For psychoneuroendocrinology, accurate measurement of progesterone (P4) and estradiol (E2) in saliva is crucial for understanding the menstrual cycle phase and its impact on physiology and behavior. Although enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) are the preferred methods and are more often found in research labs, liquid chromatography-mass spectrometry (LC-MS/MS) may provide a more valid salivary steroids assessment. In this study, we aimed to compare the performance of LC-MS/MS, ELISA, and RIA to measure salivary P4 and E2. Samples were collected from 120 participants, 81 men and 39 women, in the morning and evening. Additionally, women provided samples during the early follicular (cycle days 3-5) and luteal cycle phases (cycle days 21-23) using the forward-count method. The study considered natural hormone fluctuations (e.g., the diurnal and menstrual cycles) and quality control samples as validity criteria for method evaluation. A total of 336 samples and quality control samples were analyzed using one RIA, two ELISA, and two LC-MS/MS methods across four labs. Correlational analyses were performed to assess inter-lab x inter-method reliability, intra-lab x inter-method reliability, and inter-lab x intra-method reliability. For P4, natural hormone fluctuations in menstrual cycles were detected by all methods. However, in contrast to both LC-MS/MS methods, all immunoassays (IAs) detected an unexpected diurnal decline of P4. For E2, they were found only by LC-MS/MS and RIA. In terms of means, for P4 and E2, ELISA and RIA produced higher values compared to LC-MS/MS. For P4, the inter- and intra-method convergence was r ≥ .92. As for E2, inter-method correlations were between r = -.12 and r = .23, while the ELISA intra-method comparison showed a correlation coefficient of r = .85. Our findings suggest that while LC-MS/MS, RIA, and ELISA were capable of meeting most of the specified criteria for P4, only LC-MS/MS and RIA were able to perform similarly sufficiently at low E2 levels. LC-MS/MS provided more accurate and reliable measurements for P4 and E2, especially at low concentrations, but encountered challenges, too. Although RIA showed comparable performance to LC-MS/MS, it still suffered partly from cross-reactivity. ELISA often overestimated hormone levels and exhibited greater divergence. These differences highlight the importance of carefully selecting methods and considering their limitations in research applications.

The main challenge in congenital adrenal hyperplasia newborn screening (CAH-NBS) is the high rate of false-positive (FP) results, emphasizing the need for specific serum confirmatory tests. Data on the effectiveness of serum 17-hydroxyprogesterone (17OHP) measurements using available methodologies remain limited. to evaluate the efficacy of confirmatory tests using measurements of serum steroids by radioimmunoassay (RIA) and liquid chromatography tandem mass spectrometry (LC-MS/MS) methodologies. A prospective longitudinal cohort study. data of 708,437 newborns (NBs) were evaluated. neonatal-17OHP (N17OHP) results ≥ 2x 99.5th were recalled for serum dosages by RIA (17OHP), by LC-MS/MS (17OHP, 21-deoxycortisol-21DF, androstenedione-Δ4, cortisol) and positive predictive values (PPV) were determined. Altered hormonal results were submitted to CYP21A2 genotyping. Non-parametric tests and ROC-curves were used. Recall rate of N17OHP (first tier) was 0.03% and serum 17OHP levels remained increased in 26% (RIA) and 11% (LC-MS/MS) of NBs, yielding PPV of 30% and 49%, respectively. Fifty-eight NBs were diagnosed with classical forms, and 32 asymptomatic-NBs persisted with increased serum 17OHP levels. ROC-curves between false and true-positive tests disclosed highest diagnostic performance for 17OHP/LC-MS/MS [cutoff 48.3 ng/mL (146.3 nmol/L) = 100%-sensitivity]. The (17OHP+Δ4)/cortisol ratio presented the highest discriminative ability (cutoff 24.9 = 100%-PPV); serum 21DF presented a good performance and was comparable to 17OHP measured by LC-MS/MS (99%-specificity). Measurement of 17OHP by LC-MS/MS provides the best performance for serum confirmatory tests and may be complemented by the (17OHP+Δ4)/cortisol ratio analysis. Genotyping is useful mainly to resolve persistently indeterminate biochemical results.

