Content area
High-performance TLC and (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-oxo-butyl)-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 (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 (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 (31)P NMR were less variable, and so a library of standards may not be necessary for peak identification. Another advantage of (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.
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 high mobility on silica gel TLC in solvent systems generally used in analysis of major phospholipids. Low polarity phospholipids are naturally existing or artefact lipids produced mostly by modification of polar moieties of phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylglycerol (PG) (Figs. 1-3).
Low polarity phospholipids include phosphatidylalcohols, mostly artefact lipids produced predominantly from PC when samples containing active phospholipase D (e.g., plant samples) are extracted with appropriate alcohols [2]. This process is usually accompanied by formation of phosphatidic acid (PA) due to the presence of water in extraction media (Fig. 1). Of all phosphatidylalcohols the most important seems to be phosphatidyledianol, currently considered as a biochemical marker of alcoholism [3], which was reported to form from phospholipids in mammalian cell membranes affected by ethanol [4]. Other phosphatidylalcohols (e.g., PM [5], PB [6], PP [7]) were found to be useful markers in phospholipase D activity assays due to the ease of their chromatographic separation from the bulk of phospholipids.
PE is able to form adducts with aldehydes and ketones. Acetone (more correctly-diacetone) adduct (DOBPE, Fig. 2) can be formed even from an acetone solution of PE stored in a fridge [8]. A recent study suggested that this lipid may also be formed in diabetic blood [9]. Still, the most important of the PE-derived low polarity phospholipids seems to be NAPE, the immediate precursor of endogenous bioactive N-acylethanolamines [10].
One of N-acylphosphatidylethanolamines, namely Nacetyl PE was allegedly found in the mammalian brain and placenta [11], but reliably confirmed only in zygomycete Absidia corymbifera [12].
Two major low polarity phospholipids based on PG structure are BPA and SLBPA (Fig. 3). BPA was reported for marine bacteria [13], developing soybeans [14], fish brain [15], and degenerating cultured hamster fibroblasts [16]. BPA was also tentatively identified in some fish flesh [17]. SLBPA, also known as acyl phosphatidylglycerol, was found in a number of micro organisms, and also in plant and animal tissues [18].
The concern mat some unnatural phospholipids, like PE adducts, may be produced during the course of industrial processing of food components, and as such may contribute to development of a pathology (e.g., diabetes) at epidemic or even pandemic scale was expressed recently [19]. However, it seems that low polarity phospholipids are not considered for quantitation by the majority of food quality control laboratories.
One of the reasons for that is the lack of analytical approaches to quantifying low polarity phospholipids. Attempts to identify low polarity lipids with chromatographic methods often resulted in misidentification. For example, low polarity phospholipids found in infarcted dog heart [20] and, later, in the brain of bony fish Amia calva [15] were identified as BPA. It took some time to find out that they were not BPA, but another low polarity phospholipid, NAPE [21], the structure already established for unusual dog heart phospholipid in another laboratory [22]. Similarly, controversial identifications were reported for other low polarity lipids. A phospholipid with high mobility on TLC found in developing soybean cotyledons was identified as NAPE [23], and as PM [24]. A phospholipid with chromatographic mobility higher than that of PE and containing nitrogen was found in beef brain lipids after prolonged contact wim acetone. This lipid was identified as PE-acetone adduct [25] and as N-acetyl PE [11].
Application of MS and NMR-based techniques significantly improves reliability of low polarity phospholipids identification. For example, ^sup 1^H-^sup 13^C 2D NMR experiments allowed distinguishing between SLBPA and NAPE in oats while HPLC-MS was useful in assessing dieir fatty acid composition [26]. A drawback of this approach is that a combination of SPE and preparative TLC was required to isolate pure compounds for NMR.
It would be desirable to develop a non-discriminative technique that does not require isolation of individual lipids to distinguish between numerous low polarity phospholipids. In this publication we describe application of ^sup 31^P-NMR for analysis of low polarity phospholipids and compare the results wim the data provided by high-performance TLC.
Materials and Methods
Egg yolk PE and PC were prepared according to Barsukov et al. [27].
Brain from three frozen and thawed red gurnards CAelidonichthys kumu was excised (total weight 839 mg) and extracted according to Bligh and Dyer [28]. The yield was 85 mg.
