Isolation of lipids from biological samples

Figures & data

Figure 1. Relative polarities of biological lipid species, (A) hexadecanol/cetyl alcohol, (B) trioleoyl glycerol, (C) cholesterol, (D) dioleoyl phosphatidylcholine, (E) phosphatidylinositol-3,4,5-tris-phosphate, (F) a generic lipopolysaccharide. The blue and red dashed lines show an approximate delineation between the head group region, the connecting region, and the hydrophobic fraction. The black dotted lines show an approximate delineation between lipophilic and intermediate (left) and intermediate and hydrophilic (right) lipid species.

Scheme 1. Two types of chemical modification that may be observed in handling lipids (Garrett et al., 2011). Upper, acid-mediated electrophilic substitution of the phosphate di-ester that produces non-endogenous lipids such as phosphatidylmethanol. Lower, the degradation of chloroform on storage can produce phosgene that in turn may lead to carbamoylation of PE.

Table I. Planning steps and considerations for selecting a lipid extraction protocol.

Planning steps and example questions (Q)	Options/Considerations
1. Size/form/ content of lipid-containing sample(s)
Q1. Is mechanical disruption required before  isolation? (e.g. nuts, seeds)	If yes, options are to use pestle and mortar, kitchen blender, shear mixer, ball mill, cold press, glass beads etc. In addition, it may help to freeze the sample before processing to make it more brittle, and/or to add some extraction solvent to facilitate lubrication.
Q2. Does the lipid-containing material need to be  stored before it is delipified/ defatted?	If yes, freeze, or freeze-dry samples after mechanical disruption. Note: this may also afford protection from endogenous lipase activity (see Q4).
Q3. Does the sample need concentrating before  processing?	If yes, consider e.g. freeze-drying, centrifugation, removal of fibrous material by filtration.
Q4. Does the sample contain endogenous lipases,  phospholipases or phosphatases?	If yes, consider (i) small-molecule inhibitors, (ii) optimising conditions to minimize their effects (e.g. low temperature (<4 °C)), or (iii) use of broad-spectrum denaturation, e.g. early application of an organic solvent such as iso-propanol.
	If in doubt: run test isolations and check for example for increases in lyso-lipids and/or decreases in species sensitive to phosphatase activity (e.g. PI, PC)
2. Choice of extraction process
Q5. Is the isolation aimed at a single type of  lipid head group or isoform?	If yes, choose^a a solvent system that is compatible with the lipid(s) of interest, and where possible, not with others
	If not, consider (i) broad-spectrum solvent systems (see §4), or (ii) sequential extractions using several solvent systems (see §5)
Q6. Is the isolation aimed at lipidic material from  one locus of the sample?	If yes, preparation steps may be required. For example, membranes may need to be separated from lipid droplets (see §4) or cellular compartments may need to be separated from one another (see §5).
3. Amount of material required
Q7. Is the lipid fraction being isolated for  analytical purposes only?	Analysis by MS (HR and/or MS/MS) or TLC requires only small quantities of lipid (typically <1 °mg). Complex analysis, including e.g. GC (3–5 °mg starting material) or NMR (>5 °mg, non-destructive) requires more material, e.g. >20 °mg.In either case, excess material can be stored at −20 °C or below.
Q8. Is the lipid fraction required for other study/use?	Typically, larger sample sizes are required for (i) the study of the physical behaviour of lipids^b (often >10 mg per sample), (ii) use of lipids as a raw material for synthetic chemistry (e.g. long-chain fatty acids) or (iii) structural studies. It is important here to consider the quality requirement of the intended use (see Q4).

^aSee relevant section, §3 Lipophilic species, §4 Intermediate species, etc. for examples

^bIncluding but not limited to phase behaviour studies (Cullis & de Kruijff, 1979, Seddon, 1990).

Garrett TA, Raetz CRH, Son JD, Richardson TD, Bartling C, Guan Z. 2011. Non-enzymatically derived minor lipids found in Escherichia coli lipid extracts. Biochim Biophys Acta 1811:827–837

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