We compiled published data on hydrogen isotope values for leaf wax n-alkanes (C 27 , C 29 ,

Quantitative (2)H NMR spectroscopy has been used to study the site-specific natural occurrence of (2)H in common unsaturated fatty acids. A marked nonstatistical isotopic distribution of (2)H is observed in both methyl oleate and methyl linoleate. By chemical modification, the internal isotopic (2)H 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 (2)H 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.

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.

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 2 H incorporation was determined by quantitative 2 H-{ 1 H} NMR in isotropic and chiral oriented solvents. Thus, the individual ( 2 H/ 1 H) i ratio at each pro-R and pro-S hydrogen position of the CH 2 groups along the chain can be analyzed. These values allow the isotope redistribution coefficients (a ij ) 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 CH 2 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 CH 2 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 ⌬ 9 -and ⌬ 12 -desaturases are pro-R, and that during these desaturation steps ␣-secondary kinetic isotope effects occur at the 9 and 12 positions and not at the 10 and 13 positions.Fatty acids are ubiquitous natural products involved in many key biological processes, including acting as components of membranes, as lipophilic modifiers, as fuel stores, and as precursors of intracellular messengers (1-3). Their biosynthesis, which is strictly conserved throughout higher organisms, can be separated into two parts as follows: the formation of the basic C16 unit, palmitoylCoA (C16:1), by the fatty-acid synthase complex (FAS) 2 and the subsequent modification of this chain by a range of elongases, desaturases, conjugases, hydroxylases, and epoxidases. Many of these steps involve stereoselective enzymatic reactions during which hydrogen atoms are inserted or eliminated.We have previously shown that this process leads to a distribution of 2 H in long chain fatty acids that is nonstatistical (4 -7). Thus, isotopic fractionation is seen to be introduced. Two features general to all organisms so far examined can be noted. First, the methylenic groups at even positions tend to be richer than those at odd positions. Second, one of the ethylenic groups present at the positions of desaturation is consistently impoverished relative to the methylenic sites, whereas the other is not.Thus, each hydrogen atom in the final product will have a ( 2 H/ 1 H) ratio that is representative of its initial origin, its passage through the FAS elongation steps, and its potential participation in post-elongation modification of the chain. To date, it has proved possible to exp...

The deuterium/hydrogen (D/H)(i) ratio measurement by quantitative (2)H NMR spectroscopy is a method of choice for the analysis of kinetic isotopic effects associated with enzyme-catalyzed reactions during a biosynthetic pathway. However, the efficiency of the current isotropic (2)H-[(1)H] NMR can be limited by the rather small chemical shift dispersion of deuterium nuclei. In addition, this method does not allow the enantiotopic deuterons in prochiral molecules to be spectrally discriminated, hence precluding the quantification of isotopic fractionation on methylene prostereogenic sites. In this work, we explore another analytical strategy able to circumvent these disadvantages. This approach is based on the use of natural abundance (2)H 2D NMR experiments on solutes embedded in polypeptidic, chiral liquid crystalline solvent. Thus, we show that NMR in these oriented phases is a powerful way to separate deuterium signals on the basis of the quadrupolar interactions, providing a promising alternative to overcrowded (2)H NMR spectra obtained in liquid state. To illustrate our purpose, we have experimentally investigated the case of 1,1'-bis(phenylthio)hexane derived by cleavage from methyl linoleate of safflower. The (2)H NMR results in chiral liquid crystals are presented and discussed. We show, for the first time, that (D/H)(pro-R) and (D/H)(pro-S) can be measured at the same methylene position of a fatty acid chain.

The site-specific natural hydrogen isotope ratios of plant metabolites determined by 2H nuclear magnetic resonance (SNIF-NMR method) can provide powerful criteria for inferring mechanistic and environmental effects on biosynthetic pathways. This work examines the potential of isotopic profiles for the main constituents of carbohydrates, glucose and fructose, to distinguish different photosynthetic pathways. An appropriate analytical strategy, involving three suitable isotopic probes, has been elaborated with a view to measuring simultaneously, in conditions devoid of isotopic perturbations, all (or nearly all) of the carbon-bound hydrogen isotope ratios. It is shown that the type of photosynthetic metabolism, either C3 (sugar beet, orange, and grape), C4 (maize and sugar cane), or CAM (pineapple), and the physiological status of the precursor plant exert strong influences on the deuterium distribution in the sugar molecules. Consequently, this isotopic fingerprint may be a rich source of information for the comparison of mechanisms in metabolic pathways. In addition, it can provide complementary criteria to ethanol as a probe for the origin of sugars.

The 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 ((2)H/(1)H) ratios. However, the absence of accessible isotopic data on the enantiotopic hydrogen sites (CH(2) 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 (2)H 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.

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.