Publications by authors named "Ravinder J Singh"

171 Publications

Divergent PGD and leukotriene C metabolite excretion following aspirin therapy: Ten patients with systemic mastocytosis.

Prostaglandins Other Lipid Mediat 2021 Aug 21;155:106563. Epub 2021 May 21.

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United States.

Aspirin-exacerbated respiratory disease and some cases of chronic idiopathic urticaria are disorders in which increased baseline urinary excretion of leukotriene(LT)E4 further increases following aspirin administration. Increased urinary excretion of the metabolites of prostaglandin D2, 11β-prostaglandin(PG)F2α and (2,3-dinor)-11β-PGF2α, have been documented in systemic mastocytosis (SM) and in mast cell activation syndrome (MCAS). Symptoms due to increased baseline and/or episodic release of PGD2 can be prevented with aspirin, an inhibitor of cyclooxygenase (COX)1 and COX2. Here by retrospective chart review we discovered 8 of 10 patients with SM in whom normalization of an elevated urinary (2,3-dinor)-11β-PGF2α occurred with aspirin therapy also had a parallel increased excretion of LTE4 by an average of nearly 13-fold. How widespread this phenomenon occurs in SM is unknown; however, this occurrence needs to be considered when interpreting changes in these urinary mast cell mediator metabolites during aspirin therapy.
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http://dx.doi.org/10.1016/j.prostaglandins.2021.106563DOI Listing
August 2021

Vitamin D3 Dose Requirement that Raises 25-Hydroxyvitamin D to Desirable Level in Overweight and Obese Elderly.

J Clin Endocrinol Metab 2021 May 6. Epub 2021 May 6.

Department of Internal Medicine, Division of Endocrinology, Calcium Metabolism and Osteoporosis Program, Beirut, Lebanon.

Purpose: To investigate the impact of two vitamin D doses, bracketed between the IOM recommended dietary allowance (RDA) and the upper tolerable limit, on vitamin D nutritional status in elderly individuals.

Methods: This is a post-hoc analysis on data collected from a 12-month, double-blinded, randomized control trial. 221 ambulatory participants (≥ 65 years), with a mean BMI of 30.2 kg/m 2, and a mean baseline serum 25-hydroxyvitamin D [25(OH)D] level of 20.4 ± 7.4 ng/ml, were recruited from 3 out-patient centers in Lebanon. They all received 1,000 mg of elemental calcium from calcium citrate daily, and the daily equivalent of 600 IU or 3,750 IU, of vitamin D3.

Results: Mean 25-hydroxyvitamin D [25(OH)D] level at 12 months was 26.0 ng/ml with low dose and 36.0 ng/ml with high dose, of vitamin D3. The proportion of participants reaching a value ≥ 20 ng/ml was 86% in the low dose, and 99% in the high-dose arms, with no differences between genders. The increment of 25(OH)D per 100 IU/day was 1ng/ml with the low dose, and 0.41 ng/ml with the high dose. Serum 25 (OH)D levels at 1 year were highly variable in both treatment arms. Baseline 25(OH)D level and vitamin D dose, but not age, BMI, gender, nor season, were significant predictors of serum 25(OH)D level post-intervention.

Conclusion: The IOM RDA of 600 IU/day does not bring 97.5% of ambulatory elderly individuals above the desirable threshold of 20 ng/ml. Country-specific RDAs are best derived taking into account the observed variability and predictors of achieved 25(OH)D levels.
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http://dx.doi.org/10.1210/clinem/dgab296DOI Listing
May 2021

Development and Validation of Test for "Leaky Gut" Small Intestinal and Colonic Permeability Using Sugars in Healthy Adults.

Gastroenterology 2021 Apr 16. Epub 2021 Apr 16.

Clinical Enteric NeuroscienceTranslational and Epidemiological Research (CENTER), Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota. Electronic address:

Background: Oral monosaccharides and disaccharides are used to measure in vivo human gut permeability through urinary excretion.

Aims: The aims were as follows: (1) to obtain normative data on small intestinal and colonic permeability; (2) to assess variance on standard 16 g fiber diet performed twice; (3) to determine whether dietary fiber influences gut permeability measurements; and (4) to present pilot data using 2 selected probes in patients with diarrhea-predominant irritable bowel syndrome (IBS-D).

Methods: Sixty healthy female and male adults, age 18-70 years, participated in 3 randomized studies (2 studies on 16.25 g and 1 study on 32.5 g fiber) in otherwise standardized diets. At each test, the following sugars were ingested: C-mannitol, C-mannitol, rhamnose (monosaccharides), sucralose, and lactulose (disaccharides). Standardized meals were administered from 24 hours before and during 24 hours post-sugars with 3 urine collections: 0-2, 2-8, and 8-24 hours. Sugars were measured using high-performance liquid chromatography-tandem mass spectrometry. Eighteen patients with IBS-D underwent 24-hour excretion studies after oral C-mannitol and lactulose.

Results: Baseline sugars (>3-fold above lower limits of quantitation) were identified in the 3 studies: C-mannitol in all participants; sucralose in 4-8, and rhamnose in 1-3. Median excretions/24 h (percentage of administered dose) for C-mannitol, rhamnose, lactulose, and sucralose were ∼30%, ∼15%, 0.32%, and 2.3%, respectively. C-mannitol and rhamnose reflected mainly small intestinal permeability. Intraindividual saccharide excretions were consistent, with minor differences with 16.25 g vs 32.5 g fiber diets. Median interindividual coefficient of variation was 76.5% (10-90 percentile: 34.6-111.0). There were no significant effects of sex, age, or body mass index on permeability measurements in health. C-mannitol measurements are feasible in IBS-D.

Conclusions: Baseline C-mannitol excretion precludes its use; C-mannitol is the preferred probe for small intestinal permeability.
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http://dx.doi.org/10.1053/j.gastro.2021.04.020DOI Listing
April 2021

Hyperphosphatemia with elevated serum PTH and FGF23, reduced 1,25(OH)D and normal FGF7 concentrations characterize patients with CKD.

BMC Nephrol 2021 Mar 30;22(1):114. Epub 2021 Mar 30.

Division of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic, 200 1st Street SW, MN, 55905, Rochester, USA.

Background: Hyperphosphatemia confers adverse cardiovascular outcomes, and commonly occurs in late-stage CKD. Fibroblast growth factor 7 (FGF7) is a phosphaturic peptide which decreases renal phosphate transport in vitro and in vivo. Serum FGF7 concentrations are reduced in hyperphosphatemic patients with hypophosphatasia and are elevated in some hypophosphatemic patients with tumor-induced osteomalacia. No data, however, are available on whether circulating FGF7 concentrations increase to compensate for phosphate retention in CKD patients.

Methods: This was a cross-sectional study performed among 85 adult patients with varying estimated glomerular filtration rates (eGFR). We measured serum intact FGF7 (iFGF7) concentration using an iFGF7 immunoassay and determined its associated factors. Relationships between eGFR and mineral metabolism biomarkers [phosphate, iFGF7, iFGF23, parathyroid hormone (PTH), and 1,25-dihydroxyvitamin D (1,25(OH)D)] were explored.

Results: For eGFRs of ≥ 60 (n = 31), 45-59 (n = 16), 30-44 (n = 11), 15-29 (n = 15), and < 15 mL/min/1.73 m (n = 12), median (IQ25-75) iFGF7 concentrations were 46.1 (39.2-56.9), 43.1 (39.0-51.5), 47.3 (38.3-66.5), 47.7 (37.7-55.8), and 49.6 (42.5-65.6) pg/mL, respectively (P = 0.62). Significant increases in serum iFGF23, PTH, and phosphate were observed at eGFRs of < 33 (95 % CI, 26.40-40.05), < 29 (95 % CI, 22.51-35.36), and < 22 mL/min/1.73 m (95 % CI, 19.25-25.51), respectively, while significant decreases in serum 1,25(OH)D were observed at an eGFR of < 52 mL/min/1.73 m (95 % CI, 42.57-61.43). No significant correlation was found between serum iFGF7 and phosphate, iFGF23, PTH or 1,25(OH)D. In multivariable analyses, body mass index (per 5 kg/m increase) was independently associated with the highest quartile of serum iFGF7 concentration (OR, 1.20; 95 % CI, 1.12-1.55).

