Isolation and analysis of membrane lipids and lipid rafts in common carp (Cyprinus carpio L.) Graham Brogden a , Marcus Propsting a,b , Mikolaj Adamek a , Hassan Y. Naim b , Dieter Steinhagen a, ⁎ a Fish Disease Research Unit, Institute of Parasitology, University of Veterinary Medicine, Bünteweg 17, 30599 Hannover, Germany b Department of Physiological Chemistry, University of Veterinary Medicine, Bünteweg 17, 30599 Hannover, Germany abstract article info Article history: Received 1 August 2013 Received in revised form 3 December 2013 Accepted 3 December 2013 Available online 8 December 2013 Keywords: Carp Lipid rafts Lipid analysis DRM DSM Cell membranes act as an interface between the interior of the cell and the exterior environment and facilitate a range of essential functions including cell signalling, cell structure, nutrient uptake and protection. It is composed of a lipid bilayer with integrated proteins, and the inner leaflet of the lipid bilayer comprises of liquid ordered (L o ) and liquid disordered (L d ) domains. L o microdomains, also named as lipid rafts are enriched in cholesterol, sphingomyelin and certain types of proteins, which facilitate cell signalling and nutrient uptake. Lipid rafts have been extensively researched in mammals and the presence of functional lipid rafts was recently demon- strated in goldfish, but there is currently very little knowledge about their composition and function in fish. Therefore a protocol was established for the analysis of lipid rafts and membranous lipids in common carp (Cyprinus carpio L.) tissues. Twelve lipids were identified and analysed in the L d domain of the membrane with the most predominant lipids found in all tissues being; triglycerides, cholesterol, phosphoethanolamine and phosphatidylcholine. Four lipids were identified in lipid rafts in all tissues analysed, triglycerides (33–62%) always found in the highest concentration followed by cholesterol (24–32%), phosphatidylcholine and sphingomyelin. Isolation of lipid rafts was confirmed by identifying the presence of the lipid raft associated protein flotillin, present at higher concentrations in the detergent resistant fraction. The data provided here build a lipid library of important carp tissues as a baseline for further studies into virus entry, protein trafficking or environmental stress analysis. © 2013 Elsevier Inc. All rights reserved. 1. Introduction The cell membrane acts as an interface between the interior machinery of the cell and the exterior environment. Cell membranes are composed of a lipid bilayer with integrated proteins, which facilitate the movements of ions or serve as adhesion and signalling molecules. In the outer leaflet of the lipid bilayer, both a liquid ordered phase, L o and a liquid disordered phase L d are present (Rietveld and Simons, 1998). The L o and L d phases consist of proteins and lipids, of which a higher percentage of cholesterol and sphingolipids are found in the L o phase. The L o phase is currently defined as dynamic, nanoscale, sterol– sphingolipid-enriched, ordered assemblies of proteins and lipids and these microdomains are associated with a range of important functions including cell signalling and nutrient transport (Simons and Gerl, 2010). Experimentally, L o and L d membrane fractions can be isolated with a non-ionic detergent such as Triton X-100 under ice-cold conditions using a protocol described by Alfalah et al. (2005). Therefore, L o membrane fractions were also described as detergent resistant membranes (DRMs) or lipid rafts, and thus the L d fraction as detergent soluble membrane (DSM) fraction (Brown, 2006). Although these membrane fractions were first discovered over 15 years ago, there is still a lot of contention surrounding these membrane microdomains, including the size and longevity. The in vitro methodology used has for instance led to discussions questioning if the DRM fractions are indeed real and not artefacts produced from the methodology (Munro, 2003), whereas data produced from in vivo studies could not always be reproduced in vitro (Munro, 2003). Nevertheless, lipid rafts have been extensively studied in mammals and meanwhile the field has grown rapidly (Lingwood and Simons, 2010). The lipid raft model is now used to investigate a large range of processes including virus entry (Nguyen and Hildreth, 2000; Ono and Freed, 2001), protein trafficking (Brown and London, 1998), metabolic diseases (Maalouf et al., 2009) and cell signalling (Varma and Mayor, 1998). In fish, lipid rafts were extracted from the brush border mem- branes (BBMs) of enterocytes from Atlantic cod (Gylfason et al., 2010), rainbow trout hepatocyte membranes (Zehmer and Hazel, 2003, 2004, 2005), and recently functional lipid rafts were demonstrated in the cell membrane of goldfish leukocytes (Garcia-Garcia et al., 2012). Compared to mammals, however very little is known about lipid rafts in aquatic species. There is currently no published work on the lipid composition of different fish tissues and of the composition of lipid rafts isolated from these tissues in fish. Comparative Biochemistry and Physiology, Part B 169 (2014) 9–15 ⁎ Corresponding author. Tel.: +49 511 953 8560; fax: +49 511 953 8587. E-mail address: dieter.steinhagen@tiho-hannover.de (D. Steinhagen). 1096-4959/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpb.2013.12.001 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb