Theophylline in Treatment of COVID-19 Induced Sinus Bradycardia.

This is a retrospective case series of two patients with laboratory-confirmed coronavirus 2 (SARS-CoV-2) infection, presented to the University of Arkansas for Medical Sciences in January 2021. Medical records of these patients were reviewed using the EPIC electronic health record system. Clinical, laboratory, and treatment data were reviewed against periods of bradycardia in each patient. Both of the patients presented with dizziness and presyncope related to sinus bradycardia in which they received treatment with 1 mg of IV atropine and theophylline 200 mg orally. We share these two cases of theophylline treatment in COVID-19 induced sinus bradycardia. The first patient was a 39-year-old female, with a past medical history of polycystic ovarian syndrome, who presented to the emergency department with lightheadedness and dizziness. Two weeks prior to her presentation, she was tested positive for COVID-19 infection that was treated with azithromycin, dexamethasone and aspirin. Upon presentation, her ECG showed sinus bradycardia at a rate of 48 bpm. The second patient, a 21-year-old female with no significant past medical history, presented with presyncope. Three weeks prior to her presentation, she tested positive for COVID-19 infection that was treated symptomatically at her home. Upon presentation, her ECG showed junctional rhythm at a heart rate of 51 bpm.

Isotope tracing in adult zebrafish reveals alanine cycling between melanoma and liver.

The cell-intrinsic nature of tumor metabolism has become increasingly well characterized. The impact that tumors have on systemic metabolism, however, has received less attention. Here, we used adult zebrafish harboring BRAFV600E-driven melanoma to study the effect of cancer on distant tissues. By applying metabolomics and isotope tracing, we found that melanoma consume ~15 times more glucose than other tissues measured. Despite this burden, circulating glucose levels were maintained in disease animals by a tumor-liver alanine cycle. Excretion of glucose-derived alanine from tumors provided a source of carbon for hepatic gluconeogenesis and allowed tumors to remove excess nitrogen from branched-chain amino acid catabolism, which we found to be activated in zebrafish and human melanoma. Pharmacological inhibition of the tumor-liver alanine cycle in zebrafish reduced tumor burden. Our findings underscore the significance of metabolic crosstalk between tumors and distant tissues and establish the adult zebrafish as an attractive model to study such processes.

Targeting unique biological signals on the fly to improve MS/MS coverage and identification efficiency in metabolomics.

When using liquid chromatography/mass spectrometry (LC/MS) to perform untargeted metabolomics, it is common to detect thousands of features from a biological extract. Although it is impractical to collect non-chimeric MS/MS data for each in a single chromatographic run, this is generally unnecessary because most features do not correspond to unique metabolites of biological relevance. Here we show that relatively simple data-processing strategies that can be applied on the fly during acquisition of data with an Orbitrap ID-X, such as blank subtraction and well-established adduct or isotope calculations, decrease the number of features to target for MS/MS analysis by up to an order of magnitude for various types of biological matrices. We demonstrate that annotating these non-biological contaminants and redundancies in real time during data acquisition enables comprehensive MS/MS data to be acquired on each remaining feature at a single collision energy. To ensure that an appropriate collision energy is applied, we introduce a method using a series of hidden ion-trap scans in an Orbitrap ID-X to find an optimal value for each feature that can then be applied in a subsequent high-resolution Orbitrap scan. Data from 100 metabolite standards indicate that this real-time optimization of collision energies leads to more informative MS/MS patterns compared to using a single fixed collision energy alone. As a benchmark to evaluate the overall workflow, we manually annotated unique biological features by independently subjecting E. coli samples to a credentialing analysis. While credentialing led to a more rigorous reduction in feature number, on-the-fly annotation with blank subtraction on an Orbitrap ID-X did not inappropriately discard unique biological metabolites. Taken together, our results reveal that optimal fragmentation data can be obtained in a single LC/MS/MS run for >90% of the unique biological metabolites in a sample when features are annotated during acquisition and collision energies are selected by using parallel mass spectrometry detection.

Supraventricular tachycardia of the sick, a unique entity.

OBJECTIVE: This study explored a unique form of atrioventricular nodal reentrant tachycardia (AVNRT) in which certain acutely ill patients have a first episode of supraventricular tachycardia (SVT) with a short RP interval. METHODS: A retrospective chart review was conducted of patients at a single institution who developed SVT with short RP and yielded 19 patients. RESULTS: None of the 19 patients had a prior history of AVNRT or any other arrhythmia. The mean age was 58 years, the majority of patients were male (13/19), and there was a presence of hypertension (10/19), diabetes mellitus (5/19), hyperlipidemia (7/19), congestive heart failure (2/19), coronary artery disease (3/19), obstructive sleep apnea (2/19), and active cancer (8/19). The reasons for admission were planned surgery (8/19), sepsis (8/19), drug abuse (2/19), and neurological disorder (2/19). The AVNRT either terminated spontaneously or following the administration of adenosine. The patients were treated with amiodarone (12/19), metoprolol (6/19), or diltiazem (1/19). Follow-up (mean: 370 days) details revealed that patients were on amiodarone (3/19), metoprolol (6/19), were not taking any cardiac medication (5/19), or had passed away (5/19). Only 1 patient had a recurrence of AVNRT, and none of the patients required ablation therapy. CONCLUSION: 'AVNRT of the sick' has not been previously described in the medical literature, to our knowledge. It can be successfully treated with medications and the chance of recurrence after resolution of the acute illness is small.