Salivary steroid assessment has become an essential part of human social neuroendocrinology, offering non-invasive, easy, and cost-effective measurements. Despite researchers' preference for methods like enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) over the complex and costly liquid chromatography-mass spectrometry (LC-MS/MS), concerns about the validity of immunoassays (IAs) remain. The present study examines the convergence of LC-MS/MS, ELISA, and RIA in measuring salivary cortisol (C) and testosterone (T) and explores the contributions of intra-lab and inter-lab factors. Samples were collected from 81 men and 39 women in the morning and evening. Moreover, women provided samples for both the follicular and the luteal cycle phases. Natural hormone fluctuations (e.g., diurnal C decrease in the evening, T male-to-female ratio, influence of hormonal cycle phase, and hormonal contraceptive intake) and quality control samples were used as validity criteria for method evaluation. Over 336 samples and quality control samples were assayed by one RIA, two ELISA, and two LC-MS/MS methods across four labs. Correlational analyses were conducted to examine inter-lab x inter-method reliability, intra-lab x inter-method reliability, and inter-lab x intra-method reliability. For C and T, all methods demonstrated sufficient validity in detecting well-known natural fluctuations, with LC-MS/MS performing consistently best across all evaluated criteria. Nevertheless, ELISA did not achieve the expected male-to-female T ratio and tended to inflate estimated C and T levels, especially in the lower concentration range. Further, we found for all methods highly significant correlations with r ≥ .92 for C and with r ≥ .85 for T. However, when samples were divided by sex, correlations stayed comparable for C but decreased for T to r ≥ .71 in men and r ≥ .41 in women. LC-MS/MS was the best-performing method for both C and T across all criteria examined. RIA, despite showing slightly higher variance, can still be considered a reliable analytical technique as it met most of the set criteria for C and T. On the other hand, ELISA overestimated values, especially at low T levels. Therefore, caution should be exercised when selecting an appropriate method for the specific need.

Orexin neuropeptides are central to sleep-wake regulation. However, current cerebrospinal fluid (CSF) assays primarily detect inactive orexin-A fragments using radioimmunoassay (RIA), which limits mechanistic understanding. We developed a high-sensitivity mass spectrometry (MS) assay that can comprehensively quantify human CSF orexin species, including the biologically active long orexin-A and orexin-B, as well as their metabolites and prepro-orexin that are biologically inactive. In participants with narcolepsy type 1 (NT1), all orexin species were significantly reduced compared to those with narcolepsy type 2 (NT2) or idiopathic hypersomnia (IH). MS detected all orexin peptides in NT1 samples below the RIA detection threshold. Notably, the ratio of short to long orexin-B peptides differentiated NT2 from IH, suggesting altered orexin peptide metabolism. Sleep deprivation increased all orexin species with distinct temporal dynamics across peptide forms. The stable isotope labeling kinetics method using MS revealed increased turnover of orexin-A and orexin-B, but not prepro-orexin, during sleep deprivation, indicating activity-dependent regulation. These findings demonstrate that MS enables comprehensive profiling of orexin dynamics and reveals biologically relevant differences in peptide processing and turnover under different sleep conditions. This approach is a powerful tool for advancing our understanding of orexin physiology and its role in sleep-wake pathophysiology.

Background Ventricular arrhythmias (VAs) frequently arise as complications in chronic ischemic heart failure (CIHF). Purpose We evaluated the effectiveness of resveratrol (RES) in a CIHF-induced rat model of VAs and explored the underlying mechanisms. Methods Animals received the left anterior descending coronary artery ligation to simulate CIHF and were subsequently treated daily with RES for 2 months. The subjects were categorized into five groups: normal control, VAs model, VAs model receiving simvastatin, and VAs models treated with RES at doses of 10 and 50 mg/kg. Key lipid factors, including high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG), were evaluated using a biochemical analyzer. Serum concentrations of inflammatory markers like interleukin-1β (IL-1β), interleukin-8 (IL-8), 6-keto-PGF1α, TXB2, endothelin (ET), and nitric oxide (NO) were quantified through radioimmunoassay (RIA). Real-time polymerase chain reaction (PCR) analyzed the VCAM-1, ICAM-1, and caspase-3 expression in the left anterior descending coronary artery, and Kv4.2, Cav1.2, and nerve growth factor (NGF) in the left ventricle. Results RES treatment led to significant improvements ( p < .05) in lipid profiles, characterized by decreased concentrations of LDL-C, TG, and TC, alongside an elevation in HDL-C. Additionally, RES significantly ( p < .05) lowered inflammatory cytokines and the ICAM-1 and VCAM-1 expression, while enhancing the Cav1.2, Kv4.2, and NGF expression ( p < .05). Furthermore, RES significantly ( p < .05) elevated 6-keto-PGF1α concentrations and increased ET and TXB2 levels. Conclusion RES emerges as an appropriate candidate for treating VAs.

