Development of targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry method for acyl-Coenzyme A covering short- to long-chain species in a single analytical run.

Acyl-CoAs play a significant role in numerous physiological and metabolic processes making it important to assess their concentration levels for evaluating metabolic health. Considering the important role of acyl-CoAs, it is crucial to develop an analytical method that can analyze these compounds. Due to the structural variations of acyl-CoAs, multiple analytical methods are often required for comprehensive analysis of these compounds, which increases complexity and the analysis time. In this study, we have developed a method using a zwitterionic HILIC column that enables the coverage of free CoA and short- to long-chain acyl-CoA species in one analytical run. Initially, we developed the method using an LC-QTOF instrument for the identification of acyl-CoA species and optimizing their chromatography. Later, a targeted HILIC-MS/MS method was created in scheduled multiple reaction monitoring mode using a QTRAP MS detector. The performance of the method was evaluated based on various parameters such as linearity, precision, recovery and matrix effect. This method was applied to identify the difference in acyl-CoA profiles in HepG2 cells cultured in different conditions. Our findings revealed an increase in levels of acetyl-CoA, medium- and long-chain acyl-CoA while a decrease in the profiles of free CoA in the starved state, indicating a clear alteration in the fatty acid oxidation process.

Metabolic perturbations mediated by propionyl-CoA accumulation in organs of mouse model of propionic acidemia.

Propionic acidemia (PA) is an autosomal recessive metabolic disorder after gene encoding propionyl-CoA carboxylase, Pcca or Pccb, is mutated. This genetic disorder could develop various complications which are ascribed to dysregulated propionyl-CoA metabolism in organs. However, the effect of attenuated PCC on propionyl-CoA metabolism in different organs remains to be fully understood. We investigated metabolic perturbations in organs of Pcca-/-(A138T) mice (a mouse model of PA) under chow diet and acute administration of [13C3]propionate to gain insight into pathological mechanisms of PA. With chow diet, the metabolic alteration is organ dependent. l-Carnitine reduction induced by propionylcarnitine accumulation only occurs in lung and liver of Pcca-/- (A138T) mice. [13C3]Propionate tracing data demonstrated that PCC activity was dramatically reduced in Pcca-/-(A138T) brain, lung, liver, kidney, and adipose tissues, but not significantly changed in Pcca-/-(A138T) muscles (heart and skeletal muscles) and pancreas, which was largely supported by PCCA expression data. The largest expansion of propionylcarnitine in Pcca-/-(A138T) heart after acute administration of propionate indicated the vulnerability of heart to high circulating propionate. The overwhelming propionate in blood also stimulated ketone production from the increased fatty acid oxidation in Pcca-/-(A138T) liver by lowering malonyl-CoA, which has been observed in cases where metabolic decompensation occurs in PA patients. This work shed light on organ-specific metabolic alternations under varying severities of PA.

Accumulation of Succinyl Coenzyme A Perturbs the Methicillin-Resistant Staphylococcus aureus (MRSA) Succinylome and Is Associated with Increased Susceptibility to Beta-Lactam Antibiotics.

