Multifocal, Asymmetric Bilateral Primary Aldosteronism Cannot be Excluded by Strong Adrenal Vein Sampling Lateralization: An International Retrospective Cohort Study.

BACKGROUND: Primary aldosteronism (PA) has been broadly dichotomized into unilateral and bilateral forms. Adrenal vein sampling (AVS) lateralization indices (LI) ≥2 to 4 are the standard-of-care to recommend unilateral adrenalectomy for presumed unilateral PA. We aimed to assess the rates and characteristics of residual PA after AVS-guided adrenalectomy. METHODS: We conducted an international, retrospective, cohort study of patients with PA from 7 referral centers who underwent unilateral adrenalectomy based on LI≥4 on baseline and/or cosyntropin-stimulated AVS. Aldosterone synthase (CYP11B2) immunohistochemistry and next generation sequencing were performed on available formalin-fixed paraffin-embedded adrenal tissue. RESULTS: The cohort included 283 patients who underwent AVS-guided adrenalectomy, followed for a median of 326 days postoperatively. Lack of PA cure was observed in 16% of consecutive patients, and in 22 patients with lateralized PA on both baseline and cosyntropin-stimulated AVS. Among patients with residual PA postoperatively, 73% had multiple CYP11B2 positive areas within the resected adrenal tissue (versus 23% in those cured), wherein CACNA1D mutations were most prevalent (63% versus 33% in those cured). In adjusted regression models, independent predictors of postoperative residual PA included Black versus White race (odds ratio, 5.10 [95% CI, 1.45-17.86]), AVS lateralization only at baseline (odds ratio, 8.93 [95% CI 3.00-26.32] versus both at baseline and after cosyntropin stimulation), and CT-AVS disagreement (odds ratio, 2.75 [95% CI, 1.20-6.31]). CONCLUSIONS: Multifocal, asymmetrical bilateral PA is relatively common, and it cannot be excluded by robust AVS lateralization. Long-term postoperative monitoring should be routinely pursued, to identify residual PA and afford timely initiation of targeted medical therapy.

Use of overnight metyrapone test in suspected secondary adrenal insufficiency: A retrospective single centre-study.

OBJECTIVE: Overnight metyrapone test (OMT) is a dynamic test used to diagnose secondary adrenal insufficiency (SAI). Data on OMT use and its safety are scarce. We aimed to describe the indications and safety of outpatient OMT and compare OMT to the cosyntropin stimulation test (CST). DESIGN: Single-centre retrospective study of adult patients undergoing OMT between 1 April 2018 and 27 January 2023. MEASUREMENTS: OMT-related adverse events, post-OMT diagnosis of SAI, and OMT comparison to CST. RESULTS: OMT was performed in 114 patients (81, 71% women) at a median age of 48 (interquartile range 37-58). The pretest probability for SAI was low in 52 (46%) patients, moderate in 48 (42%) patients and high in 14 (12%) patients. Adverse events were reported in 7 (6.1%) patients and were mild except for one hospitalization. No baseline or OMT-related factors were associated with the development of adverse events. Prevalence of the OMT-based SAI diagnosis was 26 (23%) and 47 (46%) using 11-deoxycortisol cutoff <7 and <10 mcg/dL, respectively. Higher pretest probability was associated with the OMT-based diagnosis of SAI. Post-OMT 11-deoxycortisol cutoff of 10 mcg/dL was used most to diagnose SAI. Compared to the OMT-based diagnosis of SAI (11-deoxycortisol cutoff of 10 mcg/dL), the specificity of CST was 100%, but the sensitivity was only 52%. CONCLUSIONS: OMT was well tolerated and used in patients with low and moderate pretest probability for SAI. CST can erroneously exclude patients with SAI. Thus, OMT should be considered in selected patients with normal CST.

Adrenocortical hemorrhage following intravenous tetracosactide in a dog with hypercortisolism.

