Genome mining in Streptomyces coelicolor: molecular cloning and characterization of a new sesquiterpene synthase

The terpene synthase encoded by the SCO5222 (SC7E4.19) gene of Streptomyces coelicolor was cloned by PCR and expressed in Escherichia coli as an N-terminal-His6-tag protein. Incubation of the recombinant protein, SCO5222p, with farnesyl diphosphate (1, FPP) in the presence of Mg(II) gave a new sesquiterpene, (+)-epi-isozizaene (2), whose structure and stereochemistry were determined by a combination of 1H, 13C, COSY, HMQC, HMBC, and NOESY NMR. The steady-state kinetic parameters were kcat 0.049 +/- 0.001 s-1 and a Km (FPP) of 147 +/- 14 nM. Individual incubations of recombinant epi-isozizaene synthase with [1,1-2H2]FPP (1a), (1R)-[1-2H]-FPP (1b), and (1S)-[1-2H]-FPP (1c) and NMR analysis of the resulting deuterated epi-isozizaenes supported an isomerization-cyclization-rearrangement mechanism involving the intermediacy of (3R)-nerolidyl diphosphate (3).     

Biosynthesis of irregular diterpenes in Anisotome lyallii by head-to-head coupling of geranyl diphosphate

Anisotomenes, bicyclic irregular diterpenes found in the genus Anisotome(family Apiaceae), are shown to be products of a unique biosynthetic pathway, involving head-to-head coupling of two geranyl diphosphate units. (13)C labelling studies with [1-(13)C]-glucose on plantlets of A. lyallii also revealed that the isoprene subunits were formed via the MEP pathway. The in vitro-cultured plant material also yielded a new irregular, linear diterpene alcohol, that shares the same biosynthetic pathway.     

Farnesyl diphosphate synthase: the art of compromise between substrate selectivity and stereoselectivity

Farnesyl diphosphate (FPP) synthase catalyzes the consecutive head-to-tail condensations of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) and geranyl diphosphate (GPP, C10) to give (E,E)-FPP (C15). The enzyme belongs to a genetically distinct family of chain elongation enzymes that install E-double bonds during each addition of a five-carbon isoprene unit. Analysis of the C10 and C15 products from incubations with avian FPP synthase reveals that small amounts of neryl diphosphate (Z-C10) and (Z,E)-FPP are formed along with the E-isomers during the C5 --> C10 and C10 --> C15 reactions. Similar results were obtained for FPP synthase from Escherichia coli, Artemisia tridentata (sage brush), Pyrococcus furiosus, and Methanobacter thermautotrophicus and for GPP and FPP synthesized in vivo by E. coli FPP synthase. When (R)-[2-2H]IPP was a substrate for chain elongation, no deuterium was found in the chain elongation products. In contrast, the deuterium in (S)-[2-2H]IPP was incorporated into all of the products. Thus, the pro-R hydrogen at C2 of IPP is lost when the E- and Z-double bond isomers are formed. The synthesis of Z-double bond isomers by FPP synthase during chain elongation is unexpected for a highly evolved enzyme and probably reflects a compromise between optimizing double bond stereoselectivity and the need to exclude DMAPP from the IPP binding site.     

An Unusual Skeletal Rearrangement in the Biosynthesis of the Sesquiterpene Trichobrasilenol from Trichoderma

The skeletons of some classes of terpenoids are unusual in that they contain a larger number of Me groups (or their biosynthetic equivalents such as olefinic methylene groups, hydroxymethyl groups, aldehydes, or carboxylic acids and their derivatives) than provided by their oligoprenyl diphosphate precursor. This is sometimes the result of an oxidative ring‐opening reaction at a terpene‐cyclase‐derived molecule containing the regular number of Me group equivalents, as observed for picrotoxan sesquiterpenes. In this study a sesquiterpene cyclase from Trichoderma spp. is described that can convert farnesyl diphosphate (FPP) directly via a remarkable skeletal rearrangement into trichobrasilenol, a new brasilane sesquiterpene with one additional Me group equivalent compared to FPP. A mechanistic hypothesis for the formation of the brasilane skeleton is supported by extensive isotopic labelling studies.     

