Deuterium-labeled Fatty Acids Research Articles

Hyperspectral CARS microscopy and quantitative unsupervised analysis of deuterated and non-deuterated fatty acid storage in human cells.

Coherent anti-Stokes Raman scattering (CARS) implemented as a vibrational micro-spectroscopy modality eradicates the need for potentially perturbative fluorescent labeling while still providing high-resolution, chemically specific images of biological samples. Isotopic substitution of hydrogen atoms with deuterium introduces minimal change to molecular structures and can be coupled with CARS microscopy to increase chemical contrast. Here, we investigate HeLa cells incubated with non-deuterated or deuterium-labeled fatty acids, using an in-house-developed hyperspectral CARS microscope coupled with an unsupervised quantitative data analysis algorithm, to retrieve Raman susceptibility spectra and concentration maps of chemical components in physically meaningful units. We demonstrate that our unsupervised analysis retrieves the susceptibility spectra of the specific fatty acids, both deuterated and non-deuterated, in good agreement with reference Raman spectra measured in pure lipids. Our analysis, using the cell-silent spectral region, achieved excellent chemical specificity despite having no prior knowledge and considering the complex intracellular environment inside cells. The quantitative capabilities of the analysis allowed us to measure the concentration of deuterated and non-deuterated fatty acids stored within cytosolic lipid droplets over a 24 h period. Finally, we explored the potential use of deuterium-labeled lipid droplets for non-invasive cell tracking, demonstrating an effective application of the technique for distinguishing between cells in a mixed population over a 16 h period. These results further demonstrate the chemically specific capabilities of hyperspectral CARS microscopy to characterize and distinguish specific lipid types inside cells using an unbiased quantitative data analysis methodology.

Deuterated stearic acid uptake and accumulation in lipid droplets of cat oocytes

Fatty acid uptake and accumulation in lipid droplets are essential processes of lipid metabolism. Oocyte in vitro culture in media enriched with fatty acid is used to modify the lipid content and composition, aiming to study the consequences of obesity and enhance cell cryotolerance. We applied Raman spectroscopy and deuterium labeling approach to quantify stearic acid uptake and investigate its incorporation within oocytes. Our data suggest that deuterium labeling does not affect oocyte maturation rates. The efficiency of deuterated stearic acid (dSA) uptake was shown to decrease with the increase of its concentration in culture medium and the duration of in vitro culture. The molar ratio between dSA and bovine serum albumin has no significant effect on the dSA uptake for 200 μM but modifies concentration dependence of the lipid uptake. dSA accumulates in all the lipid droplets inside oocytes. Different lipid droplets within the same oocyte exhibit different concentrations of dSA. The scatter in the dSA concentration in lipid droplets decreases with the culture time. Using dSA as an example, we provide a comprehensive description of how fatty acid concentration, its molar ratio versus bovine serum albumin, and culture time affect the uptake of the fatty acids in oocytes. Raman microspectroscopy of deuterium-labeled fatty acids is a nondestructive tool providing information about fatty acid uptake and heterogeneity of their accumulation between lipid droplets within the single oocyte.

Evaluation of Metabolic Defects in Fatty Acid Oxidation Using Peripheral Blood Mononuclear Cells Loaded with Deuterium-Labeled Fatty Acids

