bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Method in Performing PTH Washout for Localizing Parathyroid Adenoma: Case Report & an Institute Experience in Qatar
Authors: Mohammad Salameh Obada Khalil , Gul Wajiha , Al-Thani Noora , Alamer Zaina Abdelhalim , Elhadd Tarik A , Bashir Mohammed , Abou-Samra Abdul B , Zirie Mahmoud A , Naem Emad
Published: May 2021

Introduction: Primary hyperparathyroidism (PHPT) is a common cause of hypercalcemia and mostly caused by benign solitary benign adenoma (80 to 85 %). Definite treatment is the surgical removal. The most commonly used diagnostic modalities are Sestamibi scan and neck ultrasound (US) which can be inconclusive in some cases. Parathyroid hormone (PTH) washout obtained with ultrasound guided FNA may be useful to accurately localize the adenoma. In our study we describe a novel method for PTH washout. Methods: First, blood samples are drawn from the patient’s peripheral vein and placed in two yellow top tubes (3 ml of blood in each tube). 1 ml of normal saline (NS) will be add to Tube # 1 (Control tube). The suspected parathyroid lesion aspirate is obtained via US guided FNA. It is washed in 1 ml of NS and added to tube #2 (PTH washout tube). Both tubes are sent to our local laboratory for PTH assay. The ratio of PTH in PTH washout tube to control tube (PTH W/C ratio) is calculated and considered positive if more than 2. Results: Total 16 patients (12 females and 4 males) underwent the PTH washout procedure. All patients had PHPT. Out of 16 patients, 13 had inconclusive Sestamibi scan while 3 patients didn’t have the scan due to pregnancy. PTH W/C ratio was positive in 13 patients (ruled in) and negative (ruled out) in 3 patients. All patients underwent parathyroid surgery. The operative findings and pathology report were consistent with PTH W/C ratio findings. i.e. parathyroid hypercellularity was found in all the 13 patients ruled in by PTH W/C ratio. Post-surgery, biochemical parameters normalized in all. Conclusion: PTH washout is an important tool in localizing parathyroid lesion in PHPT when Sestamibi scan cannot be done or if it is inconclusive. PTH W/C ratio can be performed with our novel method to accurately localize the PTH lesion and improve surgical outcome.
Secondary Hyperparathyroidism Among Bariatric Patients: Unraveling the Prevalence of an Overlooked Foe
Authors: Mendonça Fernando , Neves João Sérgio , Silva Maria Manuel , Canha Marta Borges , Costa Cláudia , Cabral Pedro , Guerreiro Vanessa , Lourenço Rita , Meira Patricia , Ferreira Maria J , Salazar Daniela , Pedro Jorge , Varela Ana , Viana Sara , Belo Sandra , Lau Eva , Souto Selma , Freitas Paula , Carvalho Davide ,   CRIO group
Published: May 2021

Introduction: Bariatric surgery (BS) is an effective therapeutic approach for obese patients. It is associated with important gastrointestinal anatomic changes, predisposing these subjects to altered nutrient absorption that impact phosphocalcium metabolism. This study aims to clarify the prevalence of secondary hyperparathyroidism (SHPT) and its predictors in patients submitted to BS. Methods: Retrospective unicentric study of 1431 obese patients who underwent metabolic surgery between January/2010 and June/2017 and who were followed for, at least, a year. In this group, 185 subjects were submitted to laparoscopic adjustable gastric banding (LAGB), 830 underwent Roux-en-Y gastric bypass (RYGB) and 416 sleeve gastrectomy (SG). Data comprising 4 years of follow-up were available for 333 patients. We compared the clinical and analytical characteristics of patients with and without secondary hyperparathyroidism (considering SHPT a PTH˃69pg/mL), taking also into account the type of surgery. A multiple logistic regression was performed to study the predictors of SHPT after BS. Results: The overall prevalence of SHPT before surgery was 24.9%, 11.2% one year after surgery and 21.3% four years after surgery. At 12 months after surgery, LAGB had the highest prevalence of patients with SHPT (19.4%, N=36), RYGB had 12.8% (N=274) and SG 5.3% (N=131). At 48 months after surgery, RYGB had the highest prevalence of SHPT (27.0%, N=222), LAGB had 13.2% (N=53) and SG 6.9% (N=58). Multi-variate logistic analysis showed that increased body mass index and age, decreased levels of vitamin D and RYGB were independent predictors of SHPT one year after surgery. The only independent predictor of SHPT four years after surgery was RYGB. Conclusion: The prevalence of SHPT is considerably higher before and four years after BS than 1 year after surgery. This fact raises some questions about the efficacy of the implemented follow-up plans of vitamin D supplementation on the long term, mainly among patients submitted to RYGB.
The Prevalence and Burden of Hypophosphatasia in an Ambulatory Endocrinology Practice
Authors: Quinn Hugh B , Busch Robert S , Kane Michael P
Published: May 2021