Genome mining in Streptomyces clavuligerus: expression and biochemical characterization of two new cryptic sesquiterpene synthases

Two presumptive terpene synthases of unknown biochemical function encoded by the sscg_02150 and sscg_03688 genes of Streptomyces clavuligerus ATCC 27074 were individually expressed in Escherichia coli as N-terminal-His?-tag proteins, using codon-optimized synthetic genes. Incubation of recombinant SSCG_02150 with farnesyl diphosphate (1, FPP) gave (-)-δ-cadinene (2) while recombinant SSCG_03688 converted FPP to (+)-T-muurolol (3). Individual incubations of (-)-δ-cadinene synthase with [1,1-2H?]FPP (1a), (1S)-[1-2H]-FPP (1b), and (1R)-[1-2H]-FPP (1c) and NMR analysis of the resulting samples of deuterated (-)-δ-cadinene supported a cyclization mechanism involving the intermediacy of nerolidyl diphosphate (4) leading to a helminthogermacradienyl cation 5. Following a 1,3-hydride shift of the original H-1(si) of FPP, cyclization and deprotonation will give (-)-δ-cadinene. Similar incubations with recombinant SSCG_03688 supported an analogous mechanism for the formation of (+)-T-muurolol (3), also involving a 1,3-hydride shift of the original H-1(si) of FPP.     

Geosmin biosynthesis. Streptomyces coelicolor germacradienol/germacrene D synthase converts farnesyl diphosphate to geosmin

Geosmin is responsible for the characteristic odor of moist soil. Incubation of recombinant germacradienol synthase, encoded by the SCO6073 (SC9B1.20) gene of the Gram-positive soil bacterium Streptomyces coelicolor, with farnesyl diphosphate (2, FPP) in the presence of Mg2+ gave a mixture of (4S,7R)-germacra-1(10)E,5E-diene-11-ol (3) (74%), (-)-(7S)-germacrene D (4) (10%), geosmin (1) (13%), and a hydrocarbon, tentatively assigned the structure of octalin 5 (3%). Individual incubations of recombinant germacradienol synthase with [1,1-2H2]FPP (2a), (1R)-[1-2H]-FPP (2b), and (1S)-[1-2H]-FPP (2c), as well as with FPP (2) in D2O, and GC-MS analysis of the resulting deuterated products supported a mechanism of geosmin formation involving proton-initiated cyclization and retro-Prins fragmentation of the initially formed germacradienol to give intermediate 5, followed by protonation of 5, 1,2-hydride shift, and capture of water.     

Mechanism and stereochemistry of the germacradienol/germacrene D synthase of Streptomyces coelicolor A3(2)

Incubation of farnesyl diphosphate (1, FPP) with recombinant germacradienol synthase from Streptomyces coelicolor A3(2) gave, in addition to (4S,7R)-germacra-1(10)E,5E-diene-11-ol (2), 15% of (-)-germacrene D (5). Incubations of [1,1-2H2]FPP (1a), (1R)-[1-2H]FPP (1b), and (1S)-[1-2H]FPP (1c) with germacradienol/germacrene D synthase and analysis of the resulting samples of germacradienol (2) and germacrene D (5) by a combination of 1H, 2H, and 13C NMR and mass spectrometry established that it is H-1si of FPP that is lost in the formation of germacradienol (2) and that undergoes 1,3-hydride transfer in the formation of (-)-germacrene D (5). The proportion of the two products was also sensitive to isotopic labeling, with cyclization of (1S)-[1-2H]FPP (1c) giving an increased proportion (35%) of 5. These results could be explained by a mechanism involving partitioning of a common helminthogermacradienyl cation intermediate 7.     

Biosynthetic origin of BE-10988 in Streptomyces sp. BA10988

The biosynthetic origin of the tumor-inhibitory derivative, BE-10988, was studied in Streptomyces sp . BA10988 by retrobiosynthetic NMR analysis using [U-(13)C6]glucose as a precursor. The isotopologue compositions of the indole moieties of BE-10988 and tryptophan were virtually identical. This indicates that tryptophan or a closely related metabolite served as a biosynthetic precursor of BE-10988 in analogy to the biosynthetic pathway of camalexin, a structurally related phytoalexin in Arabidopsis thaliana. Labeling experiments with [U-(13)C8(15)N]indole, L-[ring-(2)H5]tryptophan, or L-[U-(13)C3(15)N]cysteine confirmed this hypothesis. However, transfer of the label from [ring-(2)H5]indole pyruvic acid, but not from the known camalexin precursor, [ring-(2)H5]indole-3-acetaldoxime, showed that plants and bacteria have evolved independent mechanisms of tryptophan modification in the biosynthesis of thiazolylindole derivatives.     

