HIGH‐RESOLUTION MAGIC ANGLE SPINNING NMR ANALYSIS OF WHOLE CELLS OF CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE) AND COMPARISON WITH 13C‐NMR AND DISTORTIONLESS ENHANCEMENT BY POLARIZATION TRANSFER 13C‐NMR ANALYSIS OF LIPOPHILIC EXTRACTS1

Lipid composition in extracted samples of Chaetoceros muelleri Lemmermann was studied with ¹³C‐NMR and distortionless enhancement by polarization transfer (DEPT) ¹³C‐NMR, resulting in well‐resolved ¹³C‐NMR spectra with characteristic resonance signals from carboxylic, olefinic, glyceryl, methylene, and methyl groups. The application of a DEPT pulse sequence aided in the assignment of methylene and methine groups. Resonance signals were compared with literature references, and signal assignment included important unsaturated fatty acids such as eicosapentaenoic and docosahexaenoic and also phospholipids and glycerols. Results from the extracted samples were used to assign resonance signals in a high‐resolution magic angle spinning (HR MAS) DEPT ¹³C spectrum from whole cells of C. muelleri. The NMR analysis on whole cells yielded equally good information on fatty acids and also revealed signals from carbohydrates and amino acids. Broad resonance signals and peak overlapping can be a problem in whole cell analysis, but we found that application of HR MAS gave a well‐resolved spectrum. The chemical shift of metabolites in an NMR spectrum depends on the actual environment of nuclei during analysis, and some differences could therefore be expected between extracted and whole cell samples. The shift differences were small, and assignment from analysis of lipophilic extract could be used to identify peaks in the whole cell spectrum. HR MAS ¹³C‐NMR therefore offers a possibility for broad‐range metabolic profiling directly on whole cells, simultaneously detecting metabolites that are otherwise not detected in the same analytical set up and avoiding tedious extraction procedures.     

The nature of fatty acid modifies the equilibrium position in the esterification catalyzed by lipase

The equilibrium position in lipase mediated esterification of various fatty acids and butanol was studied. The influence of the chain length and the presence of unsaturations in the fatty acids on the equilibrium position was measured and predicted. To predict equilibrium position the program TREP extended (TREPEX) based on the UNIFAC group contribution method was used. Using an equilibrium constant of 35, calculated on the basis of thermodynamic activities, the equilibrium position between butanol and saturated and/or unsaturated fatty acids with different chain lengths can be predicted. The ester mole fraction at equilibrium increases with the fatty acid chain length, and for fatty acids with the same carbon number, the highest values are found for unsaturated fatty acids. For reaction systems containing two saturated fatty acids, a slightly higher mole fraction is obtained for the fatty acid with the higher chain length, while for mixtures consisting of saturated and unsaturated fatty acids, the mole fractions of the unsaturated esters are lower than those of the saturated ones, regardless the chain length of the fatty acid. These experimental results are in good agreement with the calculations with TREPEX.     

Alternative NMR method for quantitative determination of acyl positional distribution in triacylglycerols and related compounds

High-resolution 13C NMR spectroscopy has been used to analyze the positional distribution of fatty acids in model triacylglycerols. A novel method for quantitative determination of the positional distribution of unsaturated chains in triacylglycerols simultaneously with the ratio of saturated/unsaturated acyl chains has been proposed, utilizing the chemical shift differences of the aliphatic atoms C4, C5, and C6. The use of HSQC-TOCSY spectra allows unequivocal proof of the position of the unsaturated chain as well as complete assignment of the 13C NMR signals in tripalmitin.     

Effects of a mixture of a saturated with an unsaturated fatty acid on secretion of the very low density lipoprotein by the liver.