Because tandem mass spectrometry- (MS/MS-) based newborn screening identifies many suspicious cases of fatty acid oxidation and carnitine cycle disorders, a simple, noninvasive test is required to confirm the diagnosis. We have developed a novel method to evaluate the metabolic defects in peripheral blood mononuclear cells loaded with deuterium-labeled fatty acids directly using the ratios of acylcarnitines determined by flow injection MS/MS. We have identified diagnostic indices for the disorders as follows: decreased ratios of d27-C14-acylcarnitine/d31-C16-acylcarnitine and d23-C12-acylcarnitine/d31-C16-acylcarnitine for carnitine palmitoyltransferase-II (CPT-II) deficiency, decreased ratios of d23-C12-acylcarnitine/d27-C14-acylcarnitine for very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, and increased ratios of d29-C16-OH-acylcarnitine/d31-C16-acylcarnitine for trifunctional protein (TFP) deficiency, together with increased ratios of d7-C4-acylcarnitine/d31-C16-acylcarnitine for carnitine palmitoyltransferase-I deficiency. The decreased ratios of d1-acetylcarnitine/d31-C16-acylcarnitine could be indicative of β-oxidation ability in patients with CPT-II, VLCAD, and TFP deficiencies. Overall, our data showed that the present method was valuable for establishing a rapid diagnosis of fatty acid oxidation disorders and carnitine cycle disorders and for complementing gene analysis because our diagnostic indices may overcome the weaknesses of conventional enzyme activity measurements using fibroblasts or mononuclear cells with assumedly uncertain viability.

Human Studies Using Isotope Labeled Fatty Acids: Answered and Unanswered Questions

Human studies using deuterium-labeled fatty acids have answered many questions related to the metabolism and health effects of dietary fats. These studies also raised a number of unanswered questions and unresolved issues. For example, studies with cis and trans positional isomers dispelled concerns and allegations that the isomers in partially hydrogenated fats were poorly absorbed, accumulate in undesirable phospholipid acyl positions, mimic stearic acid and competed with oleic acid. Trans 18:1 isomers were metabolically intermediate between 16:0 and 18:0, so the unanswered question is why are the metabolic properties of trans fatty acids not consistent with their physiological effects? Results from ²H-18:0 studies address questions regarding stearic acid absorption and desaturation. Contrary to accepted dogma, stearic acid was well absorbed and less than 10% was desaturation to oleic acid. The still unanswered question is what is the metabolic basis for why 18:0 is less hypercholesterolemic than other saturated fatty acids? The question of whether humans convert 18:3n-3 to EPA and DHA was investigated by feeding male subjects a mixture of ²H-18:3n-3 and ²H-18:2n-6. The unequivocal answer was that 18:3n-3 is converted to EPA and DHA and the conversions for 18:3n-3 to 20:5n-3 and 18:2n-6 to 20:4n-6 were about equal. A major issue that remains unresolved is the wide variability between studies for the estimated conversion of 18:3n-3 to 20:5n-3 and 22:6n-3. The commercial availability of liquid oils hardened by interesterified with 18:0 has raised the question of whether fatty acids in the sn-2 and sn-1,3 TAG positions are metabolically equivalent. To answer this question, subjects were fed triglycerides containing ²H-16:0 and ²H-18:2n-6 at specific sn-1(3) and sn-2 acyl positions. The result was that dietary fatty acids at the sn-1(3) and sn-2 triacylglycerol positions are essentially metabolically equivalent.

Biosynthesis of unusual moth pheromone components involves two different pathways in the navel orangeworm, Amyelois transitella.