OBJECTIVE: To summarize findings from a case of adrenocortical hemorrhage following tetracosactide injection during ACTH stimulation testing for monitoring of trilostane therapy in a dog. ANIMAL: A 12-year old neutered male dog with adrenal-dependent hypercortisolism. CLINICAL PRESENTATION, PROGRESSION, AND PROCEDURES: 4 hours after ACTH stimulation testing, the patient developed vomiting, lethargy, and abdominal pain. Abdominal ultrasound was performed before and after an ACTH stimulation test. Following ACTH stimulation testing, there was progressive bilateral adrenal enlargement and free abdominal fluid had developed. This was considered to be caused by adrenocortical inflammation and hemorrhage secondary to the synthetic ACTH analog, tetracosactide, used during stimulation testing. A resting cortisol performed 5 hours after tetracosactide injection was not consistent with iatrogenic hypoadrenocorticism. TREATMENT AND OUTCOME: The patient was managed with analgesia, IV fluids, and corticosteroids and made a full recovery. CLINICAL RELEVANCE: To the authors' knowledge, this was the first reported case of adrenocortical hemorrhage following administration of a synthetic ACTH analog in a dog. This should be considered as a rare potential complication of ACTH stimulation testing.

Performance of synacthen test in chronic hemodialysis patients.

BACKGROUND: Adrenal function tests (Synacthen test) in chronic hemodialysis (HD) patients are currently performed off dialysis. The study aimed to demonstrate equivalence of serum cortisol concentrations pre- and during HD, each for standard-dose (250 µg) and low-dose (1 µg) Synacthen test. METHODS: In a single-center cross-over diagnostic equivalence study, Synacthen tests were performed in four settings, in standard- and low-dose as well as pre- and during HD. Serum cortisol concentration was measured at 30 and 60 min after Synacthen administration, and additionally at 20 min in low dose test. Based on a multivariable linear mixed model the means of cortisol concentration on log-scale were estimated in each dose and test time combination. Differences in means were calculated and the TOST approach was applied to test for equivalence. Equivalence was proven if the 90% confidence interval of the difference of two cortisol means was entirely between - 0.22 and 0.22. RESULTS: In 28 chronic HD patients, serum cortisol concentrations at 30 and 60 min after Synacthen administration in both standard- and low-dose were shown to be equivalent pre- and during HD. In 10 of 56 low-dose tests, the cortisol peak was already reached after 20 min. However, cortisol concentrations at 20 and 30 min after low-dose Synacthen test pre- and during HD showed no significant difference. CONCLUSION: These results suggest that the adrenal function test may be carried out during an ongoing HD session, leading to a more patient-friendly performance of the test, less organizational effort and potentially earlier diagnosis of adrenal insufficiency.

Altered biomarkers for cardiovascular disease and inflammation in autoimmune Addison's disease - a cross-sectional study.

OBJECTIVE: Increased prevalence of cardiovascular disease has been reported in autoimmune Addison's disease (AAD), but pathomechanisms are poorly understood. DESIGN: Cross-sectional study. METHODS: We compared serum levels of 177 cardiovascular and inflammatory biomarkers in 43 patients with AAD at >18-h glucocorticoid withdrawal and 43 matched controls, overall and stratified for sex. Biomarker levels were correlated with the frequency of adrenal crises and quality of life (QoL) by AddiQoL-30. Finally, we investigated changes in biomarker levels following 250 µg tetracosactide injection in patients without residual adrenocortical function (RAF) to explore glucocorticoid-independent effects of high ACTH. RESULTS: Nineteen biomarkers significantly differed between patients with AAD and controls; all but 1 (ST1A1) were higher in AAD. Eight biomarkers were significantly higher in female patients compared with controls (IL6, MCP1, GAL9, SPON2, DR4, RAGE, TNFRSF9, and PGF), but none differed between male patients and controls. Levels of RAGE correlated with the frequency of adrenal crises (r = 0.415, P = .006) and AddiQoL-30 scores (r = -0.347, P = .028) but not after correction for multiple testing. PDL2 and leptin significantly declined 60 min after injection of ACTH in AAD without RAF (-0.15 normalized protein expression [NPX], P = .0001, and -0.25 NPX, P = .0003, respectively). CONCLUSIONS: We show that cardiovascular and inflammatory biomarkers are altered in AAD compared with controls, particularly in women. RAGE might be a marker of disease severity in AAD, associated with more adrenal crises and reduced QoL. High ACTH reduced PDL2 and leptin levels in a glucocorticoid-independent manner but the overall effect on biomarker profiles was small.

