8
c.c. Parrish
collection and extraction stage (Berge et aI., 1995). They advocate the use of
boiling water to deactivate lipolytic enzymes.
1.2.2. Lipid Extraction
Most laboratories use some variation of the Folch et a1. (1957) extraction procedure involving chloroform and methanol (Fig. 1.3). The sample is homogenized
in a 20-fold volume of chloroform:methanol (2: I) and then washed with water to
remove nonlipid contaminants. Excessive washing should be avoided, and use of
salt in the wash may influence the chromatographic properties of some lipid
classes (Nelson, 1991). The Bligh and Dyer (1959) method is a very popular
simplification of the method of Folch et a!. (1957), which is to be recommended
for large samples (>5 g). For very small freshwater samples «20 mg), a microprocedure developed by Gardner et a1. (1985) has been used successfully by other
workers as well (Butler, 1994; Wainman et a!., 1993; Arts and Evans, 1991). A
variation on the Bligh and Dyer method that is currently popular for freshwater
fish samples (Andrade et aI., 1995; Rahman et a!., 1995) is that of Kinsella et a!.
(1977). The modifications are mainly in terms of scale, phase separation, and
sample preservation. Kinsella et a!. (1977) published the first comprehensive
analyses of lipid and fatty acid content of a variety of freshwater fish.
In most extraction procedures, the proportions of sample, water, and solvents
are critical for maximal recovery of lipids and minimal contamination by nonlipids. In our version of the Folch et a1. (1957) procedure (Fig. 1.3), it is important
to extract the sample in a solvent-to-sample ratio of at least 3 ml per 150 mg dry
weight of actual sample. The volume includes the chloroform and the methanol,
and the weight does not include any filters or water associated with the filter or
sample. It is also important that in the water washing procedure, the ratio of
chloroform:methanol:water is 8:4:3. This amount of water includes that in the
sample and filter if present (in our case, 0.5 ml) and is equivalent to a solvent-towater ratio of 4: I. We typically put our samples on precombusted 4.25-cm Whatman GF/C glass microfiber filters for ease of handling. This also makes samples
easier to grind with a rod.
There is growing concern over the potential health hazard associated with the
use of chloroform, and so there are other solvent mixtures available (Ackman,
1993; Nelson, 1991; Shaikh, 1986). However, chloroform:methanol is still considered to be the most efficient extraction mixture for general use. Chen et a!.
(1981) have indicated that dicholormethane (methylene chloride) may be substituted for chloroform in the Folch et a!. procedure. It is less toxic and more
volatile, making it easier to use when solvent removal is required, but some lipids
(e.g., tribehenin r I ,2,3-tridocosanoylglycerol]) are less soluble in it.
1.2.3. Determination of Total Lipid
Total lipid can be determined gravimetrically, colorimetric ally, or by summing
individually determined lipid classes. In each case, the extract or a known portion
c.c. Parrish
collection and extraction stage (Berge et aI., 1995). They advocate the use of
boiling water to deactivate lipolytic enzymes.
1.2.2. Lipid Extraction
Most laboratories use some variation of the Folch et a1. (1957) extraction procedure involving chloroform and methanol (Fig. 1.3). The sample is homogenized
in a 20-fold volume of chloroform:methanol (2: I) and then washed with water to
remove nonlipid contaminants. Excessive washing should be avoided, and use of
salt in the wash may influence the chromatographic properties of some lipid
classes (Nelson, 1991). The Bligh and Dyer (1959) method is a very popular
simplification of the method of Folch et a!. (1957), which is to be recommended
for large samples (>5 g). For very small freshwater samples «20 mg), a microprocedure developed by Gardner et a1. (1985) has been used successfully by other
workers as well (Butler, 1994; Wainman et a!., 1993; Arts and Evans, 1991). A
variation on the Bligh and Dyer method that is currently popular for freshwater
fish samples (Andrade et aI., 1995; Rahman et a!., 1995) is that of Kinsella et a!.
(1977). The modifications are mainly in terms of scale, phase separation, and
sample preservation. Kinsella et a!. (1977) published the first comprehensive
analyses of lipid and fatty acid content of a variety of freshwater fish.
In most extraction procedures, the proportions of sample, water, and solvents
are critical for maximal recovery of lipids and minimal contamination by nonlipids. In our version of the Folch et a1. (1957) procedure (Fig. 1.3), it is important
to extract the sample in a solvent-to-sample ratio of at least 3 ml per 150 mg dry
weight of actual sample. The volume includes the chloroform and the methanol,
and the weight does not include any filters or water associated with the filter or
sample. It is also important that in the water washing procedure, the ratio of
chloroform:methanol:water is 8:4:3. This amount of water includes that in the
sample and filter if present (in our case, 0.5 ml) and is equivalent to a solvent-towater ratio of 4: I. We typically put our samples on precombusted 4.25-cm Whatman GF/C glass microfiber filters for ease of handling. This also makes samples
easier to grind with a rod.
There is growing concern over the potential health hazard associated with the
use of chloroform, and so there are other solvent mixtures available (Ackman,
1993; Nelson, 1991; Shaikh, 1986). However, chloroform:methanol is still considered to be the most efficient extraction mixture for general use. Chen et a!.
(1981) have indicated that dicholormethane (methylene chloride) may be substituted for chloroform in the Folch et a!. procedure. It is less toxic and more
volatile, making it easier to use when solvent removal is required, but some lipids
(e.g., tribehenin r I ,2,3-tridocosanoylglycerol]) are less soluble in it.
1.2.3. Determination of Total Lipid
Total lipid can be determined gravimetrically, colorimetric ally, or by summing
individually determined lipid classes. In each case, the extract or a known portion