Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time-m/z pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, researchers employ tandem mass spectral libraries and challenging in silico fragmentation software. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) may offer an additional layer of structural information in untargeted metabolomics, especially for identifying specific unidentified metabolites that are revealed to be statistically significant. Here, we investigate the potential of hydrophilic interaction liquid chromatography (HILIC)-HDX-MS in untargeted metabolomics. Specifically, we evaluate the effectiveness of two approaches using hypothetical targets: the post-column addition of deuterium oxide (D2O) and the on-column HILIC-HDX-MS method. To illustrate the practical application of HILIC-HDX-MS, we apply this methodology using the in silico fragmentation software MS-FINDER to an unknown compound detected in various biological samples, including plasma, serum, tissues, and feces during HILIC-MS profiling, subsequently identified as N1-acetylspermidine.
- Klíčová slova
- hydrogen/deuterium exchange, liquid chromatography, mass spectrometry, metabolomics, unknown identification,
- MeSH
- chromatografie kapalinová metody MeSH
- deuterium MeSH
- hydrofobní a hydrofilní interakce MeSH
- metabolomika * metody MeSH
- vodík/deuteriová výměna a hmotnostní spektrometrie * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- deuterium MeSH
Breast milk is a complex mixture containing underexplored bioactive lipids. We performed an observational case-control study to compare the impact of delivery mode: caesarean section (CS) and vaginal birth (VB); and term (preterm and term delivery) on the levels of lipokines in human milk at different stages of lactation. Metabolomic analysis of the milk identified triacylglycerol estolides as a metabolic reservoir of the anti-inflammatory lipid mediator 5-palmitic acid ester of hydroxystearic acid (5-PAHSA). We found that triacylglycerol estolides were substrates of carboxyl ester lipase and 5-PAHSA-containing lipids were the least preferred substrates among tested triacylglycerol estolide isomers. This explained exceptionally high colostrum levels of 5-PAHSA in the VB group. CS and preterm birth negatively affected colostrum lipidome, including 5-PAHSA levels, but the lipidomic profiles normalized in mature milk. Mothers delivering term babies vaginally produce colostrum rich in 5-PAHSA, which could contribute to the prevention of intestinal inflammation in newborns.
- Klíčová slova
- Colostrum, Elective caesarean section, Human breast milk, Lipidomics, PAHSA, Preterm birth,
- MeSH
- císařský řez MeSH
- estery metabolismus MeSH
- kojenec MeSH
- kolostrum metabolismus MeSH
- kyselina palmitová metabolismus MeSH
- laktace MeSH
- lidé MeSH
- lipasa metabolismus MeSH
- mateřské mléko * metabolismus MeSH
- novorozenec MeSH
- předčasný porod * metabolismus MeSH
- studie případů a kontrol MeSH
- těhotenství MeSH
- triglyceridy metabolismus MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- novorozenec MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- CEL protein, human MeSH Prohlížeč
- estery MeSH
- kyselina palmitová MeSH
- lipasa MeSH
- triglyceridy MeSH
Fatty acid esters of hydroxy fatty acids (FAHFAs) represent a complex lipid class that contains both signaling mediators and structural components of lipid biofilms in humans. The majority of endogenous FAHFAs share a common chemical architecture, characterized by an estolide bond that links the hydroxy fatty acid (HFA) backbone and the fatty acid (FA). Two structurally and functionally distinct FAHFA superfamilies are recognized based on the position of the estolide bond: omega-FAHFAs and in-chain branched FAHFAs. The existing variety of possible HFAs and FAs combined with the position of the estolide bond generates a vast quantity of unique structures identified in FAHFA families. In this review, we discuss the anti-diabetic and anti-inflammatory effects of branched FAHFAs and the role of omega-FAHFA-derived lipids as surfactants in the tear film lipid layer and dry eye disease. To emphasize potential pharmacological targets, we recapitulate the biosynthesis of the HFA backbone within the superfamilies together with the degradation pathways and the FAHFA regioisomer distribution in human and mouse adipose tissue. We propose a theoretical involvement of cytochrome P450 enzymes in the generation and degradation of saturated HFA backbones and present an overview of small-molecule inhibitors used in FAHFA research. The FAHFA lipid class is huge and largely unexplored. Besides the unknown biological effects of individual FAHFAs, also the enigmatic enzymatic machinery behind their synthesis could provide new therapeutic approaches for inflammatory metabolic or eye diseases. Therefore, understanding the mechanisms of (FA)HFA synthesis at the molecular level should be the next step in FAHFA research.