Conclusions: Compensatory decreases in circulating 1,25(OH)D and increases in circulating iFGF23 and PTH, but not iFGF7, facilitate normalization of serum phosphate concentration in early stages of CKD. Whether other circulating phosphaturic peptides change in response to phosphate retention in CKD patients deserves further study.
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http://dx.doi.org/10.1186/s12882-021-02311-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011073PMC
March 2021

Chemical Characterization and Quantification of Circulating Intact PTH and PTH Fragments by High-Resolution Mass Spectrometry in Chronic Renal Failure.

Clin Chem 2021 Jun;67(6):843-853

Division of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA.

Background: The precise concentrations of full-length parathyroid hormone (PTH1-84) and the identity and concentrations of PTH fragments in patients with various stages of chronic renal failure are unknown.

Methods: We developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method to characterize and quantify PTH1-84 and PTH fragments in serum of 221 patients with progressive renal dysfunction. Following capture by matrix-bound amino-terminal or carboxyl-terminal region-specific antibodies and elution from matrix, PTH1-84 and PTH fragments were identified and quantitated using LC-HRMS. PTH was simultaneously measured using an intact PTH (iPTH) immunoassay.

Results: Full-length PTH1-84 and 8 PTH fragments (PTH28-84, 34-77, 34-84, 37-77, 37-84, 38-77, 38-84, and 45-84) were unequivocally identified and were shown to increase significantly when an eGFR declined to ≤17-23 mL/min/1.73m2. Serum concentrations of PTH1-84 were similar when measured by LC-HRMS following capture by amino-terminal or carboxyl-terminal immunocapture methods. In patients with an eGFR of <30 mL/min/1.73 m2, serum PTH concentrations measured using LC-HRMS were significantly lower than PTH measured using an iPTH immunoassay. PTH7-84 and oxidized forms of PTH1-84 were below the limit of detection (30 and 50 pg/mL, respectively).

Conclusions: LC-HRMS identifies circulating PTH1-84, carboxyl-terminal PTH fragments, and mid-region PTH fragments, in patients with progressive renal failure. Serum PTH1-84 and its fragments markedly rise when an eGFR decreases to ≤17-23 mL/min/1.73 m2. PTH concentrations measured using LC-HRMS tend to be lower than those measured using an iPTH immunoassay, particularly in severe chronic renal failure. Our data do not support the existence of circulating PTH7-84 and oxidized PTH1-84.
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http://dx.doi.org/10.1093/clinchem/hvab013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167341PMC
June 2021

1α,25-Dihydroxyvitamin D Encapsulated in Nanoparticles Prevents Venous Neointimal Hyperplasia and Stenosis in Porcine Arteriovenous Fistulas.

J Am Soc Nephrol 2021 Feb 24. Epub 2021 Feb 24.

Department of Radiology, Vascular and Interventional Translational Laboratory, Mayo Clinic, Rochester, Minnesota

Background: Few therapies prevent venous neointimal hyperplasia (VNH) and venous stenosis (VS) formation in arteriovenous fistulas (AVF). Expression of the immediate early response gene X-1 (), also known as is associated with VNH and stenosis in murine AVFs. The study aimed to determine if local release of long-acting inhibitor 1α,25(OH)D from poly(lactic-co-glycolic acid) (PLGA) nanoparticles embedded in a thermosensitive Pluronic F127 hydrogel (1,25 NP) could affect VNH/VS formation in a large animal model.

Methods: Immediately after AVF creation in a porcine model of renal failure, 1,25 NP or vehicle control was injected into the adventitia space of AVF outflow veins. Scanning electron microscopy and dynamic light scattering characterized drug and control nanoparticles. Animals were sacrificed 3 and 28 days later for gene expression, immunohistologic, magnetic resonance imaging and angiography, and ultrasound analyses. Whole transcriptome RNA sequencing with differential gene expression analysis was performed on outflow veins of AVF.

Results: Encapsulation of 1α,25(OH)D in PLGA nanoparticles formed nanoparticles of uniform size that were similar to nanoparticles without 1α,25(OH)D. The 1,25 NP-treated AVFs exhibited lower VNH/VS, gene expression, and IER-3, MCP-1, CD68, HIF-1α, and VEGF-A immunostaining, fibrosis, and proliferation. Blood flow and lumen area increased significantly, whereas peak systolic velocity and wall shear stress decreased. Treatment increased Young's modulus and correlated with histologic assessment of fibrosis and with no evidence of vascular calcification. RNA sequencing analysis showed changes in the expression of genes associated with inflammatory, TGFβ1, and apoptotic pathways.

Conclusions: Local release of 1,25 NP improves AVF flow and hemodynamics, and reduces stenosis in association with reduction in inflammation, apoptosis, and fibrosis in a porcine model of arteriovenous fistula.
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http://dx.doi.org/10.1681/ASN.2020060832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8017547PMC
February 2021

Steroidogenesis in patients with adrenal incidentalomas: Extended steroid profile measured by liquid chromatography-mass spectrometry after ACTH stimulation and dexamethasone suppression.

Clin Endocrinol (Oxf) 2021 Jul 2;95(1):29-40. Epub 2021 Mar 2.

Adrenal and Hypertension Unit, Division of Endocrinology and Metabolism, Department of Medicine, UNIFESP- EPM, Federal University of São Paulo, São Paulo, Brasil.

Objective: Describe the secretion and profile of adrenal steroids in patients with adrenal incidentalomas compared to control subjects.

Design, Setting And Participants: A prospective study, 73 patients with adrenal incidentalomas, 21 bilateral and 52 unilateral and 34 matched controls in University Hospital.

Methods: Collect fasting blood sample before and 60 min after ACTH test (250 µg IV). One week later, perform overnight 1 mg dexamethasone test. The following steroids were measured by liquid chromatography-mass spectrometry (LC-MS): pregnenolone, 17-OH pregnenolone, 17-OH progesterone, 11-deoxycorticosterone, 11-deoxyortisol, 21-deoxycortisol, corticosterone, cortisol, androstenedione and aldosterone.

Results: Mean baseline serum cortisol was higher in incidentalomas, bilateral 361 ± 124, (range 143-665) nmol/L,(p < .0001), unilateral 268 ± 89 3.2 (range 98-507) nmol/L (p < .019) compared to controls 207 ± 100 (range 72-502) nmol/L. ACTH stimulation showed significantly higher levels in bilateral and unilateral cases compared to controls. After dexamethasone, mean serum cortisol levels suppressed in bilaterals 89 ± 69 (range 30-3) nmol/L (p < .0001), 58 ± 52 (range 16-323) nmol/L in unilateral (p < .01) compared to 26 ± 9 (range 7-46) nmol/L in controls. Mean baseline serum corticosterone was higher in bilateral 9.3 ± 4.8 (range 2.4-18.4) nmol/L (p < .005) and unilateral 7.3 ± 5.7 (range 0.1-30.3) nmol/L (p < .01) compared to controls 4.2 ± 2.4 (range 1.1-10.2) nmol/L, after ACTH stimulation significantly increased to higher levels in bilateral (p < .0002) and unilateral cases (p < .044) compared to controls. After dexamethasone, mean levels were 2.5 ± 2.6 (range 0.5-12.5) nmol/L in bilateral (p < .0006), 1.5 ± 1.6 (range 0.3-9.3) nmol/L in unilateral (p < .09) and 0.75 ± 0.46 (range 0.1-2.1) nmol/L in controls. Mean baseline serum 11-deoxycorticosterone (DOC) was higher in bilaterals 0.32 ± 0.23 (range 0.08-1.1) nmol/L (p < .03) compared to controls 0.15 ± 0.21 (range 0.08-1.1) nmol/L. ACTH stimulation increased levels to 3.27 ± 1.72 (range 0.5-7.4) nmol/L in bilateral cases compared to controls 1.369 ± 1.53 (range 0.1-7.1) nmol/L (p < .0001). Dexamethasone decreased levels to baseline (p ns). There were significant differences in serum 21-deoxycortisol (p < .0002) and serum pregnenolone (p < .004) only after ACTH stimulation.