Penicillin binding protein 2a (PBP2a)-dependent resistance to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is regulated by the activity of the tricarboxylic acid (TCA) cycle via a poorly understood mechanism. We report that mutations in sucC and sucD, but not other TCA cycle enzymes, negatively impact ß-lactam resistance without changing PBP2a expression. Increased intracellular levels of succinyl coenzyme A (succinyl-CoA) in the sucC mutant significantly perturbed lysine succinylation in the MRSA proteome. Suppressor mutations in sucA or sucB, responsible for succinyl-CoA biosynthesis, reversed sucC mutant phenotypes. The major autolysin (Atl) was the most succinylated protein in the proteome, and increased Atl succinylation in the sucC mutant was associated with loss of autolytic activity. Although PBP2a and PBP2 were also among the most succinylated proteins in the MRSA proteome, peptidoglycan architecture and cross-linking were unchanged in the sucC mutant. These data reveal that perturbation of the MRSA succinylome impacts two interconnected cell wall phenotypes, leading to repression of autolytic activity and increased susceptibility to ß-lactam antibiotics. IMPORTANCEmecA-dependent methicillin resistance in MRSA is subject to regulation by numerous accessory factors involved in cell wall biosynthesis, nucleotide signaling, and central metabolism. Here, we report that mutations in the TCA cycle gene, sucC, increased susceptibility to ß-lactam antibiotics and was accompanied by significant accumulation of succinyl-CoA, which in turn perturbed lysine succinylation in the proteome. Although cell wall structure and cross-linking were unchanged, significantly increased succinylation of the major autolysin Atl, which was the most succinylated protein in the proteome, was accompanied by near complete repression of autolytic activity. These findings link central metabolism and levels of succinyl-CoA to the regulation of ß-lactam antibiotic resistance in MRSA through succinylome-mediated control of two interlinked cell wall phenotypes. Drug-mediated interference of the SucCD-controlled succinylome may help overcome ß-lactam resistance.

Techniques for the Measurement of Molecular Species of Acyl-CoA in Plants and Microalgae.

The acyl-CoA pool is pivotal in cellular metabolism. The ability to provide reliable estimates of acyl-CoA abundance and distribution between molecular species in plant tissues and microalgae is essential to our understanding of lipid metabolism and acyl exchange. Acyl-CoAs are typically found in low abundance and require specific methods for extraction, separation and detection. Here we describe methods for acyl-CoA extraction and measurement in plant tissues and microalgae, with a focus on liquid chromatography hyphenated to detection techniques including ultraviolet (UV), fluorescence and mass spectrometry (MS). We address the resolution of isobaric species and the selection of columns needed to achieve this, including the analysis of branched chain acyl-CoA thioesters. For MS analyses, we describe diagnostic ions for the identification of acyl-CoA species and how these can be used for both discovery of new species (data dependent acquisition) and routine quantitation (triple quadrupole MS with multiple reaction monitoring).

Suspect screening and targeted analysis of acyl coenzyme A thioesters in bacterial cultures using a high-resolution tribrid mass spectrometer.

Analysis of acyl coenzyme A thioesters (acyl-CoAs) is crucial in the investigation of a wide range of biochemical reactions and paves the way to fully understand the concerned metabolic pathways and their superimposed networks. We developed two methods for suspect screening of acyl-CoAs in bacterial cultures using a high-resolution Orbitrap Fusion tribrid mass spectrometer. The methods rely on specific fragmentation patterns of the target compounds, which originate from the coenzyme A moiety. They make use of the formation of the adenosine 3',5'-diphosphate key fragment (m/z 428.0365) and the neutral loss of the adenosine 3'-phosphate-5'-diphosphate moiety (506.9952) as preselection criteria for the detection of acyl-CoAs. These characteristic ions are generated either by an optimised in-source fragmentation in a full scan Orbitrap measurement or by optimised HCD fragmentation. Additionally, five different filters are included in the design of method. Finally, data-dependent MS/MS experiments on specifically preselected precursor ions are performed. The utility of the methods is demonstrated by analysing cultures of the denitrifying betaproteobacterium "Aromatoleum" sp. strain HxN1 anaerobically grown with hexanoate. We detected 35 acyl-CoAs in total and identified 24 of them by comparison with reference standards, including all 9 acyl-CoA intermediates expected to occur in the degradation pathway of hexanoate. The identification of additional acyl-CoAs provides insight into further metabolic processes occurring in this bacterium. The sensitivity of the method described allows detecting acyl-CoAs present in biological samples in highly variable abundances. Graphical abstract.

Quantification of lactoyl-CoA (lactyl-CoA) by liquid chromatography mass spectrometry in mammalian cells and tissues.