Clinical relevance of the Maglumi immunoassay for anti-GAD65 in diabetes and neurological disorders: Analytical challenges and diagnostic insights.

Controlled ovarian hyperstimulation (COH) is a cornerstone of assisted reproductive technologies, yet its effects on endometrial function and embryo implantation remain poorly understood, particularly regarding the role of hypoxia-inducible factor 2α (HIF-2α) signaling. Therefore, the objective of this study was to investigate whether COH-induced endometrial dysfunction impairs mouse embryo implantation through the HIF-2α pathway. A COH mouse model was established using gonadotropin-releasing hormone agonist (GnRH-a)/human menopausal gonadotropin (hMG)/human chorionic gonadotropin (hCG) administration. Embryo implantation status was evaluated on gestational days 5, 6, and 20; endometrial tissues were analyzed for HIF-2α pathway activity via immunohistochemistry (IHC), immunofluorescence (IF), Western blot (WB) and quantitative real-time PCR (qRT-PCR); histological changes were assessed by Jones silver staining and transmission electron microscopy (TEM); serum estradiol (E2), progesterone (P4), and prolactin (PRL) levels were measured by radioimmunoassay (RIA). COH mice exhibited reduced total embryo implantation rates (on day 5, 6 and 20), together with decreased serum E2, P4, and PRL levels. COH mice exhibited preserved luminal epithelium integrity with increased microvillus density and continuous basement membrane structure. qRT-PCR and WB demonstrated significantly downregulated HIF-2α expression at both mRNA and protein levels, accompanied by reduced downstream RAB27B (member of RAS oncogene family)/matrix metalloproteinase 9 (MMP9) and lysyl oxidase (LOX)/adrenomedullin (ADM) signaling, which associated with impaired luminal epithelium detachment and compromised trophoblast invasion. Together, these findings identify HIF-2α as a potential key mediator of COH-induced endometrial microenvironment alterations, revealing molecular mechanisms underlying implantation failure. Importantly, the HIF-2α-RAB27B/MMP9 and HIF-2α-LOX/ADM axes are highlighted as promising therapeutic targets to optimize assisted reproductive outcomes.

Background: Calcitonin Gene-Related Peptide (CGRP), Vasoactive Intestinal Peptide (VIP), and Pituitary Adenylate Cyclase-Activating Peptide (PACAP) play a crucial role in migraine pathophysiology. In recent years, anti-CGRP(R) monoclonal antibodies (mAbs) have emerged as the first targeted and highly effective therapy for migraine. This study aimed to assess plasma levels of these neuropeptides before and after prophylactic treatment with anti-CGRP(R) mAbs, evaluating potential changes after therapy and identifying predictors of treatment response Methods: Between February 2022 and February 2023, we enrolled 56 migraine patients who initiated prophylaxis with either erenumab (26 patients), galcanezumab (16 patients), or fremanezumab (14 patients). Responders were defined as those achieving a ≥50% reduction in monthly migraine days (MMDs) after six months. Blood samples were collected at baseline and at each follow-up visit (baseline T0, 3 months T1, 6 months T2, 12 months T3). Plasma levels of CGRP, VIP, and PACAP were measured using a validated radioimmunoassay (RIA) and commercially available enzyme-linked immunosorbent assay (ELISA) kits. Results: Overall, 80.3% (45 out of 56) of patients responded to anti-CGRP(R) mAbs. In patients treated with erenumab, CGRP levels did not show a significant change over the treatment period; however, responders exhibited a decreasing trend from T0 to T2 compared to non-responders. In the galcanezumab group, CGRP levels significantly decreased as early as T1 (p < 0.01). Conversely, in the fremanezumab group, we observed a rapid increase in CGRP plasma levels above 3000 pg/ml, which was deemed unreliable due to assay interference from fremanezumab. VIP and PACAP levels remained stable over time, with no significant differences between responders and non-responders Conclusion: Anti-CGRP(R) mAbs are very effective migraine prophylaxes. Galcanezumab reduced CGRP plasmatic levels already after three months, while erenumab did not affect significantly CGRP plasmatic levels. VIP and PACAP levels were not influenced by the therapy.

