Citations

Abstract Context Prior reports of plasma estradiol concentrations and their reference ranges in postmenopausal women identified marked variation, in part due to assay method and body mass index (BMI). Objective To develop an international reference range for plasma estradiol in postmenopausal women, support accurate assessment of concentrations during systemic and local (eg, vaginal) estradiol therapy, and improve risk prediction for diseases, including osteoporosis, breast, and endometrial cancers. Methodology Estradiol concentrations were measured using primarily liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays in 5 international cohorts of community-dwelling, postmenopausal women of primarily European ancestry aged 38-100 years who were not using exogenous estrogens (n = 7206). Measurements were harmonized to the Centers for Disease Control and Prevention (CDC) reference measurement procedure (RMP) by re-assaying a subset of samples for each cohort and generating recalibration equations. Reference intervals were determined for all women, non-obese women, and specific BMI categories. Results Estradiol concentrations from separate LC-MS/MS assays correlated closely with the CDC RMP, and harmonization minimized inter-assay variability. Estradiol concentrations correlated with BMI but not with chronological age. The reference range (2.5th-97.5th percentile) was 1.1-18.2 pg/mL (4.0-66.8 pmol/L) with median of 4.9 pg/mL (18.0 pmol/L) for all postmenopausal women and 1.1-12.5 pg/mL (4.0-45.9 pmol/L) with median of 4.1 pg/mL (15.1 pmol/L) for non-obese women (BMI < 30). Reference ranges increased progressively with higher BMI categories. Conclusion Cross-calibration of estradiol measurements with the CDC RP enabled the development of harmonized plasma estradiol reference ranges for postmenopausal women who were not using exogenous estrogens, suitable for future studies to establish clinical utility. Increasing obesity in postmenopausal women was associated with increasing estradiol concentration and BMI category-specific reference ranges.

Abstract Background Historically, 25-hydroxyvitamin D (25OHD) assays have under- or over-recovered 25-hydroxyvitamin D2 (25OHD2), but assay manufacturers have modified their reagents to address this problem. In this study, we compared the second- and third-generation Roche assays as well as two contemporary offerings from Diasorin and Beckman against liquid chromatography–tandem mass spectrometry (LC-MS/MS). Methods We identified 50 remnant serum samples with 25OHD concentrations from across the analytical range of the second-generation Roche assay. To increase 25OHD2 representation, we identified 25 additional samples from individuals prescribed high-dose vitamin D2 supplements. We tested samples on Roche assays and circulated to laboratories performing Beckman and Diasorin 25OHD assays. We tested samples by LC-MS/MS to obtain concentrations for 25OHD2 and 25-hydroxyvitamin D3. Results Mean overall bias for each assay was 5.1 ng/mL or less against the LC-MS/MS measurement; mean proportional bias was 8.7% to 12.1%. Some individual specimens had much larger bias. 25OHD2 was under-recovered on average, but the bias for the third-generation Roche assay represents a significant improvement over the previous assay, and mean bias for current generation assays was no worse than −3.2 ng/mL. In most cases, clinical classification by automated assay values agreed with clinical classification by LC-MS/MS; where present, disagreements occurred near classification thresholds. Conclusions Automated 25OHD assays continue to improve, and 25OHD2 recovery no longer appears to be a significant concern for the assays evaluated here. All assays evaluated were adequate for clinical classification of vitamin D nutritional status and are suitable for routine use, including in patients prescribed high-dose vitamin D2.