Conclusions: There is increased activity in several steroid biosynthesis pathways and higher steroid levels in bilateral compared to unilateral cases and evidence of hypercortisolism in 30% unilateral and 62% of bilateral incidentalomas.
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http://dx.doi.org/10.1111/cen.14445DOI Listing
July 2021

Total, Bioavailable, and Free 25(OH)D Relationship with Indices of Bone Health in Elderly: A Randomized Controlled Trial.

J Clin Endocrinol Metab 2021 Jan;106(2):e990-e1001

Department of Internal Medicine, Division of Endocrinology, Calcium Metabolism and Osteoporosis Program, Beirut, Lebanon.

Context: Questions regarding the superiority of free and bioavailable 25-hydroxyvitamin D [25(OH)D] in predicting health outcomes remain unresolved.

Objective: This study investigates the impact of vitamin D variables-total, bioavailable, or free 25(OH)D-on indices of bone and mineral metabolism, at baseline and in response to 2 vitamin D doses.

Design: Our objectives are implemented as exploratory analyses on data collected in a 1-year, double-blind, randomized controlled trial completed in July 2014.

Setting: Participants were recruited from 3 major hospitals in an ambulatory setting.

Participants: Participants were >65 years of age, overweight, and had a baseline serum 25(OH)D between 10 and 30 ng/mL. A total of 221 participants completed the study.

Intervention: Subjects were randomized to receive calcium and oral vitamin D3 (600 IU/day or 3750 IU/day) supplementation.

Results: Participants who received the higher vitamin D dose had levels that were 1.3- to 1.4-fold higher than those taking the lower dose, for all variables (P value < 0.001). Serum values of bioavailable and free 25(OH)D were associated with total 25(OH)D, with r values of 0.942 and 0.943, respectively (P value < 0.001). Parathyroid hormone (PTH) was negatively associated with all vitamin D variables, with correlation coefficients ranging from -0.22 to -0.25, while calcium and bone turnover markers (carboxy-terminal collagen crosslinks and osteocalcin) did not. Only total 25(OH)D had a positive relationship with % change bone mineral density (BMD) at the femoral neck at 12 months, while only free and bioavailable 25(OH) had a positive relationship with % change total body BMD at 12 months.

Conclusion: Calculated free and bioavailable 25(OH)D do not appear to be superior to total 25(OH)D in predicting indices of bone health in an elderly population.
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http://dx.doi.org/10.1210/clinem/dgaa780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823248PMC
January 2021

Development of a pregnancy-specific reference material for thyroid biomarkers, vitamin D, and nutritional trace elements in serum.

Clin Chem Lab Med 2020 Oct 26. Epub 2020 Oct 26.

National Institute of Standards and Technology, Hollings Marine Laboratory, Charleston, SC, USA.

Objectives: Matrix differences among serum samples from non-pregnant and pregnant patients could bias measurements. Standard Reference Material 1949, Frozen Human Prenatal Serum, was developed to provide a quality assurance material for the measurement of hormones and nutritional elements throughout pregnancy.

Methods: Serum from non-pregnant women and women in each trimester were bottled into four levels based on pregnancy status and trimester. Liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were developed and applied to the measurement of thyroid hormones, vitamin D metabolites, and vitamin D-binding protein (VDBP). Copper, selenium, and zinc measurements were conducted by inductively coupled plasma dynamic reaction cell MS. Thyroid stimulating hormone (TSH), thyroglobulin (Tg), and thyroglobulin antibody concentrations were analyzed using immunoassays and LC-MS/MS (Tg only).

Results: Certified values for thyroxine and triiodothyronine, reference values for vitamin D metabolites, VDBP, selenium, copper, and zinc, and information values for reverse triiodothyronine, TSH, Tg, and Tg antibodies were assigned. Significant differences in serum concentrations were evident for all analytes across the four levels (p≤0.003).TSH measurements were significantly different (p<0.0001) among research-only immunoassays. Tg concentrations were elevated in research-only immunoassays vs. Federal Drug Administration-approved automated immunoassay and LC-MS/MS. Presence of Tg antibodies increased differences between automated immunoassay and LC-MS/MS.

Conclusions: The analyte concentrations' changes consistent with the literature and the demonstration of matrix interferences in immunoassay Tg measurements indicate the functionality of this material by providing a relevant matrix-matched reference material for the different stages of pregnancy.
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http://dx.doi.org/10.1515/cclm-2020-0977DOI Listing
October 2020

Drug Tissue Distribution of TUDCA From a Biodegradable Suprachoroidal Implant versus Intravitreal or Systemic Delivery in the Pig Model.

Transl Vis Sci Technol 2020 05 15;9(6):11. Epub 2020 May 15.

Department of Civil Engineering, University of Minnesota, Minneapolis, MN, USA.

Purpose: To determine local ocular tissue levels of the bile acid, tauroursodeoxycholic acid (TUDCA), in the pig model using oral, intravenous (IV), intravitreal injection (IVitI) and low- and high-dose suprachoroidal, sustained-release implants (SCI-L or SCI-H).

Methods: Forty-six pigs (92 globes) were included in the study. TUDCA was delivered orally in 5 pigs, IV in 4, IVitI in 6, SCI-L in 17, and SCI-H in 14. Testing timeframes varied from the same day (within minutes) for IV; 1 to 6 days, oral; and 1 to 4 weeks, IVitI and SCI. Enucleated globes were dissected, specimens from specific tissues were separated, and TUDCA was extracted and quantified using mass spectrometry.

Results: The highest TUDCA tissue levels occurred after IV delivery in the macula (252 ± 238 nM) and peripheral retina (196 ± 171 nM). Macular choroid and peripheral choroid levels were also high (1032 ± 1269 and 1219 ± 1486 nM, respectively). For IVitI delivery, macular levels at day 6 were low (0.5 ± 0.5 nM), whereas peripheral choroid was higher (15.3 ± 16.7 nM). Neither the SCI-L nor SCI-H implants delivered meaningful macular doses (≤1 nM); however, peripheral retina and choroid levels were significantly higher. Bile acid isoforms were found in the serum specimens.

Conclusions: The highest TUDCA tissue levels in the pig model were obtained using IV delivery. Oral delivery was associated with reasonable tissue levels. Local delivery (IVitI and SCI) was able to achieve measurable local ocular tissue levels.

Translational Relevance: Diffusional kinetics from the suprachoroidal space follow the choroidal blood flow, away from the macula and toward the periphery.
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http://dx.doi.org/10.1167/tvst.9.6.11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408862PMC
May 2020

Low-Dose Testosterone Augmentation for Antidepressant-Resistant Major Depressive Disorder in Women: An 8-Week Randomized Placebo-Controlled Study.

Am J Psychiatry 2020 10 14;177(10):965-973. Epub 2020 Jul 14.

Neuroendocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston (Dichtel, Kimball, Miller); Depression Clinical and Research Program, Department of Psychiatry, Massachusetts General Hospital and Harvard Medical School, Boston (Nyer, Mischoulon, Deckersbach, Dougherty, Yeung, Cassano, Hahn, Farabaugh, Pedrelli, Trinh, Dording, Cusin, Papakostas, Chang, Fisher, Shapero, Chen, Fava); Department of Psychiatry, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston (Brady); Biostatistics Center, Massachusetts General Hospital, Boston (Schoenfeld); Butler Hospital and Department of Psychiatry and Human Behavior, Warren Alpert Medical School of Medicine, Brown University, Providence, R.I. (Carpenter, Tyrka, Price); Neuroendocrine Unit, Department of Medicine, Massachusetts General Hospital, Boston (Rao); Mayo Clinic Endocrine Laboratory, Rochester, Minn. (Singh).