Lysine lactoylation is a recently described protein post-translational modification (PTM). However, the biochemical pathways responsible for this acylation remain unclear. Two metabolite-dependent mechanisms have been proposed: enzymatic histone lysine lactoylation derived from lactoyl-coenzyme A (lactoyl-CoA, also termed lactyl-CoA), and non-enzymatic lysine lactoylation resulting from acyl-transfer via lactoyl-glutathione. While the former has precedent in the form of enzyme-catalysed lysine acylation, the lactoyl-CoA metabolite has not been previously quantified in mammalian systems. Here, we use liquid chromatography-high-resolution mass spectrometry (LC-HRMS) together with a synthetic standard to detect and validate the presence of lactoyl-CoA in cell and tissue samples. Conducting a retrospective analysis of data from previously analysed samples revealed the presence of lactoyl-CoA in diverse cell and tissue contexts. In addition, we describe a biosynthetic route to generate 13C315N1-isotopically labelled lactoyl-CoA, providing a co-eluting internal standard for analysis of this metabolite. We estimate lactoyl-CoA concentrations of 1.14 × 10-8 pmol per cell in cell culture and 0.0172 pmol mg-1 tissue wet weight in mouse heart. These levels are similar to crotonyl-CoA, but between 20 and 350 times lower than predominant acyl-CoAs such as acetyl-, propionyl- and succinyl-CoA. Overall our studies provide the first quantitative measurements of lactoyl-CoA in metazoans, and provide a methodological foundation for the interrogation of this novel metabolite in biology and disease.

Exclusive neuronal detection of KGDHC-specific subunits in the adult human brain cortex despite pancellular protein lysine succinylation.

The ketoglutarate dehydrogenase complex (KGDHC) consists of three different subunits encoded by OGDH (or OGDHL), DLST, and DLD, combined in different stoichiometries. DLD subunit is shared between KGDHC and pyruvate dehydrogenase complex, branched-chain alpha-keto acid dehydrogenase complex, and the glycine cleavage system. Despite KGDHC's implication in neurodegenerative diseases, cell-specific localization of its subunits in the adult human brain has never been investigated. Here, we show that immunoreactivity of all known isoforms of OGDHL, OGDH, and DLST was detected exclusively in neurons of surgical human cortical tissue samples identified by their morphology and visualized by double labeling with fluorescent Nissl, while being absent from glia expressing GFAP, Aldhl1, myelin basic protein, Olig2, or IBA1. In contrast, DLD immunoreactivity was evident in both neurons and glia. Specificity of anti-KGDHC subunits antisera was verified by a decrease in staining of siRNA-treated human cancer cell lines directed against the respective coding gene products; furthermore, immunoreactivity of KGDHC subunits in human fibroblasts co-localized > 99% with mitotracker orange, while western blotting of 63 post-mortem brain samples and purified recombinant proteins afforded further assurance regarding antisera monospecificity. KGDHC subunit immunoreactivity correlated with data from the Human Protein Atlas as well as RNA-Seq data from the Allen Brain Atlas corresponding to genes coding for KGDHC components. Protein lysine succinylation, however, was immunohistochemically evident in all cortical cells; this was unexpected, because this posttranslational modification requires succinyl-CoA, the product of KGDHC. In view of the fact that glia of the human brain cortex lack succinate-CoA ligase, an enzyme producing succinyl-CoA when operating in reverse, protein lysine succinylation in these cells must exclusively rely on propionate and/or ketone body metabolism or some other yet to be discovered pathway encompassing succinyl-CoA.

Targeted Determination of Tissue Energy Status by LC-MS/MS.