Rapid preparation of isotopolog libraries by in vivo transformation of 1)C-glucose. Studies on 6,7-dimethyl-8-ribityllumazine, a biosynthetic precursor of vitamin B2

An Escherichia coli strain engineered for expression of the ribABGH genes of Bacillus subtilis was shown to produce 100 mg of the riboflavin precursor 6,7-dimethyl-8-ribityllumazine per liter of minimal medium. Growth of the recombinant strain in medium supplemented with [U-13C6]glucose and/or 15NH4Cl as single sources of carbon and/or nitrogen afforded 6,7-dimethyl-8-ribityllumazine universally labeled with 13C and/or 15N. The yield of [U-13C13]-6,7-dimethyl-8-ribityllumazine based on [U-13C6]glucose was 25 mg/g. Fermentation with [1-13C1]-, [2-13C1]-, or [3-13C1]glucose afforded mixtures of 6,7-dimethyl-8-ribityllumazine isotopologs, predominantly with 13C enrichment of single carbon atoms. The isotope-labeled samples enabled a comprehensive NMR analysis of 6,7-dimethyl-8-ribityllumazine. Isotopolog libraries of a wide variety of microbial metabolites can be produced by the same experimental approach.     

Structures and Biosynthesis of Corvol Ethers—Sesquiterpenes from the Actinomycete Kitasatospora setae

Here we present the functional characterization of a sesquiterpene cyclase from Kitasatospora setae. The enzyme converts the sesquiterpene precursor farnesyl diphosphate (FPP) into two previously unknown and unstable sesquiterpene ethers for which we propose the trivial names corvol ethers A and B. Both compounds were purified and their structures were determined by one‐ and two‐dimensional NMR spectroscopy. A biosynthetic mechanism for the FPP cyclization by the corvol ether synthase was proposed. The results from the incubation experiments of the corvol ether synthase with isotopically labeled precursors were in line with this mechanism, while alternative mechanisms could clearly be ruled out.     

Microdetermination of glucose content of plasma and its isotopic enrichment using capillary gas chromatography/ammonia chemical-ionization mass spectrometry

A new sensitive and precise method for the determination of the isotopic enrichment of [6,6-D2]glucose and concentration of glucose in plasma microsamples (20 microL) has been developed. Glucose was extracted from plasma samples by anion-cation column-exchange with absolute ethanol, derivatized as 1,2:3,5-bis(butylboronate)-6-acetyl-alpha-D-glucofuranose, and analysed by capillary gas chromatography/ammonia chemical-ionization mass spectrometry. This method gives a better reproducibility and precision (variation coefficient below 1%) than methods using isobutane chemical ionization. Stable isotopes are being used increasingly to investigate energy metabolism in vivo. Recent work has involved the development of methodologies, especially mass spectrometry, to perform tracer experiments using the stable isotopes 3H, 13C, or 13N(1-4). Chemical-ionization mass spectrometry is extensively used for the analysis of isotopically labelled amino acids. In neonates and children, "true" glucose production can be measured by the continuous infusion of the stable isotopically labelled tracer 6,6-dideutero-glucose (6,6-D2-glucose), and analytical measurement is performed using gas chromatography/electron-ionization mass spectrometry (GC/EIMS). Herein, we present a new, simple and sensitive method for the determination of the isotopic enrichment of [6,6-D2]glucose and measurement of the concentration of glucose in plasma microsamples (20 microL), based on the use of capillary gas-chromatography/ammonia chemical-ionization mass spectrometry of 1,2:3,5-bis(butylboronate)-6-acetyl-alpha-D-glucofuranose.     

Biosynthesis of isoprenoids in Escherichia coli: stereochemistry of the reaction catalyzed by farnesyl diphosphate synthase

[formula: see text] Farnesyl diphosphate (FPP) synthase from Escherichia coli catalyzes the condensation of isopentenyl diphosphate (IPP) and geranyl diphosphate (GPP) with selective removal of the pro-R hydrogen at C2 of IPP, the same stereochemistry observed for the pig liver, yeast, and avian enzymes.     