Palmitic acid (16:0) and palmitoleic acid (16:1), as the complex with bovine serum albumin, were infused at rates of 62 and 124 μmoles/hr into an albumin-buffer medium perfusing livers isolated from normal fed male rats. In other experiments, equimolar mixtures (124 μmoles/hr, total) of 16:0 + 16:1, or myristate (14:0) + 16:1 were infused. The output of triglyceride when 16:1 was infused was greater than when equivalent amounts of 14:0 or 16:0 were infused; output with equimolar mixtures of 14:0 and 16:1, or 16:0 and 16:1 was intermediate between that of saturated and unsaturated fatty acids alone. Rate-zonal mobility of the VLDL in the ultracentrifuge was more rapid as the quantity of 16:1 available to the liver increased, but did not change with increasing amounts of 16:0. The rate-zonal mobility of the mixtures of 14:0 and 16:1, or of 16:0 and 16:1, was not different than that of 16:1 alone. The ratios of phospholipid and cholesterol relative to triglyceride in the VLDL decreased with increasing output of triglyceride and with unsaturation of the fatty acid. Ratios resulting from mixtures of the fatty acids appeared to be in an intermediate position. The composition and properties of the secreted VLDL clearly are dependent on the structure and quantity of FFA available to the liver; with mixtures of saturated and unsaturated fatty acids, the unsaturated fatty acid seems to exert a dominant effect.     

Evidence for cross-linking in tomato cutin using HR-MAS NMR spectroscopy

Cutin is a polyester biopolymer component of plant leaf and fruit cuticles, most often associated with waxes and cuticular polysaccharides, and sometimes with another aliphatic biopolymer called cutan. Insolubility of these cuticular biopolymers has made it difficult to apply traditional analytical techniques for structure determination, because most techniques providing molecular level details require solubility. By using the relatively new technique of one and two-dimensional high-resolution magic angle spinning (HR-MAS) NMR spectroscopy, with added information from solid-state 13C NMR spectroscopy, detailed through-bond connectivities and assignments are made for cutin from Lycopersicon esculentum (tomato) fruit. Based on the data obtained, tomato cutin is found to be predominantly an aliphatic polyester with some olefinic and aromatic moieties, consistent with previous studies that employed various degradative approaches. Aside from esters, there are free primary and secondary alcohol groups, as well as free fatty acids. A significant finding is the presence of alpha-branched fatty acids/esters. Mid-chain hydroxyls appear to be generally unesterified, but esters of mid-chain hydroxyls have been identified. The alpha-branched fatty acids/esters and esters of mid-chain hydroxyls could point towards cross-linking.     

Non-invasive histochemistry of plant materials by magnetic resonance microscopy

Summary We have combined nuclear magnetic resonance (NMR) imaging on the microscopic scale with chemical shift selection to demonstrate the application of magnetic resonance imaging (MRI) to plant histochemistry. As an example of the method we have obtained separate images of the distribution of reserve oil and anethole in dried fennel mericarps. The technique can be employed to separately image the distribution of aromatics, carbohydrates, oils, water and possibly fatty acids in suitable plant materials.Abbreviations NMR nuclear magnetic resonance - MRI magnetic resonance imaging - COSY correlation spectroscopy - TMS tetramethylsilane     

Nondestructive analyses of unsaturated fatty acid species in dietary oils by attenuated total reflectance with Fourier transform IR spectroscopy

The aim of this study was to develop a nondestructive method to quantitate relative amounts of n-3 and n-6 polyunsaturated fatty acid (PUFA) species in vegetable oils and oil seeds using Fourier transform IR spectroscopy (FTIR). The alkene Cbond;H stretching vibrations of unsaturated fatty acids in oils showed IR absorption bands with various peak positions and intensities at around 3010 cm(-1), depending on the extent of unsaturation and PUFA species. With the aid of partial least-squares regression analysis, the FTIR measurement could practically predict the content of each PUFA species in the oil to be tested. A calculation method was also presented to directly find PUFA species in oils from the FTIR spectra. This technique was applied to dried soybean seeds to demonstrate a nonhomogenous distribution of saturated fatty acids and PUFAs, as well as glycans, in soybean cross sections.     

Fast NMR evaluation of lipids in human tissues

1H and 13C NMR spectroscopy was used to evaluate the degree of unsaturation and the cholesterol/cholesteryl ester ratio on the total lipid fractions obtained from human renal and cerebral tissues. The unsaturated/saturated fatty acid ratio was determined in the 13C NMR spectra from the ratio of the integrated areas of the resonances at 14.13 and 14.17 ppm assigned to the terminal methyl groups of saturated and unsaturated FA, respectively, and is validated by the traditional but time consuming gas-chromatographic analysis. Cholesteryl esters are easily discriminated in the total lipid fraction extracted from human tissues by means of the well-resolved component at 0.99 ppm (1H NMR spectra) of the resonance at about 1.00 ppm generally assigned to free cholesterol. The role of NMR spectroscopy in the study of lipidic biochemistry of human tissues is confirmed.     