A 65-Year-Old Man with Bilateral Adrenal Hemorrhage Following Prophylaxis for Postoperative Deep Vein Thrombosis with Rivaroxaban.

BACKGROUND Direct oral anticoagulant (DOAC) agents, such as rivaroxaban, treat and prevent venous thrombosis. Although adrenal hemorrhage due to DOACs has previously been reported, this is a rare condition that can present as an emergency. In this case report, we present a 65-year-old man who recently had bilateral knee arthroplasty and was started on rivaroxaban 10 mg daily for deep vein thrombosis (DVT) prophylaxis following the surgery. CASE REPORT Ten days after bilateral knee arthroplasty and starting rivaroxaban, the patient presented to the Emergency Department with severe, sudden abdominal pain. Abdominal computed tomography detected significantly enlarged bilateral adrenals, with ill-defined heterogeneous density extending to the upper part of perinephric and paranephric spaces, suggesting bilateral adrenal hemorrhage. A cosyntropin stimulation test was used to confirm the suspicion of adrenal insufficiency. Cortisol levels were 66 nmol/L before stimulation and 83 nmol/L 60 min after cosyntropin administration. Hydrocortisone was started intravenously at a dose of 50 mg every 8 h. After his symptoms improved, he was discharged on oral hydrocortisone at 10 mg in the morning and 5 mg in the evening. Seven weeks after discharge, follow-up abdominal ultrasonography showed that the bilateral adrenal hemorrhage had resolved. CONCLUSIONS This case supports previous cases of adrenal hemorrhage as a rare but serious association with rivaroxaban and highlights the importance of rapid diagnosis using imaging and monitoring of patients for this possible adverse effect. Practitioners must remain vigilant when prescribing anticoagulation therapy, especially in patients who are at an increased risk for adrenal hemorrhage.

Role of cosyntropin in the management of postpartum post-dural puncture headache: a two-center retrospective cohort study.

BACKGROUND: Research suggests that postpartum post-dural puncture headache (PDPH) might be prevented or treated by administering intravenous cosyntropin. METHODS: In this retrospective cohort study, we questioned whether prophylactic (1 mg) and therapeutic (7 µg/kg) intravenous cosyntropin following unintentional dural puncture (UDP) was effective in decreasing the incidence of PDPH and therapeutic epidural blood patch (EBP) after birth. Two tertiary-care American university hospitals collected data from November 1999 to May 2017. Two hundred and fifty-three postpartum patients who experienced an UDP were analyzed. In one institution 32 patients were exposed to and 32 patients were not given prophylactic cosyntropin; in the other institution, once PDPH developed, 36 patients were given and 153 patients were not given therapeutic cosyntropin. The primary outcome for the prophylactic cosyntropin analysis was the incidence of PDPH and for the therapeutic cosyntropin analysis in exposed vs. unexposed patients, the receipt of an EBP. The secondary outcome for the prophylactic cosyntropin groups was the receipt of an EBP. RESULTS: In the prophylactic cosyntropin analysis no significant difference was found in the risk of PDPH between those exposed to cosyntropin (19/32, 59%) and unexposed patients (17/32, 53%; odds ratio (OR) 1.37, 95% CI 0.48 to 3.98, P = 0.56), or in the incidence of EBP between exposed (12/32, 38%) and unexposed patients (6/32, 19%; OR 2.6, 95% CI 0.83 to 8.13, P = 0.095). In the therapeutic cosyntropin analysis, in patients exposed to cosyntropin the incidence of EBP was significantly higher (20/36, 56% vs. 43/153, 28%; OR 3.20, 95% CI 1.52 to 6.74, P = 0.002). CONCLUSIONS: Our data show no benefits from the use of cosyntropin for preventing or treating postpartum PDPH.