Abstract Disclosure: A.M. Gonzalez Gil: None. E.F. Elli: None. R.J. Singh: None. S. Peres: None. A.Y. Chang: None. Background: Treatment of hypercortisolism in primary bilateral macronodular adrenal hyperplasia (PBMAH) with mild autonomous cortisol secretion (MACS) can be challenging. Medical treatment is useful for therapeutic purposes and as a bridge for adrenalectomy. We present the case of a patient who benefited from mifepristone but developed hyperestrogenism. Clinical Case: A 64-year-old man with prediabetes, hypertension, and cardiovascular disease had incidentally discovered bilateral nodular adrenal enlargement on CT scan following coronary artery bypass surgery. A few months later, he reported increasing fatigue, brain fog, weight gain, and worsening blood pressure. Physical exam showed no features of hypercortisolism. Work-up revealed baseline AM cortisol 17.3 mcg/dL [reference range (RR) 4.8-20], ACTH 10 pg/mL (RR 7.2-63) and DHEA-S 55 mcg/dL (RR 12-227); two abnormal AM cortisols after 1 mg overnight dexamethasone (5.4 and 6.9 mcg/dL; cut-off <1.8), and a normal 24-hour urine free cortisol (23 mcg/24h, RR 3.5-45). 24-hour urine metanephrines and aldosterone/renin ratio were normal. A 2-hour 75 g oral glucose tolerance test was diagnostic for type 2 diabetes mellitus (2-hour glucose 214 mg/dL). Baseline total testosterone (TT) was 366 ng/dL (RR 264-916) with normal follicle stimulating hormone (FSH) and luteinizing hormone (LH)concentrations. After a diagnosis of MACS, adrenal vein sampling showed bilateral excess cortisol secretion (lateralization ratio 1.09). Medical management versus right adrenalectomy based on nodule burden were offered, and he started mifepristone. With initial symptom improvement, his dose was increased from 300 mg to 300 mg alternating with 600 mg daily. When he developed increased blood pressure and hypokalemia, eplerenone was added. After 6 months of mifepristone and 5 months eplerenone 50 mg daily, he developed tender gynecomastia with elevated estradiol (106 pg/mL, RR ≤39), low TT (112 ng/dL) and undetectable FSH and LH. As expected with mifepristone, 17-hydroxyprogesterone was high (933 ng/dL, RR 51-190), but androstenedione and DHEA-S were normal. Reduction in mifepristone and eplerenone decreased estradiol levels (56 pg/mL) but gonadotrophins remained undetectable and TT decreased to 72 ng/dL. Despite discontinuation of eplerenone for 1 month, estradiol again increased (91 pg/mL), TT remained low (77 ng/dL), and mastalgia persisted. A decision to stop mifepristone and proceed with right adrenalectomy was made. Conclusion: This is a previously unreported incident of symptomatic estradiol-induced secondary hypogonadism on mifepristone therapy. Hyperestrogenism with gynecomastia and secondary hypogonadism with mifepristone suggests that some individuals may convert cortisol precursors to estradiol. Presentation: Saturday, July 12, 2025

Abstract Background The lack of standardization and harmonization among ferritin assays results in significant variability in clinical values. These assays have different reference ranges and numerous clinical cut-offs for interpreting anemia and iron toxicity. This inconsistency is due to manufacturers using various calibrators. Such variability complicates the interpretation and comparison of results across different clinical settings. Methods Ferritin was ordered for 7815 patient samples in 3 months and assays were performed by DXI. Similarly, 5818 patients’ samples were run by Roche in two months. Both assays are FDA cleared and are commonly used by hundreds of clinical laboratories. Frequency of no of patients flagged low or flagged were calculated with respective reference ranges of Dxi and Cobas. Results 12 percent of the patients were flagged low by the Beckman assay and would have gotten both physician and patients attention. However, by the Roche assay only 4 percent were flagged low. This was alarming to our physicians and lab was told to investigate to find for this shift. Manufacturer/Time Range Total Volume Total High Total Low %High %Low Dxi-May-24 2617 368 326 14.1% 12% Dxi-Jun-24 2391 345 278 14% 12% Dxi-Jul-24 2807 500 308 17.8% 11% Cobas-801 August 2024 - Ferritin Go Live Cobas-801-Sep-24 2545 572 105 22% 4% Cobas-801-Oct-24 2841 695 121 24% 4% Conclusion Lack of Standardization and harmonization has serious consequences for the patients being tested for ferritin to diagnose deficiency of iron or high iron situations. We are planning to do a prospective study to determine clinical cut-offs by correlating Ferritin lab results with other gold standard biomarkers including bone marrow biopsy.