Increased unsaturation of lipids in cytoplasmic lipid droplets in DAOY cancer cells in response to cisplatin treatment

Increases in ¹H nuclear magnetic resonance spectroscopy (NMR) visible lipids are a well-documented sign of treatment response in cancers. Lipids in cytoplasmic lipid droplets (LDs) are the main contributors to the NMR lipid signals. Two human primitive neuroectodermal tumour cell lines with different sensitivities to cisplatin treatment were studied. Increases in NMR visible saturated and unsaturated lipids in cisplatin treated DAOY cells were associated with the accumulation of LDs prior to DNA fragmentation due to apoptosis. An increase in unsaturated fatty acids (UFAs) was detected in isolated LDs from DAOY cells, in contrast to a slight decrease in UFAs in lipid extracts from whole cells. Oleic acid and linoleic acid were identified as the accumulating UFAs in LDs by heteronuclear single quantum coherence spectroscopy (HSQC). ¹H NMR lipids in non-responding PFSK-1 cells were unchanged by exposure to 10 μM cisplatin. These findings support the potential of NMR detectable UFAs to serve as a non-invasive marker of tumour cell response to treatment.     

Effects of various non-esterified fatty acids on the transfer of cholesteryl esters from HDL to LDL induced by the cholesteryl ester transfer protein

The effects of saturated, monounsaturated and polyunsaturated non-esterified fatty acids on the rate of transfer of radiolabeled cholesteryl esters from high density lipoproteins (HDL) to low density lipoproteins (LDL), induced by the cholesteryl ester transfer protein (CETP), have been studied. Human high-density lipoproteins-subfraction 3 (HDL3) containing radiolabeled cholesteryl esters were incubated with LDL at 37 degrees C with or without CETP and in the absence or in the presence of non-esterified fatty acids. Less than 6% of the total radioactivity was recovered in the LDL fraction after incubation of HDL3, and LDL for 3 h at 37 degrees C in the absence of CETP, regardless of whether or not non-esterified fatty acids were added. The addition of CETP to the incubation mixture induced a time-dependent redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. Non-esterified fatty acids were found to alter the rate of transfer of cholesteryl esters induced by CETP. While short chain saturated non-esterified fatty acids (caprylic and capric acids) had no effect on the rate of transfer of cholesteryl esters, the medium and long chain ones (lauric, myristic, palmitic and stearic acids) significantly increased the CETP-mediated transfers from HDL3 to LDL. At low concentrations, unsaturated fatty acids also stimulated the CETP-mediated redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. As the concentration of either oleic, linoleic or arachidonic acids increased to higher levels, a significant proportion of fatty acids remained unassociated with lipoprotein particles. Under these circumstances the transfer process was inhibited. These results show that non-esterified fatty acids can modulate the CETP-mediated transfer of cholesteryl esters from HDL to LDL and that this effect is dependent on both the length and the degree of unsaturation of their monomeric carbon chain.     

Lipids in plant tissue cultures. II. Unusual fatty acids in lipids of Hydnocarpus anthelminthica cultures

The lipids in callus cultures of Hydnocarpus anthelminthica were studied after 60, 160 and 460 days of growth. In each of the cultures the lipid classes usually found in plant tissue cultures were detected. With increasing age of the cultures the total lipid content as well as the proportions of triglycerides decreased. The major constituent fatty acids of the total lipids were palmitic and linoleic acids. Small amounts of cyclopentenyl fatty acids were also present. The proportions of saturated straight-chain fatty acids increased with the age of the cultures whereas the proportions of monounsaturated straight-chain fatty acids decreased. Only small changes were observed with polyunsaturated fatty acids. The content of cyclopentenyl fatty acids rose with the age of the cultures. The monounsaturated straight-chain fatty acids consisted of mixtures of isomers whose composition changed with the age of the cultures. In contrast, the polyunsaturated straight-chain fatty acids belonged exclusively to the Δ9 series, regardless of the age of the cultures.     

Triester wax: a novel neutral lipid from the skin of the rhino mutant mouse.