Introduction: Hypophosphatasia (HPP) is an autosomal disease resulting from loss-of-function mutations in the ALPL gene encoding tissue-nonspecific alkaline phosphatase (TNSALP). The presentation and severity of the disease is highly variable ranging from perinatal-onset HPP with mortality rates as high as 100%, to adult onset with little mortality but with high disease-burden. Overall estimated prevalence of HPP in the general population is 1:100,000 though it may be significantly higher in specific populations. Hypophosphatasia is a heterogeneous disease that can reveal itself at any age, presenting within a wide range of symptoms. Adult HPP typically presents during middle age and is often misdiagnosed or missed in practice. The objective of this study was to determine the prevalence and burden of hypophosphatasia in an ambulatory care endocrinology practice. Methods: Potential subjects were identified via a computerized text search of the laboratory fields of patient electronic medical records (EMR). Search terms included serum ALP levels of less than or equal to 40 mg/dL. Records of patients with at least two low ALP levels were reviewed manually to identify potential patients with a history consistent with HPP. Results: A total of 315 patients with ALP levels < 40 mg/dL were identified via text search from an estimated 20,000 patient records. Fifty-six patients with a single low level were not considered for further review. The remaining 259 patients were reviewed for histories consistent with hypophosphatasia. These patients were predominantly white (64.9%), with an average age of 55 (+ 15) years, and an average BMI of 28 (+ 7) kg/m2. Ten of these patients had histories consistent with hypophosphatasia including musculoskeletal pain requiring scheduled use of pain medications, polyarthropathy, chondrocalcinosis, deformity secondary to fractures, low BMD, a history of nontraumatic fracture, delayed or incomplete fracture healing, a history of multiple orthopedic surgeries, fatigue, impaired mobility, impaired gait, impairment of daily activities, a history of renal stones or nephrocalcinosis, and/or high serum B6 levels. None of the identified ten patients were currently being treated or had previously been treated for hypophosphatasia and have subsequently been recommended for genetic testing. Conclusions: Hypophosphatasia is an uncommon condition with a highly variable presentation often resulting in a missed diagnosis. Surveillance of practices by identifying patients with low ALP levels is a reasonable screening approach to identifying potential patients with hypophosphatasia.
Causal Effect of Fibroblast Growth Factor 23 on Osteoporosis and Cardiometabolic Disorders: A Mendelian Randomization Study
Authors: Yokomoto-Umakoshi Maki , Umakoshi Hironobu , Miyazawa Takashi , Ogata Masatoshi , Sakamoto Ryuichi , Ogawa Yoshihiro
Published: May 2021

Pathological excess of fibroblast growth factor 23 (FGF23) causes mineral and bone disorders. However, the causality of FGF23 in the development of osteoporosis remains unknown. Whether FGF23 has systemic effects on cardio-metabolic disorders beyond regulating mineral metabolism is also controversial. In this study, we evaluated the causal effect of FGF23 on osteoporosis and cardiometabolic disorders using Mendelian randomization (MR) analysis. Summary statistics for single-nucleotide polymorphisms with traits of interest were obtained from the relevant genome-wide association studies. As a result, FGF23 was found to be inversely associated with femoral neck-BMD (odds ratio [OR] 0.682, 95% confidence interval [CI] 0.546–0.853, p = 8e-04) and heel estimated BMD (eBMD) (OR 0.898, 95%CI 0.820–0.985, p = 0.022) in the inverse-variance-weighted analysis, but not lumbar spine-BMD and fractures. The results were supported by the weighted-median analysis, and there was no evidence of pleiotropy in the MR-Egger analysis. FGF23 was directly associated with FN-BMD and eBMD after adjustment for estimated glomerular filtration rate, height, and body mass index in multivariable MR analysis. On the other hand, there was no association between FGF23 and cardiometabolic traits including cardio artery disease, brachial-ankle pulse wave velocity, intima-media thickness of carotid arteries, systolic and diastolic blood pressure, fasting glucose, high and low-density lipoprotein cholesterol, and triglycerides. Therefore, FGF23 has been causally associated with bone loss. In contrast, FGF23 has not been causally associated with cardiometabolic disorders. The data of this study provides important insights into the role of FGF23 in the pathogenesis of osteoporosis and cardiometabolic disorders.
Effect of Cholecalciferol Supplementation on Bone Turnover Markers in Children and Adolescents With Type 1 Diabetes Mellitus
Authors: Pankiv Volodymyr I , Pankiv Ivan V
Published: May 2021

Background: Cholecalciferol is known to play an important role in bone mineral metabolism. Its deficiency may affect growth and status of bone markers in children. Aim of the study: to evaluate the correlation between serum 25(OH)D and bone markers and impact of vitamin D supplementation on serum bone formation [procollagen type 1 amino-terminal propeptide (P1NP)] and bone resorption [β-cross laps (CTx)] markers among children and adolescents with type 1 diabetes mellitus (DM). Materials and Methods: Total 58 children and adolescents with type 1 DM, who were given 2000 UI of cholecalciferol supplementation, were included in the study. These 58 children with available anthropometry, serum biochemistry, 25-hydroxyvitamin D ([25(OH)D]), and parathormone (PTH) were evaluated for bone formation (procollagen type 1 amino-terminal propeptide [P1NP]) and resorption (β-cross laps [CTx]) markers. Results: The mean age and body mass index of these children were 11.6 ± 2.3 years (boys: 11.7 ± 2.4; girls: 12.2 ± 1.4 years; p = 0.04) and 18.2 ± 3.9 kg/m2 (boys: 18.1 ± 3.8; girls: 17.8 ± 3.4 kg/m2; p = 0.206), respectively. Baseline serum P1NP levels were positively correlated with serum phosphates (r = 0.281, p < 0.001), PTH (r = 0.291, p < 0.001), and CTx (r = 0.425, p < 0.001) but not with age (r = -0.016, p = 0.404), BMI (r = -0.080, p = 0.032), serum calcium (r = -0.038, p = 0.107), and baseline 25(OH)D (r = -0.069, p = 0.035). Postsupplementation serum P1NP and CTx levels maintained similar correlations. There was a significant decline in serum P1NP (from 681 ± 223 ng/ml to 630 ± 279 ng/ml, p < 0.01) and CTx (from 1.63 ± 0.51 ng/ml to 1.37 ± 0.53 ng/ml, p < 0.01) following supplementation. Though decline in serum P1NP and CTx levels was observed in both boys and girls, among all supplementation patients, the effect was more marked in serum CTx than P1NP levels. Conclusions: Vitamin D supplementation in children resulted in decrease in both bone formation (P1NP) and resorption (CTx). The impact, however, was more marked on bone resorption than bone formation.
Endocrinology, Vitamin D and the UK’s Covid-19 Disaster
Authors: Anderson David Coussmaker , Grimes David S
Published: May 2021