Role of cosyntropin in the prevention of post-dural puncture headache: a propensity-matched retrospective analysis.

BACKGROUND: Post-dural puncture headache (PDPH) is a well-documented complication of accidental dural puncture in obstetric patients. Reports have shown successful treatment with adrenocorticotropic hormone (ACTH) but evidence remains low and limited. In this retrospective analysis, we assessed whether prophylactic administration of cosyntropin, a synthetic derivative of ACTH, reduced the incidence of PDPH after accidental dural puncture in parturients. METHOD: The study population included 132 women with an accidental dural puncture over a three-year period (June 1, 2018 to Oct 31, 2021) at a large tertiary-care center. Patient electronic medical records were reviewed for patient characteristics, prophylactic administration of cosyntropin, PDPH diagnosis, and need for epidural blood patch. Typically, 1 mg of cosyntropin was administered as an intravenous bolus or infusion post-delivery. The propensity score was calculated based on the following factors: age, body mass index, and placement of an intrathecal catheter. Patients were matched allowing 10% variation in scores to reduce potential treatment assignment bias. RESULTS: A total of 115 patients were included in the final analysis. Intravenous cosyntropin was administered to 65 patients (55.6%). Among those who received cosyntropin, 37 (56.9%) developed PDPH compared with 29 patients (58%) in the no-cosyntropin group (P = 0.08). Epidural blood patch was performed in 21 patients (56.8%) who received cosyntropin and 13 patients (61.7%) who did not (P = 0.70). CONCLUSION: Prophylactic administration of cosyntropin is not associated with a reduced incidence of PDPH.

Serum 21-Deoxycortisol for Diagnosis of Nonclassic Congenital Adrenal Hyperplasia in Women With Androgen Excess.

CONTEXT: Nonclassic congenital adrenal hyperplasia (NCCAH) requires exclusion before diagnosing polycystic ovary syndrome (PCOS). Increasing use of liquid chromatography and tandem mass spectrometry (LC-MS/MS) necessitates revision of immunoassay-based criteria for NCCAH. Measurement of 21-deoxycortisol (21DF) may simplify the diagnosis of heterozygosity (HTZ), the presence of 1 affected CYP21A2 allele, which currently relies on complex molecular studies. OBJECTIVE: We aimed to determine LC-MS/MS-specific criteria for NCCAH and HTZ and compare the diagnostic accuracy of 21DF and 17-hydroxyprogesterone (17OHP). METHODS: A cross-sectional study involving 99 hyperandrogenic females was performed. We identified females who had undergone both a synacthen stimulation test (SST) and CYP21A2 genotyping from 2010 to 2017, and prospectively recruited females referred for an SST to investigate hyperandrogenic symptoms from 2017 to 2021. Steroids were compared between genetically confirmed NCCAH, HTZ, and PCOS. Optimal 17OHP and 21DF thresholds for HTZ and NCCAH were determined by receiver operating characteristic analysis. RESULTS: Basal 17OHP, stimulated 17OHP, and 21DF were measured in 99, 85, and 42 participants, respectively. Optimal thresholds for NCCAH were 3.0 nmol/L and 20.7 nmol/L for basal and stimulated 17OHP, respectively. Basal and stimulated 21DF thresholds of 0.31 nmol/L and 13.3 nmol/L provided 100% sensitivity with specificities of 96.8% and 100% for NCCAH, respectively. Diagnostic thresholds for HTZ of 8.0 nmol/L, 1.0 nmol/L, and 13.6 for stimulated 17OHP, 21DF, and the ratio (21DF + 17OHP)/cortisol each provided 100% sensitivity with specificities of 80.4%, 90.5%, and 85.0%, respectively. CONCLUSION: LC-MS/MS-specific 17OHP thresholds for NCCAH are lower than those based on immunoassay. LC-MS/MS-quantified 17OHP and 21DF accurately discriminate HTZ and NCCAH from PCOS.