Intracellular nucleotides and acyl-CoAs are metabolites that are central to the regulation of energy metabolism. They set the cellular energy charge and redox state, act as allosteric regulators, modulate signaling and transcription factors, and thermodynamically activate substrates for oxidation or biosynthesis. Unfortunately, no method exists to simultaneously quantify these biomolecules in tissue extracts. A simple method was developed using ion-pairing reversed-phase high-performance liquid chromatography-electrospray-ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to simultaneously quantify adenine nucleotides (AMP, ADP, and ATP), pyridine dinucleotides (NAD+ and NADH), and short-chain acyl-CoAs (acetyl, malonyl, succinyl, and propionyl). Quantitative analysis of these molecules in mouse liver was achieved using stable-isotope-labeled internal standards. The method was extensively validated by determining the linearity, accuracy, repeatability, and assay stability. Biological responsiveness was confirmed in assays of liver tissue with variable durations of ischemia, which had substantial effects on tissue energy charge and redox state. We conclude that the method provides a simple, fast, and reliable approach to the simultaneous analysis of nucleotides and short-chain acyl-CoAs.

Comprehensive quantitative analysis of fatty-acyl-Coenzyme A species in biological samples by ultra-high performance liquid chromatography-tandem mass spectrometry harmonizing hydrophilic interaction and reversed phase chromatography.

Fatty acyl-Coenzyme A species (acyl-CoAs) are key biomarkers in studies focusing on cellular energy metabolism. Existing analytical approaches are unable to simultaneously detect the full range of short-, medium-, and long-chain acyl-CoAs, while chromatographic limitations encountered in the analysis of limited amounts of biological samples are an often overlooked problem. We report the systematic development of a UHPLC-ESI-MS/MS method which incorporates reversed phase (RP) and hydrophilic interaction liquid chromatography (HILIC) separations in series, in an automated mode. The protocol outlined encompasses quantification of acyl-CoAs of varying hydrophobicity from C2 to C20 with recoveries in the range of 90-111 % and limit of detection (LOD) 1-5 fmol, which is substantially lower than previously published methods. We demonstrate that the poor chromatographic performance and signal losses in MS detection, typically observed for phosphorylated organic molecules, can be avoided by the incorporation of a 0.1% phosphoric acid wash step between injections. The methodological approach presented here permits a highly reliable, sensitive and precise analysis of small amounts of tissues and cell samples as demonstrated in mouse liver, human hepatic (HepG2) and skeletal muscle (LHCNM2) cells. The considerable improvements discussed pave the way for acyl-CoAs to be incorporated in routine targeted lipid biomarker profile studies.

Comprehensive Analysis of Short-, Medium-, and Long-Chain Acyl-Coenzyme A by Online Two-Dimensional Liquid Chromatography/Mass Spectrometry.

Acyl-coenzyme A (CoA) is a pivotal metabolic intermediate in numerous biological processes. However, comprehensive analysis of acyl-CoAs is still challenging as the properties of acyl-CoAs greatly vary with different carbon chains. Here, we designed a two-dimensional liquid chromatography method coupled with high-resolution mass spectrometry (2D LC/HRMS) to cover all short-, medium-, and long-chain acyl-CoAs within one analytical run. Complex acyl-CoAs were separated into two fractions according to their acyl chains by the first dimensional prefractionation. Then, two fractions containing short-chain acyl-CoAs or medium- and long-chain acyl-CoAs were further separated by the two parallel columns in the second dimension. Nineteen representative standards were chosen to optimize the analytical conditions of the 2D LC/HRMS method. Resolution and sensitivity were demonstrated to be improved greatly, and lowly abundant acyl-CoAs and acyl-CoA isomers could be detected and distinguished. By using the 2D LC/HRMS method, 90 acyl-CoAs (including 21 acyl-dephospho-CoAs) were identified from liver extracts, which indicated that our method was one of the most powerful approaches for obtaining comprehensive profiling of acyl-CoAs so far. The method was further employed in the metabolomics study of malignant glioma cells with an isocitrate dehydrogenase 1 (IDH1) mutation to explore their metabolic differences. A total of 46 acyl-CoAs (including 2 acyl-dephospho-CoAs) were detected, and 12 of them were dysregulated in glioma cells with the IDH1 mutation. These results demonstrated the practicability and the superiority of the established method. Therefore, the 2D LC/HRMS method provides a robust and reproducible approach to the comprehensive analysis of acyl-CoAs in tissues, cells, and other biological samples.