The formation of cholecalciferol (Vitamin D3) in skin depends on solar UVB to break the B ring of 7-dehydrocholesterol. Its discovery more than a century ago resulted from the identification of rickets as due to deficient sunshine in latitudes far from the equator, exacerbated by the air pollution, factory work and indoor living. Rickets resulted from defective endocrine control of blood calcium, and was accompanied by epidemic tuberculosis from failure of the D3-dependent first-line immune system. The influenza pandemic of 2018 revealed the need for D3 to fight viruses. Half a century later the systemic hormone role of 1,25(OH)D3 of renal origin, under control of PTH, was a major stimulus to understanding the mechanism of action via the VDR-RXR heterodimer. It was soon realised that 1,25(OH)D3 is also produced and acts locally in many organs and tissues provided that there are adequate reserves of the (protein-bound) blood storage form, 25(OH)D. This is the common pool for 1-hydroxylation by any cells that need local activation of VDR for induction of specific genes. In the case of the immune system, the trigger is foreign proteins recognised as ‘non-self’. Local production and action of 1,25(OH)D, and then its local destruction by 24-hydroxylation must all occur below the ‘endocrine radar’, so as not to interfere with systemic calcium control. Coronaviruses through their ‘spike’ protein are internalised by interacting with the ACE-2 receptor, which in turn is down-regulated by Vitamin D. In the process, 25(OH)D is hydroxylated to the active 1,25(OH)D, which must later be degraded to 1,24,25(OH)D. So it is to be expected that when 25(OH)D reserves are low at the onset of infection, they will fall further, allowing virus to enter the cells and trigger a cytokine storm and other damage. Blood PTH will rise to claim any residual 25(OH)D for the dominating systemic role in calcium homeostasis. It follows that intake of vitamin D3 should always be much more than the minimum claimed by the globally-active endocrine system. Unfortunately, the UK’s Specialised Advisory Committee on Nutrition (SACN), does not recognise this. It is dominated by nutritionists, even though food sources of D3 are for most non-existent, and of D2, the vegetable substitute, highly variable. The 400IU of D3 reluctantly recommended for those ‘at risk’, based on endocrinology alone, is grossly inadequate; 4,000IU daily is needed to maintain a blood 25(OH)D at more than 30 ng/ml (75 nmol/l), and provide sufficient reserve for its many autocrine and paracrine functions. The dangers of letting the dominant endocrine function of 1,25(OH)D in ionic calcium control dictate the level of D3 supplements, have once again been underlined by the Covid-19 disaster.
Factors Associated With Low Bone Mineral Density in Adults With Type 1 Diabetes: A Cross-Sectional Study
Authors: Coll Julie-Catherine , Garceau Élodie , Michou Laëtitia , Weisnagel S John , Mac-Way Fabrice , Morin Suzanne N , Rabasa-Lhoret Remi , Gagnon Claudia
Published: May 2021

Context: Individuals with type 1 diabetes (T1D) have a two- to threefold increase in fracture risk at any site, and up to a sevenfold increase in hip fracture risk compared to those without diabetes. The mechanisms accounting for this bone fragility are not yet fully understood.

Objectives: 1) To determine factors associated with low bone mineral density (BMD) in patients with T1D; 2) To assess the association between skin advanced glycation end products (AGEs) and low BMD in patients with T1D.

Methods: These are preliminary data from patients with T1D included in a cross-sectional study aiming at comparing the prevalence of vertebral fractures between adult patients with T1D from two tertiary care centers and age- and sex-matched controls without diabetes. Patients were eligible if they were aged ≥20 years and had a diagnosis of T1D for at least 5 years. Patients were classified as having a low BMD if Z-score was ≤-2.0 at any site (lumbar spine, femoral neck, total hip, radius) in patients aged <50 years or if T-score was ≤-1.0 at any site in patients aged ≥50 years or in postmenopausal women. Skin AGEs (surrogate marker of overall including bone AGEs) were measured by skin autofluorescence (AGE Reader ®). Unpaired t-tests or Chi-squared tests were used to compare characteristics between patients with or without a low BMD. Variables associated with a low BMD were determined by univariate analysis and were subsequently included in a multivariate logistic regression analysis if p<0.1 in the univariate analysis. All variables were tested for multicollinearity.

Results: 106 patients with T1D were included (mean age 45.2±15.0 years; mean BMI 26.3±5.1 kg/m2; 54.7% women; mean duration of diabetes 28.2±13.6 years; 44.3% with a microvascular complication). Mean HbA1C over the past 3 years was 7.5±0.8%. A third of the patients (31.1%) had a low BMD (3 patients using Z-score; 30 patients using T-score). Patients with a low BMD were older (58.3 vs 39.3 years, p<0.001), had a lower mean HbA1C over the past 3 years (7.3% vs 7.6%, p=0.047), a longer diabetes duration (36.1 vs 24.6 years, p<0.001), higher skin AGEs (2.50 vs 2.03, p<0.001), a higher prevalence of microvascular complications (63.6% vs 37.7%, p=0.02) and a higher prevalence of abnormal albumin to creatinine ratio (ACR ≥2.0) on the day of assessment (38.7% vs 11.8%, p=0.003). In multivariate regression analysis, age (p<0.001), abnormal ACR (p=0.003) and lower mean HbA1C over the past 3 years (p=0.02) remained significantly associated with a low BMD. Skin AGEs were correlated with age (r=0.56) and diabetes duration (r=0.47).

