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1.
Am J Physiol Cell Physiol ; 321(3): C535-C548, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34288724

RESUMO

Extracellular vesicles (EVs) contain biological molecules and are secreted by cells into the extracellular milieu. The endothelial sodium channel (EnNaC) plays an important role in modulating endothelial cell stiffness. We hypothesized EVs secreted from human aortic endothelial cells (hAoECs) positively regulate EnNaC in an autocrine-dependent manner. A comprehensive lipidomic analysis using targeted mass spectrometry was performed on multiple preparations of EVs isolated from the conditioned media of hAoECs or complete growth media of these cells. Cultured hAoECs challenged with EVs isolated from the conditioned media of these cells resulted in an increase in EnNaC activity when compared with the same concentration of media-derived EVs or vehicle alone. EVs isolated from the conditioned media of hAoECs but not human fibroblast cells were enriched in MARCKS-like protein 1 (MLP1). The pharmacological inhibition of the negative regulator of MLP1, protein kinase C, in cultured hAoECs resulted in an increase in EV size and release compared with vehicle or pharmacological inhibition of protein kinase D. The MLP1-enriched EVs increased the density of actin filaments in cultured hAoECs compared with EVs isolated from human fibroblast cells lacking MLP1. We quantified 141 lipids from glycerolipids, glycerophospholipids, and sphingolipids in conditioned media EVs that represented twice the number found in control media EVs. The concentrations of sphingomyelin, lysophosphatidylcholine and phosphatidylethanolamine were higher in conditioned media EVs. These results provide the first evidence for EnNaC regulation in hAoECs by EVs and provide insight into a possible mechanism involving MLP1, unsaturated lipids, and bioactive lipids.


Assuntos
Proteínas de Ligação a Calmodulina/genética , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Lisofosfatidilcolinas/metabolismo , Proteínas dos Microfilamentos/genética , Fosfatidiletanolaminas/metabolismo , Esfingomielinas/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Aorta/citologia , Aorta/metabolismo , Comunicação Autócrina , Proteínas de Ligação a Calmodulina/metabolismo , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Vesículas Extracelulares/química , Expressão Gênica , Glicerofosfolipídeos/metabolismo , Humanos , Lipidômica/métodos , Lisofosfatidilcolinas/farmacologia , Proteínas dos Microfilamentos/metabolismo , Fosfatidiletanolaminas/farmacologia , Cultura Primária de Células , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Esfingomielinas/farmacologia
2.
Toxicology ; 458: 152841, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34216699

RESUMO

The cardiotoxicity of various anticancer therapies, including radiotherapy, can lead to cardiovascular complications. These complications can range from damaging cardiac tissues within the irradiation field to increasing the long-term risks of developing heart failure, coronary artery disease, and myocardial infarction. We analyzed radiation-induced metabolites capable of mediating critical biological processes, such as inflammation, senescence, and apoptosis. Previously, by applying QTOF-MASS analysis to irradiated human fibroblasts, we identified that metabolite sets of lysophosphatidylcholine (LPC) were increased in these cells. In this study, radiation-induced LPC accumulation in human aortic endothelial cells (HAECs) increased reactive oxygen species (ROS) production and senescence-associated-beta-galactosidase staining, in addition to decreasing their tube-forming ability. Knockdown of lipoprotein-associated phospholipase A2 (Lp-PLA2) with small interfering RNA (siRNA) inhibited the increased LPC production induced by radiation, and reduced the radiation-induced cell damage produced by ROS and oxidized low-density lipoprotein (LDL). Lp-PLA2 depletion abolished the induction of proinflammatory factors, such as interleukin 1ß, tumor necrosis factor-alpha, matrix metalloproteinase 2, and matrix metalloproteinase 9, as well as adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and E-selection. Likewise, we showed that Lp-PLA2 expression was upregulated in the vasculature of irradiated rat, resulting in increased LPC production and LDL oxidation. Our data demonstrate that radiation-induced LPC production is a potential risk factor for cardiotoxicity that is mediated by Lp-PLA2 activity, suggesting that LPC and Lp-PLA2 offer potential diagnostic and therapeutic approaches to cardiovascular damage during radiotherapy.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase/efeitos da radiação , Células Endoteliais/patologia , Células Endoteliais/efeitos da radiação , Lisofosfatidilcolinas/metabolismo , Fosfolipases A2/metabolismo , Fosfolipases A2/efeitos da radiação , Animais , Aorta/patologia , Aorta/efeitos da radiação , Citocinas/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Inflamação/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/efeitos da radiação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/efeitos da radiação , Radiação Ionizante , Ratos , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio/metabolismo
3.
Toxicology ; 458: 152835, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34126166

RESUMO

Aflatoxin M1 (AFM1) and ochratoxin A (OTA) are pernicious mycotoxins widely co-existing in the environment. However, nephrotoxicity and underlying mechanism induced by AFM1 coupled with OTA still remain to be explored. In this study, CD-1 mice were treated with 3.5 mg/kg b.w. AFM1, OTA, and AFM1 + OTA for 35 days, and UPLC-MS-based metabolomics method was effectuated to investigate metabolomic profiles of mice kidney. Subsequent experiments on human renal proximal tubular (HK-2) cells were performed to dig out the causal connections between distinguished differential metabolites and nephrotoxicity. Compared with DMSO vehicle group, all three toxin treatments (AFM1 and OTA alone, and in combination) significantly reduced final body weight, and remarkably elevated the concentration of serum creatinine (SCr) and caused abnormal histological phenotypes (shown by histopathological slices). OTA, AFM1 + OTA but not AFM1 reduced the relative weight index of kidney. These phenotypic results indicated that AFM1 and OTA were both toxic to the body, and it seemed that OTA exhibited a notable impairment to kidney while AFM1 had similar but limited effect compared with OTA. Further metabolomics analysis showed that when AFM1 and OTA were combined together, OTA exerted dominant effect on the alteration of metabolic processes. There were few differences in the number of changed metabolites between OTA and AFM1 + OTA group. Among the differentially expressed metabolites affected by OTA, and AFM1 + OTA, lysophosphatidylcholines (LysoPCs) were identified as the main type with significant upregulation, in which LysoPC (16:0) accounted for the most prime proportion. Western blotting results of HK-2 cells showed that single OTA and AFM1 + OTA increased the apoptotic protein expressions of Bax, caspase 3 and PARP, and decreased the expression of Bcl-2; while AFM1 only raised the expression of caspase 3. LysoPC (16:0) but not LysoPC (18:1) lifted the protein level of caspase 3 and PARP in HK-2 cells, and reduced the level of Bcl-2. Taken together, this study is the first effort trying to assess nephrotoxicity of AFM1 with OTA, and we guessed that OTA had a more pronounced toxicity to kidney in contrast to AFM1. No obvious synergism between AFM1 and OTA was found to contribute to the occurrence or development of nephropathy. LysoPC (16:0) might be the pivotal metabolite in response to single OTA and combined AFM1 + OTA engendering renal injury.


Assuntos
Aflatoxina M1/toxicidade , Carcinógenos/toxicidade , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Metabolômica , Ocratoxinas/toxicidade , Animais , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Células CACO-2 , Caspase 3/metabolismo , Linhagem Celular , Humanos , Rim/patologia , Nefropatias/patologia , Lisofosfatidilcolinas/metabolismo , Masculino , Camundongos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteômica
4.
Exp Eye Res ; 207: 108601, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33910035

RESUMO

Electrical stimulation (ES) of the eye represents a therapeutic approach in various clinical applications ranging from retinal dystrophies, age-related macular degeneration, retinal artery occlusion and nonarteritic ischemic optic neuropathy. In clinical practice, ES of the eye is mainly performed with a transcorneal or transpalpebral approach. These procedures are non-invasive and well-tolerated by the patients, reporting only minimal and transient adverse events, while serious adverse effects were not observed. Despite the growing literature on animal models, only clinical parameters have been investigated in humans and few data are available about biochemical changes induced by ES of the eye. The purpose of this study is to investigate the possible mechanism that regulates the beneficial effects of ES on retinal cells function and survival in humans. 28 patients undergoing pars plana vitrectomy (PPV) for idiopathic epiretinal membrane (iERM) were randomly divided in two groups: 13 patients were treated with transpalpebral ES before surgery and 15 underwent surgery with no prior treatment. Vitreous samples were collected for biochemical analysis during PPV. ES treatment leads to a reduction in the vitreous expression of both proinflammatory cytokines, namely IL-6 and IL-8, and proinflammatory lipid mediators, such as lysophosphatidylcholine. Indeed, we observed a 70% decrease of lysophosphatidylcholine 18:0, which has been proven to exert the greatest proinflammatory activities among the lysophosphatidylcholine class. The content of triglycerides is also affected and significantly decreased following ES application. The vitreous composition of patients undergoing PPV for iERM displays significant changes following ES treatment. Proinflammatory cytokines and bioactive lipid mediators expression decreases, suggesting an overall anti-inflammatory potential of ES. The investigation of the mechanism by which this treatment alters the retinal neurons leading to good outcomes is essential for supporting ES therapeutic application in various types of retinal diseases.