"A morning cortisol is the most effective clinical predictor of short synacthen test outcome": A tertiary care centre experience.

OBJECTIVE: Increasing referrals to Endocrinology with nonspecific symptoms of suspected adrenal insufficiency (AI) has increased use of the short-synacthen test (SST). Prevailing resource and safety concerns emphasise importance of patient selection criterion to optimise SST use. This study aimed to (1) document the adverse event profile of the SST (2) identify any pretest predictors of SST outcome. DESIGN, PATIENTS AND MEASUREMENTS: Retrospective data analysis of all patients referred for SST in Oxford from 2017 to 2021. Pretest clinical variables (age, sex, BMI, blood pressure and electrolytes), symptoms (fatigue, dizziness, weight loss) and pretest morning cortisol were included in the statistical model with the aim of identifying any variables that could predict SST outcome in Group 1 primary AI, Group 2 central AI and Group 3 glucocorticoid induced AI. Symptoms and signs during and post SST were also noted with the aim of describing adverse effects to synacthen across a large cohort. RESULTS: A total 1480 SSTs (Males:38%, age 52 [39-66] years) were performed: 505 (34.1%) in Group 1, 838 (57%) in Group 2, and 137 (9.3%) in Group 3. Adverse-effects were recorded in 1.8% of tests, including one episode of anaphylaxis. Pretest morning-cortisol was the only predictor for an "SST pass" (whole cohort: B = 0.015, p < 0.001, Group 1: B = 0.018, p < .001; Group 2: B = 0.010, p < 0.012; Group 3: B = 0.018, p = <.001). A threshold of ≥343 nmol/l (receiver-operating characteristic [ROC] area under the curve [AUC] = 0.725, 95% confidence interval [CI] 0.675-0.775, p < 0.001) for the whole cohort, ≥300 nmol/L (ROC AUC = 0.763, 95% CI 0.675 to 0.850, p < 0.001) for Group 1, ≥340 nmol/L (ROC AUC = 0.688, 95% CI 0.615 to 0.761, p < 0.001) for Group2, and ≥376 nmol/L [baseline cortisol] (ROC AUC = 0.783, 95% CI 0.708 to 0.859, p < 0.001) for Group 3, predicted an 'SST pass' with 100% specificity. CONCLUSIONS: Adverse effects to synacthen are rare. Pretest morning cortisol is a reliable predictor for SST outcome and is a helpful tool to rationalise use of the SST. Predictive morning-cortisol thresholds vary according to the aetiology of AI.

Peak Serum Cortisol Cutoffs to Diagnose Adrenal Insufficiency Across Different Cortisol Assays in Children