Conclusion: In this population with T1D, a low BMD was independently associated with older age, abnormal ACR and, unexpectedly, with a lower mean HbA1C over the past 3 years, but not with skin AGEs.
Obesity, Dyslipidemia, Diabetes, and Vitamin D Levels
Authors: Moya Elena A Amoroso , Nicolalde Marcelo , Torres Herbarth
Published: May 2021

Context: There are several studies that refer to an association between vitamin D levels and the prevalence of atherogenic dyslipidemia and diabetes 2, however these studies present different non-conclusive results. Methods: A cross-sectional study was carried out with a propositional sequential sample of 110 patients at Rio and San Juan Hospital Riobamba -Ecuador 2020. The sample was divided into three groups according to BMI and vitamin D levels; 25 hydroxycalciferol, by cluster analysis, the final groups G1(56), G2(59), and G3(16) had a similar BMI average of 27 kg/m2, and significant differences in the average of vitamin D: G1 = 13.6, G2 = 24.5 and G3 = 45.3 ng/dl. Results: The prevalence of dyslipidemia increased gradually as the average level of vitamin D of the group increased as follows: G1 = 12.5%, G2 = 18.6%, G3 = 18.8%, the same behavior was observed in the prevalence of type 2 diabetes per analysis group: G1 = 30.4%, G2 = 45.8% and G3 = 50%. The differences found in the prevalence of atherogenic dyslipidemia and diabetes 2 were not statistically significant p> 0.05. Conclusion: in the group of patients studied and divided by cluster analysis into three groups with different levels of vitamin D, there were no statistically significant differences with respect to the prevalence of atherogenic dyslipidemia and diabetes 2.
Androgen and Estrogen Receptor Activation Impacts Cardiac Function in a PCOS-Prone Rodent Model
Authors: Vine Donna Faye , Raggi Paolo , Proctor Spencer , Becher Harald
Published: May 2021

Introduction: The global risk and incidence of cardiovascular disease (CVD) is increased in polycystic ovary syndrome (PCOS). Early risk factors and subclinical CVD includes atherogenic dyslipidemia, obesity, insulin resistance, blood pressure, atherosclerosis and impaired cardiac function. Androgen exposure is associated with onset of adiposity, impaired insulin-glucose and lipid metabolism, and cardiac dysfunction. The mechanisms of increased risk of CVD and cardiac dysfunction in PCOS related to hyperandrogenemia, AR and estrogen receptor (ER) activation remain unclear. Aim: The aim of this study was to investigate the effect of androgen treatment on cardiac AR and ER activation, fatty acid metabolism and cardiac function in a PCOS-prone rodent model. Methods: A PCOS-prone rodent model at 6 wks of age with obesity, apoB-remnant lipemia and insulin resistance, and controls were treated with testosterone for 12 weeks. Cardiac function was assessed using transthoracic doppler echocardiography (M-Mode 2D-imaging), lipogenic, AR, ER and other metabolic gene and protein expression were assessed using RTPCR and SDS-PAGE western blot. Results: PCOS-prone animals exhibited left ventricular (LV) hypertrophy, with increased LV mass to body weight (551.6 ± 38.85 mg vs 999 ± 96.17 mg, p<0.05), LV posterior wall diastolic diameter and LV internal diastolic diameter compared to controls. Isovolumetric relaxation time (IVRT) was prolonged (15.91 ± 1.591 msec vs 23.75 ± 0.722 msec, p<0.05). Mild systolic dysfunction was evidenced by increased isovolumetric contraction time (IVCT; 22.5 ± 1.348 msec vs 28.96 ± 1.248 msec, p<0.05) and decreased % ejection fraction and % fractional shortening in PCOS-prone compared to controls. T treatment increased LV mass, IVCT and IVRT in controls but did not exacerbate cardiac function in PCOS-prone animals. T treatment increased cardiac protein expression of PPAR-⍺ in PCOS-prone and controls, and T increased ACC in controls. AR protein expression tended to be reduced, and ER-⍺ was reduced in both T treated control and PCOS-prone animals. Conclusions: The PCOS-prone rodent model demonstrates early cardiac LV hypertrophy and diastolic-systolic dysfunction and T treatment alters fatty acid metabolism, and AR and ER activation are associated with altered cardiac morphology and function in the PCOS-prone and control conditions.
Feasibility of a Novel Nonhuman Primate Model of Age-Related Nonalcoholic Fatty Liver Disease
Authors: Azees Parveez Ahamed Abdul , Palavicini Juan Pablo , Han Xianlin , Salmon Adam , Kamat Amrita
Published: May 2021

The objective of the proposed study is to investigate the feasibility of the marmoset as an animal model to study age-associated nonalcoholic fatty liver disease (NAFLD). This chronic liver disease includes a spectrum of disorders ranging from increased triglyceride accumulation in the liver or hepatic steatosis to the more severe inflammatory form nonalcoholic steatohepatitis that can lead to cirrhosis and even hepatocellular carcinoma in individuals who do not have a history of alcohol abuse. Aging increases the prevalence of NAFLD and is strongly associated with the progression and severity of this disease. End-stage hepatic failure and liver cancer resulting from advanced NAFLD are leading indications for liver transplantation enhancing the burden on our healthcare systems. Accumulating clinical evidence also suggests that patients with NAFLD have a higher prevalence of cardiovascular disease. Pathogenetic mechanisms involved in the development and progression of NAFLD are poorly understood and as such, there is a lack of effective therapies. The common marmoset is a relatively short-lived non-human primate that recapitulates many of the physiological changes that occur in human aging. We hypothesized an age-associated increase in hepatic steatosis and alterations in serum lipid profile in the marmoset model. An increase in triglyceride levels and oil red o staining in liver tissues of old marmosets compared to young animals was observed suggesting an age-associated increase in hepatic steatosis in marmosets as observed in humans. Lipidomic studies were also performed using blood samples from male and female marmosets to investigate age-associated changes in specific lipid species, which are characteristic of aberrant lipid metabolism. Analysis of the results revealed significant decreases in several phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin species in the plasma of old marmosets compared to young marmosets. We are now performing studies to determine whether the observed changes in different lipid species could influence the development of cardiovascular disease and provide new insights underlying the mechanisms of NAFLD development with aging.
Generation of Affinity Purified Antibody Reagents for Specific Determination of Efruxifermin in Biological Matrices
Authors: Kinne Adam S , Abdeen Sanofar J , Parmer Elijah S , Thystrup Jennifer A , Tillman Erik J , Rolph Timothy P , Bowsher Ronald R
Published: May 2021

