Exploring contrast-enhancing staining agents for studying adipose tissue through contrast-enhanced computed tomography.

Contrast-enhanced computed tomography offers a nondestructive approach to studying adipose tissue in 3D. Several contrast-enhancing staining agents (CESAs) have been explored, whereof osmium tetroxide (OsO4) is the most popular nowadays. However, due to the toxicity and volatility of the conventional OsO4, alternative CESAs with similar staining properties were desired. Hf-WD 1:2 POM and Hexabrix have proven effective for structural analysis of adipocytes using contrast-enhanced computed tomography but fail to provide chemical information. This study introduces isotonic Lugol's iodine (IL) as an alternative CESA for adipose tissue analysis, comparing its staining potential with Hf-WD 1:2 POM and Hexabrix in murine caudal vertebrae and bovine muscle tissue strips. Single and sequential staining protocols were compared to assess the maximization of information extraction from each sample. The study investigated interactions, distribution, and reactivity of iodine species towards biomolecules using simplified model systems and assesses the potential of the CESA to provide chemical information. (Bio)chemical analyses on whole tissues revealed that differences in adipocyte gray values post-IL staining were associated with chemical distinctions between bovine muscle tissue and murine caudal vertebrae. More specific, a difference in the degree of unsaturation of fatty acids was identified as a likely contributor, though not the sole determinant of gray value differences. This research sheds light on the potential of IL as a CESA, offering both structural and chemical insights into adipose tissue composition.

Diacylglycerol kinase-ε is S-palmitoylated on cysteine in the cytoplasmic end of its N-terminal transmembrane fragment.

Diacylglycerol kinase-ε (DGKε) catalyzes phosphorylation of diacylglycerol to phosphatidic acid with a unique specificity toward 1-stearoyl-2-arachidonoyl-sn-glycerol, which is a backbone of phosphatidylinositol (PI). Owing to this specificity, DGKε is involved in the PI cycle maintaining the cellular level of phosphorylated PI derivatives of signaling activity and was also found crucial for lipid metabolism. DGKε dysfunction is linked with the development of atypical hemolytic uremic syndrome (aHUS) and possibly other human diseases. Despite the DGKε significance, data on its regulation by cotranslational and/or post-translational modifications are scarce. Here, we report that DGKε is S-palmitoylated at Cys38/40 (mouse/human DGKε) located in the cytoplasmic end of its N-terminal putative transmembrane fragment. The S-palmitoylation of DGKε was revealed by metabolic labeling of cells with a palmitic acid analogue followed by click chemistry and with acyl-biotin and acyl-polyethylene glycol exchange assays. The S-acyltransferases zDHHC7 (zinc finger DHHC domain containing) and zDHHC17 and the zDHHC6/16 tandem were found to catalyze DGKε S-palmitoylation, which also increased the DGKε abundance. Mouse DGKε-Myc ectopically expressed in human embryonic kidney 293 cells localized to the endoplasmic reticulum where zDHHC6/16 reside and in small amounts also to the Golgi apparatus where zDHHC7 and zDHHC17 are present. The Cys38Ala substitution upregulated, whereas hyperpalmitoylation of wild-type DGKε reduced the kinase activity, indicating an inhibitory effect of the Cys38 S-palmitoylation. In addition, the substitution of neighboring Pro31 with Ala also diminished the activity of DGKε. Taken together, our data indicate that S-palmitoylation can fine-tune DGKε activity in distinct cellular compartments, possibly by affecting the distance between the kinase and its substrate in a membrane.

Spatial lipidomics of coronary atherosclerotic plaque development in a familial hypercholesterolemia swine model.