Objective: Low-dose testosterone has been shown to improve depression symptom severity, fatigue, and sexual function in small studies in women not formally diagnosed with major depressive disorder. The authors sought to determine whether adjunctive low-dose transdermal testosterone improves depression symptom severity, fatigue, and sexual function in women with antidepressant-resistant major depression. A functional MRI (fMRI) substudy examined effects on activity in the anterior cingulate cortex (ACC), a brain region important in mood regulation.

Methods: The authors conducted an 8-week randomized double-blind placebo-controlled trial of adjunctive testosterone cream in 101 women, ages 21-70, with antidepressant-resistant major depression. The primary outcome measure was depression symptom severity as assessed by the Montgomery-Åsberg Depression Rating Scale (MADRS). Secondary endpoints included fatigue, sexual function, and safety measures. The primary outcome of the fMRI substudy (N=20) was change in ACC activity.

Results: The participants' mean age was 47 years (SD=14) and their mean baseline MADRS score was 26.6 (SD=5.9). Eighty-seven (86%) participants completed 8 weeks of treatment. MADRS scores decreased in both study arms from baseline to week 8 (testosterone arm: from 26.8 [SD=6.3] to 15.3 [SD=9.6]; placebo arm: from 26.3 [SD=5.4] to 14.4 [SD=9.3]), with no significant difference between groups. Improvement in fatigue and sexual function did not differ between groups, nor did side effects. fMRI results showed a relationship between ACC activation and androgen levels before treatment but no difference in ACC activation with testosterone compared with placebo.

Conclusions: Adjunctive transdermal testosterone, although well tolerated, was not more effective than placebo in improving symptoms of depression, fatigue, or sexual dysfunction. Imaging in a subset of participants demonstrated that testosterone did not result in greater activation of the ACC.
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http://dx.doi.org/10.1176/appi.ajp.2020.19080844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7748292PMC
October 2020

Improved Recognition of 25-Hydroxyvitamin D2 by 2 Automated Immunoassays.

J Appl Lab Med 2020 11;5(6):1287-1295

Department of Pathology and Laboratory Medicine, Dartmouth-Hitchcock Health System, Lebanon, NH; The Geisel School of Medicine at Dartmouth, Hanover, NH.

Background: Despite recommendations to limit vitamin D testing to specific clinical scenarios, test volume remains high in many clinical laboratories. Automated total vitamin D immunoassays frequently under- or over-recover 25-hydroxyvitamin D2 [25(OH)D2], making accurate assessment of vitamin D status difficult in patients taking high-dose 25(OH)D2 supplements. Mass spectrometry-based methods offer excellent recovery of 25(OH)D2 but are not practical for use in all laboratories. In this study, we evaluated 2 automated immunoassays against an LC-MS/MS method performed at a national reference laboratory.

Methods: A method comparison against LC-MS/MS was performed for the Roche Elecsys Vitamin D total II assay and the IDS-iSYS 25 VitDS immunoassays using 49 patient specimens submitted for clinical 25(OH)D measurement. Mean bias was calculated, and vitamin D status was determined for each specimen according to the 2011 Endocrine Society clinical practice guidelines.

Results: Theil-Sen regression lines relative to LC-MS/MS were y = 0.88x + 2.94 for Roche and y = 1.03x + 2.48 for IDS. Mean bias (±SD) in samples with 25(OH)D2 concentrations less than 5 ng/mL was -0.25 ng/mL (±6.30) for Roche and -1.45 ng/mL (±6.82) for the IDS. Mean bias (±SD) in samples with 25(OH)D2 concentrations greater than 5 ng/mL was -3.19 ng/mL (±6.61) for Roche and 5.52 ng/mL (±6.36) for IDS. Median percentage recovery of 25(OH)D2 was 87.1% (interquartile range 76.0-111.3) for Roche and 120.6% (interquartile range: 105.3-133.4) for IDS. Vitamin D status was misclassified in 7 samples by the Roche assay and 3 by the IDS assay. For all but one of the discordant pairs, the immunoassay result was within 1.7 ng/mL of the diagnostic cutoff.

Conclusions: The automated immunoassays evaluated here demonstrate improved recovery of 25(OH)D2 relative to previous generations. Both are acceptable for use in the determination of vitamin D status.
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http://dx.doi.org/10.1093/jalm/jfaa070DOI Listing
November 2020

Risk of Iodine Deficiency in Extremely Low Gestational Age Newborns on Parenteral Nutrition.

Nutrients 2020 Jun 1;12(6). Epub 2020 Jun 1.

Department of Pediatrics, Division of Neonatology, Case Western Reserve University, Metro Health Medical Center, Rm C.G75, 2500 MetroHealth Drive, Cleveland, OH 44109, USA.

is an essential component of thyroid hormones, which play a critical role in neurodevelopment. The iodine status of pregnant women and their newborns is not checked routinely. Extremely Low Gestational Age Newborns do not receive Iodine supplementation while on parenteral nutrition (PN). We measured urine iodine levels and thyroid function tests in 50 mother-infant dyads at birth, at 1 week, 1, 2, 3 months and near discharge. We correlated maternal and neonatal urine iodine levels with thyroid functions and measured iodine levels in milk and PN. In our study, 64% of mothers were iodine deficient at the time of delivery, their free T4 levels were 0.48 (0.41-0.54) ng/dL with normal thyroid-stimulating hormone (TSH). Iodine levels were thirty-fold higher in extremely low gestational age newborns (ELGAN) exposed to iodine comparing to full terms ( < 0.001), but this effect lasted <1 week. At 1 month of age, ELGAN on PN developed iodine deficiency ( = 0.017) and had high thyroglobulin levels of 187 (156-271) ng/mL. Iodine levels improved with enteral feeds by 2 months of age ( = 0.01). Iodine deficiency is prevalent among pregnant women and ELGAN; in particular, those on PN are at risk of hypothyroidism. Iodine supplementation during pregnancy and postnatally should be considered to avoid iodine deficiency.
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http://dx.doi.org/10.3390/nu12061636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352251PMC
June 2020

Development and Application of Mass Spectroscopy Assays for Nε-(1-Carboxymethyl)-L-Lysine and Pentosidine in Renal Failure and Diabetes.

J Appl Lab Med 2020 05;5(3):558-568

Mayo Clinic College of Medicine, Rochester, MN.

Background: Advanced glycation end products (AGEs) are formed via the nonenzymatic glycation of sugars with amino acids. Two AGEs, Nε-(1-carboxymethyl)-L-Lysine (CML) and pentosidine, have been observed to be elevated in subjects suffering from a multitude of chronic disease states, and accumulation of these compounds may be related to the pathophysiology of disease progression and aging.

Methods: We describe here the development and validation of a specific and reproducible LC-MS/MS method to quantify CML and pentosidine in human serum with lower limits of quantitation of 75 ng/mL and 5 ng/mL, respectively. The analyte calibration curve exhibited excellent linearity at a range of 0-10 900 ng/mL for CML and 0-800 ng/mL for pentosidine. High-low linearity of 5 serum pairs was assessed, with a mean recovery of 103% (range 94-116%) for CML, and 104% (range 97-116%) for pentosidine.

Results: Serum concentrations of CML and pentosidine were quantified in 30 control and 30 subjects with chronic renal insufficiency. A significant increase in both analytes was observed in renal failure compared to control subjects (2.1-fold and 8.4-fold, respectively; P < 0.001 for both). In a separate cohort of 49 control versus 95 subjects with type 2 diabetes mellitus (T2DM), serum CML but not serum pentosidine, was significantly elevated in the T2DM patients, and CML was also correlated with glycemic control, as assessed by hemoglobin A1c (r = 0.34, P < 0.001).