A methanolic solution of HCl (5%, 5 ml) was added to a portion of gurnard brain lipids for 5 min at 50 °C to remove vinyl ethers. Chloroform (0.5 ml) and water (0.5 ml) was added, and the mixture centrifuged. The lower layer was collected and solvent removed from the lipid under a stream of argon.
A cabbage (Brassica oleracea var. capitata) stem was extracted as follows:
1. 37.5 g of fresh stem was homogenised in the 100 ml Waring blender cup on high speed for two bursts of 30 s each, men extracted according to Bligh and Dyer method with 113 ml of chloroform-methanol (1:2, by v/v), followed by an extraction with 38 ml of chloroform, yielding 57.9 mg of lipids;
2. 37.6 g of fresh cabbage stem was boiled in water for 5 min, then extracted as above, yielding 89.4 mg lipids;
3. 34.7 g of fresh cabbage stem was homogenised in the 100 ml Waring blender cup on high speed for two bursts of 30 s each, than extracted as follows: 70 ml of methanol was added, the mixture was homogenised again, and allowed to stay at room temperature for 20 min. After that, 35 ml of chloroform was added, the mixture was homogenised and filtered. The residue was extracted with 35 ml of chloroform. Combined extracts were rotary evaporated, yielding 72.8 mg lipids.
PG, PM, PEt, PP and PB were prepared by transphosphatidylation of corresponding alcohols (at 4% of media) and phosphatidylcholine by cabbage phospholipase D [2]. PG was produced by the same approach and purified by column chromatography on silica gel.
Bis-phosphatidic acid (BPA) and semi-lyso bis-phosphatidic acid (SLBPA) were prepared by acylation of PG by palmitoyl chloride using the method of EIlingson [29], modified by addition of few crystals of catalyst, 4-N, Ndimethylaminopyridine.
N-palmitoyl PE (NAPE) and N-acetyl PE were prepared by acylation of PE by palmitoyl chloride and acetyl chloride, correspondingly, using the method of EIlingson [29], modified as above.
N-(1,1-dimethyl-3-oxo-butyl)-derivative of phosphatidylethanolamine (acetone adduct of PE, DOBPE) was prepared by storing PE in acetone in a fridge for 2 weeks [8]. Other phospholipids were from Industrial Research Limited's collection of lipid standards.
Solvent systems for TLC were taken from Vaskovsky and Vysotskii [30]. To develop plates in the 1st direction the system used was chloroform-methanol-benzene-25% aqueous ammonia (60:15:10:1, by v/v/v/v). For 2nd direction the system acetone-benzene-glacial acetic acidwater (20:30:4:1, by v/v/v/v) was used. Development was performed in filter paper-lined tanks saturated with solvent systems for at least 1 h prior to development. Solvent systems in tanks were refreshed after each plate developed. TLC plates (10 × 10 cm glass-backed HPTLC-Plates Nano-Sil 20, Macherey-Nagel, Germany) were activated at 110 °C at least for 90 min and left to cool to room temperature in a vacuum desiccator prior to sample loading. Samples were loaded using l-µl capillary tubes (Billbate Ltd, England). Between developments plates were ambient air-dried with a faint stream of argon applied.
Model mixtures of low polarity phospholipids were prepared as chloroform solutions with concentrations of individual components varying from 1 to 5 mg/ml.
Phospholipids were detected on TLC by phosphomolybdate spray prepared according to Vaskovsky et al. [31].
Complete removal of O-linked fatty acids from glycerolipids was achieved using monomethylamine [32]. Sample (10 mg) was weighed into a stoppered test tube and dispersed in ethanol (1.5 ml). Monomethylamine (40% solution in water, 4.5 ml) was added and the test tube heated in a water bath at 55 °C for 1 h. Solvent was removed under a stream of argon on a heating block (60 °C). The residue was redispersed in ethanol (0.5 ml) and ethyl formate (100 µl) added to neutralise any residual monomethylamine. The solvent was removed under argon and the sample dissolved in the NMR detergent as outlined below.
^sup 31^P-NMR analysis was based on the method of Lehnhardt et al. [33]. A detergent solution was prepared containing: sodium cholate (10% w/w), EDTA (1% w/w) and phosphonomethylglycine (PMG) as an internal standard for quantification (0.3 g/L), pH was adjusted to 7.3 using sodium hydroxide. The detergent solution was an aqueous solution containing 20% D^sub 2^O for deuterium fieldfrequency lock capability. Sample (10 mg) was mixed with detergent solution (750 µl) by vortexing, and then dispersed by ultrasonication at 60 °C for 10 min. The solution was then transferred to a 5-mm NMR tube for analysis.