The sex pheromone of the navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), consists of two different types of components, one type including (11Z,13Z)-11,13-hexadecadienal (11Z,13Z-16:Ald) with a terminal functional group containing oxygen, similar to the majority of moth pheromones reported, and another type including the unusual long-chain pentaenes, (3Z,6Z,9Z,12Z,15Z)-3,6,9,12,15-tricosapentaene (3Z,6Z,9Z,12Z,15Z-23:H) and (3Z,6Z,9Z,12Z,15Z)- 3,6,9,12,15-pentacosapentaene (3Z,6Z,9Z,12Z,15Z-25:H). After decapitation of females, the titer of 11Z,13Z-16:Ald in the pheromone gland decreased significantly, whereas the titer of the pentaenes remained unchanged. Injection of a pheromone biosynthesis activating peptide (PBAN) into the abdomens of decapitated females restored the titer of 11Z,13Z-16:Ald and even increased it above that in intact females, whereas the titer of the pentaenes in the pheromone gland was not affected by PBAN injection. In addition to common fatty acids, two likely precursors of 11Z,13Z-16:Ald, i.e., (Z)-11-hexadecenoic and (11Z,13Z)-11,13-hexadecadienoic acid, as well as traces of (Z)-6-hexadecenoic acid, were found in gland extracts. In addition, pheromone gland lipids contained (5Z,8Z,11Z,14Z,17Z)-5,8,11,14,17-icosapentaenoic acid, which also was found in extracts of the rest of the abdomen. Deuterium-labeled fatty acids, (16,16,16-D3)-hexadecanoic acid and (Z)-[13,13,14,14,15,15,16,16,16-D9]-11-hexadecenoic acid, were incorporated into 11Z,13Z-16:Ald after topical application to the sex pheromone gland coupled with abdominal injection of PBAN. Deuterium label was incorporated into the C23 and C25 pentaenes after injection of (9Z,12Z,15Z)- [17,17,18,18,18-D5]-9,12,15-octadecatrienoic acid into 1–2 d old female pupae. These labeling results, in conjunction with the composition of fatty acid intermediates found in pheromone gland extracts, support different pathways leading to the two pheromone components. 11Z,13Z-16:Ald is probably produced in the pheromone gland by Δ11 desaturation of palmitic acid to 11Z-16:Acid followed by a second desaturation to form 11Z,13Z-16:Acid and subsequent reduction and oxidation. The production of 3Z,6Z,9Z,12Z,15Z-23:H and 3Z,6Z,9Z,12Z,15Z-25:H may take place outside the pheromone gland, and appears to start from linolenic acid, which is elongated and desaturated to form (5Z,8Z,11Z,14Z,17Z)-5,8,11,14,17-icosapentaenoic acid, followed by two or three further elongation steps and finally reductive decarboxylation.

Whole body distribution of deuterated linoleic and α-linolenic acids and their metabolites in the rat

Little is known about the uptake or metabolism of essential fatty acids (EFAs) in various mammalian organs. Thus, the distribution of deuterated alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) and their metabolites was studied using a stable isotope tracer technique. Rats were orally administered a single dose of a mixture (20 mg each) of ethyl D5-18:3n-3 and D5-18:2n-6, and 25 tissues per animal were analyzed for D5-labeled PUFAs at 4, 8, 24, 96, 168, 240, 360, and 600 h after dosing. Plasma, stomach, and spleen contained the highest concentrations of labeled precursors at the earliest time points, whereas other internal organs and red blood cells reached their maximal concentrations at 8 h. The time-course data were consistent with liver metabolism of EFAs, but local metabolism in other tissues could not be ruled out. Brain, spinal cord, heart, testis, and eye accumulated docosahexaenoic acid with time, whereas skin accumulated mainly 20:4n-6. On average, approximately 16-18% of the D5-18:3n-3 and D5-18:2n-6 initial dosage was eventually accumulated in tissues, principally in adipose, skin, and muscle. Approximately 6.0% of D5-18:3n-3 and 2.6% of D5-18:2n-6 were elongated/desaturated and stored, mainly in muscle, adipose, and the carcass. The remaining 78% of both precursors was apparently catabolized or excreted.

Rapid measurement of deuterium-labeled long-chain fatty acids in plasma by HPLC-ESI-MS

Imbalanced fatty acid metabolism contributes significantly to the increased incidence of metabolic disorders. Isotope-labeled fatty acids (2H, 13C) provide efficient means to trace fatty acid metabolism in vivo. This study reports a new and rapid method for the quantification of deuterium-labeled fatty acids in plasma by HPLC-MS. The sample preparation protocol developed required only hydrolysis, neutralization, and quenching steps followed by high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis in negative ion mode using single ion monitoring. Deuterium-labeled stearic acid (d7-C18:0) was synthesized to reduce matrix interference observed with d5 analog, which improved the limit of detection (LOD) significantly, depending on the products analyzed. Linearity > 0.999 between the LOD (100 nM) and 30 microM, accuracy > 90%, precision > 88%, and adequate recovery in the dynamic range were obtained for d7-C18:0 and d7-oleic acid (C18:1). Upon oral dosing of d7-C18:0 in rats, the parent compound and its desaturation and beta-oxidation products, d7-C18:1 and d7-C16:0, were circulating with a maximal concentration ranging from 0.6 to 2.2 microM, with significant levels of d7-fatty acids detected for up to 72 h.