Conclusions: These mass spectroscopy-based assays for serum CML and pentosidine should be useful in accurately evaluating circulating levels of these key AGEs in various disease states.
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http://dx.doi.org/10.1093/jalm/jfaa023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192546PMC
May 2020

Baseline estrogen levels in postmenopausal women participating in the MAP.3 breast cancer chemoprevention trial.

Menopause 2020 06;27(6):693-700

Division of Medical Oncology, Mayo Clinic, Rochester, MN.

Objective: The aim of the study was to quantify baseline estradiol (E2) and estrone (E1) concentrations according to selected patient characteristics in a substudy nested within the MAP.3 chemoprevention trial.

Methods: E2 and E1 levels were measured in 4,068 postmenopausal women using liquid chromatography-tandem mass spectrometry. Distributions were described by age, years since menopause, race, body mass index (BMI), smoking status, and use and duration of hormone therapy using the Kruskal-Wallis test. Multivariable linear regression was also used to identify characteristics associated with estrogen levels.

Results: After truncation at the 97.5th percentile, the mean (SD)/median (IQR) values for E2 and E1 were 5.41 (4.67)/4.0 (2.4-6.7) pg/mL and 24.7 (14.1)/21 (15-31) pg/mL, respectively. E2 and E1 were strongly correlated (Pearson correlation [r] = 0.8, P < 0.01). The largest variation in E2 and E1 levels was by BMI; mean E2 and E1 levels were 3.5 and 19.1 pg/mL, respectively for women with BMI less than 25 and 7.5 and 30.6 pg/mL, respectively, for women with BMI greater than 30. E2 and E1 varied by age, BMI, smoking status, and prior hormone therapy in multivariable models (P < 0.01).

Conclusions: There was large interindividual variability observed for E2 and E1 that varied significantly by participant characteristics, but with small absolute differences except in the case of BMI. Although the majority of participant characteristics were independently associated with E1 and E2, together, these factors only explained about 20% of the variation in E1 and E2 levels.
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http://dx.doi.org/10.1097/GME.0000000000001568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469568PMC
June 2020

Cavernous Malformation Hemorrhagic Presentation at Diagnosis Associated with Low 25-Hydroxy-Vitamin D Level.

Cerebrovasc Dis 2020 29;49(2):216-222. Epub 2020 Apr 29.

Department of Neurosurgery, Mayo Clinic, Rochester, Minnesota, USA.

Background: Cavernous malformations (CM) are angiographically occult vascular malformations that may be incidental or present with intracerebral or spinal hemorrhage, seizures, or nonhemorrhagic focal neurologic deficit (FND). Recently in vitro data have suggested vitamin D may play a role in stabilizing CCM2 endothelial cells. Little is known about the effect of vitamin D in human CM disease.

Methods: Beginning in 2015, consecutive patients at our institution with radiologically confirmed CM were recruited to participate in a prospective clinical registry as well as 25-hydroxy-vitamin D study. A structured interview, survey, and examination were performed at baseline. Medical records and magnetic resonance imaging studies were reviewed and data collected included comorbid conditions, medication use, and location of CM. Standard definition of clinical hemorrhage, FND, and seizures was used. Univariate and multivariate logistic regression models were used, and OR, 95% CIs, and likelihood-ratio p values were calculated to determine the influence of the 25-hydroxy-vitamin D level on clinical presentation with hemorrhage.

Results: Of 213 patients enrolled in the clinical registry between January 2015 and October 2018, 70 participated in the vitamin D study (median age: 38.3 years; 51.4% female). Of the 70 participants, 30 (42.9%) presented with hemorrhage. 25-Hydroxy-vitamin D levels were performed within 1 year of symptoms in 64.1% of patients. Patients presenting with hemorrhage had a lower 25-hydroxy-vitamin D level compared to those presenting with seizure without hemorrhage, FND, or as an incidental finding (median 25.5 ng/mL; range 11-59 hemorrhage vs. median 31.0; range 14-60, no hemorrhage; p = 0.04). After adjusting for age, month of blood draw, and body mass index, 25-hydroxy-vitamin D remained a significant predictor of hemorrhagic presentation. Brainstem location also predicted hemorrhage at presentation.

Conclusion: Low 25-hydroxy-vitamin D level was more common in patients with CM presenting with hemorrhage. This study supports the potential role of modifiable factor in the initial clinical presentation of CM. Further study is needed to determine the role of vitamin D on prospective hemorrhage risk and whether supplementation may be beneficial.
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http://dx.doi.org/10.1159/000507789DOI Listing
September 2020

Accelerated osteocyte senescence and skeletal fragility in mice with type 2 diabetes.

JCI Insight 2020 05 7;5(9). Epub 2020 May 7.

Division of Endocrinology.

The worldwide prevalence of type 2 diabetes (T2D) is increasing. Despite normal to higher bone density, patients with T2D paradoxically have elevated fracture risk resulting, in part, from poor bone quality. Advanced glycation endproducts (AGEs) and inflammation as a consequence of enhanced receptor for AGE (RAGE) signaling are hypothesized culprits, although the exact mechanisms underlying skeletal dysfunction in T2D are unclear. Lack of inducible models that permit environmental (in obesity) and temporal (after skeletal maturity) control of T2D onset has hampered progress. Here, we show in C57BL/6 mice that a onetime pharmacological intervention (streptozotocin, STZ) initiated in adulthood combined with high-fat diet-induced (HFD-induced) obesity caused hallmark features of human adult-onset T2D, including prolonged hyperglycemia, insulin resistance, and pancreatic β cell dysfunction, but not complete destruction. In addition, HFD/STZ (i.e., T2D) resulted in several changes in bone quality that closely mirror those observed in humans, including compromised bone microarchitecture, reduced biomechanical strength, impaired bone material properties, altered bone turnover, and elevated levels of the AGE CML in bone and blood. Furthermore, T2D led to the premature accumulation of senescent osteocytes with a unique proinflammatory signature. These findings highlight the RAGE pathway and senescent cells as potential targets to treat diabetic skeletal fragility.
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http://dx.doi.org/10.1172/jci.insight.135236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253018PMC
May 2020

Anastrozole has an Association between Degree of Estrogen Suppression and Outcomes in Early Breast Cancer and is a Ligand for Estrogen Receptor α.

Clin Cancer Res 2020 06 25;26(12):2986-2996. Epub 2020 Feb 25.

Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota.

Purpose: To determine if the degree of estrogen suppression with aromatase inhibitors (AI: anastrozole, exemestane, letrozole) is associated with efficacy in early-stage breast cancer, and to examine for differences in the mechanism of action between the three AIs.

Experimental Design: Matched case-control studies [247 matched sets from MA.27 (anastrozole vs. exemestane) and PreFace (letrozole) trials] were undertaken to assess whether estrone (E1) or estradiol (E2) concentrations after 6 months of adjuvant therapy were associated with risk of an early breast cancer event (EBCE). Preclinical laboratory studies included luciferase activity, cell proliferation, radio-labeled ligand estrogen receptor binding, surface plasmon resonance ligand receptor binding, and nuclear magnetic resonance assays.

Results: Women with E1 ≥1.3 pg/mL and E2 ≥0.5 pg/mL after 6 months of AI treatment had a 2.2-fold increase in risk ( = 0.0005) of an EBCE, and in the anastrozole subgroup, the increase in risk of an EBCE was 3.0-fold ( = 0.001). Preclinical laboratory studies examined mechanisms of action in addition to aromatase inhibition and showed that only anastrozole could directly bind to estrogen receptor α (ERα), activate estrogen response element-dependent transcription, and stimulate growth of an aromatase-deficient T47D breast cancer cell line.

Conclusions: This matched case-control clinical study revealed that levels of estrone and estradiol above identified thresholds after 6 months of adjuvant anastrozole treatment were associated with increased risk of an EBCE. Preclinical laboratory studies revealed that anastrozole, but not exemestane or letrozole, is a ligand for ERα. These findings represent potential steps towards individualized anastrozole therapy.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299827PMC
June 2020

Correlation Between Size and Function of Unilateral and Bilateral Adrenocortical Nodules: An Observational Study.

