Predictors of lack of glycemic control in persons with type 2 diabetes.

BACKGROUND: Professional guidelines recommend an HbA1c < 7% for most people with diabetes and < 8.5% for those with relaxed glycemic goals. However, many people with type 2 diabetes mellitus (T2DM) are unable to achieve the desired HbA1c goal. This study evaluated factors associated with lack of improvement in HbA1c over 3 years. METHODS: All patients with T2DM treated within a major academic healthcare system during 2015-2020, who had at least one HbA1c value > 8.5% within 3 years from their last HbA1c were included in analysis. Patients were grouped as improved glycemic control (last HbA1c ≤ 8.5%) or lack of improvement (last HbA1c > 8.5%). Multivariate logistic regression analysis was performed to assess independent predictors of lack of improvement in glycemic control. RESULTS: Out of 2,232 patients who met the inclusion criteria, 1,383 had an improvement in HbA1c while 849 did not. In the fully adjusted model, independent predictors of lack of improvement included: younger age (odds ratio, 0.89 per 1-SD [12 years]; 95% CI, 0.79-1.00), female gender (1.30, 1.08-1.56), presence of hypertension (1.29, 1.08-1.55), belonging to Black race (1.32, 1.04-1.68, White as reference), living in low income area (1.86,1.28-2.68, high income area as reference), and insurance coverage other than Medicare (1.32, 1.05-1.66). Presence of current smoking was associated with a paradoxical improvement in HbA1c (0.69, 0.47-0.99). In a subgroup analysis, comparing those with all subsequent HbA1c values > 8.5% (N = 444) to those with all subsequent HbA1c values < 8.5% (N = 341), similar factors were associated with lack of improvement, but smoking was no longer significant. CONCLUSION: We conclude that socioeconomic factors like race, type of insurance coverage and living in low-income areas are associated with lack of improvement in HbA1c over a period of 3-years in people with T2DM. Intervention strategies focused on low-income neighborhoods need to be designed to improve diabetes management.

Insulin resistance probability score and incident cardiovascular disease.

BACKGROUND: Insulin resistance (IR) is associated with cardiovascular disease (CVD). However, insulin immunoassay variability and scarce research of the elderly have hindered the adoption of IR assessment for CVD prevention. We asked whether the probability of having IR [p(IR)]-derived from insulin and C-peptide mass-spectrometry assays-was associated with CVD in the elderly. METHODS: A random cohort was drawn from MPP, a population-based study of the elderly. After excluding those with missing data, CVD, or diabetes, 3645 participants (median age = 68) remained. RESULTS: During follow-up (13.3 years), 794 incident CVD events were observed. p(IR) > 80% (n = 152) compared with p(IR) ≤ 80% was associated with incident CVD (HR = 1.51, 95% CI 1.12-2.05, p = 0.007) and CVD or all-cause mortality (HR = 1.43, 95% CI 1.16-1.77, p = 0.0009) after adjusting for age, sex, hypertension, smoking, HDL-cholesterol, total cholesterol, triglycerides, BMI, and prediabetes. CONCLUSION: High p(IR) was associated with >50% greater risk of incident CVD. IR assessment in the elderly may be warranted.

Differences in HDL-Bound Apolipoproteins in Patients With Advanced Liver Fibrosis Due to Nonalcoholic Fatty Liver Disease.

CONTEXT: The mechanisms leading to increased cardiovascular disease in patients with nonalcoholic fatty liver disease (NAFLD) and advanced liver fibrosis remain incompletely understood. OBJECTIVE: This study assessed HDL-bound proteins in patients with NAFLD with or without advanced fibrosis. METHODS: This cross-sectional study at a university hospital included 185 patients with or without type 2 diabetes (T2D). Patients underwent liver proton magnetic resonance spectroscopy to measure intrahepatic triglyceride accumulation and those with NAFLD underwent a percutaneous liver biopsy. Advanced lipid testing with lipoprotein subfraction measurements and targeted proteomics of HDL-bound proteins was performed. RESULTS: Patients with and without advanced fibrosis had similar clinical characteristics, except for lower HDL-C (34 ± 8 vs 38 ± 9 mg/dL, P = 0.024) and higher prevalence of T2D in advanced fibrosis. Patients with advanced fibrosis had lower HDL particle number. A panel of 28 HDL-bound proteins were targeted and quantified by multiple reaction monitoring liquid chromatography-tandem mass spectrometry. Five proteins were found to be decreased in patients with advanced fibrosis (ApoC-I [P < 0.001], ApoC-IV [P = 0.012], ApoM [P = 0.008], LCAT [P = 0.014], and SAA4 [P = 0.016]). No differences were observed in these proteins in patients with vs without NAFLD or steatohepatitis. The pCAD index, associated with coronary artery disease and cardiovascular mortality, was significantly higher in patients with advanced fibrosis (97 ± 5 vs 86 ± 25, P = 0.04). CONCLUSION: Patients with NAFLD with advanced fibrosis showed significant differences in HDL-bound protein levels; this translated into increased cardiovascular risk based on pCAD index. Different lipoprotein composition and function may explain the link between liver disease and increased cardiovascular mortality in these patients.