- Klíčová slova
- Cytochrome P450, FAHFA, Inhibitors, Meibum, OAHFA, PAHSA,
- MeSH
- diabetes mellitus * MeSH
- estery * chemie metabolismus MeSH
- lidé MeSH
- mastné kyseliny metabolismus MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- estery * MeSH
- mastné kyseliny MeSH
Glucose tolerance represents a complex phenotype in which many tissues play important roles and interact to regulate metabolic homeostasis. Here, we perform an analysis of 13C6-glucose tissue distribution, which maps the metabolome and lipidome across 12 metabolically relevant mouse organs and plasma, with integrated 13C6-glucose-derived carbon tracing during oral glucose tolerance test (OGTT). We measure time profiles of water-soluble metabolites and lipids and integrate the global metabolite response into metabolic pathways. During the OGTT, glucose use is turned on with specific kinetics at the organ level, but fasting substrates like β-hydroxybutyrate are switched off in all organs simultaneously. Timeline profiling of 13C-labeled fatty acids and triacylglycerols across tissues suggests that brown adipose tissue may contribute to the circulating fatty acid pool at maximal plasma glucose levels. The GTTAtlas interactive web application serves as a unique resource for the exploration of whole-body glucose metabolism and time profiles of tissue and plasma metabolites during the OGTT.
- Klíčová slova
- 13C, brown adipose tissue, de novo lipogenesis, glucose tolerance, lipidomics, metabolite cycling, metabolomics, metabolomics atlas, pathway analysis, tracer analysis,
- MeSH
- biologické markery krev MeSH
- časové faktory MeSH
- chromatografie kapalinová MeSH
- energetický metabolismus * MeSH
- glukózový toleranční test * MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- krevní glukóza metabolismus MeSH
- lipidomika MeSH
- lipidy krev MeSH
- metabolom * MeSH
- metabolomika * MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- tandemová hmotnostní spektrometrie MeSH
- tkáňová distribuce MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- biologické markery MeSH
- krevní glukóza MeSH
- lipidy MeSH
Branched esters of palmitic acid and hydroxy stearic acid are antiinflammatory and antidiabetic lipokines that belong to a family of fatty acid (FA) esters of hydroxy fatty acids (HFAs) called FAHFAs. FAHFAs themselves belong to oligomeric FA esters, known as estolides. Glycerol-bound FAHFAs in triacylglycerols (TAGs), named TAG estolides, serve as metabolite reservoir of FAHFAs mobilized by lipases upon demand. Here, we characterized the involvement of two major metabolic lipases, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), in TAG estolide and FAHFA degradation. We synthesized a library of 20 TAG estolide isomers with FAHFAs varying in branching position, chain length, saturation grade, and position on the glycerol backbone and developed an in silico mass spectra library of all predicted catabolic intermediates. We found that ATGL alone or coactivated by comparative gene identification-58 efficiently liberated FAHFAs from TAG estolides with a preference for more compact substrates where the estolide branching point is located near the glycerol ester bond. ATGL was further involved in transesterification and remodeling reactions leading to the formation of TAG estolides with alternative acyl compositions. HSL represented a much more potent estolide bond hydrolase for both TAG estolides and free FAHFAs. FAHFA and TAG estolide accumulation in white adipose tissue of mice lacking HSL argued for a functional role of HSL in estolide catabolism in vivo. Our data show that ATGL and HSL participate in the metabolism of estolides and TAG estolides in distinct manners and are likely to affect the lipokine function of FAHFAs.