Objective: Current peak serum cortisol cutoffs for the diagnosis of adrenal insufficiency (AI) after Cosyntropin stimulation have been established using polyclonal antibody (pAb) immunoassays. However, new and highly specific cortisol monoclonal antibody (mAb) immunoassays are being used more widely, which can potentially yield higher false positive rates. Thus, this study aimed to redefine the biochemical diagnostic cutoff points for AI in children when using a highly specific cortisol mAb immunoassay and liquid chromatography tandem mass spectrometry (LC/MS) to avoid unnecessary steroid use. Methods: Cortisol levels from 36 children undergoing 1 mcg Cosyntropin stimulation tests to rule out AI were measured using pAb immunoassay (Roche Elecsys Cortisol I), mAB immunoassay (Roche Elecsys Cortisol II), and LC/MS. Logistic regression was used to predict AI using the pAB as the reference standard. A receiver operator characteristic curve, area under the curve (AUC), sensitivity, specificity, and kappa agreement were also calculated. Results: Using a peak serum cortisol cutoff value of 12.5 µg/dL for the mAb immunoassay provided 99% sensitivity and 94% specificity for diagnosing AI, when compared to the historical pAb immunoassay cutoff of 18 µg/dL (AUC=0.997). Likewise, a cutoff of value of 14 µg/dL using the LC/MS, provided 99% sensitivity and 88% specificity when compared to the pAb immunoassay (AUC=0.995). Conclusion: To prevent overdiagnosis of AI in children undergoing 1 mcg Cosyntropin stimulation test, our data support using a new peak serum cortisol cutoff of 12.5 µg/dL and 14 µg/dL to diagnose AI when using mAb immunoassays and LC/MS in children, respectively.

Acthar Gel Inhibits the Activation of CD4+ and CD8+ T Cells.

Several inflammatory diseases are characterized by elevated T cell counts and high pro-inflammatory cytokine levels. Inhibiting T cell activity may reduce tissue damage associated with these diseases. Acthar® Gel has potent anti-inflammatory properties, yet little is known about its effect on T cells. This study compared the effects of Acthar, synthetic adrenocorticotropic hormone 1-24 (ACTH1-24) depot, and prednisolone in a murine model of T cell activation. Assessments of CD4+ helper and CD8+ cytotoxic T cells and plasma concentrations of interferon-γ (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α) were made following anti-CD3-activation. Acthar significantly reduced the number of activated CD4+ and CD8+ T cells at amounts comparable to synthetic ACTH1-24 depot or prednisolone. However, Acthar reduced production of IFN-γ, IL-2, and TNF-α significantly more than the other drugs, suggesting that the in vivo immunomodulatory effects of Acthar on T cells are distinct from synthetic ACTH1-24 depot or prednisolone.

The utility of a random cortisol level in determining neonatal central adrenal insufficiency.

OBJECTIVES: While it has been established within the first 4 months of life that there is no circadian rhythm, what is unclear is the usefulness of a random serum cortisol (rSC) level in determining neonatal central adrenal insufficiency (CAI). The objective of the study is to determine the utility of using rSC in infants less than 4 months old in the evaluation of CAI. DESIGN AND PATIENTS: Retrospective chart review of infants who underwent a low dose cosyntropin stimulation test ≤4 months of life with rSC taken as baseline cortisol before stimulation. Infants were divided into three groups: those diagnosed with CAI, those at risk for CAI (ARF-CAI) and a non-CAI group. Mean rSC for each group was compared, and ROC analysis was used to identify rSC cut-off for the diagnosis of CAI. RESULTS: Two hundred and fifty one infants with the mean age of 50.5 ± 38.08 days, and 37% of these were born at term gestation. The mean rSC were lower in the CAI group (1.98 ± 1.88 mcg/dl) as compared to the ARF-CAI (6.27 ± 5.48 mcg/dl, p = .002) and non-CAI (4.6 ± 4.02 mcg/dl, p = .007) groups. ROC analysis identified a cut-off of rSC level of 5.6 mcg/dL is associated with 42.6% sensitivity and 100% specificity for the diagnosis of CAI in term infants. CONCLUSIONS: This study demonstrates that though an rSC can be used within the first 4 months of life, its value is best when done ≤30 days of life. Moreover, a diagnostic cut-off for CAI using rSC levels was identified for term infants.

Prednisolone or tetracosactide depot for infantile epileptic spasms syndrome? A prospective analysis of data embedded within two randomised controlled trials.