High prevalence of subnormal testosterone in obese adolescent males: reversal with bariatric surgery.

OBJECTIVE: Obesity in adolescent males is associated with the lowering of total and free testosterone concentrations. Weight loss may increase testosterone concentrations. DESIGN AND METHODS: We evaluated the changes in sex hormones following bariatric surgery in 34 males (age range: 14.6-19.8 years) with obesity. These participants were part of a prospective multicenter study, Teen-Longitudinal Assessment of Bariatric Surgery. The participants were followed up for 5 years after surgery. Total testosterone, total estradiol, luteinizing hormone, follicle-stimulating hormone, sex hormone-binding globulin, C-reactive protein, insulin and glucose were measured at baseline, 6 months and annually thereafter. Free testosterone, free estradiol and HOMA2-IR were calculated. RESULTS: Study participants lost one-third of their body weight after bariatric surgery, with maximum weight loss achieved at 24 months for most participants. Free testosterone increased from 0.17 (95% CI: 0.13 to 0.20) at baseline to 0.34 (95% CI: 0.30 to 0.38) and 0.27 nmol/L (95% CI: 0.23 to 0.32) at 2 and 5 years (P < 0.001 for both), respectively. Total testosterone increased from 6.7 (95% CI: 4.7 to 8.8) at baseline to 17.6 (95% CI: 15.3 to 19.9) and 13.8 (95% CI: 11.0 to 16.5) nmol/L at 2 and 5 years (P < 0.001), respectively. Prior to surgery, 73% of the participants had subnormal free testosterone (<0.23 nmol/L). After 2 and 5 years, only 20 and 33%, respectively, had subnormal free testosterone concentrations. Weight regain was related to a fall in free testosterone concentrations. CONCLUSIONS: Bariatric surgery led to a robust increase in testosterone concentrations in adolescent males with severe obesity. Participants who regained weight had a decline in their testosterone concentrations.

Association of changes in lipid levels with changes in vitamin D levels in a real-world setting.

In clinical trials, vitamin D supplementation has been reported to reduce serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG) but not high-density lipoprotein cholesterol (HDL-C). In this cohort study we evaluated the association between changes in vitamin D (25-hydroxyvitamin D) and changes in lipid levels in a real-world setting. Changes in lipid levels over a 1-year period were evaluated among individuals whose vitamin D levels increased (group 1) or decreased (group 2) by ≥ 10 ng/mL in year 2018 versus 2017 (cohort 1; n = 5580), in 2019 versus 2018 (cohort 2, n = 6057), or in 2020 versus 2019 (cohort 3, n = 7249). In each cohort, levels of TC, LDL-C, and TG decreased in group 1 and increased in group 2. Between-group differences in average changes in the 3 cohorts ranged from 10.71 to 12.02 mg/dL for TC, from 7.42 to 8.95 mg/dL for LDL-C, and from 21.59 to 28.09 mg/dL for TG. These differences were significant after adjusting for age, sex, race, education, body mass index, blood pressure, smoking status, geographical location, and baseline levels of vitamin D and lipids (P < 0.001). Changes in vitamin D levels were not significantly associated with changes in HDL-C levels.

Antibody-Free Quantification of Serum Chromogranin A by Targeted Mass Spectrometry.