Validation of deuterium-labeled fatty acids for the measurement of dietary fat oxidation during physical activity

Measurement of 13C-labeled fatty acid oxidation is hindered by the need for acetate correction, measurement of the rate of CO2 production in a controlled environment, and frequent collection of breath samples. The use of deuterium-labeled fatty acids may overcome these limitations. Herein, d31-palmitate was validated against [1-13C]palmitate during exercise. Thirteen subjects with body mass index of 22.9 +/- 3 kg/m2 and body fat of 19.6 +/- 11% were subjected to 2 or 4 h of exercise at 25% maximum volume oxygen consumption (VO2max). The d31-palmitate and [1-13C] palmitate were given orally in a liquid meal at breakfast. The d3-acetate and [1-13C]acetate were given during another visit for acetate sequestration correction. Recovery of d31-palmitate in urine at 9 h after dose was compared with [1-13C] palmitate recovery in breath. Cumulative recovery of d31-palmitate was 10.6 +/- 3% and that of [1-13C]palmitate was 5.6 +/- 2%. The d3-acetate and [1-13C]acetate recoveries were 85 +/- 4% and 54 +/- 4%, respectively. When [1-13C]acetate recovery was used to correct 13C data, the average recovery differences were 0.4 +/- 3%. Uncorrected d31-palmitate and acetate-corrected [1-13C]palmitate were well correlated (y=0.96x + 0; P <0.0001) when used to measure fatty acid oxidation during exercise. Thus, d31-palmitate can be used in outpatient settings as it eliminates the need for acetate correction and frequent sampling.

Sex pheromone biosynthesis in the pine caterpillar moth, Dendrolimus punctatus (Lepidoptera: Lasiocampidae): pathways leading to Z5-monoene and 5,7-conjugated diene components

Biosynthesis of the sex pheromone components ( Z)-5-dodecenol and ( Z,E)-5,7-dodecadienol in Dendrolimus punctatus was studied by topical application of deuterium-labeled fatty acids to pheromone glands and subsequent analysis of fatty acyl groups and pheromone components by gas chromatography-mass spectrometry. Our studies suggest that both ( Z)-5-dodecenol and ( Z,E)-5,7-dodecadienol can be biosynthetically derived from chain elongation of palmitate to stearate in the gland, and its subsequent Δ11 desaturation to produce ( Z)-11-octadecenoate. After three cycles of 2-carbon chain-shortening, the pheromone glands produce ( Z)-5-dodecenoate, which is then converted to ( Z)-5-dodecenol by reduction. A second Δ11 desaturation of ( Z)-9-hexadecenoate produces ( Z,E)-9,11-hexadecadienoate, which is then chain shortened in two cycles of beta-oxidation and finally converted to ( Z,E)-5,7-dodecadienol by reduction.

A study of resonance electron capture ionization on a quadrupole tandem mass spectrometer

Procedures that allow the realization of resonance electron capture (REC) mode on a commercial triple-quadrupole mass spectrometer, after some simple modifications, are described. REC mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments were performed and spectra for some compounds were recorded. In particular, the charge-remote fragmentation (CRF) spectra of [M − H]− ions of docosanoic and docosenoic acids under low-energy collisionally activated dissociation (CAD) conditions were obtained, and showed that there were no significant differences for [M − H]− ions produced at different resonances (i.e. for [M − H]− ions with different structures). This observation was explained on the basis of results obtained from deuterium-labeled fatty acids, which showed that different CRF ions (but with the same m/z value in the absence of labels) could be produced by different mechanisms, and all of them were obviously realized under CAD conditions that made spectra practically indistinguishable. The other example, which compared the REC-MS/MS spectrum of [M − H]− ions and EI-MS/MS spectrum of M+· ions of daidzein, demonstrated the potential of the REC-MS/MS technique for more complex structure elucidation. Copyright © 2000 John Wiley & Sons, Ltd.