Assuntos
Citocinas/metabolismo , Terapia por Estimulação Elétrica , Membrana Epirretiniana/terapia , Lisofosfatidilcolinas/metabolismo , Triglicerídeos/metabolismo , Corpo Vítreo/metabolismo , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática , Membrana Epirretiniana/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas por Ionização por Electrospray , Vitrectomia
5.
Protein J ; 40(2): 192-204, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33665770

RESUMO

Cytosolic estrogen sulfotransferase (SULT1E) mainly catalyzes the sulfate conjugation of estrogens, which decrease atherosclerosis progression. Recently we reported that a YKEG sequence in human SULT1E1 (hSULT1E1) corresponding to residues 61-64 can bind specifically to oxidized low-density lipoprotein (Ox-LDL), which plays a major role in the pathogenesis of atherosclerosis; its major oxidative lipid component lysophosphatidylcholine (LPC), and its structurally similar lipid, platelet-activating factor (PAF). In this study, we investigated the effect of Ox-LDL on the sulfating activity of hSULT1E1. In vivo experiments using a mouse model of atherosclerosis showed that the protein expression of SULT1E1 was higher in the aorta of mice with atherosclerosis compared with that in control animals. Results from a sulfating activity assay of hSULT1E1 using 1-hydroxypyrene as the substrate demonstrated that Ox-LDL, LPC, and PAF markedly decreased the sulfating activity of hSULT1E1, whereas native LDL and 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) as one of the oxidized phosphatidylcholines showed the opposite effect. The sulfating activity greatly changed in the presence of LPC, PAF, and POVPC in their concentration-dependen manner (especially above their critical micelle concentrations). Moreover, Ox-LDL specifically recognized dimeric hSULT1E1. These results suggest that the effects of Ox-LDL and native LDL on the sulfating activity of hSULT1E1 might be helpful in elucidating the novel mechanism underlying the pathogenesis of atherosclerosis, involving the relationship between estrogen metabolism, LDL, and Ox-LDL.


Assuntos
Lipoproteínas LDL , Sulfotransferases , Animais , Aterosclerose , Humanos , Lipoproteínas LDL/química , Lipoproteínas LDL/metabolismo , Lisofosfatidilcolinas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fator de Ativação de Plaquetas/metabolismo , Ligação Proteica , Sulfotransferases/química , Sulfotransferases/metabolismo
6.
J Biomed Sci ; 28(1): 20, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33722220

RESUMO

BACKGROUND AND AIMS: Previous study disclosed Fucosyltransferase 2 (Fut2) gene as a IBD risk locus. This study aimed to explore the mechanism of Fut2 in IBD susceptibility and to propose a new strategy for the treatment of IBD. METHODS: Intestinal epithelium-specific Fut2 knockout (Fut2△IEC) mice was used. Colitis was induced by dextran sulfate sodium (DSS). The composition and diversity of gut microbiota were assessed via 16S rRNA analysis and the metabolomic findings was obtained from mice feces via metabolite profiling. The fecal microbiota transplantation (FMT) experiment was performed to confirm the association of gut microbiota and LPC. WT mice were treated with Lysophosphatidylcholine (LPC) to verify its impact on colitis. RESULTS: The expression of Fut2 and α-1,2-fucosylation in colonic tissues were decreased in patients with UC (UC vs. control, P = 0.036) and CD (CD vs. control, P = 0.031). When treated with DSS, in comparison to WT mice, more severe intestinal inflammation and destructive barrier functions in Fut2△IEC mice was noted. Lower gut microbiota diversity was observed in Fut2△IEC mice compared with WT mice (p < 0.001). When exposed to DSS, gut bacterial diversity and composition altered obviously in Fut2△IEC mice and the fecal concentration of LPC was increased. FMT experiment revealed that mice received the fecal microbiota from Fut2△IEC mice exhibited more severe colitis and higher fecal LPC concentration. Correlation analysis showed that the concentration of LPC was positively correlated with four bacteria-Escherichia, Bilophila, Enterorhabdus and Gordonibacter. Furthermore, LPC was proved to promote the release of pro-inflammatory cytokines and damage epithelial barrier in vitro and in vivo. CONCLUSION: Fut2 and α-1,2-fucosylation in colon were decreased not only in CD but also in UC patients. Gut microbiota in Fut2△IEC mice is altered structurally and functionally, promoting generation of LPC which was proved to promote inflammation and damage epithelial barrier.


Assuntos
Bactérias/metabolismo , Colite/microbiologia , Fucosiltransferases/deficiência , Microbioma Gastrointestinal , Lisofosfatidilcolinas/metabolismo , Animais , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Transgênicos
7.
mBio ; 12(1)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33622719

