Isabelle Billault

Determination of origin of Atlantic salmon (Salmo salar): the use of multiprobe and multielement isotopic analyses in combination with fatty acid composition to assess wild or farmed origin.

Variability within the stable isotope ratios in various lipidic fractions and the fatty acid composition of muscle oil has been analyzed for a large sample (171 fish) of wild and farmed Atlantic salmon ( Salmo salar) from 32 origins within Europe, North America, and Tasmania. Sampling was extended over all seasons in 2 consecutive years and included fish raised by different practices, in order to maximize the range of variation present. It is shown that two readily measured parameters, delta 15N measured on choline and delta18 O measured on total oil, can be successfully used to discriminate between fish of authentic wild and farmed origin. However, the certainty of identification of mislabeling in market-derived fish is strengthened by including the percentage of linoleic acid C18:2n-6 in the lipidic fraction. Thus, several apparent misidentifications were found. The combination of these three analytical parameters and the size of the database generated makes the method practical for implementation in official laboratories as a tool of labeling verification.

Measurement of 2H distribution in natural products by quantitative 2H NMR: An approach to understanding metabolism and enzyme mechanism?

During the biosynthesis of natural products, the intra-molecular distribution of isotopes is introduced as a result of different isotope effects associated with the reactions involved. Due to the sensitivity of certain enzymes to the presence of a heavy isotope, the isotope selection effects related to some transformations can be high, especially for hydrogen. The effect of a series of isotope effects specific to each enzyme-catalysed step are additive during a biosynthetic pathway, leading to fractionation of the isotopes between the starting substrate and the final product. As the individual reactions are acting on different positions in the substrate, the net effect is a non-statistical distribution of isotope within the final product. Quantitative 2H NMR spectroscopy can be used to measure the distribution of 2H at natural abundance in natural products. In the first example, the fermentation of glucose is examined. Glucose can act as a primary carbon source for a wide range of fermentation products, produced by a variety of pathways. In many cases, competing pathways are active simultaneously. The relative fluxes are influenced by both environmental and genetic parameters. Quantitative 2H NMR spectroscopy is being used to obtain mechanistic and regulatory information about isotopic fractionation from glucose during such fermentations. Quantitative 2H NMR spectroscopy can also be used to examine the fractionation in 2H that occurs in long-chain fatty acids during chain elongation and oxygenation. It has been found that the (2H/1H) ratio shows an alternating pattern along the length of the chain and that the residual hydrogen atoms at the sites of desaturation are asymmetrically impoverished. The extent to which the non-statistical distribution of isotopes can be related to the mechanism of enzymes involved in the biosynthetic pathway via kinetic isotopic effects will be discussed.

Investigation of Fatty Acid Elongation and Desaturation Steps in Fusarium lateritium by Quantitative Two-dimensional Deuterium NMR Spectroscopy in Chiral Oriented Media

The origin of hydrogen atoms during fatty acid biosynthesis in Fusarium lateritium has been quantified by isotope tracking close to natural abundance. Methyl linoleate was isolated from F. lateritium grown in natural abundance medium or in medium slightly enriched with labeled water, glucose, or acetate, and the 2H incorporation was determined by quantitative 2H-{1H} NMR in isotropic and chiral oriented solvents. Thus, the individual (2H/1H)i ratio at each pro-R and pro-S hydrogen position of the CH2 groups along the chain can be analyzed. These values allow the isotope redistribution coefficients (aij) that characterize the specific source of each hydrogen atom to be related to the nonexchangeable hydrogen atoms in glucose and to the medium water. In turn, these can be related to the stereoselectivity that operates during the introduction or removal of hydrogens along the fatty acid chain. First, at even CH2 the pro-S hydrogen comes only from water by protonation, whereas the pro-R hydrogen is introduced partly via acetate but principally from water. Second, the nonexchangeable hydrogens of glucose (positions H-6,6 and H-1) are shown to be introduced to the odd CH2 via the NAD(P)H pool used by both reductases involved in the elongation steps of the fatty acid chain. Third, it is proved that hydrogens removed at sites 9,10 and 12,13 during desaturation by Δ9- and Δ12-desaturases are pro-R, and that during these desaturation steps α-secondary kinetic isotope effects occur at the 9 and 12 positions and not at the 10 and 13 positions.