This study evaluated the effectiveness of a single intramuscular administration of follicle-stimulating hormone (FSH) dissolved in 1% hyaluronic acid (HA) for ovarian stimulation in Italian Mediterranean buffaloes (IMB) undergoing ovum pick-up (OPU). The aim was to simplify hormonal protocols, reduce handling stress, and improve oocyte competence. In Experiment 1, multiparous IMB (n=24) were enrolled in a crossover design comparing the standard six-dose FSH protocol (FSH-6) with a single FSH-HA injection. Transrectal ultrasonography was used to record the number and the size of follicles, in vitro embryo production to assess oocyte developmental competence, and an in-house radioimmunoassay (RIA) method to measure plasma cortisol levels. In Experiment 2, plasma FSH profile during follicular growth was evaluated in IMB (n=12) subjected to FSH-HA, FSH-6, or no stimulation (control) protocols. The FSH-HA treatment resulted in a higher (P<0.05) proportion of medium-sized follicles compared to FSH-6 (3.4±2.7 and 2.0±2.3, respectively) as well as a significantly greater number of cleaved oocytes (3.3±0.4 and 2.4±0.4 respectively), although the total embryo yield remained similar (30.1±7.2 and 29. 2±4.5, respectively). Cortisol concentrations increased after OPU in all groups, but the FSH-HA group showed a trend (P<0.10) toward lower stress levels compared to FSH-6. In Experiment 2, FSH-HA maintained higher plasma FSH concentrations for a longer period, with peak values observed between 30 and 72h post-administration, suggesting the importance of optimizing the timing of OPU in HA-based protocols.

Background: The study aimed to appraise the clinical findings and thyroid profile changes in hypothyroid dogs associated with neuromuscular disorders. Methods: The study comprised seven (7) healthy dogs (Group I) and 21 hypothyroid dogs with neuromuscular disorders (Group II). The serum concentration of Total Triiodothyronine (TT3), Total Thyroxine (TT4) and free Thyroxine (fT4) was assessed using Radioimmunoassay (RIA), while Canine thyroid-stimulating hormone concentration (cTSH) was measured using a canine-specific TSH ELISA kit. Result: The study reported that the neuromuscular signs associated with hypothyroidism were nystagmus (n=1/21, 4.76%), seizures (n=2/21, 9.52%), proprioceptive ataxia (n=3/21, 14.29%), head tilt (n=3/21, 14.29%), paraparesis (n=3/21, 14.29%), facial asymmetry (n=5/21, 23.81%), regurgitation due to megaesophagus (n=5/21, 23.81%) and tetraparesis (n=6/21, 28.57%). The mean serum concentration of TT3, TT4, fT4 and cTSH in hypothyroid dogs associated with neuromuscular disorders was 0.89±0.04 (nmol/L), 9.45±0.94 (nmol/L), 7.72±0.37 (pmol/L) and 1.59±0.20 (ng/mL), respectively. The study found significantly (p≤0.01) lower TT4 and fT4 concentrations in hypothyroid dogs associated with neuromuscular disorders and significantly (p≤0.01) elevated cTSH levels compared to healthy dogs.

Disclosure: A.P. Jackson: None. H.S. Bose: None.In the United States a large number of women die due to breast cancer mostly because of environmental factors and genetic predispositions which cannot be avoided due to varying social issues. Breast cancer is usually hormonally regulated where receptors like Estrogen (Er+), Progesterone (Pr+), and Human Epidermal Growth Factor Receptor (Her2-) expression are associated with tumorigenesis. Triple-negative breast cancer (TNBC, Er-/Pr-/Her2-) minimally expresses these receptors or remains absent in these patients resulting in high mortality. TNBC patients are usually ineffective against chemotherapies. We identified and cloned a cDNA coding for a 22-kDa protein expressing in unaffected and affected breast tissue, Aromatase Interacting Partner in Breast (AIPB). Next, we subcloned AIPB in a vector under the control of Tet promoter and developed a stable MDA-MB-231-AIPB cells to have a uniform expression upon induction with tetracycline. Antihelminth drug Pyrvinium pamoate (PvR) is used to reduce various cancers including TNBC and its interaction with AIPB are of interest. AIPB expression is maximal in TNBC and minimal in unaffected breast tissue. We hypothesize that AIPB expression is directly proportional to Estradiol synthesis. Therefore, following induction of TNBC MDA-MB-231 stable cells with doxycycline, AIPB should decrease in Estradiol synthesis with a reduction in AIPB expression. We incubated these stable cells with and without doxycycline with 50,000-fold difference in concentrations of PvR (1.0 ng to 50,000 ng) for 48 hours and determined its effects by measuring a change in Estradiol synthesis by radioimmunoassay (RIA). We also determined AIPB expression by Immunoblotting with a cholesterol trafficker (CT) antibody, because 42 amino acids of AIPB have amino acid identity with the CT protein. We observed a decrease in Estradiol synthesis when the cells were induced with increasing concentrations of PvR in the presence of Doxycycline. Furthermore, we observed the lowest expression of Estradiol after induction with 10.0 microgram of PvR. Immunoblotting of the PvR induced cells in presence of doxycycline showed increased AIPB expression from 1.0 ng up to 20.0 microgram, followed by decrease in expression. We conclude AIPB expression and Estradiol synthesis is directly related in TNBC and AIPB is possibly a key role of aromatase independent Estradiol synthesis.Presentation: Monday, July 14, 2025

