Françoise Mabon

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.

Rate of carbon dioxide production and energy expenditure in fed and food-deprived adult dogs determined by indirect calorimetry and isotopic methods

OBJECTIVE To evaluate energy expenditure (EE) in dogs by estimating rate of CO2 production (rCO2). ANIMALS 15 Beagles. PROCEDURE Food was withheld for 24 hours, and all dogs received an IV infusion of 13C sodium bicarbonate for 8 hours. Breath samples were collected before infusion and at 30-minute intervals from 4 to 8 hours, and 13C enrichment in breath CO2 was measured, using gas chromatography-isotopic ratio mass spectrometry. Food was withheld from 6 dogs, and rCO2 and O2 consumption were measured, using a conventional indirect calorimeter. The CO2 production and O2 consumption were measured by use of indirect calorimetry in 6 other fed dogs that were injected with 2H2O and H2(18)O. Blood samples were collected before tracer injection, 4 hours later, and on days 4, 7, and 11. Deuterium and 18O enrichments in plasma water were determined. RESULTS Mean rCO2 measured by indirect calorimetry was 516 +/- 34 and 410 +/- 16 micromol/kg(0.75)/min in 6 fed and 6 food-deprived dogs, respectively. The rCO2 calculated from 13C-bicarbonate dilution was 482 +/- 30 micromol/kg(0.75)/min. Mean rCO2 determined by use of the double-labeled water method was 1,036 +/- 46 mmol/kg(0.75)/d. Mean energy expenditure calculated from rCO2 determined by infusion of 13C bicarbonate, indirect calorimetry in fed and food-deprived dogs, and infusion of double-labeled water was 386 +/- 39, 379 +/- 25, 338 +/- 14, and 552 +/- 25 kJ/kg(0.75)/d, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Energy expenditure calculated by indirect calorimetry in unfed dogs can be considered representative of basal metabolic rate.

High‐resolution 1H and 2H NMR spectroscopy of pure essential fatty acids for plants and animals

High‐resolution 1H and 2H NMR spectroscopy were used to study the most important saturated (C14:0, C16:0, C18:0), monounsaturated (C16:1n‐7, C18:1n‐7, C18:1n‐9, C20:1n‐9, C22:1n‐9) and polyunsaturated [C18:3n‐3, C20:5n‐3 (EPA), C22:6n‐3 (DHA)] fatty acids which may be found in fish lipids, in order to prepare the tools for a subsequent study of the recognition of the origin of fish oils by isotopic analysis. The interpretation of the 1H and 2H NMR spectra of the different acids is reported. Additionally, the overall D/H and the 13C/12C isotope ratios are given. The overall D/H ratio ranges from 114.0 to 137.2 ppm/V.SMOW (V.SMOW=water standard) and the 13C/12C ratio from ‐23.5 to ‐33.2‰. From the 2H NMR spectra, an investigation of the internal deuterium distribution in the saturated and mono‐ and polyunsaturated fatty acids was made. The deuterium distribution can be given for nearly all the chemical sites, except for the different double bonds, in EPA and DHA. The precision of the measurements was of the order of 0.5–4% depending on the nature of the cluster considered.

Biosynthesis of nornicotine in root cultures of Nicotiana alata does not involve oxidation at C-5′ of nicotine

Abstract The mechanism of the demethylation of nicotine [3-(1-methyl-2-pyrrolidinyl)pyridine] to nornicotine [3-(2-pyrrolidinyl)pyridine] has been studied in root cultures of Nicotiana alata using nicotine selectively labelled with 2 H. Administered [4′,4′,5′,5′- 2 H 4 ]nicotine was effectively incorporated into [4′,4′,5′,5′- 2 H 4 ]nornicotine without a significant alteration in the molar fractions of 2 H at the four substituted positions, as determined by quantitative 2 H NMR. Incorporation into cotinine- d 2 , and myosmine- d 4 was also observed. In contrast, no incorporation into either nornicotine or nicotine was found when [4′,4′- 2 H 2 ]cotinine was administered. Taken together, these findings provide direct evidence that neither of the protons on C-5′ of nicotine takes part in the demethylation reaction.

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.

A relation between the site-specific natural deuterium contents in α-pinenes and their optical activity

Abstract Correlations are observed between the optical rotation of α-pinenes and site- specific isotope ratios, (D/H) i , determined by natural abundance deuterium NMR (SNIF-NMR). The optical purity can therefore be estimated by NMR without the need for spectral separation of the enantiomers.

Natural selective 2H labelling applied to the study of chemical mechanisms; labelling without enrichment

It is shown that the internal distribution deuterium may considerably differ from the statistical distribution and that this property is exploitable, by quantitative 2H n.m.r. measurements, for investigating reaction mechanisms using isotopic labeling without the need for selective enrichment.

Etude par RMN 1H et 13C, de la stereochimie de [3] ferrocenophanols α,γ-disubstitues et de l'isomerisation conformationnelle des cations formes au cours de substitutions nucleophiles

Abstract The analysis of the 1 H and 13 C spectra combined with INDOR experiments has allowed the identification of the predominant conformations of α,γ-disubstituted [3] ferrocenophanols. In trifluoracetic acid the hydroxylic group leaves the molecule from the less hindered side and the corresponding cation is obtained. The cation I′ resulting from the syn alcool is thermodynamically unstable and is transformed slowly into its stable conformational isomer I″. The stereochemistry of the cations was identified through analysis of the bridge group spectra and kinetics were quantitatively studied.