A study of resonance electron capture ionization on a quadrupole tandem mass spectrometer.

Procedures that allow the realization of resonance electron capture (REC) mode on a commercial triple-quadrupole mass spectrometer, after some simple modifications, are described. REC mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments were performed and spectra for some compounds were recorded. In particular, the charge-remote fragmentation (CRF) spectra of [M - H](-) ions of docosanoic and docosenoic acids under low-energy collisionally activated dissociation (CAD) conditions were obtained, and showed that there were no significant differences for [M - H](-) ions produced at different resonances (i.e. for [M - H](-) ions with different structures). This observation was explained on the basis of results obtained from deuterium-labeled fatty acids, which showed that different CRF ions (but with the same m/z value in the absence of labels) could be produced by different mechanisms, and all of them were obviously realized under CAD conditions that made spectra practically indistinguishable. The other example, which compared the REC-MS/MS spectrum of [M - H](-) ions and EI-MS/MS spectrum of M(+.) ions of daidzein, demonstrated the potential of the REC-MS/MS technique for more complex structure elucidation.

Biosynthesis of straight-chain ester volatiles in red delicious and granny smith apples using deuterium-labeled precursors.

Biosynthesis of straight-chain ester volatiles by Granny Smith and Red Delicious apples was investigated using deuterium-labeled fatty acids, C-6 aldehydes, and alcohols. Perdeuterated saturated and monounsaturated fatty acids were metabolized to hexyl-d(11), hexanoate-d(11), heptanoate-d(13), and octanoate-d(15) esters, whereas perdeuterated linoleic acid produced only hexyl-d(11) and hexanoate-d(11) esters. Exposure of fruit to vapors of deuterated 3Z-hexenal, 2E-hexenal, and hexanal identified the following biosynthetic processes: (1) isomerization between 3E, 3Z, and 2E-hexenals; (2) reduction to 3E, 3Z, and 2E-hexenyl esters; (3) reduction to hexanol and hexyl esters; (4) oxidation to hexanoic acid and formation of hexanoate esters; (5) beta-oxidation of hexanoic acid leading to butyl and butanoate esters; and (6) alpha-oxidation of hexanoic acid leading to pentyl and pentanoate esters. Unsaturated straight-chain ester volatiles appear to arise only by the lipoxygenase pathway and may be useful indicators of lipoxygenase activity in fruit.

Measurement of the metabolic interconversion of deuterium‐labeled fatty acids by gas chromatography/mass spectrometry

An analytical method that was developed to analyze deuterium-labeled fatty acids in human blood has been extended to identify labeled fatty acids from C14 to C24 chain length which are formed by metabolic processes such as desaturation, elongation, or shortening of the labeled fatty acids fed. A new computer and a hardware adder have been utilized to assure reliable data acquisition. Relative standard deviations for the analysis of labeled fatty acids were measured at 0.02, 0.03, and 0.04 at the 5%, 1%, and 0.2% levels of the labeled fatty acid methyl esters, respectively. The method makes extensive use of standards and computer processing for accuracy and high productivity. Data from a chylomicron triacylglycerol fraction are included to demonstrate the sensitivity of detection of metabolites formed by desaturation and elongation.

Sex pheromone biosynthetic pathway in Spodoptera littoralis and its activation by a neurohormone.

Deuterium-labeled fatty acids have been used to elucidate the sex pheromone biosynthetic pathway in Spodoptera littoralis. Label from palmitic acid was incorporated during the scotophase into all the pheromone acetates and their corresponding fatty acyl intermediates. (Z,E)-9,11-tetradecadienyl acetate, the major component of the pheromone blend, is synthesized from palmitic acid via tetradecanoic acid, which, by the action of a specific (E)-11 desaturase and subsequently a (Z)-9 desaturase, is converted into (Z,E)-9,11-tetradecadienoate. By further reduction and acetylation, this compound leads to the dienne acetate. Deuterated precursors applied to the pheromone gland during the photophase were also incorporated into the pheromone. The percentage of labeled (Z,E)-9,11-tetradecadienyl acetate relative to natural compound was significantly higher during the light period. Label incorporation from different intermediates into the pheromone was stimulated by injection of brain-subesophageal ganglion extract during the photophase. The influence of the pheromone biosynthesis-activating neuropeptide on the biosynthetic pathway is discussed.