Quantitative phosphorus NMR spectra using inverse gate proton decoupling for suppression of nuclear Overhauser effect were recorded on the two-channel Bruker Avance300 with the following instrument settings: spectrometer frequency for ^sup 31^P 121.498 MHz, sweep width 6,067 Hz, 65,536 data points, 90 degree excitation pulse, 192 transients were normally taken, each with an 3.5 s delay time and free induction decay acquisition time of 5.4 s. Spectra were processed with a standard exponential weighting function with 0.2 Hz line broadening before Fourier transformation.
Chemical shifts were measured relative to the PMG internal standard, and also relative to SM [present naturally in samples which had been prepared from egg yolk lipids, or as an introduced standard (Avanti)].
Lecitase Ultra (5 µl) was added to a NMR detergent containing fresh cabbage stem lipids followed by incubation at 40 °C for 20 min. The mixture was placed in boiling water for 10 min to deactivate the enzyme then reanalysed to confirm the peak positions of LPM regioisomers.
Results
Thin-Layer Chromatography
The use of the solvent systems developed for one-dimensional TLC separation of low polarity phospholipids [30] resulted in good separation for some lipids (Fig. 4).
Effect of chain length of polar heads of low polarity phospholipids on their TLC mobility was clearly observed: N-palmitoyl PE (NAPE) and N-acetyl PE were easily separated; PM, PEt and PP were also resolved from each other. Further increase in the substitute's chain length had less pronounced effect: PB was clearly resolved from PP only at lower loadings.
SLBPA and PB were not resolved, while SLBPA and PP could be resolved only at low loadings. SLBPA and NAPE were poorly resolved even at low loadings under onedimensional TLC.
Two-dimensional TLC produced significantly better separation of low polarity phospholipids (Fig. 5). All of the low polarity phospholipids were well resolved, including SLBPA and PB which could not be separated by onedimensional TLC.
^sup 31^P NMR
Results for the chemical shifts of the low polarity phospholipids relative to PMG and SM both as intact and deacylated lipids are presented in Table 1 with example spectra of mixtures of the nine less polar phospholipid classes in Figs. 6 and 7. Unlike with the TLC separation there is no clear trend in the chemical shifts based on the alcohol chain length of the phosphatidylalcohols. PM is well separated from the other phosphatidylalcohols, but PE, PP and PB are all within 0.03 ppm. The other low polarity phospholipids are well separated from each other.
Each of the low polarity phospholipids studied, except for BPA, produced a single signal. BPA produced two signals, split by 0.09 ppm, which possibly reflects the existence of two diastereomeric forms. This view is supported by the finding that PG we used as starting material for BPA synthesis was produced from PC via transphosphatidylation by cabbage phospholipase D. It was demonstrated that such PG is, in fact, a racemic mixture of 1-phosphatidyl-D-glycerol and 1-phosphatidyl-L-glycerol [2], and, therefore, BPA used in our experiments was a diastereomeric mixture of sn-3/sn-3' and sn-3/sn-l' isomers (Fig. 3). Differences in chemical shifts have been observed previously between the 31P NMR of mixtures containing L-PS compared to D-PS [34]. However, these differences were noted in phospholipid aggregates, whereas the Na cholate of the NMR detergent used in the current study is present to disrupt such aggregates and achieve sharper signals for individual phospholipids. Preparation of pure diastereomers of BPA is required to confirm that the peak splitting is due to diastereomeric differences, and for correct assignment of each peak. However, if this is the cause of the peak splitting then the ability to differentiate between the diastereomers is an advantage of ^sup 31^P NMR over that of 2D TLC.
Unlike TLC where separation is based on polarity and thus the low polarity phospholipids by definition are well separated from other phospholipids, there is some overlap of the ^sup 31^P-NMR chemical shifts of the low polarity phospholipids and some other phospholipids. The examples of this are: BPA(a) (-0.33 ppm relative to SM) and ILPC (-0.34 ppm); BPA(b) (-0.24 ppm) and PS (-0.25 ppm); DOBPE (0.10 ppm) and dihydrosphingomyelin (0.09 ppm); NAPE (0.37 ppm) and CL (0.36) and 2LPE (0.38 ppm) [35].