Efruxifermin (EFX) is a novel Fc-fusion analog of human fibroblast growth factor 21 (FGF21), currently in clinical development as a potential treatment for non-alcoholic steatohepatitis (NASH). Each molecule of EFX consists of two modified FGF21 molecules, each attached at their N-termini to a human IgG1 Fc domain by a short polyglycine-serine linker. The FGF21 moiety of EFX incorporates three amino acid substitutions (L98R, P171G, and A180E relative to native FGF21). Two of these are proximal to the C-terminus (P171G and A180E), and reduce cleavage and inactivation by an endogenous protease, fibroblast activation protein (FAP), thereby prolonging its half-life. Fusion to human IgG1 Fc domain further extends circulating half-life, enabling once-weekly subcutaneous dosing. Accordingly, to support on-going clinical development of EFX, a specific assay is needed to distinguish intact EFX from both endogenous FGF21 and any in vivo biotransformation products of EFX that display reduced pharmacology. To maximize the antigenicity of EFX, FGF21 amino acid sequences were compared across species. Based on this, an antibody generation campaign was initiated in both rabbits and chickens. Comparison of titer responses against EFX and human FGF21 suggested that antisera from chickens was superior to rabbit antisera. Following a scaled-up, 12-week antibody campaign, antisera were purified by a combination of batch and column chromatographic procedures. By exploiting differences in structure and amino acid sequence of EFX relative to human FGF21, a purification strategy was designed to isolate chicken antibodies with increased specificity for EFX unique sequences. This reagent is being used as a capture antibody in the development of a noncompetitive ECLIA employing chemiluminescence detection. Presently, a number of different antibodies are being evaluated for potential pairing with the specific capture. We conclude that application of affinity purified chicken anti-EFX IgY will enable sensitive and specific determination of EFX in biological matrices with decreased cross-reactivity from endogenous hFGF21 and EFX metabolites.
Lipoprotein Insulin Resistance Score: Validation and Utility in African Ancestry Populations
Authors: Sharma Vandhna Rani , Cravalho Celeste K L , Dawson Joshua , Villalobos-Perez Alfredo , Mabundo Lilian , Matta Samantha , DuBose Christopher , Sumner Anne E , Chung Stephanie Therese
Published: May 2021

Lipoprotein insulin resistance (LPIR) is an emerging biomarker of insulin resistance (IR), and a score of >48 is a strong predictor of incident cardiometabolic disease disease in a predominantly European ancestry population. LPIR is derived from a composite score of nuclear magnetic resonance (NMR) lipoprotein (Lp) parameters: triglyceride-rich (TRLp), low density (LDLp), and high density (HDLp). Yet, there is a paucity of data in African ancestry population, in whom there is low-normal TRLp despite high rates of IR and diabetes. Therefore, we examined Lp profiles and LPIR in a large African ancestry cohort, stratified by sex to determine the relationship of LPIR with established markers of IR. This is a secondary analysis from 2 studies (The Africans in America and Federal Women’s Study) designed to evaluate the genetic, biological and socio-environmental factors of diabetes risk in those of African ancestry. All participants self-identified as healthy and lived in the DC metro area, n= 518: 87.7% African immigrant,12.3% African American; age 39±10 (20-65y); BMI 28.1±4.8 (18.2–45.2 kg/m2); 58% male; 31% with obesity, and 37% with abnormal glucose tolerance; mean±SD (range); median (25th-75th percentile). Fasting measures of IR (LPIR, triglyceride/HDL (TG/HDL) ratio and homeostasis model of insulin resistance (HOMA-IR)) were compared with the whole-body insulin sensitivity index (WBISI) obtained during a multi-sample 75g OGTT, using spearman correlations. Lp particle size and subclass concentrations were measured by the amplitudes of the lipid-methyl group signals (NMR LipoProfile®). Men had lower BMI (27.1±3.9 vs 29.3±5.6 kg/m2), fat mass (23.5±5.5 vs 37.9±6.8 %), insulin resistance (WBISI: 6.2 (3.7–10.1) vs 4.9 (3.2–8.6), HOMA-IR: 1.3 (0.7–2.0) vs 1.6 (0.9–2.4), TG/HDL: 1.4 (1.0–2.2) vs 1.1 (0.8–1.5)), all P<0.001. LDLp (1226 (959–1531) vs 1239 (981–1553) nmol/L) and HDLp (17.6 (16.2–19) vs 17.5 (15.9–19.7) µmol/L) were similar by sex, P>0.6, while small LDLp 734 (523–1039) vs 541 (370–805) nmol/L and TRLp 80.5 (52.2–116.4) vs 53.6 (28.7 -89.3) nmol/L were higher in men. The total mean LPIR score was 28.9±18.7 and was higher in men (34±19 vs. 23±17), P<0.001. LPIR and TG/HDL ratio correlated with WBISI (r≥-0.40) and HOMA-IR (r≥0.40), P<0.001 with no differences by sex. HOMA-IR correlated with WBISI (r=-0.95, P<0.001). Overall, African ancestry individuals had high rates of abnormal glucose tolerance, obesity and LDLp but LPIR was 20 points lower than the established score for predicting cardiometabolic disease. It’s utility for detecting IR was modest but it may be an important adjunct for early cardiometabolic risk stratification in African ancestry populations in whom traditional screening methods have lower sensitivity.
Long Term Effects of Leptin on Hepatic Fibrosis in Generalized Lipodystrophy
Authors: Lightbourne Marissa , Okawa Marinna , Koh Christopher , Brown Rebecca J
Published: May 2021