Recent advances in the analysis of the site-specific isotopic fractionation of metabolites such as fatty acids using anisotropic natural-abundance 2H NMR spectroscopy: application to conjugated linolenic methyl esters

AbstractThe full elucidation of the enzymatic mechanisms leading to polyunsaturated ω-3 to ω-5 fatty acids (PUFAs) occurring in plants or microorganisms by analyzing their site-specific isotopic fractionation profiles is a challenging task. Isotropic SNIF-NMR® method is an historical, powerful tool for the determination of (2H/1H) ratios. However, the absence of accessible isotopic data on the enantiotopic hydrogen sites (CH2 groups) prevents the study of the enzymatic reaction stereoselectivity. Natural-abundance deuterium (NAD) 2D NMR experiment using chiral liquid crystals (CLC) as solvent is a new tool in this field, overcoming this limitation. In this work, we have explored various possibilities for optimizing the enantio-discrimination properties of CLC by changing the nature of the polypeptide and/or increasing the polarity of the organic co-solvents. We report also the first applications of TMU as co-solvent for preparing enantio-discriminating, homogenous polypeptide mesophases. The various experimental NAD NMR results recorded at an optimal sample temperature are discussed and compared in terms of number of discriminated 2H sites and magnitude of spectral separation for different PUFAs such as the linoleic and linolenic acids. The comparison of all NMR results shows that optimal results are obtained when CLC mixtures made of poly-γ-benzyl-l-glutamate (PBLG) and high polarity co-solvents are used. As new challenging examples of applications, we report the preliminary analytical results obtained from two ω-5 conjugated linolenic acids: the α-eleostearic acid (9Z, 11E, 13E) and the punicic acid (9Z, 11E, 13Z). NMR data are discussed in terms of molecular orientational ordering parameters and isotopic distribution. FigureThe new challenges of the site-specific isotopic fractionation analysis of fatty acids using the natural-abundance deuterium NMR in polypeptide aligned media

Combined analysis of C-18 unsaturated fatty acids using natural abundance deuterium 2D NMR spectroscopy in chiral oriented solvents.

The quantitative determination of isotopic (2H/1H)i ratios at natural abundance using the SNIF-NMR protocol is a well-known method for understanding the enzymatic biosynthesis of metabolites. However, this approach is not always successful for analyzing large solutes and, specifically, is inadequate for prochiral molecules such as complete essential unsaturated fatty acids. To overcome these analytical limitations, we use the natural abundance deuterium 2D NMR (NAD 2D NMR) spectroscopy on solutes embedded in polypeptide chiral liquid crystals. This approach, recently explored for measuring (2H/1H)i ratios of small analytes (Lesot, P.; Aroulanda, C.; Billault, I. Anal. Chem. 2004, 76, 2827-2835), is a powerful way to separate the 2H signals of all nonequivalent enantioisotopomers on the basis both of the 2H quadrupolar interactions and of the 2H chemical shift. Two significant advances over our previous work are presented here and allow the complete isotopic analysis of four mono- and polyunsaturated fatty acid methyl esters: methyl oleate (1), methyl linoleate (2), methyl linolenate (3), and methyl vernoleate (4). The first consists of using NMR spectrometers operating at higher magnetic field strength (14.1 T) and equipped with a selective cryoprobe optimized for deuterium nuclei. The second is the development of Q-COSY Fz 2D NMR experiments able to produce phased 2H 2D maps after a double Fourier transformation. This combination of modern hardware and efficient NMR sequences provides a unique tool to analyze the (2H/1H)i ratios of large prochiral molecules (C-18) dissolved in organic solutions of poly(gamma-benzyl-L-glutamate) and requires smaller amounts of solute than previous study on fatty acids. For each compound (1-4), all 2H quadrupolar doublets visible in the 2D spectra have been assigned on the basis of 2H chemical shifts, isotopic data obtained from isotropic quantitative NAD NMR, and by an interspectral comparison of the anisotropic NAD spectra of four fatty acids. The NMR results are discussed in terms of (2H/1H)i isotopic distribution and molecular orientation in the mesophase. For the first time, we show that the investigation of natural isotopic fractionation of complete fatty acids is possible without the need of chemical modifications, hence providing an alternative method to probe the mechanisms of enzymes implied in the biosynthetic pathway of unsaturated fatty acids.

Natural Deuterium Distribution in Long-Chain Fatty Acids Is Nonstatistical: A Site-Specific Study by Quantitative 2H NMR Spectroscopy

Quantitative 2H NMR spectroscopy has been used to study the site‐specific natural occurrence of 2H in common unsaturated fatty acids. A marked nonstatistical isotopic distribution of 2H is observed in both methyl oleate and methyl linoleate. By chemical modification, the internal isotopic 2H signatures of these products have been partially accessed. Notably, it can be seen that (1) the sites of desaturation show a strong impoverishment at only one ethylenic position of each desaturation; (2) the level of impoverishment reflects the source of the hydrogen atoms present; and (3) a gradient of 2H content occurs along the chain. These effects can in part be related to the mechanisms proposed for the enzymes responsible for the various steps of biosynthesis of unsaturated fatty acids in plants.