Impact of a High-fat Diet on Tissue Acyl-CoA and Histone Acetylation Levels.

Cellular metabolism dynamically regulates the epigenome via availability of the metabolite substrates of chromatin-modifying enzymes. The impact of diet on the metabolism-epigenome axis is poorly understood but could alter gene expression and influence metabolic health. ATP citrate-lyase produces acetyl-CoA in the nucleus and cytosol and regulates histone acetylation levels in many cell types. Consumption of a high-fat diet (HFD) results in suppression of ATP citrate-lyase levels in tissues such as adipose and liver, but the impact of diet on acetyl-CoA and histone acetylation in these tissues remains unknown. Here we examined the effects of HFD on levels of acyl-CoAs and histone acetylation in mouse white adipose tissue (WAT), liver, and pancreas. We report that mice consuming a HFD have reduced levels of acetyl-CoA and/or acetyl-CoA:CoA ratio in these tissues. In WAT and the pancreas, HFD also impacted the levels of histone acetylation; in particular, histone H3 lysine 23 acetylation was lower in HFD-fed mice. Genetic deletion of Acly in cultured adipocytes also suppressed acetyl-CoA and histone acetylation levels. In the liver, no significant effects on histone acetylation were observed with a HFD despite lower acetyl-CoA levels. Intriguingly, acetylation of several histone lysines correlated with the acetyl-CoA: (iso)butyryl-CoA ratio in liver. Butyryl-CoA and isobutyryl-CoA interacted with the acetyltransferase P300/CBP-associated factor (PCAF) in liver lysates and inhibited its activity in vitro This study thus provides evidence that diet can impact tissue acyl-CoA and histone acetylation levels and that acetyl-CoA abundance correlates with acetylation of specific histone lysines in WAT but not in the liver.

Lipidomics analysis of long-chain fatty acyl-coenzyme As in liver, brain, muscle and adipose tissue by liquid chromatography/tandem mass spectrometry.

RATIONALE: Long-chain fatty acyl-coenzyme As (FA-CoAs) are important bioactive molecules, playing key roles in biosynthesis of fatty acids, membrane trafficking and signal transduction. Development of sensitive analytical methods for profiling theses lipid species in various tissues is critical to understand their biological activity. A high-pressure liquid chromatography/tandem mass spectrometry method has been developed for the quantitative analysis and screening of long-chain FACoAs in liver, brain, muscle and adipose tissue. METHODS: The sample preparation method consists of tissue homogenization, extraction with organic solvent and reconstitution in an ammonium hydroxide buffer. Extracts are separated by liquid chromatography (LC) on a reversed-phase column and detected by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in positive mode. An additional neutral loss scan allows for untargeted FA-CoAs screening. RESULTS: Extraction was optimized for low sample load (10 mg) of four tissue types (liver, brain, muscle and adipose tissue) with recoveries between 60-140% depending on the analyte and tissue type. Targeted quantification was validated for ten FA-CoAs in the range 0.1-500 ng/mL with accuracies between 85-120%. CONCLUSIONS: We have developed and validated a LC/MS/MS method for the quantifications and screening of long-chain FA-CoAs in four different types of mammalian tissue. The extraction method is straightforward and long-chain FA-CoA species can be quantified using only minimum amount of tissue. Copyright © 2016 John Wiley & Sons, Ltd.

Metabolome Analysis Reveals Betaine Lipids as Major Source for Triglyceride Formation, and the Accumulation of Sedoheptulose during Nitrogen-Starvation of Phaeodactylum tricornutum.