Incorporation of deuterium-labeled fatty acids into human milk, plasma, and lipoprotein phospholipids and cholesteryl esters.

Fatty acid metabolism and the contribution of dietary fatty acids to milk cholesteryl ester (CE) and phospholipid (PL) were investigated in normal lactating mothers. The approach used was to feed mixtures of triglycerides containing deuterium-labeled palmitic acid (16:0-2H2), oleic acid (18:1-2H6), and linoleic acid (18:2-2H4). Milk and plasma samples were collected for 72 hr. Triglyceride (TG), CE, and PL fractions from milk, plasma, and lipoprotein were isolated and analyzed by gas-liquid chromatography and mass spectrometry. Data for the milk CE and PL fractions showed a definite selectivity for incorporation of 16:0-2H2 and 18:1-2H6 relative to 18:2-2H4. Based on the ratios of the deuterated fatty acids incorporated into the milk CE and PL samples, their incorporation times and isotopic enrichment data, it appears that these fatty acids are supplied mainly by the TG derived from chylomicrons and very low density lipoproteins. Plasma and lipoprotein CE data showed a progressive increase in 18:2-2H4 content, with 16:0-2H2 and 18:1-2H4 remaining relatively constant over the collection period. Plasma and lipoprotein PL data showed a higher rate for incorporation of 18:2-2H4 than 16:0-2H2 and 18:1-2H6 over the course of the sampling period. Comparison to previous data from adult males indicates lactation does not have a major effect on the general metabolism of these fatty acids. An increase with time in the isotopic enrichment of 18:2-2H4 in the plasma and lipoprotein CE and PL samples was observed which is consistent with in vitro selectivities reported for lecithin:cholesterol acyltransferase and phosphatidylcholine acyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of deuterium labeled blood lipids by chemical ionization mass spectrometry.

A quantitative analytical method has been developed to analyze methyl esters of blood fatty acids derived from human subjects fed deuterium-labeled fats. The GCMS computer method provides for the analysis of the fed deuterium-labeled fatty acids, the naturally occurring blood fatty acids and new fatty acids formed by chain elongation or shortening of the fed labeled fats. Approximately 20 fatty acids including 16, 17, 18 and 20 carbon chain acids were analyzed with a relative standard deviation of 0.02 at the microgram level and a sensitivity of less than one nanogram. The method uses capillary GC to separate the fatty acid esters and isobutane chemical ionization mass spectrometry with multiple ion detection to determine the isotopic constituents of the GC peaks. The technique provides for the determination of overlapping GC peaks labeled with 2, 4 and 6 deuterium atoms and makes extensive use of computers both for data acquisition and processing.

Steric course of deuterium incorporation from [2-2H2]malonyl-CoA into fatty acids by fatty acid synthetases.

The steric course of the enoyl reduction catalyzed by fatty acid synthetase was investigated with the enzymes from bakers' yeast, rat liver and Brevibacterium ammoniagenes. The non-enzymic hydrogen-deuterium exchange of the methylene group of malonyl-CoA was studied by NMR spectroscopy. The half-life period of the methylene protons was 4.8 min at 37 degrees C and 12.2 min at 23 degrees C at p2H 7.5. Deuterium-labeled fatty acids were synthesized by incubating the synthetases with [2-2H2]malonyl-CoA for 8 min. The deuterium-labeled fatty acids thus produced were extracted and subjected to the action of acyl-CoA oxidase, which had been previously shown to catalyze the anti elimination of the pro-2R and pro-3R hydrogens of acyl-CoA. The resulting products, 2,3-dehydroacyl-CoAs, were methylated and converted to 3-chlorofatty acid methyl esters by addition of hydrogen chloride. The deuterium contents of saturated fatty acids and 3-chlorofatty acids were analyzed by gas chromatography-mass spectrometry. The oleic acid produced by the enzyme from B. ammoniagenes was oxidized to nonanoic acid and azelaic acid. The resulting nonanoic acid was also subjected to the action of acyl-CoA oxidase. The deuterium contents of nonanoic acid and trans-2-nonenoic acid were analyzed. The results suggested that fatty acid synthetase from yeast and rat liver incorporated hydrogen from water via a 2-Si attack and the enzyme from B. ammoniagenes incorporated hydrogen via a 2-Re attack during enoyl reduction. The partial racemization of the C-2 position was observed and the magnitude of this racemization was correlated with the deuterium content of synthesized fatty acids. This phenomenon may be attributed to the non-stereospecific hydrogen exchange of the C-2 position of the elongating acyl residue catalyzed by fatty acid synthetases.