AJR Am J Roentgenol 2020 04 18;214(4):800-807. Epub 2020 Feb 18.

Department of Medicine, Division of Endocrinology and Metabolism, Adrenal and Hypertension Unit, Universidade Federal de São Paulo, R. Pedro de Toledo 781, 13th Fl, São Paulo 04039-032, Brazil.

Adrenal incidentalomas occur in 5% of adults and can produce autonomous cortisol secretion that increases the risk of metabolic syndrome and cardiovascular disease. The objective of our study was to evaluate the relationship between adrenal nodule size measured on CT and autonomous cortisol secretion. In a prospective study of 73 patients 22-87 years old with incidentalomas, unilateral in 52 patients and bilateral in 21 patients, we measured maximum nodule diameter on CT and serum cortisol levels at 8:00 am, 60 minutes after the adrenocorticotropic hormone stimulation test, and after the dexamethasone suppression test. We also studied 34 age-, sex-, and body mass index-matched control subjects. Statistics used were Spearman correlation coefficients, tests, ANOVA test, and multivariate analysis. The mean maximum diameter of unilateral nodules measured on CT was larger on the right (2.47 ± 0.98 [SD] cm) than on the left (2.04 ± 0.86 cm) ( = 0.01). In the bilateral cases, the mean diameter of the right nodules was 2.69 ± 0.93 cm compared with 2.13 ± 0.89 cm on the left ( = 0.06). Mean baseline serum cortisol level was significantly higher in the patients with incidentalomas (bilateral, 13.1 ± 4.5 mcg/dL [ < 0.001]; unilateral, 9.7 ± 3.2 mcg/dL [ = 0.019]) than in the control subjects (7.5 ± 3.6 mcg/dL). After dexamethasone suppression test, serum cortisol levels were suppressed to less than 1.8 mcg/dL in 100% of control subjects, 33% of patients with bilateral incidentalomas, and 62% of patients with unilateral incidentalomas ( < 0.001). There were significant correlations between maximum nodule diameter on CT and serum cortisol levels after the dexamethasone suppression test (ρ = 0.500; < 0.001) and at baseline (ρ = 0.373; = 0.003). Increasing size of adrenal nodules is associated with more severe hyper-cortisolism and less dexamethasone suppression; these cases need further evaluation and possibly surgery because of increased risks of metabolic syndrome and cardiovascular mortality.
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http://dx.doi.org/10.2214/AJR.19.21753DOI Listing
April 2020

Determinants and Measurement of Neonatal Vitamin D: Overestimation of 25(OH)D in Cord Blood Using CLIA Assay Technology.

J Clin Endocrinol Metab 2020 04;105(4)

Department of Pediatrics, Golisano Children's Hospital at Strong, University of Rochester Medical Center, Rochester, New York.

Context: Vitamin D (VD) deficiency in pregnancy and the neonatal period has impacts on childhood outcomes. Maternal VD sufficiency is crucial for sufficiency in the neonate, though the effect of early versus late pregnancy 25-hydroxy-vitamin D (25(OH)D) levels on neonatal levels is unknown. Furthermore, chemiluminescence immunoassays (CLIAs) are widely used, though their validity in measuring 25(OH)D specifically in cord blood specimens has not been established.

Objective: To assess the validity of a CLIA in the measurement of cord blood 25(OH)D and to evaluate maternal determinants of neonatal 25(OH)D, including early versus late pregnancy 25(OH)D levels.

Design: This is an ancillary analysis from the Vitamin D Antenatal Asthma Reduction Trial (VDAART), a randomized, double-blinded, placebo-controlled study.

Participants And Intervention: A total of 881 pregnant women at high risk of having offspring asthma were randomized to receive VD supplementation or placebo. Serum samples were collected from mothers in early and late pregnancy and from offspring cord blood at birth. 25(OH)D levels were assayed by CLIA in all maternal and offspring samples and by LC-MS/MS in all offspring samples and a subset of 200 maternal third trimester samples.

Results: Cord blood 25(OH)D levels were higher as measured by CLIA (mean 37.13 ng/mL [SD 18.30]) than by LC-MS/MS (mean 23.54 ng/mL [SD 11.99]), with a mean positive bias of 13.54 ng/mL (SD 12.92) by Bland-Altman analysis. This positive bias in measurement by CLIA was not observed in maternal samples. Third trimester 25(OH)D was a positive determinant of neonatal 25(OH)D levels.

Conclusion: Chemiluminescence immunoassays overestimate 25(OH)D levels in human cord blood samples, an effect not observed in maternal blood samples. The quantification of 25(OH)D by CLIA should therefore not be considered valid when assayed in cord blood samples. Third trimester, but not first trimester, maternal 25(OH)D is one of several determinants of neonatal 25(OH)D status.
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http://dx.doi.org/10.1210/clinem/dgz299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065842PMC
April 2020

Clinical and Biochemical Phenotypes in a Family With ENPP1 Mutations.

J Bone Miner Res 2020 04 16;35(4):662-670. Epub 2020 Jan 16.

Department of Medicine, Division of Endocrinology, Diabetes, Metabolism, and Nutrition, Mayo Clinic, Rochester, MN, USA.

Inactivating mutations of the ENPP1 gene are associated with generalized arterial calcification of infancy (GACI) and less often autosomal-recessive hypophosphatemic rickets type 2 (ARHR2). We aimed to investigate the spectrum of phenotypes in a family with monoallelic and biallelic mutations of ENPP1 after identification through whole exome sequencing of a 54-year-old female with biallelic mutation of ENPP1, c.323G > T; p.Cys108Phe and c.1441C > T; p.Arg481Trp. Including the proband, 2 subjects had biallelic mutations, 5 had monoallelic mutations, and 2 had no mutation of ENPP1. The maternal mutation, a known pathogenic variant associated with GACI, was found in 3 subjects with monoallelic mutations, while the paternal mutation, which was not previously reported, was present in 2 subjects with monoallelic mutations. Both subjects with biallelic mutations had bowing of bilateral femurs, periarticular mineral deposition, normocalcemic primary hyperparathyroidism with multigland parathyroidectomy, increased carotid intima-media thickness, and enthesopathy was also noted in one subject. Intact FGF23 was elevated in both subjects with biallelic mutations, while C-terminal FGF23 was only elevated in one and PPi was reduced in one. Subjects with monoallelic mutations did not have periarticular calcifications or bone deformities. To conclude, patients with biallelic GACI causing mutations can survive well into adulthood, and despite the same biallelic ENPP1 pathogenic variants, clinical and biochemical manifestations can significantly differ, and include enthesopathy and primary hyperparathyroidism, which have not been previously described. Although carriers of monoallelic ENPP1 variants appear unaffected by classic disease manifestations, there may be subtle biochemical and clinical findings that warrant further investigation. © 2019 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771569PMC
April 2020

Center of Mass Calculation in Combination with MS/MS Allows Robust Identification of Single Amino Acid Polymorphisms in Clinical Measurements of Insulin-Like Growth Factor-1.

J Proteome Res 2020 01 9;19(1):186-193. Epub 2019 Dec 9.

Division of Clinical Biochemistry and Immunology, Department of Laboratory Medicine and Pathology , Mayo Clinic , Rochester , Minnesota 55905 , United States.