O-deacylation of the sample produced sharper peaks, which are better resolved both from other low polarity phospholipids and other phospholipids. GPB and GPP are not resolved, but all other O-deacylated low polarity phospholipids are separated from each other. However, deacylation converts BPA and SLBPA (as well as PG) to the single compound - glycerophosphoglycerol (GPG). Deacylation of DOBPE produced a single phosphoruscontaining component, with chemical shift identical to that of glycerophosphoethanolamine, suggesting that acetone adduct is destroyed during deacylation.
To demonstrate the applicability of the described techniques to the real life samples analysis, a number of extracts were prepared. To illustrate a formation of an artefact phosphatidylmethanol during extraction of plant samples, a cabbage (Brassica oleracea var. capitata) stem was extracted as follows:
1. Fresh stem was extracted by a standard Bligh and Dyer method;
2. Boiled stem was extracted by a standard Bligh and Dyer method;
3. To demonstrate a danger of adding methanol first (as recommended sometimes, since methanol mixes better with tissues than chloroform), the methanol portion of Bligh and Dyer first extraction solvent was added first to the stem and allowed to stay for 20 min prior to addition of chloroform.
One-dimensional TLC demonstrated a formation of PM during extraction of fresh stem, especially when methanol was added prior to chloroform; no PM was formed during extraction of the boiled stem (Fig. 8).
^sup 31^P NMR measured high levels of PM in both extracts from fresh cabbage stem (Table 2). There was measurable PM (0.4 mol %) even in the extract from boiled cabbage indicating some residual phospholipase D activity. Peaks due to ILPM and 2LPM (-5.79 and -5.63 ppm relative to PMG respectively) were confirmed by a partial phospholipase Al hydrolysis of a fresh cabbage stem extract.
The presence of low polarity phospholipid in red gurnard (Chelidonichthys kumu) brain was demonstrated by two-dimensional TLC (Fig. 9, left pane), and the fact that it differs from SLBPA was demonstrated by spiking the sample with SLBPA standard (Fig. 9, right pane).
A comparison with the reference standard, SLBPA, and a known low polarity lipid two-dimensional TLC pattern (Fig. 5) suggests that the low polarity phospholipid from red gurnard brain is NAPE.
As discussed above, the ^sup 31^P-NMR peak position of NAPE is very close to that of 2LPE and CL. NAPE was not observed in the spectrum of intact gurnard brain lipids (Fig. 10). Analysis of deacylated red gurnard brain lipids did not result in a significant NAGPE peak, but a peak did appear at 0.77 ppm relative to SM. Acid treatment to destroy vinyl ethers, followed by deacylation resulted in the disappearance of this peak, and presence of a NAGPE peak. This indicates that most of the NAPE present in red gurnard brain is in the plasmalogen form.
Discussion
In our search for NAPE in bony and cartilaginous fish we found that it is difficult to distinguish SLBPA and NAPE even by high-performance TLC, and chemical transformations are required to attribute an unknown lipid to one of these without employing physico-chemical methods of structure elucidation [30]. Later we demonstrated that reliable separation of SLBPA and NAPE requires the use of homemade HPTLC-plates coated with silica sol-bound microfractionated silica gel KSK, and low humidity [36].
One-dimensional TLC is helpful in screening of significant number of samples for low polarity phospholipids, but it has shortcomings, like poor resolution of NAPE, SLBPA, PB and PP.
In contrast, 2D TLC, while requiring significantly longer procession times, allows the separation of all the above mentioned low polarity phospholipids.
TLC separation of low polarity phospholipids requires a careful selection of solvent systems. An inappropriate solvent system seemed to be used in the analysis of NAPE from kenaf seeds with iV-linked acids being predominantly linoleic, palmitic and oleic [37], since it was reported to have the same chromatographic mobility as N-acetyl PE, while these are clearly separable as it was demonstrated in the current work.
Apart from NAPE, an unknown lipid identified as BPA was reported for developing soybeans and soybean suspension cultures [14]. The authors mentioned that chromatographic mobility of the latter lipid is lower than that of NAPE, which allows separation of these two lipids by preparative TLC. Considering the previous findings that SLBPA (e.g., partially deacylated BPA, thus more polar than BPA) has a mobility on TLC similar or equal to that of NAPE [29], it seems rather unlikely for BPA to have lower mobility than that of NAPE in normal phase chromatography.