RESUMO

Lipids are biologically active molecules involved in a variety of cellular processes and immunological functions, including inflammation. It was recently shown that phospholipids and their derivatives, lysophospholipids, can reactivate latent (dormant) tumor cells, causing cancer recurrence. However, the potential link between lipids and HIV latency, persistence, and viral rebound after cessation of antiretroviral therapy (ART) has never been investigated. We explored the links between plasma lipids and the burden of HIV during ART. We profiled the circulating lipidome from plasma samples from 24 chronically HIV-infected individuals on suppressive ART who subsequently underwent an analytic treatment interruption (ATI) without concurrent immunotherapies. The pre-ATI viral burden was estimated as time-to-viral-rebound and viral load set points post-ATI. We found that higher pre-ATI levels of lysophospholipids, including the proinflammatory lysophosphatidylcholine, were associated with faster time-to-viral-rebound and higher viral set points upon ART cessation. Furthermore, higher pre-ATI levels of the proinflammatory by-product of intestinal lysophosphatidylcholine metabolism, trimethylamine-N-oxide (TMAO), were also linked to faster viral rebound post-ART. Finally, pre-ATI levels of several phosphatidylcholine species (lysophosphatidylcholine precursors) correlated strongly with higher pre-ATI levels of HIV DNA in peripheral CD4+ T cells. Our proof-of-concept data point to phospholipids and lysophospholipids as plausible proinflammatory contributors to HIV persistence and rapid post-ART HIV rebound. The potential interplay between phospholipid metabolism and both the establishment and maintenance of HIV latent reservoirs during and after ART warrants further investigation.IMPORTANCE The likelihood of HIV rebound after stopping antiretroviral therapy (ART) is a combination of the size of HIV reservoirs that persist despite ART and the host immunological and inflammatory factors that control these reservoirs. Therefore, there is a need to comprehensively understand these host factors to develop a strategy to cure HIV infection and prevent viral rebound post-ART. Lipids are important biologically active molecules that are known to mediate several cellular functions, including reactivating latent tumor cells; however, their role in HIV latency, persistence, and post-ART rebound has never been investigated. We observed significant links between higher levels of the proinflammatory lysophosphatidylcholine and its intestinal metabolic by-product, trimethylamine-N-oxide, and both faster time-to-viral-rebound and higher viral load set point post-ART. These data highlight the need for further studies to understand the potential contribution of phosphatidylcholine and lysophosphatidylcholine metabolism in shaping host immunological and inflammatory milieu during and after ART.


Assuntos
Antirretrovirais/uso terapêutico , Infecções por HIV/tratamento farmacológico , Fosfolipídeos/sangue , Fosfolipídeos/metabolismo , Carga Viral , Latência Viral , Suspensão de Tratamento , Adulto , Linfócitos T CD4-Positivos/virologia , Estudos de Coortes , DNA Viral/análise , Feminino , Infecções por HIV/virologia , Humanos , Lisofosfatidilcolinas/sangue , Lisofosfatidilcolinas/metabolismo , Masculino , Pessoa de Meia-Idade , Fosfatidilcolinas/sangue , Fosfatidilcolinas/metabolismo , Fosfolipídeos/classificação , Estudo de Prova de Conceito , Adulto Jovem
8.
Nutr Res ; 85: 119-134, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33482601

RESUMO

Docosahexaenoic acid (DHA) is one of the most important fatty acids that plays a critical role in maintaining proper brain function and cognitive development. Deficiency of DHA leads to several neurodegenerative disorders and, therefore, dietary supplementations of these fatty acids are essential to maintain cognitive health. However, the complete picture of how DHA is incorporated into the brain is yet to be explored. In general, the de novo synthesis of DHA is poor, and targeting the brain with specific phospholipid carriers provides novel insights into the process of reduction of disease progression. Recent studies have suggested that compared to triacylglycerol form of DHA, esterified form of DHA (i.e., lysophosphatidylcholine [lysoPC]) is better incorporated into the brain. Free DHA is transported across the outer membrane leaflet of the blood-brain barrier via APOE4 receptors, whereas DHA-lysoPC is transported across the inner membrane leaflet of the blood-brain barrier via a specific protein called Mfsd2a. Dietary supplementation of this lysoPC specific form of DHA is a novel therapy and is used to decrease the risk of various neurodegenerative disorders. Currently, structured glycerides of DHA - novel nutraceutical agents - are being widely used for the prevention and treatment of various neurological diseases. However, it is important to fully understand their metabolic regulation and mechanism of transportation to the brain. This article comprehensively reviews various studies that have evaluated the bioavailability of DHA, mechanisms of DHA transport, and role of DHA in preventing neurodegenerative disorders, which provides better insight into the pathophysiology of these disorders and use of structured DHA in improving neurological health.


Assuntos
Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácidos Docosa-Hexaenoicos/metabolismo , Lisofosfatidilcolinas/administração & dosagem , Lisofosfatidilcolinas/metabolismo , Doenças Neurodegenerativas/prevenção & controle , Animais , Disponibilidade Biológica , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Gorduras Insaturadas na Dieta/administração & dosagem , Ácidos Docosa-Hexaenoicos/biossíntese , Ácidos Docosa-Hexaenoicos/química , Ácidos Graxos Insaturados/administração & dosagem , Humanos , Lisofosfatidilcolinas/química , Doenças Neurodegenerativas/fisiopatologia , Obesidade/metabolismo
9.
Int J Mol Sci ; 21(22)2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-33233525

