In matrix derivatization combined with LC-MS/MS results in ultra-sensitive quantification of plasma free metanephrines and catecholamines

Plasma-free metanephrines and catecholamines are essential markers in the biochemical diagnosis and follow-up of neuroendocrine tumors and inborn errors of metabolism. However, their low circulating concentrations (in the nanomolar range) and poor fragmentation characteristics hinder facile simultaneous quantification by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Here, we present a sensitive and simple matrix derivatization procedure using propionic anhydride that enables simultaneous quantification of unconjugated l-DOPA, catecholamines, and metanephrines in plasma by LC-MS/MS. Dilution of propionic anhydride 1:4 (v/v) in acetonitrile in combination with 50 μL of plasma resulted in the highest mass spectrometric response. In plasma, derivatization resulted in stable derivatives and increased sensitivity by a factor of 4-30 compared with a previous LC-MS/MS method for measuring plasma metanephrines in our laboratory. Furthermore, propionylation increased specificity, especially for 3-methoxytyramine, by preventing interference from antihypertensive medication (β-blockers). The method was validated according to international guidelines and correlated with a hydrophilic interaction LC-MS/MS method for measuring plasma metanephrines (R2 > 0.99) and high-performance liquid chromatography with an electrochemical detection method for measuring plasma catecholamines (R2 > 0.85). Reference intervals for l-DOPA, catecholamines, and metanephrines in n = 115 healthy individuals were established. Our work shows that analytes in the subnanomolar range in plasma can be derivatized in situ without any preceding sample extraction. The developed method shows improved sensitivity and selectivity over existing methods and enables simultaneous quantification of several classes of amines

Genetic and Microbial Associations to Plasma and Fecal Bile Acids in Obesity Relate to Plasma Lipids and Liver Fat Content

Bile acids (BAs) have been implicated in obesity-related conditions such as NAFLD and hyperlipidemia. Different human BAs exert variable biological activities. Chen et al. define genetic and microbial associations to plasma and fecal BA concentrations and composition in persons with obesity and establish their relationships with liver fat and lipid phenotypes

Repurposing Metformin in Nondiabetic People With HIV:Influence on Weight and Gut Microbiota

Background. People with HIV (PWH) taking antiretroviral therapy (ART) may experience weight gain, dyslipidemia, increased risk of non-AIDS comorbidities, and long-term alteration of the gut microbiota. Both low CD4/CD8 ratio and chronic inflammation have been associated with changes in the gut microbiota of PWH. The antidiabetic drug metformin has been shown to improve gut microbiota composition while decreasing weight and inflammation in diabetes and polycystic ovary syndrome. Nevertheless, it remains unknown whether metformin may benefit PWH receiving ART, especially those with a low CD4/CD8 ratio. Methods. In the Lilac pilot trial, we recruited 23 nondiabetic PWI I receiving ART for more than 2 years with a low CD4/CD8 ratio ( Results. Metformin decreased weight in PWH, and weight loss was inversely correlated with plasma levels of the satiety factor GDF-15. Furthermore, metformin changed the gut microbiota composition by increasing the abundance of anti-inflammatory bacteria such as butyrate-producing species and the protective Akkermansia muciniphila. Conclusions. Our study provides the first evidence that a 12-week metformin treatment decreased weight and favored anti-inflammatory bacteria abundance in the microbiota of nondiabetic ART-treated PWH. Larger randomized placebo-controlled clinical trials with longer metformin treatment will be needed to further investigate the role of metformin in reducing inflammation and the risk of non-AIDS comorbidities in ART-treated PWH

The Microbiota Mediates Pathogen Clearance from the Gut Lumen after Non-Typhoidal Salmonella Diarrhea

Many enteropathogenic bacteria target the mammalian gut. The mechanisms protecting the host from infection are poorly understood. We have studied the protective functions of secretory antibodies (sIgA) and the microbiota, using a mouse model for S. typhimurium diarrhea. This pathogen is a common cause of diarrhea in humans world-wide. S. typhimurium (S. tmatt, sseD) causes a self-limiting gut infection in streptomycin-treated mice. After 40 days, all animals had overcome the disease, developed a sIgA response, and most had cleared the pathogen from the gut lumen. sIgA limited pathogen access to the mucosal surface and protected from gut inflammation in challenge infections. This protection was O-antigen specific, as demonstrated with pathogens lacking the S. typhimurium O-antigen (wbaP, S. enteritidis) and sIgA-deficient mice (TCRβ−/−δ−/−, JH−/−, IgA−/−, pIgR−/−). Surprisingly, sIgA-deficiency did not affect the kinetics of pathogen clearance from the gut lumen. Instead, this was mediated by the microbiota. This was confirmed using ‘L-mice’ which harbor a low complexity gut flora, lack colonization resistance and develop a normal sIgA response, but fail to clear S. tmatt from the gut lumen. In these mice, pathogen clearance was achieved by transferring a normal complex microbiota. Thus, besides colonization resistance ( = pathogen blockage by an intact microbiota), the microbiota mediates a second, novel protective function, i.e. pathogen clearance. Here, the normal microbiota re-grows from a state of depletion and disturbed composition and gradually clears even very high pathogen loads from the gut lumen, a site inaccessible to most “classical” immune effector mechanisms. In conclusion, sIgA and microbiota serve complementary protective functions. The microbiota confers colonization resistance and mediates pathogen clearance in primary infections, while sIgA protects from disease if the host re-encounters the same pathogen. This has implications for curing S. typhimurium diarrhea and for preventing transmission

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase 1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age  6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score  652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc = 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N = 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in Asia and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701

Methods and kits for the derivatization of a biogenic amine

The invention relates to analytical chemistry, in particular to means and methods for the quantitative determination of derivatized biogenic amines, precursors or metabolites thereof. Provided is a method for the in situ derivatization of at least one biogenic amine, precursor or metabolite thereof in an isolated aqueous sample, the derivatization comprising the steps of: (i) contacting said sample with a propionic anhydride / acetonitril solution in the presence of a phosphate buffer having a pH in the range of 7.0 to 9.0 and allowing the conversion of amine and/or hydroxyl moieties of said biogenic amine or metabolite thereof to form a propionyl derivative of said biogenic amine; followed by (ii) adding to the reaction mixture obtained in step (i) a carbodiimide compound and an electrophilic amine-containing compound, and allowing the carbodiimide-mediated derivatization of carboxylic acid moieties of a biogenic amine, precursor or metabolite thereof