Rivard, Ann L

Abstract Disclosure: J. Saini: None. S. Singh: None. A.L. Ebbehøj: None. C.D. Zhang: None. R. Nathani: None. V. Fell: None. E. Atkinson: None. S. Achenbach: None. A. Rivard: None. R.J. Singh: None. S.K. Grebe: None. I. Bancos: None. Abstract Context: Mild autonomous cortisol secretion (MACS) is diagnosed based on post-dexamethasone cortisol>1.8 mcg/dL. Scarce evidence exists on steroid circadian secretion and steroid metabolome in MACS. Objective: To characterize 24-hour urine steroid metabolome in patients with MACS and determine circadian differences in urine steroid profiling and cortisol concentrations in patients with MACS versus referent subjects. Design: Cross-sectional study, 2018-2023. Setting: Referral center Participants: 72 patients with MACS and 72 age-, sex-, BMI-, and menopausal status-matched referent subjects. Measurements: Urine was collected over a 24-hour (h) period in separate day- and night-time collections. High-resolution mass spectrometry assay was used to measure 25 steroids. A subgroup of patients and referent subjects were admitted for every 2-hour serum measurement of free and total cortisol. Outcomes: Steroids, sums, and ratios Results: Patients with MACS had lower mcg/24h median androgens (2084 vs 3283, P<0.001), higher glucocorticoids (15754 vs 12936, P<0.001), and higher glucocorticoid/androgen ratio (8.7 vs 3.9, P<0.001). Steroid day/night ratios were lower in patients when compared to referent subjects, reflecting a higher relative nocturnal steroid production in MACS. In a subgroup of 12 patients with MACS and 10 referent subjects, the 24-hour area under the curves for total and free cortisol were similar. However, nocturnal mean total (5.3 vs 4.0 mcg/dL, P=0.056) and free (0.2 vs 0.1 mcg/dL, P=0.035) cortisol were higher in patients vs referent subjects. Conclusion: Patients with MACS demonstrate an abnormal urine steroid metabolome, with a high glucocorticoid to androgen ratio, and a higher nocturnal steroid production. Presentation: 6/3/2024

Abstract Context Mild autonomous cortisol secretion (MACS) is diagnosed based on postdexamethasone cortisol >1.8 µg/dL. Scarce evidence exists on steroid circadian secretion and steroid metabolome in MACS. Objective To characterize 24-hour (h) urine steroid metabolome in patients with MACS and determine circadian differences in urine steroid profiling and cortisol concentrations in patients with MACS vs referent subjects. Methods Cross-sectional study, 2018-2023, at a referral center. Patients with MACS and age-, sex-, body mass index–, and menopausal status–matched referent subjects were included. Urine was collected over a 24 hour period as separate daytime and nighttime collections. High-resolution mass spectrometry assay was used to measure 25 steroids. A subgroup of patients and referent subjects was admitted for serum measurements of free and total cortisol every 2 hours. Outcomes were steroids, steroid sums, and ratios. Results Patients with MACS (n = 72) had lower µg/24 hour median androgens (2084 vs 3283, P < .001), higher glucocorticoids (15 754 vs 12936, P < .001), and higher glucocorticoid/androgen ratio (8.7 vs 3.9, P < .001) than referent subjects. Patients also had lower steroid day/night ratios than referent subjects, reflecting a higher relative nocturnal steroid production in MACS. In a subgroup of 12 patients with MACS and 10 referent subjects, the 24-hour areas under the curve for total and free serum cortisol were similar. However, evening mean total (5.3 vs 4.0 µg/dL, P = .056) and free (0.2 vs 0.1 µg/dL, P = .035) cortisol was higher in patients vs referent subjects. Conclusion Patients with MACS demonstrate an abnormal urine steroid metabolome, with a high glucocorticoid to androgen ratio, and a higher nocturnal steroid production.

Abstract Background: Frailty is characterized by an increased vulnerability and a decline in physiological reserve. Frailty has been previously linked to cortisol concentrations and blunted diurnal cortisol secretion. Our objective was to determine the association of urine steroid metabolome and its diurnal variation with frailty and prefrailty in older adults. Methods: Cross-sectional study of community-dwelling adults ≥ 50 years. Participants with adrenal disorders, end-stage renal or liver disease, on exogenous steroids or drugs affecting steroid metabolism were excluded. All participants completed day and night separate urine collection. Frailty was assessed using a phenotype model (weight loss, exhaustion, grip strength, low physical activity, and slow walking pace). Participants were characterized as frail if they met at least three criteria, pre-frail if they fulfilled one or two criteria, and robust if no criteria were met. Urine samples were analyzed with the liquid-chromatography, high-resolution, accurate-mass mass spectrometry for 25 urine steroid metabolites. Results: Of 119 participants, 60 (50.4%) were women, without sex differences in age or education status. On frailty assessment, 5 (4.2%) participants were frail and 33 (27.7%) were prefrail, with equal sex distribution. Urine steroid metabolome analysis demonstrated 21/25 steroids were higher in men vs women. In an age adjusted model, presence of prefrailty or frailty was associated with a higher ratio of total cortisol metabolites/total androgen metabolites (TCM/TAM) in men (estimate 0.64, P-value= 0.0004), but not in women. In men, after adjusting for age, among cortisol metabolites, lower day to night ratio of 5α-Tetrahydrocortisol (estimate -0.36, P-value= 0.0419) and β-Cortol (estimate -0.35, P-value= 0.0238) were associated with frail or prefrail phenotype. After adjusting for age, higher ratio of TCM/TAM was associated lower gait speed in men (estimate -1.2, P-value= 0.046) and women (estimate -3.9, P-value= 0.012); and lower hand grip strength in men (estimate -0.04, P-value= 0.046) but not in women. Conclusion: We showed that a higher glucocorticoid to androgen ratio and a flattened circadian steroid variation were associated with presence of frail or prefrail phenotype in men. Further studies should examine the role of steroid metabolism and HPA axis impairment, and the associated sex differences, in the functional decline in aging population.