Coronary atherosclerosis is caused by plaque build-up, with lipids playing a pivotal role in its progression. However, lipid composition and distribution within coronary atherosclerosis remain unknown. This study aims to characterize lipids and investigate differences in lipid composition across disease stages to aid in the understanding of disease progression. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to visualize lipid distributions in coronary artery sections (n = 17) from hypercholesterolemic swine. We performed histology on consecutive sections to classify the artery segments and to investigate colocalization between lipids and histological regions of interest in advanced plaque, including necrotic core and inflammatory cells. Segments were classified as healthy (n = 6), mild (n = 6), and advanced disease (n = 5) artery segments. Multivariate data analysis was employed to find differences in lipid composition between the segment types, and the lipids' spatial distribution was investigated using non-negative matrix factorization (NMF). Through this process, MALDI-MSI detected 473 lipid-related features. NMF clustering described three components in positive ionization mode: triacylglycerides (TAG), phosphatidylcholines (PC), and cholesterol species. In negative ionization mode, two components were identified: one driven by phosphatidylinositol(PI)(38:4), and one driven by ceramide-phosphoethanolamine(36:1). Multivariate data analysis showed the association between advanced disease and specific lipid signatures like PC(O-40:5) and cholesterylester(CE)(18:2). Ether-linked phospholipids and LysoPC species were found to colocalize with necrotic core, and mostly CE, ceramide, and PI species colocalized with inflammatory cells. This study, therefore, uncovers distinct lipid signatures correlated with plaque development and their colocalization with necrotic core and inflammatory cells, enhancing our understanding of coronary atherosclerosis progression.

A high-resolution 13C NMR approach for profiling fatty acid unsaturation in lipid extracts and in live Caenorhabditiselegans.

Unsaturated fatty acids (UFA) play a crucial role in central cellular processes in animals, including membrane function, development, and disease. Disruptions in UFA homeostasis can contribute to the onset of metabolic, cardiovascular, and neurodegenerative disorders. Consequently, there is a high demand for analytical techniques to study lipid compositions in live cells and multicellular organisms. Conventional analysis of UFA compositions in cells, tissues, and organisms involves solvent extraction procedures coupled with analytical techniques such as gas chromatography, MS and/or NMR spectroscopy. As a nondestructive and nontargeted technique, NMR spectroscopy is uniquely capable of characterizing the chemical profiling of living cells and multicellular organisms. Here, we use NMR spectroscopy to analyze Caenorhabditis elegans, enabling the determination of their lipid compositions and fatty acid unsaturation levels both in cell-free lipid extracts and in vivo. The NMR spectra of lipid extracts from WT and fat-3 mutant C. elegans strains revealed notable differences due to the absence of Δ-6 fatty acid desaturase activity, including the lack of arachidonic and eicosapentaenoic acyl chains. Uniform 13C-isotope labeling and high-resolution 2D solution-state NMR of live worms confirmed these findings, indicating that the signals originated from fast-tumbling lipid molecules within lipid droplets. Overall, this strategy permits the analysis of lipid storage in intact worms and has enough resolution and sensitivity to identify differences between WT and mutant animals with impaired fatty acid desaturation. Our results establish methodological benchmarks for future investigations of fatty acid regulation in live C. elegans using NMR.

Human COQ10A and COQ10B are distinct lipid-binding START domain proteins required for coenzyme Q function.

Coenzyme Q (CoQ or ubiquinone) serves as an essential redox-active lipid in respiratory electron and proton transport during cellular energy metabolism. CoQ also functions as a membrane-localized antioxidant protecting cells against lipid peroxidation. CoQ deficiency is associated with multiple human diseases; CoQ10 supplementation in particular has noted cardioprotective benefits. In Saccharomyces cerevisiae, Coq10, a putative START domain protein, is believed to chaperone CoQ to sites where it functions. Yeast coq10 deletion mutants (coq10Δ) synthesize CoQ inefficiently during log phase growth and are respiratory defective and sensitive to oxidative stress. Humans have two orthologs of yeast COQ10, COQ10A and COQ10B Here, we tested the human co-orthologs for their ability to rescue the yeast mutant. We showed that expression of either human ortholog, COQ10A or COQ10B, rescues yeast coq10Δ mutant phenotypes, restoring the function of respiratory-dependent growth on a nonfermentable carbon source and sensitivity to oxidative stress induced by treatment with PUFAs. These effects indicate a strong functional conservation of Coq10 across different organisms. However, neither COQ10A nor COQ10B restored CoQ biosynthesis when expressed in the yeast coq10Δ mutant. The involvement of yeast Coq10 in CoQ biosynthesis may rely on its interactions with another protein, possibly Coq11, which is not found in humans. Coexpression analyses of yeast COQ10 and human COQ10A and COQ10B provide additional insights to functions of these START domain proteins and their potential roles in other biologic pathways.