A novel class of neutral lipids has been isolated from the skin of the rhino mutant mouse and has been characterized as a triester wax. The lipid, on saponification and transesterification, yielded fatty acids, omega-hydroxy fatty acids and 1,2-alkane diols. These products were identified by gas-liquid and thin-layer chromatography, infrared, nuclear magnetic resonance and mass spectroscopy, combined gas-liquid chromatography and mass spectrometry and chemical methods. Fatty acids were found to be predominantly of even chain length between C14 and C36 with highest concentration at C22 : 1. Hydroxy fatty acid methyl esters (trimethylsilyl ether derivatives) showed the presence of only three components in the relative abundance of 9: 70 : 21. The structure of the major component was established as 34-hydroxytetratricont-25-enoic acid and the other two components were characterized as 32-hydroxyditricont-23-enoic and 36-hydroxyhexatricont-27-enoic acids. In addition to these omega-9 unsaturates, other isomers having unsaturation at omega-7 and omega-8 were also present in small amounts. The 1,2-alkane diols were predominantly saturated in the range of C16-C24.     

Changes in hepatic lipids of mice infected with cysticerci of Taenia crassiceps

Nuclear magnetic resonance (NMR) spectroscopy was employed to investigate the effect of infection with Taenia crassiceps cysticerci on the lipid profile of mouse liver. Chloroform/methanol extracts of livers from infected mice showed lower concentrations of phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, total glycerophospholipid, triacylglycerol, total fatty acid (FA) and all measured FA components than those from controls. Furthermore, the ratios obtained on dividing concentrations of the FA components by that of total FA demonstrate that the concentration decreases caused by infection are less for polyunsaturated fatty acids (FAs) than for other FAs. Extracts of T. crassiceps displayed a similar lipid profile to that of host liver but contained a lower lipid content and a shorter average FA chain length.     

A single column packing for the gas-liquid chromatographic separation of various biologically important compounds.

The use of a single, commercially available column packing, TabsorbR, is described for the g.l.c. separation of a large number of different compounds. The resolution of the homologous members of the following series of compounds was achieved: (1) saturated fatty acids (C1-C18), (2) normal aliphatic saturated dicarboxylic acids (C2-C14), (3) normal aliphatic saturated alcohols (C1-C24), (4) normal aliphatic saturated amines (C1-C12), (5) the common amino acids except arginine, histidine and cysteine, (6) aliphatic hydrocarbons (C10-C20) and (7) monosaccharides. It should be noted that twenty-two monosaccharides including three hexosamines and two anhydrohexoses, could be resolved as alditol acetates in a single run. In addition, galacturonic, glucuronic and iduronic acids could be separated from one another as their 1,4-lactones. The resolution achieved in these series of compounds was found to be consistent and highly reproducible. It is of further interest that certain isomers of the higher fatty acids and hydrocarbons with one double bond could also be separated from the normal and saturated compounds, respectively. The applicability of "Tabsorb" for the g.l.c. separation, although noted above to be considerably broad, is by far not yet exhausted. These procedures which form the basis for the quantitative determinations of the various compounds studied as demonstrated by analysis of glycopeptides for neutral hexoses and proteins for the amino acids, can readily be adapted to preparative methods. From the biochemical point of view "Tabsorb" is an extremely versatile column packing in that it can be used for the identification of many of the common building blocks of natural products.     

Formation of lysophospholipids from unsaturated phosphatidylcholines under the influence of hypochlorous acid

The formation of lysophosphatidylcholines from unsaturated phosphatidylcholines upon treatment with hypochlorous acid was evaluated by means of MALDI-TOF mass spectrometry and 31P NMR spectroscopy. With an increasing number of double bonds in a fatty acid residue, the yield of lysophosphatidylcholines with a saturated fatty acid residue increased considerably in comparison to the total amount of higher molecular weight products like chlorohydrins and glycols. High amounts of lysophosphatidylcholines were formed from phospholipids containing arachidonic or docosahexaenoic acid residues. In phospholipids with monounsaturated fatty acid residues, the position of the double bond did not influence the yield of lyso-products. Besides the exclusive formation of chlorohydrin and glycol, hypochlorous acid caused the cleavage of the unsaturated fatty acid residue independent of its location at the first or second position of the glycerol backbone. In contrast, strong alkaline conditions, i.e. saponification led also to a hydrolysis of the saturated fatty acid residue from phosphatidylcholines. It is concluded that both MALDI-TOF mass spectrometry and 31P NMR spectroscopy are able to detect the formation of lysophosphatidylcholines. We conclude also that the formation of lysophospholipids from unsaturated phosphatidylcholines by hypochlorous acid can be relevant in vivo under acute inflammatory conditions.     