Radioimmunoassay (RIA)-serum trypsin serves as a pancreas function biomarker because its blood concentration correlates with duodenal trypsin output. With the unavailability of RIA tests and global availability of dried blood spot immunoreactive trypsin (DBS-IRT) testing as part of cystic fibrosis newborn-screening programs, IRT can be made accessible for routine clinical use. We aimed to validate DBS-IRT for its use as a biomarker of exocrine pancreas function in children older than the newborn age group. Analytical accuracy of DBS-IRT was evaluated in 28 children (mean [SD] age: 13 [4.5] years) with known pancreas function status and simultaneous RIA-based serum trypsin testing. Reference ranges were established based on 134 metabolically stable children. Clinical validation was performed in 164 children with well-established pancreas phenotypes. Available cross-sectional imaging was reviewed to assess for pancreas atrophy to measure acinar cell loss. Total imprecision of DBS-IRT ranged from 7.1% to 16.4%; samples were stable samples at -20C for 28 days and showed excellent correlation to RIA-serum trypsin (r = 0.95, P < 0.001). A reference range of 6.9-21.2 ng/mL was newly established. DBS-IRT replicated serum trypsin in discriminating exocrine pancreatic insufficiency (EPI) in the cystic fibrosis group with high sensitivity (90.7%; 95% CI: 79.7%-96.9%) and specificity (94.7%; 95% CI: 73.9%-99.8%). Children with pancreatitis and EPI had significantly lower median DBS-IRT level compared with those without EPI ( P = 0.01). Low DBS-IRT was associated with pancreas atrophy ( P = 0.001). DBS-IRT was successfully validated as a biomarker of exocrine pancreas function which allows its use in clinical setting, in addition to fecal elastase to determine pancreas function.

Abstract Background Given that the conventional aldosterone cut-offs for primary aldosteronism (PA) screening are derived from immunoassays and vary from study to study, the applicability of traditional cut-offs in patients with aldosterone detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method remains to be investigated. Thus, the study aims to compare the differences in PA screening based on traditional aldosterone cut-offs using LC-MS/MS and radioimmunoassay (RIA), respectively. Intra-individual variability and seasonal variations in aldosterone were also assessed based on cohort data. Methods Proportions of patients below traditionally adopted aldosterone cut-offs (10, 12, 15, and 20 ng/dL), intra-individual variability, and seasonal changes in aldosterone were calculated among patients with aldosterone detected using LC-MS/MS and RIA assays. The Bland-Altman plot and the kappa were used to assess the agreement around the cut-offs (15, and 20ng/dL) and the continuous measurements. To explore the intra-individual variability of aldosterone, the coefficient of variation (CV), measured as the ratio of standard deviation to the mean value, was calculated in patients with at least two aldosterone measurements. Results This retrospective cohort study included 608 patients with PA, 208 of whom 208 had at least two aldosterone measurements based on the LC-MS/MS assay. The median values of aldosterone measured for the first time by LC-M/MS and RIA were 14.53 and 21.48 ng/dL. The proportion of patients with at least one aldosterone measurement below 5, 10, 12, 15, and 20 ng/dL was 12.8%, 36.5%, 45.6%, 56.7%, and 70.7% by LC-MS/MS, and only 0.2%, 3.7%, 7.5%, 18.2% and 48.4% by RIA. The Bland-Altman plot and the kappa value 0.127 showed a low agreement between LC/MS/MS and RIA assays. High intra-individual variability was found in LC-MS/MS-detected plasma aldosterone, with a median CV of 30.3%. The plasma aldosterone concentrations were significantly lower in spring and summer than in autumn and winter (median, 13.1, 14.0 vs 15.4, 16.2 ng/dL, p=0.028). Conclusion Significant discrepancies in plasma aldosterone measurements between assays have been observed, and the conventional cut-offs derived from immunoassays are not applicable in patients with aldosterone measured by LC-MS/MS. An assay-specific cut-off for aldosterone is required to achieve accurate PA screening.