Enzymatic synthesis and chemical inversion provide both enantiomers of bioactive epoxydocosapentaenoic acids.

Epoxy PUFAs are endogenous cytochrome P450 (P450) metabolites of dietary PUFAs. Although these metabolites exert numerous biological effects, attempts to study their complex biology have been hampered by difficulty in obtaining the epoxides as pure regioisomers and enantiomers. To remedy this, we synthesized 19,20- and 16,17-epoxydocosapentaenoic acids (EDPs) (the two most abundant EDPs in vivo) by epoxidation of DHA with WT and the mutant (F87V) P450 enzyme BM3 from Bacillus megaterium WT epoxidation yielded a 4:1 mixture of 19,20:16,17-EDP exclusively as (S,R) enantiomers. Epoxidation with the mutant (F87V) yielded a 1.6:1 mixture of 19,20:16,17-EDP; the 19,20-EDP fraction was ∼9:1 (S,R):(R,S), but the 16,17-EDP was exclusively the (S,R) enantiomer. To access the (R,S) enantiomers of these EDPs, we used a short (four-step) chemical inversion sequence, which utilizes 2-(phenylthio)ethanol as the epoxide-opening nucleophile, followed by mesylation of the resulting alcohol, oxidation of the thioether moiety, and base-catalyzed elimination. This short synthesis cleanly converts the (S,R)-epoxide to the (R,S)-epoxide without loss of enantiopurity. This method, also applicable to eicosapentaenoic acid and arachidonic acid, provides a simple, cost-effective procedure for accessing larger amounts of these metabolites.

Cytotoxic 1-deoxysphingolipids are metabolized by a cytochrome P450-dependent pathway.

The 1-deoxysphingolipids (1-deoxySLs) are atypical sphingolipids (SLs) that are formed when serine palmitoyltransferase condenses palmitoyl-CoA with alanine instead of serine during SL synthesis. The 1-deoxySLs are toxic to neurons and pancreatic ß-cells. Pathologically elevated 1-deoxySLs cause the inherited neuropathy, hereditary sensory autonomic neuropathy type 1 (HSAN1), and are also found in T2D. Diabetic sensory polyneuropathy (DSN) and HSAN1 are clinically very similar, suggesting that 1-deoxySLs may be implicated in both pathologies. The 1-deoxySLs are considered to be dead-end metabolites, as they lack the C1-hydroxyl group, which is essential for the canonical degradation of SLs. Here, we report a previously unknown metabolic pathway, which is capable of degrading 1-deoxySLs. Using a variety of metabolic labeling approaches and high-resolution high-accuracy MS, we identified eight 1-deoxySL downstream metabolites, which appear to be formed by cytochrome P450 (CYP)4F enzymes. Comprehensive inhibition and induction of CYP4F enzymes blocked and stimulated, respectively, the formation of the downstream metabolites. Consequently, CYP4F enzymes might be novel therapeutic targets for the treatment of HSAN1 and DSN, as well as for the prevention of T2D.

An optimized method for measuring fatty acids and cholesterol in stable isotope-labeled cells.

Stable isotope labeling has become an important methodology for determining lipid metabolic parameters of normal and neoplastic cells. Conventional methods for fatty acid and cholesterol analysis have one or more issues that limit their utility for in vitro stable isotope-labeling studies. To address this, we developed a method optimized for measuring both fatty acids and cholesterol from small numbers of stable isotope-labeled cultured cells. We demonstrate quantitative derivatization and extraction of fatty acids from a wide range of lipid classes using this approach. Importantly, cholesterol is also recovered, albeit at a modestly lower yield, affording the opportunity to quantitate both cholesterol and fatty acids from the same sample. Although we find that background contamination can interfere with quantitation of certain fatty acids in low amounts of starting material, our data indicate that this optimized method can be used to accurately measure mass isotopomer distributions for cholesterol and many fatty acids isolated from small numbers of cultured cells. Application of this method will facilitate acquisition of lipid parameters required for quantifying flux and provide a better understanding of how lipid metabolism influences cellular function.