RESUMO

We investigated the synthesis of N-docosahexaenoylethanolamine (synaptamide) in neuronal cells from unesterified docosahexaenoic acid (DHA) or DHA-lysophosphatidylcholine (DHA-lysoPC), the two major lipid forms that deliver DHA to the brain, in order to understand the formation of this neurotrophic and neuroprotective metabolite of DHA in the brain. Both substrates were taken up in Neuro2A cells and metabolized to N-docosahexaenoylphosphatidylethanolamine (NDoPE) and synaptamide in a time- and concentration-dependent manner, but unesterified DHA was 1.5 to 2.4 times more effective than DHA-lysoPC at equimolar concentrations. The plasmalogen NDoPE (pNDoPE) amounted more than 80% of NDoPE produced from DHA or DHA-lysoPC, with 16-carbon-pNDoPE being the most abundant species. Inhibition of N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) by hexachlorophene or bithionol significantly decreased the synaptamide production, indicating that synaptamide synthesis is mediated at least in part via NDoPE hydrolysis. NDoPE formation occurred much more rapidly than synaptamide production, indicating a precursor-product relationship. Although NDoPE is an intermediate for synaptamide biosynthesis, only about 1% of newly synthesized NDoPE was converted to synaptamide, possibly suggesting additional biological function of NDoPE, particularly for pNDoPE, which is the major form of NDoPE produced.


Assuntos
Ácidos Araquidônicos/biossíntese , Ácidos Docosa-Hexaenoicos/metabolismo , Endocanabinoides/biossíntese , Etanolaminas/metabolismo , Lisofosfatidilcolinas/metabolismo , Neurônios/metabolismo , Animais , Ácidos Araquidônicos/antagonistas & inibidores , Ácidos Araquidônicos/isolamento & purificação , Bitionol/farmacologia , Isótopos de Carbono , Linhagem Celular Tumoral , Cromatografia Líquida , Endocanabinoides/antagonistas & inibidores , Endocanabinoides/isolamento & purificação , Etanolaminas/antagonistas & inibidores , Etanolaminas/isolamento & purificação , Hexaclorofeno/farmacologia , Cinética , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Plasmalogênios/antagonistas & inibidores , Plasmalogênios/biossíntese , Plasmalogênios/isolamento & purificação , Alcamidas Poli-Insaturadas/antagonistas & inibidores , Alcamidas Poli-Insaturadas/isolamento & purificação , Espectrometria de Massas em Tandem
10.
Int J Mol Sci ; 21(22)2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-33182805

RESUMO

Lipid catabolism and anabolism changes play a role in stemness acquisition by cancer cells, and cancer stem cells (CSCs) are particularly dependent on the activity of the enzymes involved in these processes. Lipidomic changes could play a role in CSCs' ability to cause disease relapse and chemoresistance. The exploration of lipid composition and metabolism changes in CSCs in the context of hepatocellular cancer (HCC) is still incomplete and their lipidomic scenario continues to be elusive. We aimed to evaluate through high-throughput mass spectrometry (MS)-based lipidomics the levels of the members of the six major classes of sphingolipids and phospholipids in two HCC cell lines (HepG2 and Huh-7) silenced for the expression of histone variant macroH2A1 (favoring stemness acquisition), or silenced for the expression of focal adhesion tyrosine kinase (FAK) (hindering aggressiveness and stemness). Transcriptomic changes were evaluated by RNA sequencing as well. We found definite lipidomic and transcriptomic changes in the HCC lines upon knockdown (KD) of macroH2A1 or FAK, in line with the acquisition or loss of stemness features. In particular, macroH2A1 KD increased total sphingomyelin (SM) levels and decreased total lysophosphatidylcholine (LPC) levels, while FAK KD decreased total phosphatidylcholine (PC) levels. In conclusion, in HCC cell lines knocked down for specific signaling/epigenetic processes driving opposite stemness potential, we defined a lipidomic signature that hallmarks hepatic CSCs to be exploited for therapeutic strategies.


Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Metabolismo dos Lipídeos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/deficiência , Quinase 1 de Adesão Focal/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Hep G2 , Histonas/antagonistas & inibidores , Histonas/deficiência , Histonas/genética , Humanos , Metabolismo dos Lipídeos/genética , Lipidômica , Neoplasias Hepáticas/genética , Lisofosfatidilcolinas/metabolismo , Fosfatidilcolinas/metabolismo , RNA-Seq , Esfingomielinas/metabolismo
11.
Cells ; 9(10)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-33003477

RESUMO

The physiological fate of cells that die by apoptosis is their prompt and efficient removal by efferocytosis. During these processes, apoptotic cells release intracellular constituents that include purine nucleotides, lysophosphatidylcholine (LPC), and Sphingosine-1-phosphate (S1P) that induce migration and chemo-attraction of phagocytes as well as mitogens and extracellular membrane-bound vesicles that contribute to apoptosis-induced compensatory proliferation and alteration of the extracellular matrix and the vascular network. Additionally, during efferocytosis, phagocytic cells produce a number of anti-inflammatory and resolving factors, and, together with apoptotic cells, efferocytic events have a homeostatic function that regulates tissue repair. These homeostatic functions are dysregulated in cancers, where, aforementioned events, if not properly controlled, can lead to cancer progression and immune escape. Here, we summarize evidence that apoptosis and efferocytosis are exploited in cancer, as well as discuss current translation and clinical efforts to harness signals from dying cells into therapeutic strategies.