Oleaginous microalgae are considered as a promising resource for the production of biofuels. Especially diatoms arouse interest as biofuel producers since they are most productive in carbon fixation and very flexible to environmental changes in the nature. Naturally, triacylglycerol (TAG) accumulation in algae only occurs under stress conditions like nitrogen-limitation. We focused on Phaeodactylum strain Pt4 (UTEX 646), because of its ability to grow in medium with low salinity and therefore being suited when saline water is less available or for wastewater cultivation strategies. Our data show an increase in neutral lipids during nitrogen-depletion and predominantly 16:0 and 16:1(n-7) accumulated in the TAG fraction. The molecular species composition of TAG suggests a remodeling primarily from the betaine lipid diacylglyceroltrimethylhomoserine (DGTS), but a contribution of the chloroplast galactolipid monogalactosyldiacylglycerol (MGDG) cannot be excluded. Interestingly, the acyl-CoA pool is rich in 20:5(n-3) and 22:6(n-3) in all analyzed conditions, but these fatty acids are almost excluded from TAG. Other metabolites most obviously depleted under nitrogen-starvation were amino acids, lyso-phospholipids and tricarboxylic acid (TCA) cycle intermediates, whereas sulfur-containing metabolites as dimethylsulfoniopropionate, dimethylsulfoniobutyrate and methylsulfate as well as short acyl chain carnitines, propanoyl-carnitine and butanoyl-carnitine increased upon nitrogen-starvation. Moreover, the Calvin cycle may be de-regulated since sedoheptulose accumulated after nitrogen-depletion. Together the data provide now the basis for new strategies to improve lipid production and storage in Phaeodactylum strain Pt4.

Analysis of coenzyme A activated compounds in actinomycetes.

Acyl-CoAs are crucial compounds involved in essential metabolic pathways such as the Krebs cycle and lipid, carbohydrate, and amino acid metabolisms, and they are also key signal molecules involved in the transcriptional regulation of lipid biosynthesis in many organisms. In this study, we took advantage of the high selectivity of mass spectrometry and developed an ion-pairing reverse-phase high-pressure liquid chromatography electrospray ionization high-resolution mass spectrometry (IP-RP-HPLC/ESI-HRMS) method to carry on a comprehensive analytical determination of the wide range of fatty acyl-CoAs present in actinomycetes. The advantage of using a QTOF spectrometer resides in the excellent mass accuracy over a wide dynamic range and measurements of the true isotope pattern that can be used for molecular formula elucidation of unknown analytes. As a proof of concept, we used this assay to determine the composition of the fatty acyl-CoA pools in Mycobacterium, Streptomyces, and Corynebacterium species, revealing an extraordinary difference in fatty acyl-CoA amounts and species distribution between the three genera and between the two species of mycobacteria analyzed, including the presence of different chain-length carboxy-acyl-CoAs, key substrates of mycolic acid biosynthesis. The method was also used to analyze the impact of two fatty acid synthase inhibitors on the acyl-CoA profile of Mycobacterium smegmatis, which showed some unexpected low levels of C24 acyl-CoAs in the isoniazid-treated cells. This robust, sensitive, and reliable method should be broadly applicable in the studies of the wide range of bacteria metabolisms in which acyl-CoA molecules participate.

LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and ¹³C-isotopic labeling of acyl-coenzyme A thioesters.

Acyl-coenzyme A (acyl-CoA) thioesters are evolutionarily conserved, compartmentalized, and energetically activated substrates for biochemical reactions. The ubiquitous involvement of acyl-CoA thioesters in metabolism, including the tricarboxylic acid cycle, fatty acid metabolism, amino acid degradation, and cholesterol metabolism highlights the broad applicability of applied measurements of acyl-CoA thioesters. However, quantitation of acyl-CoA levels provides only one dimension of metabolic information and a more complete description of metabolism requires the relative contribution of different precursors to individual substrates and pathways. Using two distinct stable isotope labeling approaches, acyl-CoA thioesters can be labeled with either a fixed [(13)C3(15)N1] label derived from pantothenate into the CoA moiety or via variable [(13)C] labeling into the acyl chain from metabolic precursors. Liquid chromatography-hybrid quadrupole/Orbitrap high-resolution mass spectrometry using parallel reaction monitoring, but not single ion monitoring, allowed the simultaneous quantitation of acyl-CoA thioesters by stable isotope dilution using the [(13)C3(15)N1] label and measurement of the incorporation of labeled carbon atoms derived from [(13)C6]-glucose, [(13)C5(15)N2]-glutamine, and [(13)C3]-propionate. As a proof of principle, we applied this method to human B cell lymphoma (WSU-DLCL2) cells in culture to precisely describe the relative pool size and enrichment of isotopic tracers into acetyl-, succinyl-, and propionyl-CoA. This method will allow highly precise, multiplexed, and stable isotope-resolved determination of metabolism to refine metabolic models, characterize novel metabolism, and test modulators of metabolic pathways involving acyl-CoA thioesters.