Mutating a conserved cysteine in GPIHBP1 reduces amounts of GPIHBP1 in capillaries and abolishes LPL binding.

Mutation of conserved cysteines in proteins of the Ly6 family cause human disease-chylomicronemia in the case of glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1) and paroxysmal nocturnal hemoglobinuria in the case of CD59. A mutation in a conserved cysteine in CD59 prevented the protein from reaching the surface of blood cells. In contrast, mutation of conserved cysteines in human GPIHBP1 had little effect on GPIHBP1 trafficking to the surface of cultured CHO cells. The latter findings were somewhat surprising and raised questions about whether CHO cell studies accurately model the fate of mutant GPIHBP1 proteins in vivo. To explore this concern, we created mice harboring a GPIHBP1 cysteine mutation (p.C63Y). The p.C63Y mutation abolished the ability of mouse GPIHBP1 to bind LPL, resulting in severe chylomicronemia. The mutant GPIHBP1 was detectable by immunohistochemistry on the surface of endothelial cells, but the level of expression was ∼70% lower than in WT mice. The mutant GPIHBP1 protein in mouse tissues was predominantly monomeric. We conclude that mutation of a conserved cysteine in GPIHBP1 abolishes the ability of GPIHBP1 to bind LPL, resulting in mislocalization of LPL and severe chylomicronemia. The mutation reduced but did not eliminate GPIHBP1 on the surface of endothelial cells in vivo.

Localization of 1-deoxysphingolipids to mitochondria induces mitochondrial dysfunction.

1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids that are elevated in the plasma of patients with type 2 diabetes and hereditary sensory and autonomic neuropathy type 1 (HSAN1). Clinically, diabetic neuropathy and HSAN1 are very similar, suggesting the involvement of deoxySLs in the pathology of both diseases. However, very little is known about the biology of these lipids and the underlying pathomechanism. We synthesized an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, to trace the metabolism and localization of deoxySLs. Our results indicate that the metabolism of these lipids is restricted to only some lipid species and that they are not converted to canonical sphingolipids or fatty acids. Furthermore, exogenously added alkyne-doxSA [(2S,3R)-2-aminooctadec-17-yn-3-ol] localized to mitochondria, causing mitochondrial fragmentation and dysfunction. The induced mitochondrial toxicity was also shown for natural doxSA, but not for sphinganine, and was rescued by inhibition of ceramide synthase activity. Our findings therefore indicate that mitochondrial enrichment of an N-acylated doxSA metabolite may contribute to the neurotoxicity seen in diabetic neuropathy and HSAN1. Hence, we provide a potential explanation for the characteristic vulnerability of peripheral nerves to elevated levels of deoxySLs.

Mobility of "HSPG-bound" LPL explains how LPL is able to reach GPIHBP1 on capillaries.

In mice lacking glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1), the LPL secreted by adipocytes and myocytes remains bound to heparan sulfate proteoglycans (HSPGs) on all cells within tissues. That observation raises a perplexing issue: Why isn't the freshly secreted LPL in wild-type mice captured by the same HSPGs, thereby preventing LPL from reaching GPIHBP1 on capillaries? We hypothesized that LPL-HSPG interactions are transient, allowing the LPL to detach and move to GPIHBP1 on capillaries. Indeed, we found that LPL detaches from HSPGs on cultured cells and moves to: 1) soluble GPIHBP1 in the cell culture medium; 2) GPIHBP1-coated agarose beads; and 3) nearby GPIHBP1-expressing cells. Movement of HSPG-bound LPL to GPIHBP1 did not occur when GPIHBP1 contained a Ly6 domain missense mutation (W109S), but was almost normal when GPIHBP1's acidic domain was mutated. To test the mobility of HSPG-bound LPL in vivo, we injected GPIHBP1-coated agarose beads into the brown adipose tissue of GPIHBP1-deficient mice. LPL moved quickly from HSPGs on adipocytes to GPIHBP1-coated beads, thereby depleting LPL stores on the surface of adipocytes. We conclude that HSPG-bound LPL in the interstitial spaces of tissues is mobile, allowing the LPL to move to GPIHBP1 on endothelial cells.