On the incorporation of geraniol and farnesol into cantharidin (author's transl)

On the incorporation of geraniol and farnesol into cantharidin Earlier investigations [1] have shown that cantharidin (1) is biosynthesized by the male Lytta vesicatoria L. (Meloidae, Coleoptera) from the common terpenoid precursors mevalonate and farnesol (3) . To prove if geraniol (2) is incorporated via farnesol (3) into cantharidin (1) the following geraniols have been synthesized and injected into either larvae or male adult Lytta vesicatoria, partly in a mixture with synthetic 11′, 12-[³H]-farnesol as an internal standard: 2-[¹⁴C]-, 7-[¹⁴C]-, 7′, 8-[¹⁴C]-, 7′, 8-[³H]-geraniol. Unexpectedly, geraniol (2) was not specifically incorporated into cantharidin (1) perhaps due to its higher toxicity or its faster degradation relative to the other precursors before incorporation. The incorporation of U-[¹⁴C]-leucine, U-[¹⁴C]-isoleucine and 1-[¹⁴C]-glucose into cantharidin (1) via their metabolites is evident by degradation studies, whereas 1-[¹⁴C]- and 2-[¹⁴C]-glycine do not serve as precursors for cantharidin (1) .     

Isolation and identification of impurities in L-696, 229 drug substance

Two low level impurities in 3-[2-(2-benzoxazolyl)ethyl]-5-ethyl-6-methyl-2(1H)-pyridinone drug substance (L-696,229) have been isolated by a combination of preparative HPLC, solid-phase extraction and liquid-liquid extraction. They were identified as 3-[2-(2-benzoxazolyl)ethyl]-5-ethyl-6-(2-phenylethyl)-2(1H)-pyridinone (I) and 6,6'-(2-phenyl-1,3-propanediyl)bis[3-[2-(2-benzoxazolyl)ethyl]-5-ethyl-2(1H)-pyridinone] (II) by mass spectrometry and by their (13)C and (1)H-NMR spectra.     

Late stages in the biosynthesis of abnormal erythrina alkaloids

The incorporation of (±)-, N-norprotosinomenine, N-nor-orientaline, N-nor-reticuline, norlaudanosoline, protosinomenine, and N-[2-(3-hydroxy-4-methoxyphenyl)ethyl]-2-(4-hydroxyphenyl) ethylamine into coccuvine has been studied, and the specific utilisation of the (±)-norprotosinomenine demonstrated. A double labelling experiment with (±)-[1-³H,4'-methoxy-¹⁴C]-N-norprotosinomenine showed that the 4'-O-Me group of the precursor is retained in the bioconversion and the erythrinan ring system is not formed by addition of secondary amino function onto an ortho-quinone system. Feeding of (±)-[1-³H, 7-methoxy-¹⁴C]norprotosinomenine established that O-demethylation is the terminal step in the biosynthesis of coccuvine. Feeding of labelled abnormal Erythrina alkaloids revealed that isococculidine is converted into coccoline via coccuvinine and isococculine into coccolinine via coccuvine.     

Differential EI fragmentation pathways for peracetylated C-glycoside ketones as a consequence of bicyclic ketal ring structures

Several C-glycoside ketones and peracetylated C-glycoside ketones have been synthesized from 13 structurally-diverse aldoses sugars (including isotope labeled [1-(13)C]Glc, [U-(13)C]Glc, and [6, 6'-(2)H(2)]Glc) via an aqueous-based Knoevanagel condensation with aliphatic 1,3-diketones. Sodium adduct molecular ions observed by MALDI-TOF MS confirmed that the reactions are essentially quantitative, and that the acetylation products are the expected peracetylated C-glycoside ketones, rather than cyclized ketofurans. Analysis of the peracetylated C-glycoside ketones by gas chromatography-EI-MS show characteristic fragment ions that have been assigned to four distinct fragmentation pathways. Peracetylated aldohexose-, aldopentose-, and 6-deoxyaldohexose-C-glycoside ketones fragment via gas phase furanoid intermediates. These data, and DFT calculations, indicate that the furanoid intermediates arise because the peracetylated C-glycoside ketones adopt a bicyclic structure containing a 5-member ketal ring. This ketal ring is the precursor of the furanoid rings in the gas phase. The 2-deoxyhexose-C-glycoside ketones are unable to form an intramolecular 2-ketal bond, and therefore undergo ion fragmentations via nonfuranoid pathways.     