Insulin-like growth factor-1 (IGF-1) measurement by high-resolution accurate mass-mass spectrometry (HRAM-MS) is replacing IGF-1 immunoassays and allows for identification of single amino acid variants; by contrast, both normal and deleterious sequence variants might be missed by immunoassays or non-HRAM-MS methods. We have developed an intact molecule HRAM-MS method to identify IGF-1 variants, distinguishing them by a center of mass (COM) calculation, followed by various tandem-MS activation techniques (HCD, ETD, ETciD, EThcD, UVPD). We found single amino acid variants in 841 of 146 620 patient samples (0.57%). Most were benign (A67T, A70T). We also observed a pathogenic variant (V44M), likely pathogenic variants (A38V, V17M), and a likely benign variant (A67V). For 207 samples from unique patients with residual serum, the MS variant results were confirmed by cell-free DNA sequencing. Our approach allows accurate quantitative reporting of functional IGF-1 in the presence of single amino acid variants. The COM approach potentially enables omission of tandem-MS for known, common variants, while the combination of COM and tandem-MS allows accurate identification in all cases we encountered. This approach should be applicable to qualitative and quantitative analyses of other peptides/proteins in clinical and research settings and might lend itself to the characterization of other protein variations.
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http://dx.doi.org/10.1021/acs.jproteome.9b00494DOI Listing
January 2020

Adrenal Cortical Carcinoma Associated With Lynch Syndrome: A Case Report and Review of Literature.

J Endocr Soc 2019 Apr 5;3(4):784-790. Epub 2019 Mar 5.

Division of Endocrinology, Department of Medicine, Mayo Clinic, Rochester, Minnesota.

Objective: Adrenocortical carcinoma (ACC) is a rare malignancy with poor prognosis. ACC was reported in 3.2% patients with Lynch syndrome (LS), however no particular case-detection strategies have been recommended.

Participants: We report a case of a 65-year-old woman who was incidentally discovered with a large adrenal mass during work-up of postmenopausal uterine bleeding. She was recently diagnosed with germline mutation after her sister presented with uterine carcinoma in the setting of LS.

Results: Whereas the patient was asymptomatic for overt hormonal excess, biochemical work-up confirmed glucocorticoid autonomy and androgen and estrogen excess. Urine steroid profiling was suggestive of ACC. Adrenalectomy confirmed an oncocytic ACC with focal extracapsular extension into the periadrenal adipose tissue with a Ki-67 of 15% and a peak mitotic count of 40/50 high-power fields.

Conclusion: ACC can be the only manifestation of LS. A best case-detection approach for ACC in the asymptomatic patient with LS is unclear, however urine steroid profiling could be considered.
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http://dx.doi.org/10.1210/js.2019-00050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446885PMC
April 2019

Catecholamine and Metanephrine Levels in Breast Milk Before and After Paraganglioma Resection.

Breastfeed Med 2019 05 15;14(4):276-277. Epub 2019 Mar 15.

1 Department of Anesthesiology and Perioperative Medicine, Mayo Clinic, Rochester, Minnesota.

Catecholamine and metanephrine transfer into breast milk in the setting of secreting paraganglioma or pheochromocytoma has not been previously described. We present an investigation in which we measured catecholamine and metanephrine levels in the breast milk in a single patient undergoing resection of a paraganglioma at 5 weeks postpartum. As expected, levels were elevated preoperatively and decreased rapidly after resection. This information may be clinically relevant for patient management when pheochromocytoma or paraganglioma resection is delayed with respect to the delivery of the infant or in the postoperative monitoring of the patient's status.
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http://dx.doi.org/10.1089/bfm.2019.0018DOI Listing
May 2019

Anogenital distance is determined during early gestation in humans.

Hum Reprod 2018 09;33(9):1619-1627

Division of Neonatology, MetroHealth Medical Centre (Case Western Reserve University), 2500 MetroHealth Dr, Cleveland, OH, USA.

Study Question: Does cord blood androgen level obtained at birth affect the AGD in human newborns?

Summary Answer: In human newborns, though males have a significantly longer AGD compared to females (as early as 22 weeks of gestation) the AGD is not affected by androgen levels at birth in both the sexes.

What Is Known Already: Animal studies have reported a critical time period in early fetal life, termed the masculinization programming window (MPW) during which AGD is fixed by in utero androgen action and is unaffected by testosterone levels later during gestation. Thus, AGD may serve as a lifelong biomarker of androgen exposure during this window. This MPW is hypothesized to occur in humans at 8-14 weeks of gestation during which AGD is fixed. The effect of androgens (testosterone) on AGD after the MPW in humans is not known. Furthermore, altered AGD has been associated with various human reproductive health disorders in both males and females.

Study Design, Size, Duration: A prospective descriptive cohort study was performed using data from randomly selected neonates (n = 205) born at a single center over a period of 1 year (August 2015 to August 2016).

Participants/materials, Setting, Methods: AGDs in male (n = 117) and female infants (n = 88) together with penile width, glans girth and stretched penile length were measured by trained caregivers. Gestation ranged from 22 to 41 weeks and infants were examined within 24 h of birth (within 48-72 h in very sick preterm infants after clinical stabilization). AGD-1 was measured from the center of the anus to the posterior base of scrotum in males or to the posterior fourchette in females. AGD-2 was measured from the center of the anus to the anterior base of the penis in males or to the clitoris in females. Sex steroid hormones (testosterone, 17-OH progesterone (17-OHP) and androstenedione) were measured in serum prepared from umbilical cord blood samples taken at birth, using liquid chromatography-tandem mass spectrometry.

Main Results And The Role Of Chance: Males had a significantly lower gestational age (mean ± SD; 34.6 ± 4.9 versus 36.1 ± 4.1 weeks, P = 0.04), and a significantly longer AGD-1 (mean ± SD; 21.6 ± 6.0 versus 12.7 ± 3.8 mm, P < 0.001) and AGD-2 (41.9 ± 8.7 versus 33.9 ± 7.1 mm, P = 0.004) compared to female infants, respectively. The cord serum testosterone levels were significantly higher for male than female infants [median, interquartile range; 13.0 (7.3, 20.5) versus 4.1 (2.5, 5.9), ng/dl, P < 0.001]. There was no difference in levels of 17-OHP (P = 0.697) or androstenedione (P = 0.601) between the two sexes. On multiple regression analysis after adjusting for potential confounders, none of the AGD's in both males and females correlated with any sex steroid hormonal levels. We also provide normative charts for penile length, penile width and glans girth in preterm and term infants.

Limitations, Reasons For Caution: No data were collected on family history of genital malformation, infertility or hormonal disorders, parental endocrine-disrupting chemical exposure or diet pattern, any of which might have influenced the AGD and/or sex steroid hormone levels in the offspring.

Wider Implications Of The Findings: Our results suggest that AGD in humans, like animals, is fixed in early gestation (likely during the hypothesized MPW) and is unaffected by androgen levels thereafter. Thus, AGD can serve as a biomarker of in utero androgen action during early gestation (likely 8-14 weeks) in humans. As such, causes of human newborn and adult reproductive health disorders, such as endocrine disruptors, should be explored during early gestation. However, further larger studies are needed to help corroborate these findings.

Study Funding/competing Interests: No specific funding was obtained for this study, and all authors have no conflict of interest to declare.
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http://dx.doi.org/10.1093/humrep/dey265DOI Listing
September 2018

Diagnostic Thresholds for Androgen-Producing Tumors or Pathologic Hyperandrogenism in Women by Use of Total Testosterone Concentrations Measured by Liquid Chromatography-Tandem Mass Spectrometry.

Clin Chem 2018 11 1;64(11):1636-1645. Epub 2018 Aug 1.

Division of Endocrinology, Diabetes and Nutrition, Mayo Clinic, Rochester MN;

Background: Previously defined thresholds for total testosterone (TT) concentrations to screen for androgen-producing tumors (APTs) have used RIA, which can be less accurate in women. We aimed to define diagnostic thresholds to screen for APTs or postmenopausal pathologic hyperandrogenism using TT concentrations measured by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Methods: We performed a retrospective cohort study on all women with TT ≥3.5 nmol/L and all postmenopausal women presenting with hyperandrogenism between 2004 and 2014 at the Mayo Clinic in Rochester, MN.