An HPLC-CAD/fluorescence lipidomics platform using fluorescent fatty acids as metabolic tracers.

Fluorescent lipids are important tools for live imaging in cell culture and animal models, yet their metabolism has not been well-characterized. Here we describe a novel combined HPLC and LC-MS/MS method developed to characterize both total lipid profiles and the products of fluorescently labeled lipids. Using this approach, we found that lipids labeled with the fluorescent tags, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene [BODIPY(558/568)], and dipyrrometheneboron difluoride undecanoic acid (TopFluor) are all metabolized into varying arrays of polar and nonpolar fluorescent lipid products when they are fed to larval zebrafish. Quantitative metabolic labeling experiments performed in this system revealed significant effects of total dietary lipid composition on fluorescent lipid partitioning. We provide evidence that cholesterol metabolism in the intestine is important in determining the metabolic fates of dietary FAs. Using this method, we found that inhibitors of dietary cholesterol absorption and esterification both decreased incorporation of dietary fluorescent FAs into cholesterol esters (CEs), suggesting that CE synthesis in enterocytes is primarily responsive to the availability of dietary cholesterol. These results are the first to comprehensively characterize fluorescent FA metabolism and to demonstrate their utility as metabolic labeling reagents, effectively coupling quantitative biochemistry with live imaging studies.

In-depth sphingomyelin characterization using electron impact excitation of ions from organics and mass spectrometry.

Electron impact excitation of ions from organics (EIEIO), also referred to as electron-induced dissociation, was applied to singly charged SM molecular species in the gas phase. Using ESI and a quadrupole TOF mass spectrometer equipped with an electron-ion reaction device, we found that SMs fragmented sufficiently to identify their lipid class, acyl group structure, and the location of double bond(s). Using this technique, nearly 200 SM molecular species were found in four natural lipid extracts: bovine milk, porcine brain, chicken egg yolk, and bovine heart. In addition to the most common backbone, d18:1, sphingosines with a range of carbon chain lengths, sphingadienes, and some sphinganine backbones were also detected. Modifications in natural SMs were also identified, including addition of iodine/methanol across a carbon-carbon double bond. This unparalleled new approach to SM analysis using EIEIO-MS shows promise as a unique and powerful tool for structural characterization.

Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics (EIEIO).

Electron-induced dissociation or electron impact excitation of ions from organics (EIEIO) was applied to triacylglycerols (TAGs) for in-depth molecular structure analysis using MS. In EIEIO, energetic electrons (∼10 eV) fragmented TAG ions to allow for regioisomeric assignment of identified acyl groups at the sn-2 or sn-1/3 positions of the glycerol backbone. In addition, carbon-carbon double bond locations within the acyl chains could also be assigned by EIEIO. Beyond the analysis of lipid standards, this technique was applied to edible oils and natural lipid extracts to demonstrate the power of this method to provide in-depth structural elucidation of TAG molecular species.

Synthesis of a novel photoactivatable glucosylceramide cross-linker.

