63
ences the lipidomic data analysis. Lipids in tissues and cells are relatively protected by natural antioxidant systems and compartmentalization. But after sample
homogenization, the cellular content is mixed and unavoidably diluted, which
renders lipids more prone to chemical or enzymatic modification. Enzymatic
modifications can be minimized by extracting lipids at temperatures close to 0 °C
or by adding a small percentage of organic solvent to the homogenization buffer.
After extraction, lipids become more prone to chemical oxidation, and thus the
lipid extracts should preferably be stored in glass vials and solubilized in sufficient organic solvent at −80 °C, and air/oxygen should be eliminated by flushing
with inert gasses (Vaz et al. 2015).
4.4.2 Analytical Platforms for Lipidomics
The lipid metabolite analysis of any sample can be achieved by several methods
such as those for separation (TLC, LC, GC) and those methods used for detection (MS, ESI, NMR), stand-alone or in combination such as LC-MS, GC-MS,
ESI-MS, and TLC with GC-MS/LC-MS/NMR. Traditionally, TLC (both one
and two dimensional) has been utilized for decades to separate lipid classes in
seaweeds using silica as the stationary phase and different elution solvents
depending on the polarity of the lipid classes to be isolated. Nonpolar lipids
(glycolipids, free fatty acyls, and sterols) have been separated using hexane and
diethyl ether and polar lipids (glycerophospholipids, saccharolipids, and sphingolipids) using chloroform, methanol, acetone, benzene, water, and triethylamine in different combinations in seaweeds (Khotimchenko and Vaśkovsky
2004; Kulikova and Khotimchenko 2000; Sanina et al. 2008). The different lipid
classes have been detected by observing the intensity of the spots after spraying
with a solution of primuline in acetone and visualizing under a UV lamp, or by
placing the plate in iodine vapor, and identified based on the comparison with
migration of pure lipid standards applied to the same TLC plate and the relative
quantification achieved by densitometry. The analysis of the molecular species
has been achieved by scraping the spots of each lipid class, with organic solvents
and then analyzing by GC-MS (Dembitsky and Rozentsvet 1990; Khotimchenko
and Vaśkovsky 2004; Sanina et al. 2004, 2008; Vaśkovsky et al. 1996).
However, TLC is not a suitable method for comprehensive lipidomic studies, as
it is a time-consuming method limited to detection of lipid classes, requires a
large sample size, and has low resolution and sensitivity.
GC has been used for analyzing subsets of lipid compounds such as fatty acids,
oxylipins, and sterols in a large number of seaweeds and has been typically coupled
with MS (GC-MS) or flame-ionization detection (GC-FID) (Barbosa et al. 2015;
Bouarab et al. 2004; Galloway et al. 2012; Gaquerel et al. 2007; Kamenarska et al.
2004; Kumari et al. 2010, 2011, 2013a, b; Küpper et al. 2006; Lion et al. 2006;
Wiesemeier et al. 2008). One of its prerequisites is the capacity of lipid compound to
4 Seaweed Lipidomics in the Era of ‘Omics’ Biology: A Contemporary Perspective
ences the lipidomic data analysis. Lipids in tissues and cells are relatively protected by natural antioxidant systems and compartmentalization. But after sample
homogenization, the cellular content is mixed and unavoidably diluted, which
renders lipids more prone to chemical or enzymatic modification. Enzymatic
modifications can be minimized by extracting lipids at temperatures close to 0 °C
or by adding a small percentage of organic solvent to the homogenization buffer.
After extraction, lipids become more prone to chemical oxidation, and thus the
lipid extracts should preferably be stored in glass vials and solubilized in sufficient organic solvent at −80 °C, and air/oxygen should be eliminated by flushing
with inert gasses (Vaz et al. 2015).
4.4.2 Analytical Platforms for Lipidomics
The lipid metabolite analysis of any sample can be achieved by several methods
such as those for separation (TLC, LC, GC) and those methods used for detection (MS, ESI, NMR), stand-alone or in combination such as LC-MS, GC-MS,
ESI-MS, and TLC with GC-MS/LC-MS/NMR. Traditionally, TLC (both one
and two dimensional) has been utilized for decades to separate lipid classes in
seaweeds using silica as the stationary phase and different elution solvents
depending on the polarity of the lipid classes to be isolated. Nonpolar lipids
(glycolipids, free fatty acyls, and sterols) have been separated using hexane and
diethyl ether and polar lipids (glycerophospholipids, saccharolipids, and sphingolipids) using chloroform, methanol, acetone, benzene, water, and triethylamine in different combinations in seaweeds (Khotimchenko and Vaśkovsky
2004; Kulikova and Khotimchenko 2000; Sanina et al. 2008). The different lipid
classes have been detected by observing the intensity of the spots after spraying
with a solution of primuline in acetone and visualizing under a UV lamp, or by
placing the plate in iodine vapor, and identified based on the comparison with
migration of pure lipid standards applied to the same TLC plate and the relative
quantification achieved by densitometry. The analysis of the molecular species
has been achieved by scraping the spots of each lipid class, with organic solvents
and then analyzing by GC-MS (Dembitsky and Rozentsvet 1990; Khotimchenko
and Vaśkovsky 2004; Sanina et al. 2004, 2008; Vaśkovsky et al. 1996).
However, TLC is not a suitable method for comprehensive lipidomic studies, as
it is a time-consuming method limited to detection of lipid classes, requires a
large sample size, and has low resolution and sensitivity.
GC has been used for analyzing subsets of lipid compounds such as fatty acids,
oxylipins, and sterols in a large number of seaweeds and has been typically coupled
with MS (GC-MS) or flame-ionization detection (GC-FID) (Barbosa et al. 2015;
Bouarab et al. 2004; Galloway et al. 2012; Gaquerel et al. 2007; Kamenarska et al.
2004; Kumari et al. 2010, 2011, 2013a, b; Küpper et al. 2006; Lion et al. 2006;
Wiesemeier et al. 2008). One of its prerequisites is the capacity of lipid compound to
4 Seaweed Lipidomics in the Era of ‘Omics’ Biology: A Contemporary Perspective