International Ring Trial of a High Resolution Targeted Metabolomics and Lipidomics Platform for Serum and Plasma Analysis.

A challenge facing metabolomics in the analysis of large human cohorts is the cross-laboratory comparability of quantitative metabolomics measurements. In this study, 14 laboratories analyzed various blood specimens using a common experimental protocol provided with the Biocrates AbsoluteIDQ p400HR kit, to quantify up to 408 metabolites. The specimens included human plasma and serum from male and female donors, mouse and rat plasma, as well as NIST SRM 1950 reference plasma. The metabolite classes covered range from polar (e.g., amino acids and biogenic amines) to nonpolar (e.g., diacyl- and triacyl-glycerols), and they span 11 common metabolite classes. The manuscript describes a strict system suitability testing (SST) criteria used to evaluate each laboratory's readiness to perform the assay, and provides the SST Skyline documents for public dissemination. The study found approximately 250 metabolites were routinely quantified in the sample types tested, using Orbitrap instruments. Interlaboratory variance for the NIST SRM-1950 has a median of 10% for amino acids, 24% for biogenic amines, 38% for acylcarnitines, 25% for glycerolipids, 23% for glycerophospholipids, 16% for cholesteryl esters, 15% for sphingolipids, and 9% for hexoses. Comparing to consensus values for NIST SRM-1950, nearly 80% of comparable analytes demonstrated bias of <50% from the reference value. The findings of this study result in recommendations of best practices for system suitability, quality control, and calibration. We demonstrate that with appropriate controls, high-resolution metabolomics can provide accurate results with good precision across laboratories, and the p400HR therefore is a reliable approach for generating consistent and comparable metabolomics data.

Acute Influenza Infection Presenting with Cardiac Tamponade: A Case Report and Review of Literature.

INTRODUCTION: Cardiac complications associated with influenza infection can occur either via a direct effect of the virus on the heart or through exacerbation of preexisting cardiovascular disease. We present a case of a 57-year-old man with acute influenza infection complicated by pericardial effusion and cardiac tamponade. CASE PRESENTATION: A 57-year-old white man presented to the Emergency Department with sudden onset of severe, nonexertional, retrosternal, pressure-like chest pain for a few hours and with fever and muscle aches for 2 days. The patient was initially admitted because of suspected acute coronary syndrome. The next morning, he complained of acute-onset shortness of breath and had hypotension and tachycardia. On examination, his peripheral extremities were cold and heart sounds were distant. Pulsus paradoxus was 20 mmHg. The electrocardiogram showed low-voltage QRS complex with electrical alternans. An urgently performed bedside echocardiogram showed moderate pericardial effusion with a small right ventricular cavity with diastolic collapse. Emergent pericardiocentesis was performed, with removal of 250 mL of fluid from the pericardial space. The patient's hemodynamic status immediately improved. Analyses of pericardial fluid demonstrated no bacteria, acid-fast bacilli, or malignant cells. The result of a rapid influenza diagnostic test with polymerase chain reaction was positive for influenza A virus, with other viral panels yielding normal results. The patient was treated with oseltamivir for 5 days. DISCUSSION: Pericardial involvement is a rare and perhaps underreported complication of influenza infection. Early recognition of cardiac symptoms and appropriate diagnostic workup in a patient presenting with influenza-like symptoms is important to avoid life-threatening complications.

A Protocol to Compare Methods for Untargeted Metabolomics.

There are thousands of published methods for profiling metabolites with liquid chromatography/mass spectrometry (LC/MS). While many have been evaluated and optimized for a small number of select metabolites, very few have been assessed on the basis of global metabolite coverage. Thus, when performing untargeted metabolomics, researchers often question which combination of extraction techniques, chromatographic separations, and mass spectrometers is best for global profiling. Method comparisons are complicated because thousands of LC/MS signals (so-called features) in a typical untargeted metabolomic experiment cannot be readily identified with current resources. It is therefore challenging to distinguish methods that increase signal number due to improved metabolite coverage from methods that increase signal number due to contamination and artifacts. Here, we present the credentialing protocol to remove the latter from untargeted metabolomic datasets without having to identify metabolite structures. This protocol can be used to compare or optimize methods pertaining to any step of the untargeted metabolomic workflow (e.g., extraction, chromatography, mass spectrometer, informatic software, etc.).