The biosynthesis of glucosylceramide (GlcCer) is a key rate-limiting step in complex glycosphingolipid (GSL) biosynthesis. To further define interacting partners of GlcCer, we have made a cleavable, biotinylated, photoreactive GlcCer analog in which the reactive nitrene is closely apposed to the GlcCer head group, by substituting the native fatty acid with d, l-2-aminohexadecanoic acid. Two amino-GlcCer diastereomer cross-linkers (XLA and XLB) were generated. XLB proved an effective lactosylceramide (LacCer) synthase substrate while XLA was inhibitory. Both probes specifically bound and cross-linked the GlcCer binding protein, glycolipid transfer protein (GLTP), but not other GSL binding proteins (Shiga toxin and cholera toxin). GlcCer inhibited GLTP cross-linking. Both GlcCer cross-linkers competed with microsomal nitrobenzoxadiazole (NBD)-GlcCer anabolism to NBD-LacCer. GLTP showed marked, ATP-dependent enhancement of cell-free intact microsomal LacCer synthesis from endogenous or exogenous liposomal GlcCer, supporting a role in the transport/membrane translocation of cytosolic and extra-Golgi GlcCer. GLTP was specifically labeled by either XLA or XLB GlcCer cross-linker during this process, together with a (the same) small subset of microsomal proteins. These cross-linkers will serve to probe physiologically relevant GlcCer-interacting cellular proteins.

An LPL-specific monoclonal antibody, 88B8, that abolishes the binding of LPL to GPIHBP1.

LPL contains two principal domains: an amino-terminal catalytic domain (residues 1-297) and a carboxyl-terminal domain (residues 298-448) that is important for binding lipids and binding glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1) (an endothelial cell protein that shuttles LPL to the capillary lumen). The LPL sequences required for GPIHBP1 binding have not been examined in detail, but one study suggested that sequences near LPL's carboxyl terminus (residues ∼403-438) were crucial. Here, we tested the ability of LPL-specific monoclonal antibodies (mAbs) to block the binding of LPL to GPIHBP1. One antibody, 88B8, abolished LPL binding to GPIHBP1. Consistent with those results, antibody 88B8 could not bind to GPIHBP1-bound LPL on cultured cells. Antibody 88B8 bound poorly to LPL proteins with amino acid substitutions that interfered with GPIHBP1 binding (e.g., C418Y, E421K). However, the sequences near LPL's carboxyl terminus (residues ∼403-438) were not sufficient for 88B8 binding; upstream sequences (residues 298-400) were also required. Additional studies showed that these same sequences are required for LPL binding to GPIHBP1. In conclusion, we identified an LPL mAb that binds to LPL's GPIHBP1-binding domain. The binding of both antibody 88B8 and GPIHBP1 to LPL depends on large segments of LPL's carboxyl-terminal domain.

Open and closed states of Candida antarctica lipase B: protonation and the mechanism of interfacial activation.

Lipases (EC 3.1.1.3) are ubiquitous hydrolases for the carboxyl ester bond of water-insoluble substrates, such as triacylglycerols, phospholipids, and other insoluble substrates, acting in aqueous as well as in low-water media, thus being of considerable physiological significance with high interest also for their industrial applications. The hydrolysis reaction follows a two-step mechanism, or "interfacial activation," with adsorption of the enzyme to a heterogeneous interface and subsequent enhancement of the lipolytic activity. Among lipases, Candida antarctica lipase B (CALB) has never shown any significant interfacial activation, and a closed conformation of CALB has never been reported, leading to the conclusion that its behavior was due to the absence of a lid regulating the access to the active site. The lid open and closed conformations and their protonation states are observed in the crystal structure of CALB at 0.91 Å resolution. Having the open and closed states at atomic resolution allows relating protonation to the conformation, indicating the role of Asp145 and Lys290 in the conformation alteration. The findings explain the lack of interfacial activation of CALB and offer new elements to elucidate this mechanism, with the consequent implications for the catalytic properties and classification of lipases.

Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis.