- Klíčová slova
- ATGL, FAHFA, HSL, lipokine,
- MeSH
- bílá tuková tkáň metabolismus MeSH
- estery chemie MeSH
- HEK293 buňky MeSH
- kyselina palmitová metabolismus MeSH
- kyseliny stearové metabolismus MeSH
- lidé MeSH
- lipasa metabolismus MeSH
- lipolýza fyziologie MeSH
- mastné kyseliny metabolismus MeSH
- metabolismus fyziologie MeSH
- myši knockoutované MeSH
- myši MeSH
- sterolesterasa metabolismus MeSH
- triglyceridy metabolismus MeSH
- tuková tkáň metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- estery MeSH
- kyselina palmitová MeSH
- kyseliny stearové MeSH
- lipasa MeSH
- mastné kyseliny MeSH
- stearic acid MeSH Prohlížeč
- sterolesterasa MeSH
- triglyceridy MeSH
The discovery of branched fatty acid esters of hydroxy fatty acids (FAHFAs) in humans draw attention of many researches to their biological effects. Although FAHFAs were originally discovered in insects and plants, their introduction into the mammalian realm opened new horizons in bioactive lipid research. Hundreds of isomers from different families have been identified so far and their role in (patho) physiological processes is currently being explored. The family of palmitic acid esters of hydroxy stearic acids (PAHSAs), especially 5-PAHSA and 9-PAHSA regioisomers, stands out in the crowd of other FAHFAs for their anti-inflammatory and anti-diabetic effects. Beneficial effects of PAHSAs have been linked to metabolic disorders such as type 1 and type 2 diabetes, colitis, and chronic inflammation. Besides PAHSAs, a growing family of polyunsaturated FAHFAs exerts mainly immunomodulatory effects and biological roles of many other FAHFAs remain currently unknown. Therefore, FAHFAs represent unique lipid messengers capable of affecting many immunometabolic processes. The objective of this review is to summarize the knowledge concerning the diversity of FAHFAs, nomenclature, and their analysis and detection. Special attention is paid to the total syntheses of FAHFAs, optimal strategies, and to the formation of the stereocenter required for optically active molecules. Biosynthetic pathways of saturated and polyunsaturated FAHFAs in mammals and plants are reviewed together with their metabolism and degradation. Moreover, an overview of biological effects of branched FAHFAs is provided and many unanswered questions regarding FAHFAs are discussed.
- Klíčová slova
- Biological effects, FAHFA, Lipid mediators, PAHSA, Total synthesis,
- MeSH
- diabetes mellitus farmakoterapie metabolismus MeSH
- estery chemie metabolismus MeSH
- kolitida farmakoterapie metabolismus MeSH
- lidé MeSH
- mastné kyseliny chemie metabolismus MeSH
- molekulární struktura MeSH
- zánět farmakoterapie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- estery MeSH
- mastné kyseliny MeSH
SCOPE: The docosahexaenoic acid ester of hydroxy linoleic acid (13-DHAHLA) is a bioactive lipid with anti-inflammatory properties from the family of fatty acid esters of hydroxy fatty acids (FAHFA). METHODS AND RESULTS: To explore the biosynthesis of 13-DHAHLA from dietary oils, C57BL/6N mice are gavaged for 8 days with various corn oil/marine oil mixtures containing the same amount of DHA. Plasma levels of omega-3 FAHFAs are influenced by the lipid composition of the mixtures but do not reflect the changes in bioavailability of polyunsaturated fatty acids in plasma. Triacylglycerol-bound DHA and linoleic acid serve as more effective precursors for 13-DHAHLA synthesis than DHA bound in phospholipids or wax esters. Both 13(S)- and 13(R)-DHAHLA inhibit antigen and PGE2 -induced chemotaxis and degranulation of mast cells to a comparable extent and 13(S)-DHAHLA is identified as the predominant isomer in mouse adipose tissue. CONCLUSION: Here, the optimal nutritional source of DHA is identified, which supports production of anti-inflammatory FAHFAs, as triacylglycerol-based marine oil and also reveals a possible role of triacylglycerols in the synthesis of FAHFA lipokines.