Detection of Acyl-CoA Derivatized with Butylamide for in vitro Fatty Acid Desaturase Assay.

Membrane-bound fatty acid desaturases acting on acyl-CoA contribute to the biosynthesis of unsaturated fatty acids, such as arachidonic acid and docosahexaenoic acid in higher organisms. We propose a simplified method for measuring the desaturase activity that combines the in vitro reaction by desaturase-expressing yeast cell homogenate and the detection of acyl-CoA product as butylamide derivatives by gas chromatography. To set up the in vitro reaction, we traced the in vivo activity of rat liver ∆6 fatty acid desaturase (D6d) expressed in the yeast, Saccharomyces cerevisiae, and determined the time taken for the D6d activity to reach its maximum level. The cell homogenate of yeast expressing the maximum D6d activity was made to react in vitro with linoleoyl-CoA to generate the D6d product, γlinolenoyl-CoA. This product was successfully detected as a peak corresponding to γ-linolenoyl butylamide on gas chromatography. This procedure, with low background expression, using non-labeled acyl-CoA as substrate, will contribute toward developing a simple in vitro desaturase assay. It will also help in elucidating the functions of membrane-bound fatty acid desaturases with various substrate specificities and regioselectivities.

Genetic engineering and metabolite profiling for overproduction of polyhydroxybutyrate in cyanobacteria.

Genetic engineering and metabolite profiling for the overproduction of polyhydroxybutyrate (PHB), which is a carbon material in biodegradable plastics, were examined in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Transconjugants harboring cyanobacterial expression vectors that carried the pha genes for PHB biosynthesis were constructed. The overproduction of PHB by the engineering cells was confirmed through microscopic observations using Nile red, transmission electron microscopy (TEM), or nuclear magnetic resonance (NMR). We successfully recovered PHB from transconjugants prepared from nitrogen-depleted medium without sugar supplementation in which PHB reached approximately 7% (w/w) of the dry cell weight, showing a value of 12-fold higher productivity in the transconjugant than that in the control strain. We also measured the intracellular levels of acetyl-CoA, acetoacetyl-CoA, and 3-hydroxybutyryl-CoA (3HB-CoA), which are intermediate products for PHB. The results obtained indicated that these products were absent or at markedly low levels when cells were subjected to the steady-state growth phase of cultivation under nitrogen depletion for the overproduction of bioplastics. Based on these results, efficient factors were discussed for the overproduction of PHB in recombinant cyanobacteria.

Stable isotope dilution liquid chromatography/mass spectrometry analysis of cellular and tissue medium- and long-chain acyl-coenzyme A thioesters.