OBJECTIVE: To report a prospectively planned analysis of two randomised controlled trials with embedded comparisons of prednisolone versus tetracosactide depot for the treatment of infantile epileptic spasms syndrome (IESS). METHODS: Individual patient data from patients randomly allocated to prednisolone or tetracosactide depot were analysed from two trials (UKISS, ICISS). The comparison was embedded within trials in which some patients also received vigabatrin but only patients receiving monotherapy with randomly allocated hormonal treatments are included in this analysis. The main outcome was cessation of spasms (Days 13-14 after randomisation). Lead time to treatment and underlying aetiology were taken into account. Cessation of spasms on Days 14-42 inclusive, electroclinical response (EEG Day 14), plus developmental and epilepsy outcomes (at 14 months in UKISS and 18 months in ICISS) are also reported. Minimum treatment was prednisolone 40 mg per day for two weeks or tetracosactide depot 0·5 mg IM on alternate days for two weeks, all followed by a reducing dose of prednisolone over two weeks. RESULTS: 126 infants were included in this study. On tetracosactide depot, 47 of 62 (76%) were free of spasms on Days 13-14 compared to 43 of 64 (67%) on prednisolone (difference 9%, 95% CI -7·2% to +25·2%, chi square 1·15, p = 0·28). For Day 14-42 cessation of spasms, on tetracosactide depot, 41 of 61 (67%) were free of spasms compared to 35 of 62 (56%) on prednisolone (difference 11%, 95% CI -6·4% to +28·4%, chi square 1·51, p = 0·22). There was no significant difference in mean VABS score between infants who received prednisolone compared with those who received tetracosactide depot (74·8 (SD 18·3) versus 78·0 (SD 20·2) t = -0·91 p = 0·36). The proportion with ongoing epilepsy at the time of developmental assessment was 20 of 61 (33%) in the tetracosactide group compared with 26 out of 63 (41%) in the prednisolone group (difference 8%, 95% CI -9·2% to +25·2%, Chi [2] 0·95, p = 0·33). SIGNIFICANCE: With hormone monotherapy, either prednisolone or tetracosactide depot may be recommended for infantile epileptic spasms syndrome.

Assessment of a compounded synthetic adrenocorticotropic hormone product in 17 healthy dogs.

OBJECTIVES: Synthetic adrenocorticotropic hormone or tetracosactide is routinely used in the diagnosis of hypoadrenocorticism and frequently in the diagnosis and treatment of hyperadrenocorticism. There have been repeated shortages of tetracosactide in recent years in Australia. This study investigated the agreement of serum cortisol after a compounded tetracosactide (Bova Aus), compared to commercial tetracosactide (Synacthen®) in healthy dogs. METHODS: Prospective crossover study using 20 dogs. Ten dogs received 5 µg/kg Synacthen® on day 1 and 5 µg/kg compounded tetracosactide on Day 2. The other 10 dogs received the reverse order. Cortisol concentrations in each dog 1 h after injection were compared for agreement, which was defined as the limits of agreement of the Bland-Altman ratio to be within a range of 0.8-1.25. Passing-Bablok regression analysis examined for constant and proportional biases. RESULTS: Three dogs were excluded with post-stimulation serum cortisol concentrations markedly outside reference interval. For the remaining 17 dogs, Bland-Altman ratio analysis of cortisol concentration (tetracosactide/Synacthen®) at 1 h found virtually no constant bias (mean of ratios 1.01;95% CI 0.97-1.05) and 95% limits of agreement were 0.88 (95% CI 0.78-0.90) and 1.17 (95% CI 1.13-1.25). This met our criteria for agreement between cortisol concentrations. Bias of the Bland-Altman difference was 2.8 nmol/L (95% CI -7.2 to 12.8); 95% limits of agreement -35.2 nmol/L (95% CI -57.0 to -26.1) and 40.8 nmol/L (95% CI 31.7-62.6). Passing-Bablok regression analysis did not identify bias. CONCLUSION: In healthy dogs, cortisol concentrations were in agreement after compounded tetracosactide compared to commercial tetracosactide, Synacthen®.

