Vitamin D Toxicity from an Unusual and Unexpected Source: A Report of 2 Cases.

INTRODUCTION: Hypervitaminosis D is a relatively uncommon etiology of hypercalcemia. Toxicity is usually caused by very high doses, mostly secondary to erroneous prescription or administration of vitamin D, and less commonly, contaminated foods or manufacturing errors of vitamin D-containing supplements. CASE PRESENTATION: A 16-year-old male, previously healthy, presented with 2-week history of nonspecific symptoms (fatigue, gastrointestinal complaints). Investigations showed acute kidney injury and hypercalcemia (total calcium 3.81 mmol/L). Further diagnostic workup revealed markedly elevated 25-hydroxyvitamin D levels (1,910 nmol/L). He denied taking any vitamin D supplements; however, he reported consumption of creatine and protein supplements. Mass spectrometry analysis of the creatine supplement estimated a vitamin D content of 425,000 IU per serving (100 times the upper tolerable daily dose). A few months later, another previously healthy adolescent presented with severe hypercalcemia and acute kidney injury secondary to hypervitaminosis D. He was also using a creatine supplement, from the same manufacturer brand and lot. Both patients were treated with intravenous hydration, calcitonin, and pamidronate. They maintained normocalcemia after their initial presentation but required low-calcium diets and laboratory testing for months after this exposure. DISCUSSION/CONCLUSION: We present 2 cases of hypervitaminosis D caused by a manufacturing error of a natural health product which did not claim to contain vitamin D. The use of dietary supplements is highly prevalent; this should be incorporated while taking medical history, and considered a potential source of toxicity when an alternative source cannot be found, regardless of the product label.

REEP5 depletion causes sarco-endoplasmic reticulum vacuolization and cardiac functional defects.

The sarco-endoplasmic reticulum (SR/ER) plays an important role in the development and progression of many heart diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. Here, we report a cardiac enriched, SR/ER membrane protein, REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes results in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants show sensitized cardiac dysfunction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrates cardiac dysfunction. These results demonstrate the critical role of REEP5 in SR/ER organization and function as well as normal heart function and development.

A novel Sigma metric encompasses global multi-site performance of 18 assays on the Abbott Alinity system.

OBJECTIVES: The Abbott Alinity family of chemistry and immunoassay systems recently launched with early adopters contributing imprecision and bias data, which was consolidated to assess the performance of Alinity assays across multiple sites using the Sigma metric. Multi-site Sigma metrics were determined for 3 ion-selective electrodes, 12 photometric assays, and 3 immunoassays across 11 independent laboratory sites in 9 countries. METHODS: Total allowable error (TEa) goals followed a previously defined hierarchy that used CLIA as the primary goal. Bias was calculated against the Abbott ARCHITECT system using Passing-Bablok regression analysis using individual site data or pooled aggregate data. Sigma metrics were calculated as (%TEa - |% bias|)/%CV. For individual-site analysis, the Sigma metrics for each assay were compared using the individual-site and the pooled biases. For multi-site analysis, the average CV and the pooled bias were used to generate a Pooled Sigma metric encompassing the global performance for a given assay. RESULTS: A total of 97 individual-site and 18 Pooled Sigma metrics were calculated for available assays. Individual Sigma metrics varied across sites, with 90% of assays performing 4 Sigma or higher, and 17 of 18 Pooled Sigma metrics indicated performance greater than 4 Sigma. Sigma metrics were significantly improved in 16 assays when using pooled bias rather than individual-site bias. CONCLUSIONS: This multi-center study applies a novel application of Sigma metrics to the first Alinity users and reveals analytical performance of greater than 4 Sigma for vast majority of assays. Laboratories with limited resources can leverage larger data sets for Pooled Sigma metric analysis, providing a tool to assess the consistency of analytical performance from multiple sites.

Hypoxia-Induced Changes in the Fibroblast Secretome, Exosome, and Whole-Cell Proteome Using Cultured, Cardiac-Derived Cells Isolated from Neonatal Mice.