- Klíčová slova
- calanus, fatty acids, krill, mast cells, stereoisomers,
- MeSH
- antiflogistika nesteroidní krev farmakokinetika MeSH
- biologická dostupnost MeSH
- chemotaxe účinky léků MeSH
- kyseliny dokosahexaenové farmakokinetika MeSH
- kyseliny linolové chemie MeSH
- kyseliny mastné omega-3 farmakokinetika farmakologie MeSH
- mastocyty účinky léků MeSH
- myši inbrední C57BL MeSH
- oleje chemie farmakokinetika MeSH
- stereoizomerie MeSH
- triglyceridy chemie MeSH
- vodní organismy MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 13-hydroxylinoleic acid MeSH Prohlížeč
- antiflogistika nesteroidní MeSH
- kyseliny dokosahexaenové MeSH
- kyseliny linolové MeSH
- kyseliny mastné omega-3 MeSH
- oleje MeSH
- triglyceridy MeSH
AIMS: Brittle cornea syndrome (BCS) is a rare autosomal recessive disorder. The aim of this study was to review ZNF469 mutations associated with BCS type 1 to date and to describe an additional case of Czech/Polish background. METHODS: Whole genome sequencing was undertaken to identify the molecular genetic cause of disease in the proband. Sequence variants in ZNF469 previously reported as BCS type 1-causing were searched in the literature, manually curated and aligned to the reference sequence NM_001127464.2. RESULTS: The proband has been reviewed since childhood with progressive myopia and hearing loss. Aged 13 years had been diagnosed with Stickler syndrome. Aged 16.5 years, he developed acute hydrops in the left eye managed by corneal transplantation. At the age of 26, he experienced right corneal rupture after blunt trauma, also managed by grafting. He had a number of secondary complications and despite regular follow-up and timely management, the right eye became totally blind and the left eye had light perception at the last follow-up visit, aged 42. He was found to be a compound heterozygote for two novel mutations c.1705C>T; p.(Gln569*) and c.1402_1411del; p.(Pro468Alafs*31) in ZNF469. In total 22 disease-causing variants in ZNF469 have been identified, mainly in consanguineous families or endogamous populations. Only four probands, including the case described in the current study, harboured compound heterozygous mutations. CONCLUSION: BCS occurs very rarely in outbred populations which may cause diagnostic errors due to poor awareness of the disease. Investigation into the underlying molecular genetic cause in patients with connective tissue disorders may lead to a re-evaluation of their clinical diagnosis.
- Klíčová slova
- ZNF469, blindness, brittle cornea syndrome, corneal rupture, deafness, penetrating keratoplasty,
- MeSH
- abnormality očí diagnóza genetika patofyziologie MeSH
- artritida diagnóza MeSH
- chybná diagnóza MeSH
- dospělí MeSH
- edém rohovky patofyziologie chirurgie MeSH
- glaukom patofyziologie chirurgie MeSH
- heterozygot MeSH
- keratoplastika perforující MeSH
- kožní abnormality diagnóza genetika patofyziologie MeSH
- lidé MeSH
- myopie patofyziologie MeSH
- nemoci pojiva diagnóza MeSH
- nestabilita kloubu vrozené diagnóza genetika patofyziologie MeSH
- odchlípení sítnice diagnóza patofyziologie chirurgie MeSH
- percepční nedoslýchavost diagnóza MeSH
- perforace rohovky patofyziologie chirurgie MeSH
- reoperace MeSH
- trabekulektomie MeSH
- transkripční faktory genetika MeSH
- vitrektomie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- přehledy MeSH
- Názvy látek
- transkripční faktory MeSH
- ZNF469 protein, human MeSH Prohlížeč
Branched esters of palmitic acid and hydroxystearic acid (PAHSA) are anti-inflammatory and antidiabetic lipokines that connect glucose and lipid metabolism. We aimed to characterize involvement of the 5-PAHSA regioisomer in the adaptive metabolic response of white adipose tissue (WAT) to cold exposure (CE) in mice, exploring the cross talk between glucose utilization and lipid metabolism. CE promoted local production of 5- and 9-PAHSAs in WAT. Metabolic labeling of de novo lipogenesis (DNL) using 2H2O revealed that 5-PAHSA potentiated the effects of CE and stimulated triacylglycerol (TAG)/fatty acid (FA) cycling in WAT through impacting lipogenesis and lipolysis. Adipocyte lipolytic products were altered by 5-PAHSA through selective FA re-esterification. The impaired lipolysis in global adipose triglyceride lipase (ATGL) knockout mice reduced free PAHSA levels and uncovered a metabolite reservoir of TAG-bound PAHSAs (TAG estolides) in WAT. Utilization of 13C isotope tracers and dynamic metabolomics documented that 5-PAHSA primes adipocytes for glucose metabolism in a different way from insulin, promoting DNL and impeding TAG synthesis. In summary, our data reveal new cellular and physiological mechanisms underlying the beneficial effects of 5-PAHSA and its relation to insulin action in adipocytes and independently confirm a PAHSA metabolite reservoir linked to ATGL-mediated lipolysis.