BACKGROUND: Chromogranin A (CgA) is a 48 kDa protein that serves as a diagnostically sensitive, but nonspecific, serum biomarker for neuroendocrine tumors. Immunoassays for CgA are not standardized and have a narrow dynamic range, which requires dilution of concentrated specimens. We developed and validated an antibody-free, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for CgA without these limitations. METHODS: CgA was extracted from serum using a mixed-mode anion exchange solid-phase extraction plate, digested with trypsin, and analyzed by LC-MS/MS using well-characterized CgA calibration standards. After validation, the mass spectrometry method was compared with the CISBIO immunoassay using 200 serum specimens previously submitted for CgA analysis. Specimens with discordant results were reanalyzed by high-resolution mass spectrometry- (HRMS) -based methods to assess the contribution of truncated and post-translationally modified forms of CgA. RESULTS: The assay had a linear range of 50 to 50 000 ng/mL, recoveries between 89% and 115%, and intra- and interassay imprecision <10%. LC-MS/MS assay results showed a Pearson's correlation of r = 0.953 with the CISBIO immunoassay, with CgA values being a mean 2- to 4-fold higher. Concordance for CgA between the 2 assays was 80.9% (95% CI 72.8%-89.2%), showing substantial agreement. Truncation and posttranslational modification, including 2 phosphorylation sites that had not been previously observed or predicted to our knowledge, did not appear to contribute directly to discordance between the 2 assays. CONCLUSION: Quantification of CgA by LC-MS/MS provides an analytically sensitive and reproducible alternative to commercially available immunoassays.

Isotopic Peak Index, Relative Retention Time, and Tandem MS for Automated High Throughput IGF-1 Variants Identification in a Clinical Laboratory.

Measuring insulin-like growth factor-1 (IGF-1) is useful for assessing and managing growth-related disorders, such as acromegaly and growth hormone deficiency. High-resolution liquid chromatography-mass spectrometry (LC-MS) is used for measuring IGF-1 due to its molecular specificity, quantitative performance, well-characterized reference materials, and detailed age/sex-specific reference intervals. However, polymorphisms in the IGF1 gene may cause mass shifts in the polypeptide, which can impede quantitation and cause errors in clinical interpretation. We (1) developed a concept of "isotopic peak index", which allows simultaneous monitoring of 15 IGF-1 variants by using only four m/z ratios; (2) developed a "relative retention time" parameter that allows distinction of previously unresolved variants; and (3) utilized tandem mass spectrometry (MS/MS) to distinguish between the most common pair of variants: isobaric A67T and A70T. All methods were validated with DNA sequencing. This approach identified six variants from the ExAC database, P66A, A67S, S34N, A38 V, A67T, and A70T; two previously reported V44M and A67V variants; and discovered six unreported variants, Y31H, S33P, R50Q, R56K, T41I, and A62T. Major improvements in our workflow include enhanced automation, avoiding detailed manual calculations that are prone to human error, and the ability to monitor more, and discover new, IGF-1 variants. The workflow provides a profile of a patient's IGF-1 status and can be used to explore genotype-phenotype relationships in IGF-1 variants.

Intact Fasting Insulin Identifies Nonalcoholic Fatty Liver Disease in Patients Without Diabetes.

CONTEXT: Patients with nonalcoholic fatty liver disease (NAFLD) are characterized by insulin resistance and hyperinsulinism. However, insulin resistance measurements have not been shown to be good diagnostic tools to predict NAFLD in prior studies. OBJECTIVE: We aimed to assess a newly validated method to measure intact molecules of insulin by mass spectrometry to predict NAFLD. METHODS: Patients underwent a 2-hour oral glucose tolerance test (OGTT), a liver magnetic resonance spectroscopy (1H-MRS), and a percutaneous liver biopsy if they had a diagnosis of NAFLD. Mass spectrometry was used to measure intact molecules of insulin and C-peptide. RESULTS: A total of 180 patients were recruited (67% male; 52 ± 11 years of age; body mass index [BMI] 33.2 ± 5.7 kg/m2; 46% with diabetes and 65% with NAFLD). Intact fasting insulin was higher in patients with NAFLD, irrespective of diabetes status. Patients with NAFLD without diabetes showed ~4-fold increase in insulin secretion during the OGTT compared with all other subgroups (P = 0.008). Fasting intact insulin measurements predicted NAFLD in patients without diabetes (area under the receiver operating characteristic curve [AUC] of 0.90 [0.84-0.96]). This was significantly better than measuring insulin by radioimmunoassay (AUC 0.80 [0.71-0.89]; P = 0.007). Intact fasting insulin was better than other clinical variables (eg, aspartate transaminase, triglycerides, high-density lipoprotein, glucose, HbA1c, and BMI) to predict NAFLD. When combined with alanine transaminase (ALT) (intact insulin × ALT), it detected NAFLD with AUC 0.94 (0.89-0.99) and positive and negative predictive values of 93% and 88%, respectively. This newly described approach was significantly better than previously validated noninvasive scores such as NAFLD-LFS (P = 0.009), HSI (P < 0.001), and TyG index (P = 0.039). CONCLUSION: In patients without diabetes, accurate measurement of fasting intact insulin levels by mass spectrometry constitutes an easy and noninvasive strategy to predict presence of NAFLD.