In summary, it appears that 2D TLC is superior to ^sup 31^P NMR in the analysis of low polarity phospholipids. However, some compounds which can present difficulty in separation by 2D-TLC (e.g., SLBPA and NAPE; or DOBPE and N-acetyl PE) are 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. It is therefore important in the 2D TLC analysis to have standards for confirmation of the identity of spots in analysis of real samples. The chemical shifts of ^sup 31^P NMR are less variable, and such 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 peak splitting observed in the ^sup 31^P NMR of BPA has been attributed to the presence of diastereomers. Unlike ^sup 31^P NMR, the 2D TLC method used in this study cannot distinguish between mese diastereomers.
Acknowledgments This work was supported by the New Zealand Foundation for Research, Science and Technology grant C08X0709 "High value lipids". The authors are grateful to Dr. O. Catchpole for reviewing the manuscript and to Dr. E. Nekrasov and Dr. H. Wong for help with NMR. M.V. is also grateful to Professor V. Vaskovsky for attracting his attention to low polarity phospholipids.
References
1. Nicolaou A, Kokotos G (2004) Bioactive lipids. The Oily Press, Bridgwater
2. Yang SF, Freer S, Benson AA (1967) Transphosphatidylation by phospholipase D. J Biol Chem 242:477-484
3. Hannuksela ML, Liisanantti MK, Nissinen AET, Savolainen MJ (2007) Biochemical markers of alcoholism. Clin Chem Lab Med 45:953-961
4. Alling C, Gustavsson L, Mansson JE, Benthin G, Anggard E (1984) Phosphatidyledianol formation in rat organs after ethanol treatment. Biochim Biophys Acta 793:122-199
5. Vaskovsky VE, Khotimchenko SV (1983) Micro-chromatographic test of transphosphatidylic activity of phospholipase D in algae and other plants. J Chromatogr 261:324-328
6. Randall RW, Bonser RW, Thompson NT, Garland LG (1990) A novel and sensitive assay for phospholipase D in intact cells. FEBS 264:87-90
7. Mohn H, Chalifa V, Liscovitch M (1992) Substrate specificity of neutral phospholipase D from rat brain studied by selective labelling of endogenous synaptic membrane phospholipids in vitro. J Biol Chem 267:11131-11136
8. Ando N, Ando S, Yamakawa T (1971) Structure and formation mechanism of N-acetone derivatives of phosphatidyledianolamine. J Biochem 70:341-348
9. Kuksis A, Ravandi A, Schneider M (2005) Covalent binding of acetone to aminophospholipids in vitro and in vivo. Ann N Y Acad Sci 1043:417-439
10. Schmid HHO, Schmid PC, Natarajan V (1990) N-Acylated glycerophospholipids and their derivatives. Prog Lipid Res 29:1-43
11. Debuch H, Wendt G (1967) On a new group of colaminecontaining glycerophosphatides from brain. Hoppe-Seyler's Z Physiol Chem 348:471-474
12. Batracov SG, Konova IV, Sheichenko VI, Esipov SE, Galanina LA (2001) Two unusual glycerophospholipids from a filamentous fungus, Absidia corymbifera. Biochim Biophys Acta 1531:169-177
13. McAllister DJ, De Siervo AJ (1975) Identification of bisphosphatidic acid and its plasmalogen analogues in the phospholipids of a marine bacterium. J Bacteriol 123:302-307
14. Stearns EM, Morton WT (1977) Biosynthesis of fatty acids from acetate in soybean suspension cultures. Lipids 10:597-601
15. Hack MH, Helmy FM (1975) Bis-phosphatidic acid plasmalogen in brain of Amia calva and its correlation with the infarct plasmalogen and cardiolipin (diphosphatidyl glycerol) series of phosphatides. Comp Biochem Physiol C 52(2): 139-145
16. Brodierus J, Renkonen O (1974) Isolation and characterization of bis-phosphatidic acid and its partially deacylated derivatives from cultured BHK-cells. Chem Phys Lipids 13:11-20