1H NMR Analysis of Deuterated Fatty Acid Methyl Esters

Stearic-9, 10-d2, stearic-9, 9, 10, 10-d4, oleic-9, 10-d2, decanoic-3, 3-d2, lauric-3, 3-d2, myristic-3, 3-d2, palmitic-3, 3-d2, and stearic-3, 3-d2 acids were synthesized, and 1H NMR spectra of the methyl esters of these deuterated acids were measured. The signals due to the protons which were substituted by deuterons disappeared on the 1H NMR spectra of the deuterated acid methyl esters. Estimation of the deuterium-labeled acid content in unlabeled acids was possible by measuring the relative decrease in the integrated areas of the signals for the corresponding methylene or olefinic protons which were replaced with deuterons in the deuterated acids. The shift reagents, Eu (fod) 3 and Pr (fod) 3, were effective for the NMR determination of deuterium-labeled fatty acids. Oleic-9, 10-d2 acid and saturated 3, 3-dideutero acids seem to be useful as nonradioactive tracers in the NMR analysis.

Stereochemical Course of Enoyl Reduction Catalyzed by Fatty Acid Synthetase&lt;xref ref-type="fn" rid="fn1"&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;&lt;subtitle&gt;Stereochemistry of Hydrogen Incorporation from Reduced Pyridine Nucleotide&lt;/subtitle&gt;

The stereochemical course of the enoyl reduction catalyzed by fatty acid synthetase was investigated using the enzymes from rat liver and Brevibacterium ammoniagenes. Deuterium-labeled fatty acids were synthesized by incubating the synthetases with either 4R-[4-2H1]- or 4S-[4-2H1]NADPH. The deuterium-labeled fatty acids thus produced were subjected to the action of a stereospecific enzyme, acyl-CoA oxidase. The deuterium-labeled fatty acids and 2,3-dehydroacyl thioesters, the products of acyl-CoA oxidase, were methylated and analyed for deuterium content by gas chromatography-mass spectrometry. These experiments provided information to determine the configuration at the C-3 position of fatty acids formed by fatty acid synthetases. The results suggested that the stereochemistry of hydrogen (as hydride) incorporation from reduced pyridine nucleotides during enoyl reduction was different between rat liver and B. ammoniagenes synthetases: the enoyl reduction of rat liver enzyme involved the re-attack of hydride and that of B. ammoniagenes enzyme involved the si-attack of hydride.

Measurements of fatty acid synthesis by incorporation of deuterium from deuterated water.

Fatty acid synthesis by perfused livers of rat is measured by using D2O as tracer. The newly synthesized, deuterium-labeled fatty acids are separated from unlabeled fatty acids by gas chromatography using glass capillary columns. The areas of the deuterium-labeled peaks are proportional to the amounts of fatty acids synthesized. The absolute amounts of the individual fatty acids synthesized are obtained by use of an internal standard. The number of deuterium atoms incorporated, as determined by mass spectrometry, is proportional to the D2O concentration of the perfusate, except at very high concentrations of D2O. The relative retention times of the newly synthesized, deuterium-labeled fatty acids are proportional to their deuterium content.