Natural deuterium distribution in fatty acids isolated from peanut seed oil: a site-specific study by quantitative 2H NMR spectroscopy.

Quantitative 2H NMR spectroscopy has been used to measure the distribution of deuterium at natural abundance in long‐chain fatty acids extracted from the same vegetable oil. Peanut seed oil was selected, due to its suitable oleic and linoleic acid content. The methyl esters of the fatty acids were prepared by transesterification and isolated by modified argentation column chromatography on silica. In order to measure the natural isotopic fractionation of deuterium (D) at the maximum number of positions, the purified methyl oleate and methyl linoleate were chemically cleaved and the (D/H)i values determined by quantitative 2H NMR spectroscopy. It was thus possible to demonstrate that fractionation in deuterium occurs during the desaturation of oleate to linoleate. Furthermore, the previously observed distribution of deuterium at the sites of desaturation is confirmed, as is the alternating pattern of (D/H)i, which relates to the origin of the pertinent hydrogen atoms. The data obtained are discussed in terms of the kinetic isotopic effects intrinsic to the enzymes—synthetases and desaturases—involved in the biosynthesis of fatty acids.

Natural deuterium distribution in branched-chain medium-length fatty acids is nonstatistical: a site-specific study by quantitative 2H NMR spectroscopy of the fatty acids of capsaicinoids.

Quantitative 2H NMR spectroscopy has been used to determine the natural abundance site‐specific 2H isotopic content of 6,7‐dihydrocapsaicin (1) and capsaicin (2). Prior to analysis, the fatty acyl moieties were released as methyl 8‐methylnonanoate (3) and methyl E‐8‐methylnon‐6‐enoate (4), respectively. A marked and similar nonstatisitical isotopic distribution of 2H is observed for both fatty acids. Notably, it can be seen that: 1) the isobutyl portion of 3 is more impoverished in 2H than the methylenic portion; 2) the isobutyl portion of 4 is more impoverished than that of 3; 3) an alternating pattern occurs in the (2H/1H)i between the C3 to C7 positions; and 4) the ethylenic hydrogens at C6 and C7 of 4 are, respectively, impoverished and unchanged relative to these positions in 3. These observations are compatible with the proposed biosynthetic origins of the different parts of 1 and 2, and with the view that 1 is a proximal precursor of 2. Furthermore, it can be suggested that, firstly, the hydrogen atoms at C3 to C7 originate alternatively from the substrate and from the environment and, secondly, that the Δ6‐E desaturation is introduced by a mechanism closely mimicking that of the Z desaturation of higher plants.

Analysis of NAD 2D-NMR Spectra of Saturated Fatty Acids in Polypeptide Aligning Media by Experimental and Modeling Approaches

The overall and detailed elucidation (including the stereochemical aspects) of enzymatic mechanisms requires the access to all reliable information related to the natural isotopic fractionation of both precursors and products. Natural abundance deuterium (NAD) 2D-NMR experiments in polypeptide liquid-crystalline solutions are a new, suitable tool for analyzing site-specific deuterium isotopic distribution profiles. Here this method is utilized for analyzing saturated C14 to C18 fatty acid methyl esters (FAMEs), which are challenging because of the crowding of signals in a narrow spectral region. Experiments in achiral and chiral oriented solutions were performed. The spectral analysis is supplemented by the theoretical prediction of quadrupolar splittings as a function of the geometry and flexibility of FAMEs, based on a novel computational methodology. This allows us to confirm the spectral assignments, while providing insights into the mechanism of solute ordering in liquid-crystalline polypeptide solutions. This is found to be dominated by steric repulsions between FAMEs and polypeptides.

Quantitative 2H NMR analysis of deuterium distribution in petroselinic acid isolated from parsley seed.

We have previously demonstrated that 2H distribution in fatty acids is non-statistical and can be related to isotopic discrimination during chain extension and desaturation. Petroselinic acid (C18:1 Delta(6)), a fatty acid characteristic of the seeds of the Apiaceae, has been shown to be biosynthesised from palmitoyl-ACP (C16:0) by two steps, catalysed by a dedicated Delta(4)-desaturase and an elongase. We have now demonstrated that the isotopic profile resulting from this pathway is similar to that of the classical plant fatty acid pathway but that the isotopic fingerprint from both the desaturase and elongase steps show important differences relative to oleic and linoleic acid biosynthesis.