Deazaflavin cofactor boosts earthworms Henlea bioluminescence.

The bioluminescence of Siberian earthworms Henlea sp. was found to be enhanced by two low molecular weight activators, termed ActH and ActS, found in the hot extracts. The fluorescence emission maximum of the activators matches the bioluminescence spectrum that peaks at 464 nm. We purified 4.3 and 8.8 micrograms of ActH and ActS from 200 worms and explored them using orbitrap HRMS with deep fragmentation and 1D/2D NMR equipped with cryoprobes. Their chemical structures were ascertained using chemical shift prediction services, structure elucidation software and database searches. ActH was identified as the riboflavin analoge archaeal cofactor F0, namely 7,8-didemethyl-8-hydroxy-5-deazariboflavin. ActS is a novel compound, namely ActH sulfated at the 3' ribityl hydroxyl. We designed and implemented a new four step synthesis strategy forActH that outperformed previous synthetic approaches. The synthetic ActH was identical to the natural one and activated Henlea sp. bioluminescence. The bioluminescence enhancement factor X was measured at different ActH concentrations and the Michaelis constant Km = 0.22 ± 0.01 µM was obtained by nonlinear regression. At an excess of synthetic ActH, the factor X was saturated at Xmax = 33.3 ± 0.5, thus opening an avenue to further characterisation of the Henlea sp. bioluminescence system. ActH did not produce bioluminescence without the luciferin with an as yet unknown chemical structure. We propose that ActH and the novel sulfated deazariboflavin ActS either emit the light of the Henlea sp. bioluminescence and/or accept hydride(s) donor upon luciferin oxidation.

Salivary corticosterone measurement in large-billed crows by enzyme-linked immunosorbent assay.

Salivary corticosteroid measurement, as a surrogate for plasma corticosteroid levels to evaluate an animal's stress or metabolic state, commonly used in mammals. However, the validity of salivary corticosterone (CORT) measurements in birds has not yet been reported. We aimed to measure salivary CORT in crows using a commercially available CORT enzyme-linked immunosorbent assay kit. An adrenocorticotropic hormone (ACTH) challenge experiment using synthetic cosyntropin, an ACTH analogue, was conducted to compare CORT level elevations between the serum and the saliva in a 10-60 min range. Both salivary and blood CORT was significantly elevated 10 min after injecting synthetic cosyntropin. The results supported the validation of salivary CORT as a surrogate for a blood CORT in crows.

Molecular cloning, tissue distribution, and pharmacologic function of melanocortin-3 receptor in common carp (Cyprinus carpio).

Melanocortin-3 receptor (MC3R) not only regulates energy homeostasis in animals, but also is an important regulator of inflammation. As one of the most widely farmed freshwater fish, common carp has attracted great interest for its feeding and inflammation regulation. In this study, we cloned the coding sequence (CDS) of common carp Mc3r (ccMc3r), examined its tissue expression profile, and investigated the function of this receptor in mediating downstream signaling pathways. The results showed that the CDS of ccMc3r was 975 bp, encoding a putative protein of 324 amino acids. Homology, phylogeny, and chromosomal synteny analyses revealed that ccMc3r is evolutionarily close to the orthologs of cyprinids. Quantitative real-time PCR (qPCR) indicated that ccMc3r was highly expressed in the brain and intestine. The luciferase reporter systems showed that four ligands, ACTH (1-24), α-MSH, ß-MSH, and NDP-MSH, were able to activate the cAMP and MAPK/ERK signaling pathways downstream of ccMc3r with different potencies. For the cAMP signaling pathway, ACTH (1-24) had the highest activation potency; while for the MAPK/ERK signaling pathway, ß-MSH had the greatest activation effect. In addition, we found that the four agonists were able to inhibit TNF-α-induced NF-κB signaling in approximately the same order of potency as cAMP signaling activation. This study may facilitate future studies on the role of Mc3r in common carp feed efficiency and immune regulation.