Formation of intramolecular rings in ferramonocarbollide complexes

Addition of PPh 2Cl and Tl[PF 6] to CH 2Cl 2 solutions of [N(PPh 3) 2][6,6,6-(CO) 3- closo-6,1-FeCB 8H 9] ( 1) affords the isomeric B-substituted species [6,6,6-(CO) 3- n-(PHPh 2)- closo-6,1-FeCB 8H 8] [ n = 7 ( 2a) or 10 ( 2b)]. Deprotonation (NaH) of the phosphine ligand in 2a, with subsequent addition of [IrCl(CO)(PPh 3) 2] and Tl[PF 6], yields the neutral, zwitterionic complex [6,6,6-(CO) 3-4,7-mu-{Ir(H)(CO)(PPh 3) 2PPh 2}- closo-6,1-FeCB 8H 7] ( 3), which contains a B-P-Ir- B ring. Alternatively, deprotonation using NEt 3, followed by addition of HC[triple bond]CCH 2Br, affords [6,6,6-(CO) 3-7-(PPh 2CCMe)- closo-6,1-FeCB 8H 8] ( 4). Addition of [Co 2(CO) 8] to CH 2Cl 2 solutions of the latter gives [6,6,6-(CO) 3-7-(PPh 2-{(mu-eta (2):eta (2)-CCMe)Co 2(CO) 6})- closo-6,1-FeCB 8H 8] ( 5), which contains a {C 2Co 2} tetrahedron. In the absence of added substrates, deprotonation of the PHPh 2 group in compounds 2, followed by reaction of the resulting anions with CH 2Cl 2 solvent, affords [6,6,6-(CO) 3- n-(PPh 2CH 2Cl)- closo-6,1-FeCB 8H 8] [ n = 7 ( 6a) or 10 ( 6b)] plus [6,6-(CO) 2-6,7-mu-{PPh 2CH 2PPh 2}- closo-6,1-FeCB 8H 8] ( 7, formed from 2a), of which the latter species possesses an intramolecular B-P-C-P- Fe ring. Addition of Me 3NO to CH 2Cl 2 solutions of 2a causes loss of an Fe-bound CO ligand and formation of [6,6-(CO) 2-6,7-mu-{NMe 2CH 2PPh 2}- closo-6,1-FeCB 8H 8] ( 8), which incorporates a B-P-C-N- Fe ring. A similar reaction in the presence of ligands L yields [6,6-(CO) 2-6-L-7-(PPh 2CH 2Cl)- closo-6,1-FeCB 8H 8] [L = PEt 3 ( 9) or CNBu (t) ( 10)], in addition to 8.     

Conformational equilibrium isotope effects in glucose by (13)C NMR spectroscopy and computational studies.

Anomeric equilibrium isotope effects for dissolved sugars are required preludes to understanding isotope effects for these molecules bound to enzymes. This paper presents a full molecule study of the alpha- and beta-anomeric forms of D-glucopyranose in water using deuterium conformational equilibrium isotope effects (CEIE). Using 1D (13)C NMR, we have found deuterium isotope effects of 1.043 +/- 0.004, 1.027 +/- 0.005, 1.027 +/- 0.004, 1.001 +/- 0.003, 1.036 +/- 0.004, and 0.998 +/- 0.004 on the equilibrium constant, (H/D)K(beta/alpha), in [1-(2)H]-, [2-(2)H]-, [3-(2)H]-, [4-(2)H]-, [5-(2)H]-, and [6,6'-(2)H(2)]-labeled sugars, respectively. A computational study of the anomeric equilibrium in glucose using semiempirical and ab initio methods yields values that correlate well with experiment. Natural bond orbital (NBO) analysis of glucose and dihedral rotational equilibrium isotope effects in 2-propanol strongly imply a hyperconjugative mechanism for the isotope effects at H1 and H2. We conclude that the isotope effect at H1 is due to n(p) --> sigma* hyperconjugative transfer from O5 to the axial C1--H1 bond in beta-glucose, while this transfer makes no contribution to the isotope effect at H5. The isotope effect at H2 is due to rotational restriction of OH2 at 160 degrees in the alpha form and 60 degrees in the beta-sugar, with concomitant differences in n --> sigma* hyperconjugative transfer from O2 to CH2. The isotope effects on H3 and H5 result primarily from syn-diaxial steric repulsion between these and the axial anomeric hydroxyl oxygen in alpha-glucose. Therefore, intramolecular effects play an important role in isotopic perturbation of the anomeric equilibrium. The possible role of intermolecular effects is discussed in the context of recent molecular dynamics studies on aqueous glucose.     