Association of Circulating Sex Hormones With Inflammation and Disease Severity in Patients With COVID-19.

Importance: Male sex is a risk factor for developing severe COVID-19 illness. It is not known whether sex hormones contribute to this predisposition. Objective: To investigate the association of concentrations of serum testosterone, estradiol, and insulinlike growth factor 1 (IGF-1, concentrations of which are regulated by sex hormone signaling) with COVID-19 severity. Design, Setting, and Participants: This prospective cohort study was conducted using serum samples collected from consecutive patients who presented from March through May 2020 to the Barnes Jewish Hospital in St Louis, Missouri, with COVID-19 (diagnosed using nasopharyngeal swabs). Exposures: Testosterone, estradiol, and IGF-1 concentrations were measured at the time of presentation (ie, day 0) and at days 3, 7, 14, and 28 after admission (if the patient remained hospitalized). Main Outcomes and Measures: Baseline hormone concentrations were compared among patients who had severe COVID-19 vs those with milder COVID-19 illness. RNA sequencing was performed on circulating mononuclear cells to understand the mechanistic association of altered circulating hormone concentrations with cellular signaling pathways. Results: Among 152 patients (90 [59.2%] men; 62 [40.8%] women; mean [SD] age, 63 [16] years), 143 patients (94.1%) were hospitalized. Among 66 men with severe COVID-19, median [interquartile range] testosterone concentrations were lower at day 0 (53 [18 to 114] ng/dL vs 151 [95 to 217] ng/dL; P = .01) and day 3 (19 [6 to 68] ng/dL vs 111 [49 to 274] ng/dL; P = .006) compared with 24 men with milder disease. Testosterone concentrations were inversely associated with concentrations of interleukin 6 (ß = -0.43; 95% CI, -0.52 to -0.17; P < .001), C-reactive protein (ß = -0.38; 95% CI, -0.78 to -0.16; P = .004), interleukin 1 receptor antagonist (ß = -0.29; 95% CI, -0.64 to -0.06; P = .02), hepatocyte growth factor (ß = -0.46; 95% CI, -0.69 to -0.25; P < .001), and interferon γ-inducible protein 10 (ß = -0.32; 95% CI, -0.62 to -0.10; P = .007). Estradiol and IGF-1 concentrations were not associated with COVID-19 severity in men. Testosterone, estradiol, and IGF-1 concentrations were similar in women with and without severe COVID-19. Gene set enrichment analysis revealed upregulated hormone signaling pathways in CD14+CD16- (ie, classical) monocytes and CD14-CD16+ (ie, nonclassical) monocytes in male patients with COVID-19 who needed intensive care unit treatment vs those who did not. Conclusions and Relevance: In this single-center cohort study of patients with COVID-19, lower testosterone concentrations during hospitalization were associated with increased disease severity and inflammation in men. Hormone signaling pathways in monocytes did not parallel serum hormone concentrations, and further investigation is required to understand their pathophysiologic association with COVID-19.

Performance of Plasma Biomarkers and Diagnostic Panels for Nonalcoholic Steatohepatitis and Advanced Fibrosis in Patients With Type 2 Diabetes.

OBJECTIVE: The 2019 Standards of Medical Care in Diabetes suggested that patients with nonalcoholic fatty liver disease (NAFLD) should be evaluated for liver fibrosis. However, the performance of noninvasive clinical models/scores and plasma biomarkers for the diagnosis of nonalcoholic steatohepatitis (NASH) and advanced fibrosis has not been carefully assessed in patients with type 2 diabetes mellitus (T2DM). RESEARCH DESIGN AND METHODS: In this cross-sectional study, patients (n = 213) had a liver MRS, and those with a diagnosis of NAFLD underwent a percutaneous liver biopsy. Several noninvasive clinical models/scores and plasma biomarkers were measured to identify NASH and advanced fibrosis (NASH: ALT, cytokeratin-18, NashTest 2, HAIR, BARD, and OWLiver; advanced fibrosis: AST, fragments of propeptide of type III procollagen [PRO-C3], FIB-4, APRI, NAFLD fibrosis score, and FibroTest). RESULTS: None of the noninvasive tools assessed for the diagnosis of NASH in patients with T2DM had an optimum performance (all areas under the curve [AUCs] <0.80). Of note, none of the panels or biomarkers was able to outperform plasma ALT (AUC 0.78 [95% CI 0.71-0.84]). Performance was better to diagnose advanced fibrosis, in which plasma PRO-C3, AST, and APRI showed better results than the other approaches (AUC 0.90 [0.85-0.95], 0.85 [0.80-0.91], and 0.86 [0.80-0.91], respectively). Again, none of the approaches did significantly better than plasma AST. Sequential use of plasma AST and other noninvasive tests may help in limiting the number of liver biopsies required to identify patients with advanced fibrosis. CONCLUSIONS: Performance of noninvasive clinical models/scores and plasma biomarkers for the diagnosis of NASH or advanced fibrosis was suboptimal in patients with T2DM. Combination of multiple tests may provide an alternative to minimize the need for liver biopsies to detect fibrosis in these patients.