Assuntos
Apoptose/imunologia , Morte Celular/imunologia , Terapia de Alvo Molecular/métodos , Neoplasias/imunologia , Fagocitose/imunologia , Fosfatidilserinas/metabolismo , Evasão Tumoral , Microambiente Tumoral/imunologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Humanos , Lisofosfatidilcolinas/metabolismo , Lisofosfolipídeos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fagocitose/genética , Nucleotídeos de Purina/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
12.
Toxins (Basel) ; 12(10)2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33019554

RESUMO

The Hemiscorpius lepturus scorpion and brown spider Loxosceles intermedia represent a public health problem in Asia and America, respectively. Although distinct, these organisms contain similar toxins responsible for the principal clinical signs of envenomation. To better understand the properties of these toxins, we designed a study to compare recombinant Heminecrolysin (rHNC) and rLiD1, the major phospholipase D toxins of scorpion and spider venom, respectively. Using a competitive ELISA and a hemolytic inhibition test, we come to spot a cross reaction between scorpion and spider venoms along with an epitopic similarity between rHNC and rLiD1 associated with neutralizing antibodies. Results show that the ability of the rHNC to hydrolyze lysophosphatidylcholine (LPC) is equivalent to that of rLiD1 to hydrolyze sphingomyelin and vice-versa. rHNC exclusively catalyze transphosphatidylation of LPC producing cyclic phosphatidic acid (cPA). The in-silico analysis of hydrogen bonds between LPC and toxins provides a possible explanation for the higher transphosphatidylase activity of rHNC. Interestingly, for the first time, we reveal that lysophosphatidic acid (LPA) can be a substrate for both enzymes using cellular and enzymatic assays. The finding of the usage of LPA as a substrate as well as the formation of cPA as an end product could shed more light on the molecular basis of Hemiscorpius lepturus envenomation as well as on loxoscelism.


Assuntos
Antivenenos/farmacologia , Aranha Marrom Reclusa , Fosfolipase D/toxicidade , Diester Fosfórico Hidrolases/toxicidade , Venenos de Escorpião/toxicidade , Escorpiões , Pele/efeitos dos fármacos , Venenos de Aranha/toxicidade , Animais , Antivenenos/imunologia , Aranha Marrom Reclusa/enzimologia , Aranha Marrom Reclusa/imunologia , Reações Cruzadas , Epitopos , Hemólise/efeitos dos fármacos , Mordeduras e Picadas de Insetos/enzimologia , Lisofosfatidilcolinas/metabolismo , Necrose , Fosfolipase D/imunologia , Fosfolipase D/metabolismo , Diester Fosfórico Hidrolases/imunologia , Venenos de Escorpião/enzimologia , Venenos de Escorpião/imunologia , Escorpiões/enzimologia , Escorpiões/imunologia , Pele/enzimologia , Pele/patologia , Esfingomielinas/metabolismo , Venenos de Aranha/enzimologia , Venenos de Aranha/imunologia , Especificidade por Substrato
13.
J Neurosci ; 40(47): 9137-9147, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33051352