Insulin autoantibodies (IAA) represent the major serological marker of type 1 diabetes mellitus (T1D), the disease resulting from autoimmune damage to β-cells in the pancreatic islets. Testing for IAA is used in early and differential diagnosis of T1D, as well as to perform screening for this disorder. The best foreign diagnostic labs perform IAA tests using different radioimmunoassay (RIA) formats. The RIA performance characteristics, i. e. diagnostic sensitivity (DSe), diagnostic specificity (DSp), and diagnostic accuracy (DA), are on average equal to 44%, 100%, and 81%, respectively. Unfortunately, in Russia RIA has not been used to determine IAA for a long time. All Russian labs use the enzyme-linked immunoassay (ELISA)-based test systems for this purpose. DSe, DSp, and DA of ELISA systems are on average 24%, 87%, and 62%, respectively, i.e. considerably lower compared to RIA systems. Our study aimed to reproduce IAA RIA in the diagnostic lab of the RCCH. The method is based on IAA competitive binding to insulin and 125I-labeled insulin. Serum samples from patients with new onset T1D and patients without diabetes were tested for IAA. DSe, DSp, and DA were 43%, 100%, and 73%, respectively. Thus, performance characteristics of the reproduced IAA RIA are close to those of RIAs used in foreign labs and are significantly superior to the characteristics of ELISA-based tests.

Effect of apigenin, acacetin, and genkwanin on triiodothyronine hormone measurement using radioimmunoassay.

Aromatic ring tritiation and tritium decay catastrophe.

The passive transfer model of myasthenia gravis (PTMG) reflects the effector phase of the human condition and is induced in female rats using monoclonal antibodies. The current guideline recommends administration of monoclonal antibodies (mAb) such as mAb35 via intraperitoneal (I.P.) or intravenous (I.V.) injection, offering rapid absorption, but posing challenges such as injection placement and, in the case of I.P., animal discomfort. In this study, we investigated the suitability of subcutaneous (S.C.) administration as an alternative to I.P. in the rat PTMG model. Female rats were injected with 20 pmol mAb35 /100 g body weight (BW), either I.P. or S.C. and euthanized 48 h post-immunization, or S.C. with 20 or 40 pmol mAb35 /100 g BW and euthanized 48- or 72-hours post-immunization. Control animals received 0.5 mg/kg IgG1 isotype control S.C. or I.P. Muscle weakness, weight loss and clinical manifestations were assessed daily. Additionally, decrement-inducing curare dose and AChR content in the tibialis anterior (TA) were determined using electromyography and radioimmunoassay (RIA) respectively. S.C. mAb35 administration induced body weight loss and MG symptoms comparable to I.P. administration. However, I.P. injection presented a risk of misplacement, making it potentially a less reliable administration method. While MG characteristics were observed regardless of mAb35 dose and route, variability of clinical parameters and TA AChR content differed between approaches. S.C. induction of disease was verified using a different batch of mAb35 with a dose of 40 pmol/100 g BW S.C administered. We demonstrated that S.C. injection offers a consistent induction of PTMG, establishing it as a refined, effective alternative to I.P. administration by reducing procedural invasiveness and improving consistency in symptom onset.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-15187-2.

