This article is available online at http://www.jlr.org implicated in several diverse diseases, such as type 2 diabetes, Alzheimer disease, ( 2 ) and cancer ( 3 ). Lipid analysis has been an important area of research for several decades, and due to technological advances, the fi eld has experienced a renaissance in the last decade. In a modern laboratory, a comprehensive lipid characterization can be performed that generates quantitative data of several hundreds of molecular lipids from several different lipid classes. This kind of analysis, often called lipidomics, is based on HPLC and mass spectrometry (MS) instrumentation. The analysis is performed unattended in the 96-well format and is fully automated.An important component for successful analysis is the quality of the lipid extract. It is important that the lipid extract that is injected on the HPLC or infused into the mass spectrometer is pure; therefore, it is important that interfering substances and particles are removed. Inability in removing these substances might result in a high chemical background, which will have an effect on both the sensitivity and selectivity of the analysis. In contrast to fully automated and high-throughput analysis, lipid extraction is still often performed manually, involving exhaustive and time-consuming pipetting steps and hazardous solvents such as chloroform. Thus, a fully automated, chloroformfree method that can be used with standard 96-well robots would signifi cantly improve sample throughput, as well as reduce the negative impact on health and environment. The aim of this study was to develop that method.Abstract Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profi les of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroformbased reference method. Lipid recoveries were linear from 10-100 µl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 µl butanol:methanol (BUME) mixture (3:1) followed by twophase extraction into 300 µl heptane:ethyl acetate (3:1) using 300 µl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, highthroughput BUME method can replace chloroform-based methods, saving both human and environment...

In this study we present a simple and rapid method for tissue lipid extraction. Snap-frozen tissue (15–150 mg) is collected in 2 ml homogenization tubes. 500 μl BUME mixture (butanol:methanol [3:1]) is added and automated homogenization of up to 24 frozen samples at a time in less than 60 seconds is performed, followed by a 5-minute single-phase extraction. After the addition of 500 μl heptane:ethyl acetate (3:1) and 500 μl 1% acetic acid a 5-minute two-phase extraction is performed. Lipids are recovered from the upper phase by automated liquid handling using a standard 96-tip robot. A second two-phase extraction is performed using 500 μl heptane:ethyl acetate (3:1). Validation of the method showed that the extraction recoveries for the investigated lipids, which included sterols, glycerolipids, glycerophospholipids and sphingolipids were similar or better than for the Folch method. We also applied the method for lipid extraction of liver and heart and compared the lipid species profiles with profiles generated after Folch and MTBE extraction. We conclude that the BUME method is superior to the Folch method in terms of simplicity, through-put, automation, solvent consumption, economy, health and environment yet delivering lipid recoveries fully comparable to or better than the Folch method.

To cite this article: Nylander S, Femia EA, Scavone M, Berntsson P, Aszt ely A-K, Nelander K, L€ ofgren L, Nilsson RG, Cattaneo M. Ticagrelor inhibits human platelet aggregation via adenosine in addition to P2Y 12 antagonism. J Thromb Haemost 2013; 11: 1867-76.Summary. Background: Ticagrelor, a P2Y 12 antagonist, is an antiplatelet agent approved for the treatment of acute coronary syndromes; it also inhibits adenosine uptake by erythrocytes and other cells. Objective: To test whether ticagrelor inhibits platelet aggregation (PA) in whole blood (WB) by increasing the extracellular levels of adenosine, which inhibits PA via the A 2A receptor. Methods: Collagen-induced PA was measured in WB or platelet-rich plasma (PRP) from 50 healthy subjects and two patients with inherited P2Y 12 deficiency, in presence/absence of adenosine concentrations that by themselves marginally affected PA in WB, and ZM241385 (A 2A antagonist). The effects of ticagrelor, the active metabolite of prasugrel (PAM) (P2Y 12 antagonist), and dipyridamole (adenosine uptake inhibitor) on PA and on adenosine clearance in WB were compared. Results: For PA in WB, adenosine contributed to drug-induced inhibition of PA; the adenosine contribution was similar for dipyridamole and ticagrelor but was significantly greater for ticagrelor than for PAM (P < 0.01). For PA in PRP (no adenosine uptake by erythrocytes), adenosine contributed to inhibition of PA in the presence/absence of all tested drugs. ZM241385 reversed the inhibition by adenosine in WB and PRP. Similar results were obtained with WB and PRP from P2Y 12 -deficient patients. Adenosine (7.1 lmol L -1 ) added to WB, was detectable for 0.5 min in the presence of vehicle or PAM, for 3-6 min in the presence of ticagrelor, and for > 60 min in the presence of dipyridamole. Conclusion: This study provides the first evidence of an additional antiplatelet mechanism by ticagrelor, mediated by the induced increase of adenosine levels.

Increased visceral fat, as opposed to subcutaneous/gluteal, most strongly relates to key metabolic dysfunctions including insulin resistance, hepatic steatosis, and inflammation. Mesenteric fat hypertrophy in patients with Crohn's disease and in experimental rodent models of gut inflammation suggest that impaired gut barrier function with increased leakage of gut‐derived antigens may drive visceral lipid deposition. The aim of this study was to determine whether increased intestinal permeability is associated with visceral adiposity in healthy humans. Normal to overweight female subjects were recruited from a population‐based cohort. Intestinal permeability was assessed using the ratio of urinary excretion of orally ingested sucralose to mannitol (S/M). In study 1 (n = 67), we found a positive correlation between waist circumference and S/M excretion within a time frame of urine collection consistent with permeability of the lower gastrointestinal tract (6–9 hours post‐ingestion; P = 0.022). These results were followed up in study 2 (n = 55) in which we used computed tomography and dual energy X‐ray absorptiometry to measure visceral and subcutaneous fat areas of the abdomen, liver fat content, and total body fat of the same women. The S/M ratio from the 6–12 h urine sample correlated with visceral fat area (P = 0.0003) and liver fat content (P = 0.004), but not with subcutaneous or total body fat. This novel finding of an association between intestinal permeability and visceral adiposity and liver fat content in healthy humans suggests that impaired gut barrier function should be further explored as a possible mediator of excess visceral fat accumulation and metabolic dysfunction.

Altered lipid handling and hormone secretion in the gut were demonstrated during 1-week treatment with the DGAT1 inhibitor AZD7687. However, the apparent lack of therapeutic window owing to GI side effects of AZD7687, particularly diarrhoea, makes the utility of DGAT1 inhibition as a novel treatment for diabetes and obesity questionable.