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease characterized by motor neuron (MN) death. Lipid dysregulation manifests during disease; however, it is unclear whether lipid homeostasis is adversely affected in the in the spinal cord gray matter (GM), and if so, whether it is because of an aberrant increase in lipid synthesis. Moreover, it is unknown whether lipid dysregulation contributes to MN death. Here, we show that cholesterol ester (CE) and triacylglycerol levels are elevated several-fold in the spinal cord GM of male sporadic ALS patients. Interestingly, HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, was reduced in the spinal cord GM of ALS patients. Increased cytosolic phospholipase A2 activity and lyso-phosphatidylcholine (Lyso-PC) levels in ALS patients suggest that CE accumulation was driven by acyl group transfer from PC to cholesterol. Notably, Lyso-PC, a byproduct of CE synthesis, was toxic to human MNs in vitro Elevations in CE, triacylglycerol, and Lyso-PC were also found in the spinal cord of SOD1G93A mice, a model of ALS. Similar to ALS patients, a compensatory downregulation of cholesterol synthesis occurred in the spinal cord of SOD1G93A mice; levels of sterol regulatory element binding protein 2, a transcriptional regulator of cholesterol synthesis, progressively declined. Remarkably, overexpressing sterol regulatory element binding protein 2 in the spinal cord of normal mice to model CE accumulation led to ALS-like lipid pathology, MN death, astrogliosis, paralysis, and reduced survival. Thus, spinal cord lipid dysregulation in ALS likely contributes to neurodegeneration and developing therapies to restore lipid homeostasis may lead to a treatment for ALS.SIGNIFICANCE STATEMENT Neurons that control muscular function progressively degenerate in patients with amyotrophic lateral sclerosis (ALS). Lipid dysregulation is a feature of ALS; however, it is unclear whether disrupted lipid homeostasis (i.e., lipid cacostasis) occurs proximal to degenerating neurons in the spinal cord, what causes it, and whether it contributes to neurodegeneration. Here we show that lipid cacostasis occurs in the spinal cord gray matter of ALS patients. Lipid accumulation was not associated with an aberrant increase in synthesis or reduced hydrolysis, as enzymatic and transcriptional regulators of lipid synthesis were downregulated during disease. Last, we demonstrated that genetic induction of lipid cacostasis in the CNS of normal mice was associated with ALS-like lipid pathology, astrogliosis, neurodegeneration, and clinical features of ALS.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Metabolismo dos Lipídeos , Esclerose Amiotrófica Lateral/metabolismo , Animais , Morte Celular , Ésteres do Colesterol/metabolismo , Substância Cinzenta/metabolismo , Humanos , Lisofosfatidilcolinas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Receptores Acoplados a Proteínas G/genética , Receptores da Fosfolipase A2/metabolismo , Medula Espinal/metabolismo , Superóxido Dismutase-1/genética , Triglicerídeos/metabolismo
14.
Ann Rheum Dis ; 79(12): 1644-1656, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32907805

RESUMO

OBJECTIVES: Fibromyalgia is commonly considered a stress-related chronic pain disorder, and daily stressors are known triggers. However, the relation between stress and pain development remains poorly defined by clinical approaches. Also, the aetiology remains largely unknown. METHODS: We used a newly developed mouse model and lipidomic approaches to probe the causation and explore the biological plausibility for how perceived stress translates into chronic non-inflammatory pain. Clinical and lipidomic investigations of fibromyalgia were conducted for human validation. RESULTS: Using non-painful sound stimuli as psychological stressors, we demonstrated that mice developed long-lasting non-inflammatory hyperalgesia after repeated and intermittent sound stress exposure. Elevated serum malondialdehyde level in stressed mice indicated excessive oxidative stress and lipid oxidative damage. Lipidomics revealed upregulation of lysophosphatidylcholine 16:0 (LPC16:0), a product of lipid oxidisation, in stressed mice. Intramuscular LPC16:0 injection triggered nociceptive responses and a hyperalgesic priming-like effect that caused long-lasting hypersensitivity. Pharmacological or genetic inhibition of acid-sensing ion channel 3 impeded the development of LPC16:0-induced chronic hyperalgesia. Darapladib and antioxidants could effectively alleviate the stress-induced hyperalgesia by inhibiting LPC16:0 synthesis. Clinical investigations showed that excessive oxidative stress and LPC16:0 expression also exist in patients with fibromyalgia. Moreover, LPC16:0 expression was correlated with pain symptoms in patients with high oxidative stress and disease severity. CONCLUSIONS: Our study provides experimental evidence for the causal effect of psychological stressors on chronic pain development. The findings identify a possible pathophysiological mechanism of stress-induced chronic non-inflammatory pain at molecular, behavioural and clinical levels that might indicate a new therapeutic approach for fibromyalgia.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Fibromialgia/metabolismo , Fibromialgia/psicologia , Lisofosfatidilcolinas/metabolismo , Estresse Psicológico/metabolismo , Animais , Dor Crônica/metabolismo , Dor Crônica/psicologia , Feminino , Humanos , Hiperalgesia/metabolismo , Hiperalgesia/psicologia , Lipidômica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/fisiologia , Estresse Psicológico/complicações
15.
Cells ; 9(9)2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32917053

RESUMO

The impaired spatial arrangement and connections between cells creating islets of Langerhans as well as altered expression of G protein-coupled receptors (GPCRs) often lead to dysfunction of insulin-secreting pancreatic ß cells and can significantly contribute to the development of diabetes. Differences in glucose-stimulated insulin secretion (GSIS) are noticeable not only in diabetic individuals but also in model pancreatic ß cells, e.g., ßTC3 and MIN6 ß cell lines with impaired and normal insulin secretion, respectively. Now, we compare the ability of GPCR agonists (lysophosphatidylcholines bearing fatty acid chains of different lengths) to potentiate GSIS in ßTC3 and MIN6 ß cell models, cultured as adherent monolayers and in a form of pseudoislets (PIs) with pancreatic MS1 endothelial cells. Our aim was also to investigate differences in expression of the GPCRs responsive to LPCs in these experimental systems. Aggregation of ß cells into islet-like structures greatly enhanced the expression of Gpr40, Gpr55, and Gpr119 receptors. In contrast, the co-culture of ßTC3 cells with endothelial cells converted the GPCR expression pattern closer to the pattern observed in MIN6 cells. Additionally, the efficiencies of various LPC species in ßTC3-MS1 PIs also shifted toward the MIN6 cell model.