Glucocorticoids like cortisol are widely used to assess stress in fish, but their interpretation can be limited. Incorporating dehydroepiandrosterone (DHEA), a hormone with anti-glucocorticoid and neuroprotective effects, may provide a broader understanding of hypothalamic-pituitary-inter-renal (HPI) axis activity. As DHEA data in fish remain scarce, this study examined its role in rainbow trout (Oncorhynchus mykiss) subjected to acute stress (30 min confinement), considering sex and sexual maturity. Cortisol and DHEA were quantified by optimized radioimmunoassay (RIA) in serum, muscle, fin, and scales, and gonadal histology was performed to confirm reproductive status. Both hormones were successfully measured in all matrices, with serum DHEA levels notably higher than those reported to date in fish. As expected, serum cortisol increased markedly after stress and correlated with levels in muscle and fin but not in scales, which appears to reflect chronic rather than acute exposure. In contrast, serum DHEA showed no stress-induced changes and only minor sex- and maturity-related differences, although alternative matrices displayed variable patterns, particularly in muscle and fin. The cortisol/DHEA ratio in serum mirrored cortisol dynamics, suggesting limited utility for acute stress assessment, while tissue-specific DHEA variation may integrate longer-term influences. Further research is needed to clarify the role of DHEA under chronic stress and its potential origins in inter-renal tissue, gonads, or the brain.

Cerebrospinal fluid (CSF) hypocretin-1/orexin-A quantification via radioimmunoassay (RIA) is used for diagnosing narcolepsy type 1(NT1), but its limitations require alternative methods. Using liquid chromatography-mass spectrometry (LC-MS), this study aimed to 1) fully characterize CSF orexin-A fragments detected by the gold standard RIA method, and 2) assess diagnostic relevance of measuring the most prevalent fragment, a 16-mer, in patients with NT1, narcolepsy type 2 (NT2), idiopathic hypersomnia (IH), and nonspecified hypersomnolence (NSH). CSF samples were analyzed using RIA and LC-MS in patients with hypersomnolence disorders evaluated at the French Narcolepsy National Reference Center. Fractionation techniques isolated orexin-A and its fragments, which were identified via targeted MS. Statistical analysis compared LC-MS performance against RIA. CSF samples from 115 patients (54.8% females, mean age 30.4 ± 15.8years) including 52 with NT1, 6 NT2, 13 IH, and 44 NSH were analyzed by RIA and LC-MS. A 16-mer orexin-A fragment was identified and quantified by LC-MS, with lower levels in NT1. This fragment correlated strongly with RIA-measured orexin-A (r = 0.83, p < .0001) and demonstrated high sensitivity (98.1%) and specificity (85.7%) for diagnosis of NT1. Receiver operating characteristics analyses confirmed the high performance with an area under the curve of 0.975 and an optimal diagnostic cutoff of 10.67pg/mL for LC-MS. The 16-mer orexin-A peptide represents a promising biomarker that could in the future help in the diagnosis of central hypersomnolence disorders. The transition from RIA to LC-MS methods for the quantification of 16-mer orexin-A represents a critical advance toward improving diagnostic accuracy and understanding orexinergic dysfunction in sleep disorders.

ContextIn clinical practice, plasma arginine vasopressin (AVP) concentrations have been measured with a radioimmunoassay (RIA). However, RIAs have limitations, such as long turnaround time, use of radioisotopes, and restricted antibody availability. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers a promising alternative, eliminating the need for radioisotopes and antibodies while providing faster results.ObjectiveThis study aimed to assess the usefulness of LC-MS/MS for measuring plasma AVP concentrations in diagnosing AVP deficiency (AVP-D).MethodsWe included 16 patients with AVP-D and 28 controls. All participants underwent a hypertonic saline infusion test (HST), during which plasma AVP concentrations were measured using RIA and LC-MS/MS. Regression coefficients (gradients) for serum sodium vs plasma AVP concentrations were evaluated at 90 and 120 minutes, and receiver-operating characteristic analyses were performed based on these regression coefficients.ResultsThe area under the receiver-operating characteristic curve at 90 minutes was 0.97 (95% CI, 0.83-1.00) and 0.93 (95% CI, 0.80-0.98) for LC-MS/MS and RIA, respectively. A regression gradient cutoff with optimal values distinguished AVP-D from controls with a sensitivity of 100% in LC-MS/MS and RIA, whereas the specificity was 96% and 81% with LC-MS/MS and RIA, respectively. Sensitivity or specificity did not differ in 120 minutes between the 2 methods.ConclusionLC-MS/MS demonstrated superior diagnostic accuracy for AVP-D at 90 minutes of HST, indicating that the HST time can be shortened from 120 to 90 minutes by measuring AVP with LC-MS/MS.Clinical trial registrationThe study was registered with the University Hospital Medical Information Network (UMIN) registry (UMIN000043023).