Plasma Free Cortisol in States of Normal and Altered Binding Globulins: Implications for Adrenal Insufficiency Diagnosis.

CONTEXT: Accurate diagnosis of adrenal insufficiency is critical because there are risks associated with overdiagnosis and underdiagnosis. Data using liquid chromatography tandem mass spectrometry (LC/MS/MS) free cortisol (FC) assays in states of high or low cortisol-binding globulin (CBG) levels, including cirrhosis, critical illness, and oral estrogen use, are needed. DESIGN: Cross-sectional. OBJECTIVE: Determine the relationship between CBG and albumin as well as total cortisol (TC) and FC in states of normal and abnormal CBG. Establish the FC level by LC/MS/MS that best predicts TC of <18 µg/dL (497 nmol/L) (standard adrenal insufficiency diagnostic cutoff) in healthy individuals. SUBJECTS: This study included a total of 338 subjects in four groups: healthy control (HC) subjects (n = 243), patients with cirrhosis (n = 38), intensive care unit patients (ICU) (n = 26), and oral contraceptive (OCP) users (n = 31). MAIN OUTCOME MEASURE(S): FC and TC by LC/MS/MS, albumin by spectrophotometry, and CBG by ELISA. RESULTS: TC correlated with FC in the ICU (R = 0.91), HC (R = 0.90), cirrhosis (R = 0.86), and OCP (R = 0.70) groups (all P < 0.0001). In receiver operator curve analysis in the HC group, FC of 0.9 µg/dL (24.8 nmol/L) predicted TC of <18 µg/dL (497 nmol/L; 98% sensitivity, 91% specificity; AUC, 0.98; P < 0.0001). Decreasing the cutoff to 0.7 µg/dL led to a small decrease in sensitivity (92%) with similar specificity (91%). CONCLUSIONS: A cutoff FC of <0.9 µg/dL (25 nmol/L) in this LC/MS/MS assay predicts TC of <18 µg/dL (497 nmol/L) with excellent sensitivity and specificity. This FC cutoff may be helpful in ruling out adrenal insufficiency in patients with binding globulin derangements.

Adrenal insufficiency in neonates undergoing cardiopulmonary bypass and postoperative hypothalamic-pituitary-adrenal function after prophylactic glucocorticoids.

OBJECTIVES: Determine incidence of preoperative adrenal insufficiency in neonates >35 weeks gestation with congenital heart disease undergoing cardiothoracic surgery with bypass and effects of prophylactic methylprednisolone on postoperative hypothalamic-pituitary-adrenal function and hemodynamic stability. DESIGN: Prospective observational study in 36 neonates with preoperative adrenocorticotrophic hormone stimulation tests and serial total cortisol and adrenocorticotrophic hormone measurements before and after surgery. Data analyses: analysis of variance and regression. RESULTS: Baseline circulating adrenocorticotrophic hormone and cortisol were unchanged 4-20 days postnatal (P > 0.1); however, cortisol levels rose with increasing adrenocorticotrophic hormone, P = 0.02. Ten neonates (29%) demonstrated preoperative adrenal insufficiency (∆cortisol ≤9 µg/dl); one had postoperative hemodynamic instability. Growth-restricted neonates had lower baseline cortisol, but normal stimulation tests and responded well to surgical stresses. Seventy-five percent of neonates receiving perioperative methylprednisolone demonstrated postoperative hypothalamic-pituitary-adrenal inhibition. CONCLUSION: Adrenal insufficiency appears common in neonates >35 weeks gestation with congenital heart disease, but did not contribute to postoperative hemodynamic instability despite hypothalamic-pituitary-adrenal inhibition.

Performance of the SteatoTest, ActiTest, NashTest and FibroTest in a multiethnic cohort of patients with type 2 diabetes mellitus.