Probing lactate dehydrogenase activity in tumors by measuring hydrogen/deuterium exchange in hyperpolarized l-[1-(13)C,U-(2)H]lactate

(13)C magnetic resonance spectroscopy and spectroscopic imaging measurements of hyperpolarized (13)C label exchange between exogenously administered [1-(13)C]pyruvate and endogenous lactate, catalyzed by lactate dehydrogenase (LDH), has proved to be a powerful approach for probing tissue metabolism in vivo. This experiment has clinical potential, particularly in oncology, where it could be used to assess tumor grade and response to treatment. A limitation of the method is that pyruvate must be administered in vivo at supra-physiological concentrations. This problem can be avoided by using hyperpolarized [1-(13)C]lactate, which can be used at physiological concentrations. However, sensitivity is limited in this case by the relatively small pyruvate pool size, which would result in only low levels of labeled pyruvate being observed even if there was complete label equilibration between the lactate and pyruvate pools. We demonstrate here a more sensitive method in which a doubly labeled lactate species can be used to measure LDH-catalyzed exchange in vivo. In this experiment exchange of the C2 deuterium label between injected hyperpolarized l-[1-(13)C,U-(2)H]lactate and endogenous unlabeled lactate is observed indirectly by monitoring phase modulation of the spin-coupled hyperpolarized (13)C signal in a heteronuclear (1)H/(13)C spin-echo experiment.     

Measuring long-chain acyl-coenzyme A concentrations and enrichment using liquid chromatography/tandem mass spectrometry with selected reaction monitoring

Long-chain acyl-coenzymes A (acyl-CoAs) (LCACoA) are the activated forms of long-chain fatty acids and serve as key lipid metabolites. Excess accumulation of intracellular LCACoA, diacylglycerols (DAGs) and ceramides may create insulin resistance with respect to glucose metabolism. We present a new method to measure LCACoA concentrations and isotopic enrichment of palmitoyl-CoA ([U-(13) C]16-CoA) and oleoyl-CoA ([U-(13) C]18:1-CoA) using ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14-CoA, C16-CoA, C16:1-CoA, C18-CoA, C18:1-CoA, C18:2-CoA, C20-CoA). The molecules are separated on a reversed-phase UPLC column using a binary gradient with ammonium hydroxide (NH(4) OH) in water and NH(4) OH in acetonitrile (ACN). The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate enrichment of palmitoyl-CoA ([U(-13)C]16-CoA) and oleoyl-CoA ([U(-13)C]18:1-CoA) using ultra-performance liquid chromatography/mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14-CoA, C16-CoA, C16:1-CoA, C18-CoA, C18:1-CoA, C18:2-CoA, C20-CoA). The molecules are separated on a reversed-phase UPLC column using a binary gradient with ammonium hydroxide (NH(4) OH) in water and NH(4) OH in acetonitrile. The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupolemass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate and oleate were monitored as [M+2+H](+) and [U(13)C]16-CoA and [U(13)C]18:1-CoA were monitored as [M+16+H](+) and [M+18+H](+), respectively. The method is simple, sensitive and efficient (run time as short as 5 min) and allowed us to measure the concentration and detect enrichment in intramyocellular [U(13) C]16-CoA and [U(13) C]18:1-CoA during a low dose intravenous infusion of [U(13) C]palmitate and [U(13) C]oleate in adults undergoing either a saline control experiment or an insulin/glucose infusion experiment. This technique should allow investigators to measure the trafficking of extracellular fatty acids to the intracellular LCACoA pool.