Results: Of the 369 women with TT ≥3.5 nmol/L, 89 were included and subdivided into 3 groups based on their clinical diagnosis [21 (24%), APT; 16 (18%), postmenopausal pathologic hyperandrogenism; 52 (58%), polycystic ovary syndrome]. The source of the APT was more frequently ovarian (81%, n = 17) than adrenal (19%, n = 4). The diagnostic threshold using ROC analysis for TT to identify APT in women with severe biochemical hyperandrogenemia was ≥5.1 nmol/L (sensitivity, 90%; specificity, 81%). In a second analysis of a cohort of postmenopausal women only presenting with symptoms or signs of hyperandrogenism, median TT was significantly higher in the postmenopausal pathologic hyperandrogenism group (APT and ovarian hyperthecosis) vs the idiopathic hyperandrogenism group (4.9 vs 0.8 nmol/L; < 0.01). In postmenopausal women, the diagnostic threshold for pathologic hyperandrogenism was TT ≥2.2 nmol/L (sensitivity, 100%; specificity, 86%).

Conclusions: The diagnostic threshold for TT concentration as measured by LC-MS/MS to identify APT in women with biochemical severe hyperandrogenemia was TT ≥5.1 nmol/L. In postmenopausal women, the diagnostic threshold for pathologic hyperandrogenism was lower (TT ≥2.2 nmol/L).
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http://dx.doi.org/10.1373/clinchem.2018.290825DOI Listing
November 2018

Serum 25-hydroxyvitamin D3 levels and flares of systemic lupus erythematosus: a longitudinal cohort analysis.

Clin Rheumatol 2018 Oct 16;37(10):2685-2692. Epub 2018 Jul 16.

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA.

The aim of this study is to evaluate the relationship between baseline serum 25-hydroxyvitamin D3 levels and SLE activity/flares over time. This is a longitudinal study of 276 patients who fulfilled ≥ 4 ACR criteria for SLE and recruited in the year 2011. Serum samples were collected at baseline and assayed for 25-hydroxyvitamin D3 at the end of a mean follow-up of 32.5 months. Participants were stratified into three groups according to baseline 25-hydroxyvitamin D3 levels: group I (< 15 ng/ml, deficiency), group II (15-30 ng/ml, insufficiency), and group III (> 30 ng/ml, adequate). Baseline and summated SLE disease activity index (SLEDAI) score over time and the annual incidence of lupus flares were compared among these groups. 25-hydroxyvitamin D3 levels of < 15, 15-30, and > 30 ng/ml were present in 26, 54, and 20% of the recruited patients, respectively. Group I had significantly higher baseline SLEDAI scores. After a follow-up of 32.5 ± 5.5 months, 153 mild/moderate and 91 severe flares developed. The mean summated SLEDAI was 3.2 ± 2.0 in group I, 2.4 ± 1.9 in group II and 2.7 ± 2.1 in group III patients (P = 0.02). The annual incidence of mild/moderate and severe flares was 0.26 ± 0.39 and 0.20 ± 0.45 (group I); 0.20 ± 0.33 and 0.09 ± 0.22 (group II); and 0.20 ± 0.32 and 0.14 ± 0.46 (group III), respectively (P > 0.05). In a subgroup of 73 patients who were clinically and serologically quiescent at baseline, a similar trend of more flares was observed in group I patients. Vitamin D deficiency was frequent in Chinese SLE patients and was associated with more active disease at baseline and over time, as well as a trend of more severe lupus flares.
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http://dx.doi.org/10.1007/s10067-018-4204-1DOI Listing
October 2018

Senolytics improve physical function and increase lifespan in old age.

Nat Med 2018 08 9;24(8):1246-1256. Epub 2018 Jul 9.

Department of Molecular Medicine, Center on Aging, Scripps Research Institute, Jupiter, FL, USA.

Physical function declines in old age, portending disability, increased health expenditures, and mortality. Cellular senescence, leading to tissue dysfunction, may contribute to these consequences of aging, but whether senescence can directly drive age-related pathology and be therapeutically targeted is still unclear. Here we demonstrate that transplanting relatively small numbers of senescent cells into young mice is sufficient to cause persistent physical dysfunction, as well as to spread cellular senescence to host tissues. Transplanting even fewer senescent cells had the same effect in older recipients and was accompanied by reduced survival, indicating the potency of senescent cells in shortening health- and lifespan. The senolytic cocktail, dasatinib plus quercetin, which causes selective elimination of senescent cells, decreased the number of naturally occurring senescent cells and their secretion of frailty-related proinflammatory cytokines in explants of human adipose tissue. Moreover, intermittent oral administration of senolytics to both senescent cell-transplanted young mice and naturally aged mice alleviated physical dysfunction and increased post-treatment survival by 36% while reducing mortality hazard to 65%. Our study provides proof-of-concept evidence that senescent cells can cause physical dysfunction and decreased survival even in young mice, while senolytics can enhance remaining health- and lifespan in old mice.
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http://dx.doi.org/10.1038/s41591-018-0092-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082705PMC
August 2018

10 years of 25-hydroxyvitamin-D testing by LC-MS/MS-trends in vitamin-D deficiency and sufficiency.

Bone Rep 2018 Jun 23;8:268-273. Epub 2018 May 23.

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United States.

In early 2000's vitamin-D deficiency was shown to be prevalent in several countries including the United States (US). Studies exploring the role of vitamin-D metabolism in diverse disease pathways generated an increased demand for vitamin-D supplementation and an immense public interest in measurement of vitamin-D metabolite levels. In this report, we review the role of vitamin-D metabolism in disease processes, clinical utility of measuring vitamin-D metabolites including 25-hydroxyvitamin-D (25(OH)D), 1,25-dihydroxyvitamin-D and 24,25-dihydroxyvitamin-D and discuss vitamin-D assay methodologies including immunoassays and liquid chromatography mass spectrometry (LC-MS/MS) assays. We also provide examples of vitamin-D toxicity and insight into the trends in serum 25(OH)D levels in the US population based on 10 years of data from on serum 25(OH)D values from ~5,000,000 patients who were tested at the Mayo Medical Laboratories between February 2007-February 2017.
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http://dx.doi.org/10.1016/j.bonr.2018.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020395PMC
June 2018

Sex differences in NSAID-induced perturbation of human intestinal barrier function and microbiota.

FASEB J 2018 Jun 13:fj201800560R. Epub 2018 Jun 13.

Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, USA.

Intestinal barrier function and microbiota are integrally related and play critical roles in maintenance of host physiology. Sex is a key biologic variable for several disorders. Our aim was to determine sex-based differences in response to perturbation and subsequent recovery of intestinal barrier function and microbiota in healthy humans. Twenty-three volunteers underwent duodenal biopsies, mucosal impedance, and in vivo permeability measurement. Permeability testing was repeated after administration of indomethacin, then 4 to 6 wk after its discontinuation. Duodenal and fecal microbiota composition was determined using 16S rRNA amplicon sequencing. Healthy women had lower intestinal permeability and higher duodenal and fecal microbial diversity than healthy men. Intestinal permeability increases after indomethacin administration in both sexes. However, only women demonstrated decreased fecal microbial diversity, including an increase in Prevotella abundance, after indomethacin administration. Duodenal microbiota composition did not show sex-specific changes. The increase in permeability and microbiota changes normalized after discontinuation of indomethacin. In summary, women have lower intestinal permeability and higher microbial diversity. Intestinal permeability is sensitive to perturbation but recovers to baseline. Gut microbiota in women is sensitive to perturbation but appears to be more stable in men. Sex-based differences in intestinal barrier function and microbiome should be considered in future studies.-Edogawa, S., Peters, S. A., Jenkins, G. D., Gurunathan, S. V., Sundt, W. J., Johnson, S., Lennon, R. J., Dyer, R. B., Camilleri, M., Kashyap, P. C., Farrugia, G., Chen, J., Singh, R. J., Grover, M. Sex differences in NSAID-induced perturbation of human intestinal barrier function and microbiota.
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http://dx.doi.org/10.1096/fj.201800560RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219825PMC
June 2018