Fibromax is a diagnostic tool composed of the combination of 4 non-invasive biomarker panels for the diagnosis of steatosis (SteatoTest), necrosis and inflammation (ActiTest and NashTest-2) and fibrosis (FibroTest). The purpose of this study was to assess the performance of these biomarker panels in patients with type 2 diabetes mellitus (T2DM). All patients underwent routine labs, a 75 g oral glucose tolerance test, a liver proton magnetic resonance spectroscopy (1H-MRS) to measure intrahepatic triglyceride content, and a percutaneous liver biopsy to establish the diagnosis of non-alcoholic steatohepatitis (NASH) and to grade and stage the disease in those patients with non-alcoholic fatty liver disease (NAFLD) by 1H-MRS. For determination of the scores, plasma samples were blindly provided to establish the SteatoTest, ActiTest, NashTest-2 and FibroTest scores. A total of 220 patients with T2DM were included in this study. When the ability of the SteatoTest to identify patients with T2DM with NAFLD by 1H-MRS was assessed, the overall performance expressed as the area under the receiver operating characteristic curve was 0.73 (95% CI 0.65 to 0.81). The performance of the ActiTest and NashTest-2 to diagnose definite NASH among patients with T2DM was 0.70 (95% CI 0.63 to 0.77) and 0.69 (95% CI 0.62 to 0.76), respectively. Regarding the FibroTest score, its performance to identify patients with moderate or advanced fibrosis was 0.67 (95% CI 0.58 to 0.76) and 0.72 (95% CI 0.61 to 0.83), respectively. Non-invasive panels for the diagnosis of steatosis, NASH and/or fibrosis, which were developed and validated in non-diabetic cohorts, underperformed when applied to a large cohort of patients with T2DM. Results from non-diabetic populations should not be extrapolated to patients with T2DM.

Use of a metabolomic approach to non-invasively diagnose non-alcoholic fatty liver disease in patients with type 2 diabetes mellitus.

AIMS: To assess the utility of existing metabolomics scores to classify liver disease in patients with type 2 diabetes mellitus (T2DM). MATERIALS AND METHODS: A total of 220 patients with T2DM were recruited. Patients underwent routine laboratory tests, liver proton magnetic resonance spectroscopy (1 H-MRS), a 75-g oral glucose tolerance test, and liver biopsy if 1 H-MRS findings indicated non-alcoholic fatty liver disease. A serum sample was blindly provided to OWL Metabolomics on which to run the OWLiver Care and OWLiver tests. RESULTS: When compared with liver biopsy, the OWLiver Care and OWLiver tests had a suboptimal performance in patients with T2DM (areas under the receiver-operating characteristic [AUROC] curve both <0.70). Given the discordance of these results in this heterogeneous, multiethnic cohort compared with those of a previous report in predominantly white patients without diabetes, we examined the influence of age, ethnicity and other variables on test performance. A specific subset of patients was selected to mirror the characteristics of the population used for the development of this model (ie, white patients without T2DM). Among white patients with good glycaemic control (glycated haemoglobin <53mmol/mol [or <7%]) and without cirrhosis, the AUROC curve was significantly improved (0.79 [CI 95%: 0.68-0.90]). Among white patients with lower insulin resistance (homeostatic model assessment of insulin resistance <3) and without cirrhosis, the AUROC was even higher: 0.87 (CI 95%: 0.76-0.97). CONCLUSIONS: There is a great need to develop non-invasive approaches to diagnose non-alcoholic steatohepatitis in patients with T2DM; models originally developed for patients without diabetes cannot be directly applied to patients with T2DM.

Discordance between mass spectrometry and immunometric IGF-1 assay in pituitary disease: a prospective study.

PURPOSE: Measuring IGF-1, a biomarker for GH activity, is critical to evaluating disordered hypothalamic-pituitary GH axis. Inconsistent IGF-1 measurements among different immunoassays are well documented. We switched from Immulite 2000 immunoassay to narrow-mass-extraction, high-resolution liquid chromatography mass-spectrometry (LC-MS) compliant with recent consensus recommendations on assay standardization. Comparability of these two assays in patients with pituitary disease in a clinical practice setting is not known. We sought to compare IGF-1 levels on Immulite 2000 and LC-MS in samples from naïve and treated patients with secretory and non-secretory pituitary masses. METHODS: We prospectively collected serum samples from 101 patients treated at the Cedars-Sinai Pituitary Center between February 2012 and March 2014. We intentionally recruited more patients with acromegaly or GH deficiency to ensure a clinically representative cohort. Samples were classified as in or out of the respective reference ranges. Bland-Altman analysis was used to assess agreement between assays. RESULTS: Twenty-four percent of samples were classified differently as below, in, or above range. Agreement between the assays was poor overall, with a significant bias for immunoassay reporting higher values than LC-MS. This pattern was also observed in patients with acromegaly and those with ≥ 2 pituitary hormone deficiencies. CONCLUSIONS: IGF-1 results may differ after switching from an older immunoassay to a consensus-compliant assay such as LC-MS. Clinicians should consider the potential impact of assay switching before altering treatment due to discrepant results, particularly in patients monitored over time, such as those with acromegaly and GH deficiency.