Abstract Background Vitamin D supplementation by human individuals has significantly increased in the world. In US the total stored 25-OH-Vitamin D3 and 25-OH-Vitamin D2 has significantly increased for both in summers and winters. Under sunlight exposure, 7 dehydrocholesterol is converted to Vitamin D3 and is endogenous source in mammals. However, most of the exogenous source for Vitamin D used to be Vitamin D2 which used to be extracted only from mushrooms but is now manufactured by supplement industry as well. In last decade most of the supplementation has been due to chemically synthesized Vitamin D3. Food is now supplemented by both D2 and D3. It is not clear what is the prevalence of D2 supplementation/consumption in American population. Competitive immunoassays do not consistently recover 25-OH-D2 compared to 25-OH-D3 and some antibodies used have variable or no cross reactivity with 25-OH-D2. LC-MS/MS method can easily distinguish the two and can help in providing the precise and accurate results for 25-OH-D2 and 25-OH-D3. Here we present what is the prevalence of Vitamin D2 consumption and toxicity in American population based on our LC-MS/MS methods. Methods The study measured vitamin 25-OH-D levels (total, D2, and D3) in 148,654 patients from Mayo Clinic as well as from other medical centers within the U.S. Patients age ranging from new-born babies to over 100 years, and represents Male, Female and Neutral genders. Samples were analyzed for 25-OH-Vitamin D by LC-MS/MS as published earlier. Unlike immunoassays, LC-MS/MS gave us separate results for 25-OH-D3 and 25-OH-D2 with 100 percent specificity. Results 25-OH-Vitamin D2 levels of greater than 10 was present in at least 10,358 and ranged from 10 to 205 ng/mL. Of 10,358, 51 percent had 20-50 ng/mL, 10 percent had 51-80 ng/mL, and 1.3 percent had >80 ng/mL, ranging from 81-205 ng/mL with a median of 92 ng/mL. Of the patients found at Total Vitamin D >80 ng/mL, toxic levels of both D2 and D3 were found more in females than males. There was no significant gender difference for Vitamin D3 consumption but for D2 consumption, mean and median levels were different in males and females. Conclusion Clinical lab testing should accurately measure both 25-OH-D3 and 25-OH-D2, otherwise the patient total Vitamin D values in circulation will be underestimated.

AbstractBackground and ObjectivesIn the setting of tissue hypoxia, S‐nitrosylated haemoglobin (SNO‐Hb) plays crucial roles in the control of blood flow. This is associated with decreased oxygen affinity to haemoglobin and increase in tissue oxygenation. Red blood cell (RBC) transfusion is primarily performed to improve tissue oxygenation in anaemic patients. RBCs after storage undergo a variety of biochemical and functional alterations, including deficiency of nitric oxide (NO) bioactivity. However, how donor characteristics affect NO levels during RBC storage is unclear. We sought to investigate the association of blood donor age, gender and storage duration with NO and SNO‐Hb levels in blood units.Materials and MethodsBlood samples from 42 healthy younger (≤30 years) and older (≥45 years) donors were collected and stored for up to 42 days. Total NO kits were used to detect total nitrite and nitrate levels in blood storage solution. SNO‐Hb levels in RBCs were detected and analysed by quantitative mass spectrometry.ResultsTotal NO levels in the blood storage solution significantly increased with donor age and storage duration. Proteomic analysis revealed that RBCs from older donors, particularly older females, significantly lost SNO‐Hb during storage. Our findings indicate that RBCs from older donors are associated with reduced SNO‐Hb levels and increased NO metabolites in storage solution after ≥35 days storage.ConclusionThe findings suggest stored RBCs from older donors may have reduced capacity to deliver oxygen to tissues under hypoxia. A shorter shelf life may be required for storing RBCs from older donors, particularly older females.

Background: There are few and controversial results on 24,25(OH)2D and FGF23 acute changes following supplementation with cholecalciferol. Methods: Twenty-seven subjects with 25(OH)D < 30 ng/mL were randomized into three groups to receive a single oral dose of 25,000 I.U. or 600,000 I.U. of cholecalciferol or placebo, respectively. We measured 25(OH)D, 1,25(OH)2D, 24,25(OH)2D, and FGF23 levels at baseline and after 72 h. The 1,25(OH)2D/25(OH)D, 1,25(OH)2D/24,25(OH)2D, and 24,25(OH)2D/25(OH)D ratios were also calculated. Results: There was an increase in 25(OH)D and 1,25 (OH)2D following both doses of cholecalciferol. In the group administered 600,000 I.U., there was a significant increase in the delta changes in 25(OH)D and 1,25(OH)2D compared to the placebo and in the delta 24,25(OH)D2 compared to the placebo and 25,000 I.U. groups (all p < 0.05). A decrease in both the 1,25(OH)2D/25(OH)D and 1,25(OH)2D/24,25(OH)2D ratio (all p < 0.05) was observed in the 600,000 I.U. group. FGF23 values significantly increased only in the group administered 600,000 I.U. Conclusions: 25(OH)D and 1,25(OH)D levels significantly increased following 600,000 IU cholecalciferol administration compared to 25,000 I.U. and placebo. Following the massive administration of cholecalciferol, the CYP24A1 enzyme is actively involved in catabolism, thus, avoiding toxic effects.

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&gt;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&lt;0.001), higher glucocorticoids (15754 vs 12936, P&lt;0.001), and higher glucocorticoid/androgen ratio (8.7 vs 3.9, P&lt;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