Lipodystrophy syndromes are caused by deficiency of adipose tissue leading to severe insulin resistance, hypertriglyceridemia, and non-alcoholic fatty liver disease (NAFLD), which may progress to cirrhosis. Advanced fibrosis/cirrhosis was previously thought to be irreversible; however, eradication of hepatitis C or long-term viral suppression of hepatitis B can reverse cirrhosis. Metreleptin treatment in patients with lipodystrophy improves liver transaminases and NAFLD activity score (NAS) after a mean of 2 years, but the latter improvements were due to lower inflammation and steatosis, with no change in fibrosis. The long-term effects of metreleptin in subjects with advanced fibrosis are unknown. We analyzed 24 subjects with advanced fibrosis (NAS stage 3 or 4) prior to metreleptin. Seven had liver biopsies both before and after ≥ 3 years of metreleptin with mean treatment duration 7.8±2.9 years. Five of six subjects with stage 3 fibrosis at baseline had improved fibrosis scores after metreleptin, and 0 of 1 with stage 4 fibrosis improved. 17 patients (8 with stage 3, 9 stage 4) did not have follow up biopsies after ≥3 years. Of these 17, 4 (all stage 4) died from end stage liver disease, and 1 (stage 3) from other causes. There was no clinical indication for repeat biopsy in 6 patients (2 stage 3, 4 stage 4). 6 were lost to follow up. Of the 24 patients with advanced fibrosis, 13 had congenital generalized lipodystrophy (CGL), 7 had acquired generalized lipodystrophy (AGL), 3 had familial partial lipodystrophy (FPL) and 1 had acquired partial lipodystrophy (APL). Of the 5 subjects with improvement in their fibrosis score, 2 had AGL, 2 had CGL, and one had a novel FPL mutation (1). Of the two patients that did not improve, one had FPL and one had CGL. In conclusion, subjects with stage 3 fibrosis due to lipodystrophy may have regression of fibrosis after long-term metreleptin treatment. This improvement may be secondary to near-elimination of the inciting factors (excess nutrient intake leading to ectopic lipid storage in the liver), analogous to clearance of Hepatitis C infection. However, additional data is needed to determine if metreleptin can reverse fibrosis in subjects with stage 4 fibrosis.
Modulation of Calcium Signaling by Chemogenetic Tools to Elucidate the Pathogenesis of Primary Aldosteronism
Authors: Cosentino Teresa , Fedlaoui Bakhta , Giscos-Douriez Isabelle , Fernandes-Rosa Fabio Luiz , Magnus Christopher , Sternson Scott M , Zennaro Maria-Christina , Boulkroun Sheerazed
Published: May 2021

Primary aldosteronism (PA) is the most frequent form of secondary arterial hypertension. The identification of germline or somatic mutations in different genes coding for ion channels (KCNJ5, CACNA1D, CACNA1H and CLCN2) and ATPases (ATP1A1 and ATP2B3) defines PA as a channelopathy. These mutations promote increased intracellular calcium concentrations and activation of calcium signaling, the main trigger for aldosterone biosynthesis.

The aim of our study was to elucidate the mechanisms underlying the development of PA by modulating calcium signaling using chemogenetic tools.

We have generated two different adrenocortical H295R_S2 cell lines stably expressing different chimeric ion channels generated by fusing the mutated extracellular ligand binding domain of the α7 nicotinic acetylcholine receptor to the ion pore domain of large Cys-loop receptor ion channel family; these receptors constitute PSAM (Pharmacologically Selective Actuator Modules). The mutations introduced in the ligand-binding domain allow to use synthetic ligands, PSEM (Pharmacologically Selective Effector Molecules) to activate the PSAM. We used two different PSAM: the chimeric receptor α7-5HT3 or a mutated acetylcholine receptor nAchR, allowing respectively modulation of sodium or calcium entry into the cells in response to the specific PSEM: Varenicline for α7-5HT3 or Compound 9S for mutated nAChR. The cells lines were characterized in terms of intracellular calcium concentrations, cell proliferation, aldosterone production and steroidogenic gene expression.

Cells expressing α7-5HT3 treated for 24h with increasing concentrations of Varenicline (10–9 to 10-5M) showed increased intracellular calcium concentrations and an increase in expression of steroidogenic genes such as StAR CYP17A1, CYP21A2 and CYP11B2. Cell proliferation was not affected. Calcium entry into cells expressing the mutated nAChR receptor treated for 24h with increasing concentrations of Compound 9S (10–9 to 10-5M) induced an increase in expression of steroidogenic genes such as StAR, CYP21A2 and HSD3B2, but not CYP11B2. Similarly to the results obtained in cells expressing α7-5HT3, cell proliferation was unaffected in response to Compound 9S.