17. Olley J (1956) The lipids of fish 7. Phosphate esters in the lipids of haddock and cod flesh. Biochem J 62:107-114
18. Hsu F-F, Turk J, Shi Y, Groisman EA (2004) Characterization of acylphosphatidylglycerols from Salmonella typhimurium by tandem mass spectrometry with electrospray ionization. J Am Soc Mass Spectrom 15:1-11
19. Elliott RB (2006) Diabetes-a man made disease. Med Hypotheses 67:388-391
20. Hack MH, Ferrans VJ (I960) Nitrogen-free plasmalogen observed in infarcted myocardium of the dog. Circulat Res 8(4):738-741
21. Hack MH, Helmy FM (1982) A reappraisal of the dog-heart infarct plasmalogen, its conception as a bis-phosphatidic acid and current recognition as an N-acylphosphatidyledianolamine. Comp Biochem Physiol B 73:873-879
22. Epps DE, Natarajan V, Schmid PC, Schmid HHO (1980) Accumulation of N-acylethanolamine glycerophospholipids in infarcted myocardium. Biochim Biophys Acta 618:420-430
23. Wilson RF, Rinne RW (1974) Phospholipids in the developing soybean seed. Plant Physiol 54:744-747
24. Slack CR, Roughan PG, Balasingham N (1978) Labelling of glycerolipids in the cotyledons of developing oilseeds by [1-14C] acetate and [2-3H] glycerol. Biochem J 170:421-433
25. Helmy FM, Hack MH (1966) An edianolamine plasmalogen artifact formed by acetone extraction of freeze-dried tissue. Lipids 1:279-281
26. Holmback J, Karlsson AA, Arnoldsson KC (2001) Characterization of N-acylphosphatidyledianolaniine and acylphosphatidylglycerol in oats. Lipids 36:153-165
27. Barsukov LI, Batrakov SG, Bergelson LD, Dyatlovitskaya EV, Molodcovsky JG, Prokazova NV (1980) In: Bergelson LD (ed) Lipid biochemical preparations, Elsevier/North-Holland Biomedical Press, Amsterdam-New York-Oxford
28. Bligh EG, Dyer WJ (1959) A rapid mediod of total lipid extraction and purification. Can J Biochem Physiol 37:911-917
29. Ellingson JS (1980) Identification of (N-acyl)edianolamine phosphoglycerides and acylphosphatidylglycerol as the phospholipids which disappear as Dictyostelium discoideum cells aggregate. Biochemistry 19:6176-6182
30. Vaskovsky VE, Vysotskii MV (1985) N-acylphosphatidyledianolamine in fish brain. Khim Prirodn Soedin 1985:326-329 (in Russian)
31. Vaskovsky VE, Kostetsky EY, Vasendin IM (1975) A universal reagent for phospholipid analysis. J Chromatogr 114:129-141
32. Clarke NG, Dawson RM (1981) Alkaline O [arrow right] N-transacylation. A new mediod for the quantitative deacylation of phospholipids. Biochem J 195:301-306
33. Lehnhardt F-G, Röhn G, Ernestus R-I, Grüne M, Hoehn M (2001) ^sup 1^H- and ^sup 31^P-NMR spectroscopy of primary and recurrent human brain tumors in vitro: malignancy-characteristic profiles of water soluble and lipophilic spectral components. NMR Biomed 14:307-317
34. Epand RM, Stevenson C, Bruins R, Schram V, Glaser M (1998) The chirality of phosphatidylserine and the activation of protein kinase C. Biochemistry 37:12068-12073
35. MacKenzie AD, Wong H, Braun RM (2005) Deacylation of phospholipids to improve resolution in the ^sup 31^P NMR analysis of animal phospholipids. 26m World Congress and exhibition of the International Society of Fat Research (poster presentation), Prague, Czech Republic
36. Vysotskii MV, Vaskovsky VE (1988) Micro-thin layer chromatography of low polarity phospholipids. Identification of N-acylphosphatidyletfianolamine. VINITI deposited paper No.1036-B88, 16 pp. (in Russian)
37. Tolibaev I, KhS Mukhamedova, Akramov ST (1976) N-acylated phospholipids and lysophospholipids of kenaf seeds. Chem Nat Compd 12:650-652
M. Vyssotski (*) - A. MacKenzie * D. Scott
Industrial Research Limited, Lower Hutt, New Zealand
e-mail: [email protected]
Copyright AOCS Press Apr 2009