Monoacylglycerols are components of root waxes and can be produced in the aerial cuticle by ectopic expression of a suberin-associated acyltransferase

The interface between plants and the environment is provided for aerial organs by epicuticular waxes that have been extensively studied. By contrast, little is known about the nature, biosynthesis, and role of waxes at the root-rhizosphere interface. Waxes isolated by rapid immersion of Arabidopsis (Arabidopsis thaliana) roots in organic solvents were rich in saturated C18-C22 alkyl esters of p-hydroxycinnamic acids, but also contained significant amounts of both alpha- and beta-isomers of monoacylglycerols with C22 and C24 saturated acyl groups and the corresponding free fatty acids. Production of these compounds in root waxes was positively correlated to the expression of sn-glycerol-3-P acyltransferase5 (GPAT5), a gene encoding an acyltransferase previously shown to be involved in aliphatic suberin synthesis. This suggests a direct metabolic relationship between suberin and some root waxes. Furthermore, when ectopically expressed in Arabidopsis, GPAT5 produced very-long-chain saturated monoacylglycerols and free fatty acids as novel components of cuticular waxes. The crystal morphology of stem waxes was altered and the load of total stem wax compounds was doubled, although the major components typical of the waxes found on wild-type plants decreased. These results strongly suggest that GPAT5 functions in vivo as an acyltransferase to a glycerol-containing acceptor and has access to the same pool of acyl intermediates and/or may be targeted to the same membrane domain as that of wax synthesis in aerial organs.     

Fatty acid composition ofAerobacter aerogenes

The fatty acid composition of the whole cell and cell wall ofAerobacter aerogenes was studied employing column and gas-chromatographic technique. The cell wall contained a greater percentage of total lipid, complex lipid, and free fatty acids compared to the whole cell. Palmitic acid and oleic acid were the most abundant fatty acids found in the free fatty acid and complex lipid fractions. A saturated C₁₇ fatty acid and small quantities of a branched C₁₆ and iso and anteiso C₁₂ fatty acids were detected. The glyceride fractions of the whole cell and cell wall contained very few fatty acids.     

Skin surface lipids of the mole Scalopus aquaticus

Skin surface lipids of the mole Scalopus aquaticus were found to consist principally of squalene (70%), wax esters (15%), and sterol esters (5%), together with small amounts of triglycerides, free fatty acids, free fatty alcohols, and free sterols. Analysis of the fatty acids occurring free and as wax esters and sterol esters showed these to consist of approximately equal amounts of saturated and monounsaturated compounds. The saturated fatty acids consisted predominantly of odd-carbon anteiso and even-carbon straight-chain compounds, with minor amounts of even-carbon iso-branched chains. The unsaturated fatty acids had double bond positions that would have been produced by delta 9-desaturation of C14, C16 and C18 straight chain saturated precursors. Both the free and the esterified fatty alcohols had chain structures corresponding with those of the fatty acids but of somewhat greater average chain length. Discovery of a major proportion of squalene in the sebum of this animal extends the number of non-human species that have this characteristic to four, all of which inhabit a damp environment, suggesting that squalene conveys some biological advantage under these conditions.     

Acylation of natural flavonoids using lipase of candida antarctica as biocatalyst

Several flavonoids (quercetin, hesperidin, rutin and esculin) were acylated with fatty acids using an immobilised lipase from Candida antarctica in 2-methyl-2-butanol at 60 °C. It appears that esculin with primary OH on the sugar part is the most reactive substrate. With palmitic acid as acyl donor, the conversion yields were of about 80, 71 and 38%, respectively, for esculin, rutin and hesperidin. No reaction was observed with aglycon flavonoid (quercetin). For a given flavonoid (rutin), the conversion yield increased from 42 to 76% when the carbon number of the fatty acids rose from C6 to C12. For fatty acids with higher carbon-chain length, both conversion yield and initial rate dropped slightly. Furthermore, compared to the saturated fatty acid (C18: 0), the unsaturated one (C18: 1) exhibited a lower reactivity. For all molecules studied ¹H nuclear magnetic resonance (NMR) and ¹³C NMR analyses indicated that only flavonoid monoester was produced.