Cardiac fibroblasts (CFs) represent a major subpopulation of cells in the developing and adult heart. Cardiomyocyte (CM) and CF intercellular communication occurs through paracrine interactions and modulate myocyte development and stress response. Detailed proteomic analysis of the CF secretome in normal and stressed conditions may offer insights into the role of CF in heart development and disease. Primary neonatal mouse CFs were isolated and cultured for 24 h in 21% (normoxic) or 2% (hypoxic) O2. Conditioned medium was separated to obtain exosomes (EXO) and EXO-depleted secretome fractions. Multidimensional protein identification technology was performed on secreted fractions. Whole cell lysate data were also generated to provide subcellular context to the secretome. Proteomic analysis identified 6163 unique proteins in total. Statistical (QSpec) analysis identified 494 proteins differentially expressed between fractions and oxygen conditions. Gene Ontology enrichment analysis revealed hypoxic conditions selectively increase expression of proteins with extracellular matrix and signaling annotations. Finally, we subjected CM pretreated with CF secreted factors to hypoxia/reoxygenation. Viability assays suggested altered viability due to CF-derived factors. CF secretome proteomics revealed differential expression based on mode of secretion and oxygen levels in vitro.

Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy.

Phospholamban (PLN) plays a central role in Ca2+ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca2+-ATPase 2A (SERCA2A) Ca2+ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function.

Self-renewing resident arterial macrophages arise from embryonic CX3CR1(+) precursors and circulating monocytes immediately after birth.

Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1(+) precursors and postnatally from bone marrow-derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche.

Large-scale characterization of the murine cardiac proteome.

Cardiomyopathies are diseases of the heart that result in impaired cardiac muscle function. This dysfunction can progress to an inability to supply blood to the body. Cardiovascular diseases play a large role in overall global morbidity. Investigating the protein changes in the heart during disease can uncover pathophysiological mechanisms and potential therapeutic targets. Establishing a global protein expression "footprint" can facilitate more targeted studies of diseases of the heart.In the technical review presented here, we present methods to elucidate the heart's proteome through subfractionation of the cellular compartments to reduce sample complexity and improve detection of lower abundant proteins during multidimensional protein identification technology analysis. Analysis of the cytosolic, microsomal, and mitochondrial subproteomes separately in order to characterize the murine cardiac proteome is advantageous by simplifying complex cardiac protein mixtures. In combination with bioinformatic analysis and genome correlation, large-scale protein changes can be identified at the cellular compartment level in this animal model.

The cardiovascular exosome: current perspectives and potential.

The exosome is a secreted microvesicle that has been shown to contain genetic material and proteins and is involved in multiple levels of cellular communication. The cardiovascular exosome proteome is a promising subproteome that warrants investigation since a detailed understanding of its role in the heart should improve our comprehension of intercellular communication in the heart, and may even assist in biomarker discovery. Indeed, uncovering the role of the exosome in cardiovascular physiology could be accomplished with the application of scientific approaches and insights gained from studies of exosomes in other fields, such as cancer biology and immunology, where much of the established knowledge of the exosome has been generated. In the present review, we discuss the relevant literature and examine areas of investigation that would bring the cardiovascular exosome to the forefront of intercellular communication in the heart.

Recent advances in cardiovascular proteomics.

Cardiovascular diseases (CVDs) are the major source of global morbidity and death and more people die annually from CVDs than from any other cause. These diseases can occur quickly, as seen in acute myocardial infarction (AMI), or progress slowly over years as with chronic heart failure. Advances in mass spectrometry detection and analysis, together with improved isolation and enrichment techniques allowing for the separation of organelles and membrane proteins, now allow for the indepth analysis of the cardiac proteome. Here we outline current insights that have been provided through cardiovascular proteomics, and discuss studies that have developed innovative technologies which permit the examination of the protein complement in specific organelles including exosomes and secreted proteins. We highlight these foundational studies and illustrate how they are providing the technologies and tools which are now being applied to further study cardiovascular disease; provide new diagnostic markers and potentially new methods of cardiac patient management with identification of novel drug targets. This article is part of a Special Issue entitled: From protein structures to clinical applications.