These cell lines, in which we can modulate the intracellular calcium concentration « on demand », are a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.
The Effect of Temperature on the Stability of PCSK-9 Monoclonal Antibody: An Experimental Study
Authors: Kongmalai Tanawan , Chuanchaiyakul Nalinee , Tansit Tunsuda , Sripatumtong Chattip , Srinoulprasert Yuttana , Klinsukon Nareerak , Thongtang Nuntakorn
Published: May 2021

Background: PCSK9 monoclonal antibody lowers plasma PCSK9 and LDL-cholesterol levels. The manufacturers recommend drug storage at 2–8°C, and not above 25°C. This study aimed to investigate drug stability at various temperatures that this drug could be exposed to during medication handling and transportation in tropical countries. Methods: Alirocumab and evolocumab were tested in 3 study conditions: room temperature (RT), cooler device with cold pack, and freeze-thaw for 9 and 18 hours. Heated drugs were used as negative control. Free plasma PCSK9 levels from 9 hyperlipidemia subjects were measured with ELISA. Results: Average subject age was 49.2±18.4 years. Percent PCSK9 inhibition significantly declined in heated drugs compared to baseline. Average RT during the study period was 30.4°C. Change in percent PCSK9 inhibition of PCSK9 mAb at RT from baseline was -5.8±4.4 (p=0.005) and -11.0±8.9% (p=0.006) for alirocumab at 9 hours and 18 hours, and -9.7±11.8% (p=0.04) and -15.1±14.3% (p=0.01) for evolocumab at 9 and 18 hours, respectively. In contrast, there were no significant changes in percent PCSK9 inhibition from baseline when PCSK9 mAb was stored in a cooler. In freeze-thaw condition, changes in percent PCSK9 inhibition from baseline to 9 and 18 hours were -5.2±2.9% (p=0.001) and -2.6±4.9% (p=0.16) for alirocumab, and -1.8±4.2% (p=0.24) and 0.4±6.1% (p=0.83) for evolocumab. Conclusion: Proper drug storage according to manufacturer’s recommendation is essential. Drug storage at RT in tropical climate for longer than 9 hours significantly decreased drug efficacy; however, storage in a cooler device with cold pack for up to 18 hours is safe.
Pregnancy in Familial Chylomicronemia Syndrome: Plasmapheresis as Therapeutic Approach
Authors: Leskovar Dunja , Šabić Marina , Perica Dražen , Šućur Nediljko , Hauptman Ana Godan , Merćep Iveta , Borovečki Fran , Jelaković Bojan , Reiner Željko , Pećin Ivan
Published: May 2021

Introduction: Familial Chylomicronemia Syndrome (FCS) is a rare autosomal recessive disease where functional loss of lipoprotein lipase results in severe hypertriglyceridemia. Patients often experience recurrent acute pancreatitis, a life-threatening disease. Serum triglycerides (TGs) are physiologically elevated several folds during pregnancy, which especially endanger patients with FCS to develop hypertriglyceridemic pancreatitis (HP) and complications like miscarriage. Despite the development of new drugs for FCS, their safety in pregnancy has not yet been confirmed and their treatment represents a big challenge.

Clinical Case: A 30-year women was hospitalized for planned reevaluation while 22 weeks pregnant. She was diagnosed with FCS at the age of one month with failure to thrive, jaundice and lipemic blood serum (homozygous for the LPL gene variation C.1019-2A>T). During childhood she was treated with low-fat diet with average TG levels 10–15 mmol/l (n<1,7 mmol/l). In the past five years, she was hospitalized for HP five times due to loss of compliance to diet (serum TGs > 30 mmol/l). The last episode of HP was provoked by pregnancy and resulted in miscarriage. At the admission, the patient was asymptomatic, with BMI:18,5 kg/m2, TGs: 32,2 mmol/l (n<1,7 nmol/l), lipase: 21 U/L (n<60 U/L). Gynecology status and child growth parameters were normal. Due to inability to achieve lower TG levels plasmapheresis was started two times per week. The overall mean of pre plasmapheresis values of TGs were 21,68 nmol/l (ranging from 15,9 to 26,3 nmol/l) and post plasmapheresis were 7,15 mmol/L (ranging from 4,9 to 9,9 mmol/l). Due to the frequent need for plasmapheresis and high risk pregnancy, the patient was transferred to the Gynecology department. The patient had no adverse reactions and fetal monitoring was performed before, during and after procedure with no abnormalities registered. Plasmapheresis was performed using Spectra OPTIA via peripheral veins with 5% albumin replacement, proceeded by 250-500ml of 0.9% saline infusion. At 40th gestational week Cesarean section was performed and a healthy baby boy (weight: 3540 g, lenght: 49 cm) was born with Apgar score 10/10. We followed baby boy the first five years of the childhood. So far the patient has not experienced HP and the baby boy has normal levels of TGs.

Conclusion: Our case showed a succesfull pregnancy outcome in female patient with FCS achieved by multidisciplinary approach including plasmapheresis. For the first time therapeutic plasmaferesis was used in order to prevent pancreatitis and potential complications for both mother and child.
Scientific and Regulatory Considerations for the Approval of the First Generic Glucagon
Authors: Pang Eric , Chong William , Luke Markham C
Published: May 2021

Glucagon for Injection (NDA 020928) is a polypeptide hormone identical to human glucagon approved 20 years ago for severe hypoglycemia in patients with diabetes mellitus. On Dec 28, 2020, the U.S. FDA approved the first generic version of glucagon for injection USP, 1 mg/vial packaged in an emergency kit. The generic and the reference listed drug (RLD) version, i.e., the innovator version, of glucagon were each produced through different manufacturing processes. The RLD version of glucagon is produced via recombinant DNA in yeast while the generic version of glucagon is produced by peptide synthesis. The FDA published its current thinking on how to ensure sameness between the generic and innovator peptide products prepared with different manufacturing processes in a Draft Guidance for Industry: Submission of Abbreviated New Drug Applications for Certain Highly Purified Synthetic Peptide Drug Products, which applies to five peptide drug products, including glucagon. In this presentation, we aim to provide an overview of the regulatory recommendations for submitting generic glucagon drug products for approval, as outlined in the aforementioned draft guidance. Although glucagon may be produced using different manufacturing processes, the sameness in glucagon can be adequately demonstrated using analytical methods, which involve demonstrating physicochemical properties, as well as primary and secondary structures, oligomers and aggregation states. Biological assays may also be used as part of the demonstration of active pharmaceutical ingredient sameness. Synthetic glucagon may have different impurity profiles when compared to the RLD recombinant product. As part of the ANDA review, impurities in the synthetic drugs are analyzed and controlled, in addition, the potential immunogenicity of new impurities, which are not in the RLD products, are assessed and compared using non-clinical assays. In this work, we will discuss non-clinical assays for assessing the immunogenicity risk of these impurities, for both adaptive and innate immune responses. In conclusion, the sameness of an approved generic synthetic glucagon to an RLD can be adequately established through various analytical methods and biological assays.
Sex Disparities in Thermoregulatory and Metabolic Responses to Mild Cold Exposure Largely Explained by Differences in Body Mass and Body Surface Area
Authors: Ivanova Yoanna M , Swibas Tracy , Haman François , Hildreth Kerry L , Miao Yubin , Kohrt Wendy M , Carpentier Andre , Melanson Edward L , Blondin Denis P
Published: May 2021