Coenzyme Q (Q or ubiquinone) is a redox-active polyisoprenylated benzoquinone lipid essential for electron and proton transport in the mitochondrial respiratory chain. The aromatic ring 4-hydroxybenzoic acid (4HB) is commonly depicted as the sole aromatic ring precursor in Q biosynthesis despite the recent finding that para-aminobenzoic acid (pABA) also serves as a ring precursor in Saccharomyces cerevisiae Q biosynthesis. In this study, we employed aromatic (13)C6-ring-labeled compounds including (13)C6-4HB, (13)C6-pABA, (13)C6-resveratrol, and (13)C6-coumarate to investigate the role of these small molecules as aromatic ring precursors in Q biosynthesis in Escherichia coli, S. cerevisiae, and human and mouse cells. In contrast to S. cerevisiae, neither E. coli nor the mammalian cells tested were able to form (13)C6-Q when cultured in the presence of (13)C6-pABA. However, E. coli cells treated with (13)C6-pABA generated (13)C6-ring-labeled forms of 3-octaprenyl-4-aminobenzoic acid, 2-octaprenyl-aniline, and 3-octaprenyl-2-aminophenol, suggesting UbiA, UbiD, UbiX, and UbiI are capable of using pABA or pABA-derived intermediates as substrates. E. coli, S. cerevisiae, and human and mouse cells cultured in the presence of (13)C6-resveratrol or (13)C6-coumarate were able to synthesize (13)C6-Q. Future evaluation of the physiological and pharmacological responses to dietary polyphenols should consider their metabolism to Q.

A novel unsaturated fatty acid hydratase toward C16 to C22 fatty acids from Lactobacillus acidophilus.

Hydroxy FAs, one of the gut microbial metabolites of PUFAs, have attracted much attention because of their various bioactivities. The purpose of this study was to identify lactic acid bacteria with the ability to convert linoleic acid (LA) to hydroxy FAs. A screening process revealed that a gut bacterium, Lactobacillus acidophilus NTV001, converts LA mainly into 13-hydroxy-cis-9-octadecenoic acid and resulted in the identification of the hydratase responsible, fatty acid hydratase 1 (FA-HY1). Recombinant FA-HY1 was purified, and its enzymatic characteristics were investigated. FA-HY1 could convert not only C18 PUFAs but also C20 and C22 PUFAs. C18 PUFAs with a cis carbon-carbon double bond at the Δ12 position were converted into the corresponding 13-hydroxy FAs. Arachidonic acid and DHA were converted into the corresponding 15-hydroxy FA and 14-hydroxy FA, respectively. To the best of our knowledge, this is the first report of a bacterial FA hydratase that can convert C20 and C22 PUFAs into the corresponding hydroxy FAs. These novel hydroxy FAs produced by using FA-HY1 should contribute to elucidating the bioactivities of hydroxy FAs.

Lipoxygenase-catalyzed transformation of epoxy fatty acids to hydroxy-endoperoxides: a potential P450 and lipoxygenase interaction.

Herein, we characterize a generally applicable transformation of fatty acid epoxides by lipoxygenase (LOX) enzymes that results in the formation of a five-membered endoperoxide ring in the end product. We demonstrated this transformation using soybean LOX-1 in the metabolism of 15,16-epoxy-α-linolenic acid, and murine platelet-type 12-LOX and human 15-LOX-1 in the metabolism of 14,15-epoxyeicosatrienoic acid (14,15-EET). A detailed examination of the transformation of the two enantiomers of 15,16-epoxy-α-linolenic acid by soybean LOX-1 revealed that the expected primary product, a 13S-hydroperoxy-15,16-epoxide, underwent a nonenzymatic transformation in buffer into a new derivative that was purified by HPLC and identified by UV, LC-MS, and ¹H-NMR as a 13,15-endoperoxy-16-hydroxy-octadeca-9,11-dienoic acid. The configuration of the endoperoxide (cis or trans side chains) depended on the steric relationship of the new hydroperoxy moiety to the enantiomeric configuration of the fatty acid epoxide. The reaction mechanism involves intramolecular nucleophilic substitution (SNi) between the hydroperoxy (nucleophile) and epoxy group (electrophile). Equivalent transformations were documented in metabolism of the enantiomers of 14,15-EET by the two mammalian LOX enzymes, 15-LOX-1 and platelet-type 12-LOX. We conclude that this type of transformation could occur naturally with the co-occurrence of LOX and cytochrome P450 or peroxygenase enzymes, and it could also contribute to the complexity of products formed in the autoxidation reactions of polyunsaturated fatty acids.