IST.

Inhibition of collagenase breakdown of equine corneas by tetanus antitoxin, equine serum and acetylcysteine.

OBJECTIVE: To determine whether tetanus antitoxin, equine serum, and acetylcysteine, which are currently used in the treatment of equine corneal ulcer, inhibit the digestion of equine corneal collagen when exposed to collagenase in vitro. ANIMALS STUDIED: Corneas from 40 adult horses. PROCEDURES: Sections of equine corneas were incubated with saline, a solution of bacterial collagenase in saline, bacterial collagenase in saline plus equine tetanus antitoxin, bacterial collagenase in saline plus equine serum, or bacterial collagenase in saline plus acetylcysteine. Each one of the collagenase inhibitors was tested at different concentrations. The degree of corneal collagen digestion was determined by concentrations of hydroxyproline released into the incubation media and/or by weight loss of the cornea. RESULTS: Corneas exposed to collagenase released a significant (0.05 level) large amount of hydroxyproline (43.1 +/- 2.3 microg/mL/100 mg cornea/5 h) and decreased cornea weight by up to 89%. Blood serum (200 microL/mL), purified albumin or globulin fractions of serum, tetanus antitoxin (120 units/mL), and acetylcysteine (20 mg/mL) when used at the highest concentrations blocked collagenase digestive activity by approximately 50%. Dilution of inhibitors decreased corneal protection and linearly increased corneal weight loss. Purified equine serum albumin and globulin fractions were equally effective in protecting corneas. CONCLUSIONS: This experiment indicates that tetanus antitoxin, serum and acetylcysteine equally protected corneas from collagenase digestion, in vitro. However, a clinical trial is needed to establish relative therapeutic value.

Differential modulation of signaling pathways and apoptosis of ras-transformed 10T1/2 cells by the depsipeptide FR901228.

(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone (FR901228), a natural anticancer depsipeptide, induces apoptosis of ras-transformed 10T1/2 cells whereas it induces growth arrest of nontransformed counterpart cells in G0/G1 phase of the cell cycle. Our study of the effect of FR901228 treatment on intracellular signaling pathways reveals a discriminating activity of FR901228 to regulate signaling cascades differently in ras-transformed 10T1/2 cells and nontransformed counterpart cells. Induction of apoptosis of ras-transformed cells by FR901228 correlates with suppression of the extracellular signal-regulated kinase (ERK) signaling pathway through reduction of Raf expression and deactivation of Mek and Erk, inhibition of the phosphoinositide-3 kinase (PI3-K) pathway indexed by suppression of Akt activity, suppression of p38 activity, and activation of caspase-3. Expression of p21(Cip1) is not induced in ras-transformed cultures undergoing apoptosis induced by FR901228. In contrast, FR901228 induces p21(Cip1) expression in nontransformed counterpart cultures growth-arrested in G0/G1 that is also accompanied by moderate induction of the kinase activities of Raf, Mek, Erk, and Akt, but not accompanied by activation of caspase-3 or changes in p38 activity. Our study indicates a potential value of FR901228 in the treatment of cancer cells involving aberrant regulation of Ras through preferential induction of the caspase cascade and suppression of the ERK, PI3-K, and p38 pathways.