Sex-related differences in thermoregulatory responses to cold exposure, such as differences in metabolic heat production and fuel selection, are often attributed to differences in morphology and body composition. Whether these differences persist in response to cold when comparing lean, healthy men and women with equivalent total body mass (BM, heat producing capacity) and body surface areas (BSA, heat loss capacity) remains unknown. In this study, we aimed to compare thermoregulatory and metabolic responses to cold exposure in both men and women, before and after matching for BM (± 0.6 kg) and BSA (± 0.01 m2). Data included in this study were derived from four previously published studies and an additional 13 men and 23 women who recently completed an identical 3h mild cold exposure protocol. Included in the analyses were 45 healthy men and 23 healthy women [27 years (95% CI: 25 to 28) in men vs. 34 years (95% CI: 30 to 38) in women, P = 0.0003], including 7 men and women of the same age [28 years (95% CI: 22 to 34) vs. 29 years (95% CI: 22 to 37), P = 0.78] matched for BM and BSA. Using a combination of indirect calorimetry, electromyography and positron emission tomography with 11C-acetate and 18F-fluorodeoxyglucose, we quantified mean skin temperature, whole-body energy expenditure (EE), shivering intensity, brown adipose tissue (BAT) oxidative metabolism and glucose uptake. The cold-induced decrease in mean skin temperature was greater in women than men [-6.4°C (95% CI: -6.7 to -6.0) vs. -5.4°C (95% CI: -5.8 to -5.1), P = 0.0004], whereas EE was higher in men compared to women both during room temperature and cold exposure, with the cold-induced increase in EE being slightly greater in men than women [3.8 kJ·min-1 (95% CI: 3.2 to 4.5) vs. 2.8 kJ·min-1 (95% CI: 2.0 to 3.7), P = 0.07]. In contrast, shivering intensity (%MVC) was higher in women compared to men [3.0 %MVC (95% CI: 2.1 to 3.8) vs.1.8 %MVC (95% CI: 1.5 to 2.2), P = 0.0069]. Cold exposure also increased BAT oxidative index to a similar magnitude in men and women, increasing ~4-fold in men and ~3-fold in women (effect of sex, P = 0.2067). Both fractional glucose uptake [0.022 min-1 (95%CI: 0.017 to 0.027) in men and 0.021 min-1 (95%CI: 0.013 to 0.030) in women, P = 0.02] and net glucose uptake in BAT [92 nmol.g-1.min-1 (95%CI: 69 to 115) in men and 91 nmol.g-1.min-1 (95%CI: 53 to 129) in women] were not different between the sexes without or with matching for BM and BSA. The sex differences in mean skin temperature, energy expenditure and shivering intensity were all lost once participants were matched for BM and BSA. The present results suggest that much of the sexual dimorphism in thermoregulatory and metabolic responses to mild cold exposure can be explained by differences in BM and BSA.
The SSBP3 Co-Regulator Is a Novel Driver of Islet Cell Structure and Function
Authors: Toren Eliana , Liu Yanping , Hunter Chad
Published: May 2021

The activities of transcriptional complexes drive the proper development and function of insulin producing beta-cells, ultimately required for the regulation of glucose homeostasis. Our prior work helped to establish that the LIM-homeodomain transcription factor (TF), Islet-1 (Isl1), directly interacts with the Ldb1 co-regulator in developing and adult beta-cells. We further found that a member of the Single Stranded DNA-Binding Protein (SSBP) co-regulator family, SSBP3, interacts with the Isl1:Ldb1 complex in beta-cells and primary islets to impact critical target genes MafA and Glp1R. Members of the SSBP family of co-regulators stabilize TF complexes in various tissues, ranging from brain to skin, by binding directly to Ldb1 and protecting the factors from ubiquitin-mediated turnover. Because of this, we hypothesized that SSBP3 would have similarly critical roles as Isl1 and Ldb1 for beta-cell development and function in vivo. To assess this, we first developed a novel SSBP3 floxed mouse line, where Cre-mediated recombination is predicted to impart loss of the Ldb1-interacting domain, plus an early termination. We bred this mouse into a Pax6-Cre transgenic line to recombine SSBP3 in the developing pancreatic islet, a model termed SSBP3islet. We found that SSBP3islet neonates become progressively hyperglycemic and both male and female mice are glucose intolerant as early as postnatal day (P) 21. These results are similar to previous Ldb1 and Isl1 knockouts in the embryonic islet, both of which were hyperglycemic by P10. We observed a reduction of the beta-cell maturity marker, MafA, and disruptions in islet cell architecture with an apparent increase in both glucagon+ alpha-cells and ghrelin+epsilon-cells at P10 and P28. In ongoing studies we are generating embryonic day (E)18.5 embryos to determine islet development defects and will conduct chromatin immunoprecipitation (ChIP) experiments to determine the beta-cell and islet genes directly bound by SSBP3 in vivo. These experiments will further elucidate the regulation of islet function by LIM complexes, knowledge that is central not only for our understanding of glucose homeostasis but for the development of novel diabetes therapeutics.