Identification of the Underlying Androgen Receptor Defect in the Dallas Reifenstein Family.

CONTEXT: The Dallas Reifenstein family - first described in 1965 - includes 14 known members with partial androgen insensitivity syndrome (PAIS). However, the underlying molecular defect was never identified. OBJECTIVE: To identify the underlying genetic defect for PAIS in the Dallas Reifenstein family. DESIGN: DNA was purified from scrotal skin fibroblasts, and whole exome sequencing was then performed in four affected men in the family. Additional family members - both affected and unaffected - were subsequently recruited to confirm segregation of the candidate mutations with the PAIS phenotype. PATIENTS: The affected men have PAIS with infertility associated with azoospermia, hypospadias, and gynecomastia. RESULTS: All four men harbored an intronic variant NC_000023.10:g.66788676A>C between exon 1 and exon 2 of the androgen receptor (AR) canonical transcript NM_000044 (complementary DNA position NM_000044: c.1616+22072A>C) predicted to cause an alternatively spliced AR transcript. Reverse transcription (RT) polymerase chain (PCR) experiments detected the predicted PCR product of the alternatively spliced AR transcript, and the mutation segregated with the PAIS phenotype in this family. The transcript includes the insertion of 185 nucleotides with a premature stop codon at chrX:66863131-66863133, likely resulting in a reduction in AR protein expression due to nonsense-mediated decay. CONCLUSIONS: An intronic AR mutation was identified in the Dallas Reifenstein family. The findings suggest that in cases of PAIS without identifiable AR mutations in coding regions, intronic AR mutations should be considered.

Mutational Analysis in Pediatric Thyroid Cancer and Correlations with Age, Ethnicity, and Clinical Presentation.

BACKGROUND: Well-differentiated thyroid cancer (WDTC) incidence in pediatrics is rising, most being papillary thyroid carcinoma (PTC). The objective of the study was to assess the prevalence of different mutations in pediatric WDTC and correlate the genotype with the clinical phenotype. METHODS: This is a single-center retrospective study. Thyroid tissue blocks from 42 consecutive pediatric WDTC patients who underwent thyroidectomy between 2001 and 2013 were analyzed at Quest Diagnostics for BRAF(V600E), RAS mutations (N,K,H), and RET/PTC and PAX8/PPARγ rearrangements, using validated molecular methods. Thyroid carcinomas included PTC, follicular thyroid carcinoma (FTC), and follicular variant of PTC (FVPTC). RESULTS: Thirty-nine samples (29 females) were genotyped. The mean age at diagnosis was 14.7 years (range 7.9-18.4 years), and most were Hispanic (56.4%) or Caucasian (35.9%). The mean follow-up period was 2.9 years. Mutations were noted in 21/39 (53.8%), with both BRAF(V600E) (n = 9), and RET/PTC (n = 6) detected only in PTC. Mutations were detected in 2/5 FTC (PAX8/PPARγ and NRAS) and 3/6 FVPTC cases (PAX8/PPARγ). Of 28 PTC patients, 57.1% had mutations: 32.1% with BRAF(V600E), 21.4% with RET/PTC, and 3.6% with NRAS. Of patients with BRAF(V600E), 77.8% were Hispanic and 88.9% were >15 years, while all RET/PTC-positive patients were ≤15 years (p = 0.003). Tumor size, lymph node involvement, and distant metastasis at diagnosis (or soon after (131)I ablation) did not vary significantly based on the mutation. CONCLUSIONS: BRAF(V600E) was the most common mutation, especially in older and Hispanic adolescents. A larger, ethnically diverse pediatric cohort followed long term will enable the genotypic variability, clinical presentation, and response to therapy to be better assessed.

Hepatic Steatosis and Insulin Resistance, But Not Steatohepatitis, Promote Atherogenic Dyslipidemia in NAFLD.