- MeSH
- bílá tuková tkáň metabolismus MeSH
- glukosa metabolismus MeSH
- izotopy uhlíku MeSH
- kyselina palmitová metabolismus MeSH
- kyseliny stearové metabolismus MeSH
- lipasa genetika metabolismus MeSH
- lipogeneze genetika MeSH
- lipolýza MeSH
- mastné kyseliny metabolismus MeSH
- metabolomika MeSH
- myši knockoutované MeSH
- myši MeSH
- nízká teplota MeSH
- oxid deuteria MeSH
- triglyceridy metabolismus MeSH
- tukové buňky metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 5-PAHSA MeSH Prohlížeč
- 9-PAHSA MeSH Prohlížeč
- glukosa MeSH
- izotopy uhlíku MeSH
- kyselina palmitová MeSH
- kyseliny stearové MeSH
- lipasa MeSH
- mastné kyseliny MeSH
- oxid deuteria MeSH
- PNPLA2 protein, mouse MeSH Prohlížeč
- triglyceridy MeSH
PURPOSE: To report molecular genetic findings in six probands with congenital hereditary endothelial dystrophy (CHED) variably associated with hearing loss (also known as Harboyan syndrome). Furthermore, we developed a cellular model to determine if disease-associated variants induce aberrant SLC4A11 pre-mRNA splicing. METHODS: Direct sequencing of the entire SLC4A11 coding region was performed in five probands. In one individual, whole genome sequencing was undertaken. The effect of c.2240+5G>A on pre-mRNA splicing was evaluated in a corneal endothelial-like (CE-like) cell model expressing SLC4A11. CE-like cells were derived from autologous induced pluripotent stem cells (iPSCs) via neural crest cells exposed to B27, PDGF-BB, and DKK-2. Total RNA was extracted, and RT-PCR was performed followed by Sanger and a targeted next generation sequencing (NGS) approach to identify and quantify the relative abundance of alternatively spliced transcripts. RESULTS: In total, 11 different mutations in SLC4A11 evaluated as pathogenic were identified; of these, c.1237G>A, c.2003T>C, c.1216+1G>A, and c.2240+5G>A were novel. The c.2240+5G>A variant was demonstrated to result in aberrant pre-mRNA splicing. A targeted NGS approach confirmed that the variant introduces a leaky cryptic splice donor site leading to the production of a transcript containing an insertion of six base pairs with the subsequent introduction of a premature stop codon (p.Thr747*). Furthermore, a subset of transcripts comprising full retention of intron 16 also were observed, leading to the same functionally null allele. CONCLUSIONS: This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants.
- MeSH
- antiportéry biosyntéza genetika MeSH
- buněčná diferenciace MeSH
- dědičné dystrofie rohovky genetika metabolismus patologie MeSH
- dítě MeSH
- dospělí MeSH
- indukované pluripotentní kmenové buňky cytologie metabolismus MeSH
- kultivované buňky MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- percepční nedoslýchavost genetika metabolismus patologie MeSH
- předškolní dítě MeSH
- prekurzory RNA MeSH
- proteiny přenášející anionty biosyntéza genetika MeSH
- regulace genové exprese * MeSH
- RNA genetika MeSH
- rodokmen MeSH
- rohovkový endotel metabolismus patologie MeSH
- senioři MeSH
- sestřih RNA MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antiportéry MeSH
- prekurzory RNA MeSH
- proteiny přenášející anionty MeSH
- RNA MeSH
- SLC4A11 protein, human MeSH Prohlížeč