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Abstract High-performance TLC and ^sup 31^P-NMR were assessed as methods of observing the presence of numerous low polarity phospholipids: bis-phosphatidic acid (BPA), semi-lyso bis-phosphatidic acid (SLBPA), N-acyl phosphatidylethanolamine (NAPE), N-(1,1-dimethyl-3-oxobutyl)-phosphatidylethanolamine (diacetone adduct of PE, DOBPE), N-acetyl PE, phosphatidylmethanol (PM), phosphatidylethanol (PEt), phosphatidyl-n-propanol (PP), phosphatidyl-n-butanol (PB). Both techniques are non-discriminative and do not require the prior isolation of individual lipids. It appears that 2D TLC is superior to ^sup 31^P NMR in the analysis of low polarity phospholipids. All phosphatidylalcohols were well separated by 2D TLC. However, some compounds which can present difficulty in separation by 2D-TLC (e.g., SLBPA and NAPE; or DOBPE and N-acetyl PE) were easily distinguished using ^sup 31^P NMR so the methods are complimentary. A disadvantage of 2D TLC is that Rf values can vary with different brands and batches of TLC plates. The chemical shifts of ^sup 31^P NMR were less variable, and so a library of standards may not be necessary for peak identification. Another advantage of ^sup 31^P NMR is the ease of quantification of phospholipids. The applicability of the methods was tested on natural extracts of fish brain and cabbage stem.
Keywords ^sup 31^P-NMR * TLC * Phosphatidylmethanol * Phosphatidylethanol * N-acetone adduct * Phosphatidylpropanol * Phosphatidylbutanol * Bis-phosphatidic acid * Acyl phosphatidylglycerol * N-acyl phosphatidylethanolamine
Abbreviations
BPA Bis-phosphatidic acid
DOBPE N-(1,1-dimethyl-3-oxo-butyl)phosphatidylethanolamine (diacetone adduct of PE)
EDTA Ethylenediaminetetraacetic acid
CL Cardiolipin
CPM Ceramidephosphomethanol
GPB Glycerophosphobutanol
GPE Glycerophosphoethanolamine
GPEt Glycerophosphoethanol
GPG Glycerophosphoglycerol (diglycerophosphate)
GPM Glycerophosphomethanol
GPP Glycerophosphopropanol
LPC LysoPC
LPE Lyso PE
LPM Lyso PM
N-acetyl-GPE N-acetyl glycerophosphoethanolamine
NAGPE N-acyl glycerophosphoethanolamine
NAPE N-acyl phosphatidylethanolamine
PA Phosphatidic acid
PB Phosphatidylbutanol
PC Phosphatidylcholine
PE Phosphatidylethanolamine
PEt Phosphatidylethanol
PG Phosphatidylglycerol
PI Phosphatidylinositol
Pi Inorganic phosphate
PM Phosphatidylmethanol
PMG Phosphonomethylglycine
PP Phosphatidylpropanol
PS Phosphatidylserine
SLBPA Semi-lyso bis-phosphatidic acid (acyl phosphatidylglycerol)
SM Sphingomyelin
SPE Solid-phase extraction
Introduction
Beyond a widely known group of major phospholipids (phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidic acid and sphingomyelin), a number of other phospholipid groups exist, including low polarity phospholipids, phosphonolipids, etc. While these other groups are generally represented by minor components, their importance in lipid metabolism and signalling may be high [1].
In this work we present and compare selected analytical techniques that allow identification of low polarity phospholipids, a group of phospholipids noted for their...