CONTEXT: Patients with nonalcoholic fatty liver disease (NAFLD) are at increased risk of cardiovascular disease, and atherogenic lipoproteins may play an important role. OBJECTIVE: The objective of the study was to determine the contribution of the severity of steatohepatitis to atherogenic dyslipidemia in patients with NAFLD. DESIGN: This was a cross-sectional study. SETTING: The study was conducted at a university hospital. PATIENTS: Patients were recruited from outpatient clinics or from the general population (n = 188). INTERVENTIONS: Measurement of hepatic triglyceride content by magnetic resonance spectroscopy, histology (liver biopsy), metabolic profile by means of an oral glucose tolerance test, and lipoprotein analyses were performed. OUTCOMES: Outcomes measured included standard lipids, lipoprotein subfraction analysis (apolipoprotein B/A1 levels, low-density lipoprotein (LDL) particle size/phenotype, and LDL/high-density lipoprotein subfractions), and insulin resistance. RESULTS: Patients with NAFLD had severe insulin resistance, especially at the level of the adipose tissue, when compared with patients without NAFLD. Despite small differences in triglycerides and high-density lipoprotein-cholesterol, patients with NAFLD had a significantly higher plasma apolipoprotein B to apolipoprotein A1 ratio (0.66 ± 0.02 vs 0.58 ± 0.02, P = .01) and smaller LDL particle size (216.2 ± 0.7 vs 219.4 ± 1.1 Å, P = .01). Of note, these differences between patients with/without NAFLD were independent of the presence of obesity. Severity of steatohepatitis did not significantly influence the lipoprotein profile. Worse atherogenic dyslipidemia was best predicted by the degree of liver fat accumulation and adipose tissue and systemic insulin resistance. CONCLUSIONS: NAFLD was associated with a worse atherogenic lipoprotein profile, regardless of similar body mass index and other clinical parameters. We speculate that this lipoprotein profile is driven mostly by liver fat content and insulin resistance and appears not to be worsened by obesity or the severity of liver disease (nonalcoholic steatohepatitis).

Development of the external genitalia: perspectives from the spotted hyena (Crocuta crocuta).

This review/research paper summarizes data on development of the external genitalia of the spotted hyena, a fascinating mammal noted for extreme masculinization of the female external genitalia. The female spotted hyena is the only extant mammal that mates and gives birth through a pendulous penis-like clitoris. Our studies indicate that early formation of the phallus in both males and females is independent of androgens; indeed the phallus forms before the fetal testes or ovaries are capable of synthesizing androgens. Likewise, pre- and postnatal growth in length of the penis and clitoris is minimally affected by "androgen status". Nonetheless, several internal morphologies, as well as external surface features of the phallus, are androgen-dependent and thus account for dimorphism between the penis and clitoris. Finally, estrogens play a critical role in penile and clitoral development, specifying the position of the urethral orifice, determining elasticity of the urethral meatus, and facilitating epithelial-epithelial fusion events required for proper formation of the distal urethra/urogenital sinus and prepuce. Accordingly, prenatal inhibition of estrogen synthesis via administration of letrozole (an aromatase inhibitor) leads to malformations of the glans as well as the prepuce (hypospadias). The effects of prenatal androgens, anti-androgens and impaired estrogen synthesis correlated with the tissue expression of androgen and estrogen receptors.

"Getting from here to there"--mechanisms and limitations to the activation of the androgen receptor in castration-resistant prostate cancer.

Despite the clinical regression that typifies the initial response of advanced prostate cancer to gonadal testosterone depletion, tumors eventually progress. However, evidence supports the concept that signaling via the androgen receptor (AR) is important in progression to castration-resistant prostate cancer (CRPC).Steroid hormones are synthesized from cholesterol in a series of tightly regulated steps involving the cleavage of carbon-carbon bonds, the introduction of functional groups derived from activated molecular oxygen, and the oxidation and reduction of carbon-carbon and carbon-oxygen bonds. In the adrenal cortex and gonads, steroidogenesis is tightly regulated, very efficient, and highly directional. In contrast, steroid metabolism in peripheral tissues is characterized by competing enzymes and pathways, low efficiency, and great variability. Many steps are mechanistically and functionally irreversible, but some are not, and the repertoire of specific enzymes, intracellular redox state, and access to hormone precursors all contribute to steroid flux and accumulation.The investigation of steroid metabolizing enzymes in CRPC often assumes that the pathways and the patterns of metabolism mirror those defined in the adrenals and the gonads and validated by human deficiency syndromes. Unfortunately, several potential pathways using different enzymes might contribute substantially to androgen synthesis in CRPC. Finally, a number of mechanisms have been reported by which the AR is activated independent of ligand. Recent observations have suggested that AR forms with constitutive activity occur in CRPC, stimulating transcription without a requirement for ligand. This overview outlines a broad view of how the mechanisms by which the AR may be activated, whether by alternate pathways of androgen synthesis or the production of alternate forms of the AR, with an emphasis on what aspects must be accounted for when using model systems to explore the biology of human prostate cancer.