Trace Phosphate Improves ZIC-pHILIC Peak Shape, Sensitivity, and Coverage for Untargeted Metabolomics.

Existing hydrophilic interaction liquid chromatography (HILIC) methods, considered individually, each exhibit poor chromatographic performance for a substantial fraction of polar metabolites. In addition to limiting metabolome coverage, such deficiencies also complicate automated data processing. Here we show that some of these analytical challenges can be addressed for the ZIC-pHILIC, a zwitterionic stationary phase commonly used in metabolomics, with the addition of trace levels of phosphate. Specifically, micromolar phosphate extended metabolome coverage by hundreds of credentialed features, improved peak shapes, and reduced peak-detection errors during informatic processing. Although the addition of high levels of phosphate (millimolar) as a HILIC mobile phase buffer has been explored previously, such concentrations interfere with mass spectrometric (MS) detection. We show that using phosphate as a trace additive at micromolar concentrations improves analysis by electrospray MS, increasing signal for a diverse set of polar standards. Given the small amount of phosphate needed, comparable chromatographic improvements were also achieved by direct addition of phosphate to the sample during reconstitution. Our results suggest that defects in ZIC-pHILIC performance are predominantly driven by electrostatic interactions, which can be modulated by phosphate. These findings constitute both a methodological improvement for untargeted metabolomics and an advance in our understanding of the mechanisms limiting HILIC coverage.

Sorting cells alters their redox state and cellular metabolome.

A growing appreciation of the metabolic artifacts of cell culture has generated heightened enthusiasm for performing metabolomics on populations of cells purified from tissues and biofluids. Fluorescence activated cell sorting, or FACS, is a widely used experimental approach to purify specific cell types from complex heterogeneous samples. Here we show that FACS introduces oxidative stress and alters the metabolic state of cells. Compared to unsorted controls, astrocytes subjected to FACS prior to metabolomic analysis showed altered ratios of GSSG to GSH, NADPH to NADP+, and NAD+ to NADH. Additionally, a 50% increase in reactive oxygen species was observed in astrocytes subjected to FACS relative to unsorted controls. At a more comprehensive scale, nearly half of the metabolomic features that we profiled by liquid chromatography/mass spectrometry were changed by at least 1.5-fold in intensity due to cell sorting. Some specific metabolites identified to have significantly altered levels as a result of cell sorting included glycogen, nucleosides, amino acids, central carbon metabolites, and acylcarnitines. Although the addition of fetal bovine serum to the cell-sorting buffer decreased oxidative stress and attenuated changes in metabolite concentrations, fetal bovine serum did not preserve the metabolic state of the cells during FACS. We conclude that, irrespective of buffer components and data-normalization strategies we examined, metabolomic results from sorted cells do not accurately reflect physiological conditions prior to sorting.

Two complementary reversed-phase separations for comprehensive coverage of the semipolar and nonpolar metabolome.

Although it is common in untargeted metabolomics to apply reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) methods that have been systematically optimized for lipids and central carbon metabolites, here we show that these established protocols provide poor coverage of semipolar metabolites because of inadequate retention. Our objective was to develop an RPLC approach that improved detection of these metabolites without sacrificing lipid coverage. We initially evaluated columns recently released by Waters under the CORTECS line by analyzing 47 small-molecule standards that evenly span the nonpolar and semipolar ranges. An RPLC method commonly used in untargeted metabolomics was considered a benchmarking reference. We found that highly nonpolar and semipolar metabolites cannot be reliably profiled with any single method because of retention and solubility limitations of the injection solvent. Instead, we optimized a multiplexed approach using the CORTECS T3 column to analyze semipolar compounds and the CORTECS C8 column to analyze lipids. Strikingly, we determined that combining these methods allowed detection of 41 of the total 47 standards, whereas our reference RPLC method detected only 10 of the 47 standards. We then applied credentialing to compare method performance at the comprehensive scale. The tandem method showed more than a fivefold increase in credentialing coverage relative to our RPLC benchmark. Our results demonstrate that comprehensive coverage of metabolites amenable to reversed-phase separation necessitates two reconstitution solvents and chromatographic methods. Thus, we suggest complementing HILIC methods with a dual T3 and C8 RPLC approach to increase coverage of semipolar metabolites and lipids for untargeted metabolomics. Graphical abstract Analysis of semipolar and nonpolar metabolites necessitates two reversed-phase chromatography (RPLC) methods, which extend metabolome coverage more than fivefold for untargeted profiling. HILIC hydrophilic interaction liquid chromatography.