RATIONALE: Acyl-Coenzyme A (CoA) thioesters are the principal form of activated carboxylates in cells and tissues. They are employed as acyl carriers that facilitate the transfer of acyl groups to lipids and proteins. Quantification of medium- and long-chain acyl-CoAs represents a significant bioanalytical challenge because of their instability. METHODS: Stable isotope dilution liquid chromatography/selected reaction monitoring-mass spectrometry (LC/SRM-MS) provides the most specific and sensitive method for the analysis of CoA species. However, relevant heavy isotope standards are not available and they are challenging to prepare by chemical synthesis. Stable isotope labeling by essential nutrients in cell culture (SILEC), developed originally for the preparation of stable isotope labeled short-chain acyl-CoA thioester standards, has now been extended to medium-chain and long-chain acyl-CoAs and used for LC/SRM-MS analyses. RESULTS: Customized SILEC standards with >98% isotopic purity were prepared using mouse Hepa 1c1c7 cells cultured in pantothenic-free media fortified with [(13) C3 (15) N1 ]-pantothenic acid and selected fatty acids. A SILEC standard in combination with LC/SRM-MS was employed to quantify cellular concentrations of arachidonoyl-CoA (a representative long-chain acyl-CoA) in two human colon cancer cell lines. A panel of SILEC standards was also employed in combination LC/SRM-MS to quantify medium- and long-chain acyl-CoAs in mouse liver. CONCLUSIONS: This new SILEC-based method in combination with LC/SRM-MS will make it possible to rigorously quantify medium- and long-chain acyl-CoAs in cells and tissues. The method will facilitate studies of medium- and long-chain acyl-CoA dehydrogenase deficiencies as well as studies on the role of medium- and long-chain acyl-CoAs in cellular metabolism.

Kinetics of eicosapentaenoic acid in brain, heart and liver of conscious rats fed a high n-3 PUFA containing diet.

Eicosapentaenoic acid (EPA, 20:5n-3), a precursor of docosahexaenoic acid (DHA), may benefit cardiovascular and brain health. Quantifying EPA's in vivo kinetics might elucidate these effects. [1-(14)C]EPA was infused i.v. for 5min in unanesthetized male rats fed a standard EPA-DHA diet. Plasma and microwaved tissue were analyzed. Kinetic parameters were calculated using our compartmental model. At 5min, 31-48% of labeled EPA in brain and heart was oxidized, 7% in liver. EPA incorporation rates from brain and liver precursor EPA-CoA pools into lipids, mainly phospholipids, were 36 and 2529nmol/s/g×10(-4), insignificant for heart. Deacylation-reacylation half-lives were 22h and 38-128min. Conversion rates to DHA equaled 0.65 and 25.1nmol/s/g×10(-4), respectively. The low brain concentration and incorporation rate and high oxidation of EPA suggest that, if EPA has a beneficial effect in brain, it might result from its suppression of peripheral inflammation and hepatic conversion to bioactive DHA.

Carbonylation as a key reaction in anaerobic acetone activation by Desulfococcus biacutus.

Acetone is activated by aerobic and nitrate-reducing bacteria via an ATP-dependent carboxylation reaction to form acetoacetate as the first reaction product. In the activation of acetone by sulfate-reducing bacteria, acetoacetate has not been found to be an intermediate. Here, we present evidence of a carbonylation reaction as the initial step in the activation of acetone by the strictly anaerobic sulfate reducer Desulfococcus biacutus. In cell suspension experiments, CO was found to be a far better cosubstrate for acetone activation than CO2. The hypothetical reaction product, acetoacetaldehyde, is extremely reactive and could not be identified as a free intermediate. However, acetoacetaldehyde dinitrophenylhydrazone was detected by mass spectrometry in cell extract experiments as a reaction product of acetone, CO, and dinitrophenylhydrazine. In a similar assay, 2-amino-4-methylpyrimidine was formed as the product of a reaction between acetoacetaldehyde and guanidine. The reaction depended on ATP as a cosubstrate. Moreover, the specific activity of aldehyde dehydrogenase (coenzyme A [CoA] acylating) tested with the putative physiological substrate was found to be 153 ± 36 mU mg(-1) protein, and its activity was specifically induced in extracts of acetone-grown cells. Moreover, acetoacetyl-CoA was detected (by mass spectrometry) after the carbonylation reaction as the subsequent intermediate after acetoacetaldehyde was formed. These results together provide evidence that acetoacetaldehyde is an intermediate in the activation of acetone by sulfate-reducing bacteria.