Abstract Background: Elevated urine cortisol (<1% of urinary steroid metabolome) was reported to predict future development of dementia. Our objective was to determine the association of urine steroid metabolome and its diurnal variation with cognitive function in men and women. Methods: Cross-sectional study of community-dwelling adults ≥ 50 years. Participants with adrenal disorders, end-stage renal or liver disease, on exogenous steroids or drugs affecting steroid metabolism were excluded. All participants completed day and night separate urine collection. A series of seven IPad-based tests using the National Institute of Health Toolbox Cognition Battery were administered to evaluate five key domains; performance was reported using fully corrected T-scores for age, sex, education, and race with a national normative mean of 50. T-scores were generated for the two summary measures: 1) fluid cognition (includes executive function, episodic memory, working memory, and processing speed), and 2) total composite (composite of fluid and language score). Urine samples were analyzed with the liquid-chromatography, high-resolution, accurate-mass mass spectrometry for 25 urine steroid metabolites. Results: Of 109 participants, 56 (51%) were women, and age and educational status were similar in men and women. On cognitive assessment, men and women had similar median composite cognition (T-score of 53 vs 54, p=0.74) and fluid cognition (T-score of 53 vs 51, p-value 0.96). Urine steroid metabolome analysis demonstrated 21/25 steroids were higher in men vs women. In both women and men, the ratio of total cortisol metabolites/total androgen metabolites (TCM/TAM) was associated with lower fluid cognition (women: ρ= -0.34, p=0.01, men: ρ= -0.43, p=0.001) and composite cognition (women: ρ= -0.27, p=0.04, men: ρ= -0.39, p=0.004). Higher ratio of day to night TCM were associated with a better fluid cognition in men (ρ= 0.35, p=0.01), but not in women (ρ= -0.11, p=0.41). Steroid ratios suggesting a relative enzymatic deficiency of 5α-Reductase type 2 was associated with lower fluid cognition in women (ρ= -0.29, p=0.03). In men, the fluid composite score was associated with a relative deficiency in 21-Hydroxylase (ρ= 0.42, p=0.002), 3β-Hydroxysteroid dehydrogenase (ρ= 0.43, p=0.001), and P450oxidoreductase (ρ= -0.35, p=0.01). Conclusion: We showed that a higher glucocorticoid to androgen ratio and a flattened circadian steroid variation were associated with lower global and fluid cognition score. Steroid ratios reflecting steroidogenesis enzymatic activity demonstrated sex differences in relation to cognition. Additional studies should examine whether the steroid fingerprint associated with lower cognition is predictive of a future dementia onset.

Abstract Background While quantitation methods for small-molecule and tryptic peptide bottom-up mass spectrometry (MS) have been well defined, quantitation methods for top-down or middle-up MS approaches have not been as well defined. Therapeutic monoclonal antibodies (t-mAbs) are a group of proteins that can be used to both demonstrate the advantages of top-down or middle-up detection methods over classic tryptic peptide bottom-up along with the growing need for robust quantitation strategies/software for these top-down or middle-up methods. Bottom-up proteolytic digest methods for the t-mAbs tend to suffer from challenges such as limited peptide selection due to potential interference from the polyclonal immunoglobulin background, complicated workflows, and inadequate sensitivity and specificity without laborious purification steps, and therefore have prompted the search for new detection and quantitation methods. Time-of-flight along with Orbitrap MS have recently evolved from the research and/or pharmaceutical setting into the clinical laboratory. With their superior mass measurement accuracy, resolution and scanning speeds, these are ideal platforms for top-down or middle-up characterization and quantitation. Methods We demonstrate a validated, robust, middle-up protein subunit detection and quantitation method for the IgG1 t-mAb, vedolizumab (VEDO), which takes advantage of the high resolution of the Orbitrap MS detection and quantitation software to increase specificity. Results Validated performance characteristics met pre-defined acceptance criteria with simple workflows and rapid turnaround times: characteristics necessary for implementation into a high-volume clinical MS laboratory. Conclusions While the extraction method can easily be used with other IgG1 t-mAbs, the detection and quantitation method may become an option for measurement of other proteins.