Analysis of Lipids in Lichens
In all living organisms lipids play several roles, and according to their structures they can be divided into two main groups: the neutral lipids (acylglycerols, sterols, free fatty acids, wax and steryl esters) and polar lipids (phospholipids, glycolipid
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PROTOCOL
Analysis of lipids in lichens IRINA
A.
BYCHEK-GUSCHINA
Introduction In all living organisms lipids play several roles, and according to their structures they can be divided into two main groups: the neutral lipids (acylglycerols, sterols, free fatty acids, wax and steryl esters) and polar lipids (phospholipids, glycolipids and betaine lipids). Triacylglycerols act as a compact, easily metabolised and non-hydrated energy store. Waxes are commonly extracellular components such as the surface covering, which function both to reduce water loss and to protect plants from noxious environmental conditions (Harwood 1998). Polar lipids and sterols are important structural components of all cell membranes. Also, there are many examples of what could be termed biologically active lipids (e.g., inositol lipids, sphingolipids, oxidation products). In recent years, scientists have started to realize that lipid metabolism is a key factor in the adaptation mechanisms of many organisms to environmental and anthropogenic stress. In lichens, the importance of their lipids in the response and adaptation to environmental factors such as temperature, elevation, light, high levels of radiation, and sulphur have been studied (Dertien et aL 1977, Piervittori et al. 1995, Bychek and Bychek 1996, Shapiro et al. 1998, Bychek-Guschina et aL 1999). The present chapter will describe methods of lipid analysis that are available for many laboratories and have been used by the author for extraction, separation and quantification of basic lipids.
Irina A. Bychek-Guschina, Institute of Ecology of the Volga River Basin RAS, Togliatti, 445003, Russia (phone +7-8482- 489389; fax +7-8482-489504; e-mail [email protected])
I. C. Kranner et al. (eds .), Protocols in Lichenology © Springer-Verlag Berlin Heidelberg 2002
20 Analysis of Lipids in Lichens
I Subprotocol 1
Lipid Extraction
The aim of lipid extraction procedures is to extract lipids quantitatively and without contamination by non-lipid components.
Materials -
Heating block
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Methylation tubes (16 x 125 mm, with screw caps and Teflon or silicone liners)
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Pestle and mortar
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Centrifuge
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Small volume (e.g. 1 ml) tubes with screw caps and Teflon or silicone liners
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Isopropanol (propan-2-ol)
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Methanol
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Chloroform
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Garbus solution (2 M KCl in 0.5 M potassium phosphate buffer, pH 7.4) (Garbus et al. 1963)
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Supply of nitrogen gas
Procedure 1. Cut lichen thalli (about 0.5 g fresh weight) into small pieces.
2. Put the material in a methylation tube, add 3 ml of isopropanol and heat the tissue at 70°C for 30 min. Be sure to seal the tube to prevent evaporation. 3. After cooling, homogenise the tissue using a pestle and mortar in isopropanol and filter the homogenate into a 20 ml centrifuge tube. Perform all lipid extraction procedures in glass using pure solvents, and extract at, or below, room temperature.
Equipment
Reagents
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BYCHEK-GUSCHINA
4. Re-extract the tissue homogenate in 1.5 ml of methanol and 3 ml of chloroform, and then fi
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