Assuntos
Betacelulina/metabolismo , Glucose/metabolismo , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Lisofosfatidilcolinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Humanos
16.
J Photochem Photobiol B ; 211: 111994, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32858337

RESUMO

Circadian rhythm is the most important and universal biological rhythm in marine organisms. In this research, the movement behaviour of abalone (Haliotis discus hannai) was continuously monitored under a light cycle of 12 L:12D. It was found that the cumulative movement distance and cumulative movement time of abalone reached was highest from 00:00-03:00 h. The minimum values of maximum movement velocity occurred between 21:00-00:00 h, and a significant circadian cosine rhythm was exhibited during these periods (P < 0.05). Metabolomic analysis of cerebral ganglions of abalone was conducted at 06:00 h (6 M), 14:00 h (14 M), and 22:00 h (22 M) and 380, 385, and 315 metabolites with significant differences were identified in 6 M vs 14 M, 14 M vs 22 M, and 6 M vs 22 M, respectively (P < 0.05). With the alternation of day and night, the expression levels of phosphatidylcholine, 5-HT, N-acetyl-5-hydroxytryptamine, indole-3-acetaldehyde, hypoxanthine, and deoxyinosine declined significantly, while those of Lysophosphatidylcholines (lysoPC) (20: 5 (5Z, 8Z, 11Z, 14Z, 17Z)), lysoPC (22: 4 (7Z, 10Z, 13Z, 16Z)), lysoPC (16: 1 (9Z) / 0: 0), phosphatidylethanolamine (PE) (18: 1 (11Z) 22: 2 (13Z, 16Z)), and guanosine 5'-phosphate rose significantly. These 11 metabolites can be used as differential metabolic markers. These findings not only quantitatively describe the circadian movement behaviours of abalone, but also provide an initial analysis of the circadian mechanism of the physiological metabolic conversion of abalone, which in turn provides guidelines for light control and feeding strategy for use in aquaculture production.


Assuntos
Metaboloma/fisiologia , Movimento/fisiologia , Animais , Escala de Avaliação Comportamental , Relógios Circadianos , Análise por Conglomerados , Gastrópodes , Hipoxantina/análise , Hipoxantina/metabolismo , Indóis/análise , Indóis/metabolismo , Inosina/análogos & derivados , Inosina/análise , Inosina/metabolismo , Lisofosfatidilcolinas/análise , Lisofosfatidilcolinas/metabolismo , Fosfatidilcolinas/análise , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/análise , Fosfatidiletanolaminas/metabolismo , Serotonina/análogos & derivados , Serotonina/análise , Serotonina/metabolismo , Espectrometria de Massas em Tandem , Fatores de Tempo
17.
Proc Natl Acad Sci U S A ; 117(36): 22080-22089, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32820071

RESUMO

Nonshivering thermogenesis occurs in brown adipose tissue to generate heat in response to cold ambient temperatures. Thioesterase superfamily member 1 (Them1) is transcriptionally up-regulated in brown adipose tissue upon exposure to the cold and suppresses thermogenesis in order to conserve energy reserves. It hydrolyzes long-chain fatty acyl-CoAs that are derived from lipid droplets, preventing their use as fuel for thermogenesis. In addition to its enzymatic domains, Them1 contains a C-terminal StAR-related lipid transfer (START) domain with unknown ligand or function. By complementary biophysical approaches, we show that the START domain binds to long-chain fatty acids, products of Them1's enzymatic reaction, as well as lysophosphatidylcholine (LPC), lipids shown to activate thermogenesis in brown adipocytes. Certain fatty acids stabilize the START domain and allosterically enhance Them1 catalysis of acyl-CoA, whereas 18:1 LPC destabilizes and inhibits activity, which we verify in cell culture. Additionally, we demonstrate that the START domain functions to localize Them1 near lipid droplets. These findings define the role of the START domain as a lipid sensor that allosterically regulates Them1 activity and spatially localizes it in proximity to the lipid droplet.


Assuntos
Ácidos Graxos/metabolismo , Lisofosfatidilcolinas/metabolismo , Palmitoil-CoA Hidrolase/química , Palmitoil-CoA Hidrolase/metabolismo , Acil Coenzima A/metabolismo , Tecido Adiposo Marrom/enzimologia , Tecido Adiposo Marrom/metabolismo , Regulação Alostérica , Ácidos Graxos/química , Humanos , Cinética , Gotículas Lipídicas/enzimologia , Gotículas Lipídicas/metabolismo , Lisofosfatidilcolinas/química , Palmitoil-CoA Hidrolase/genética , Domínios Proteicos
18.
Phytomedicine ; 77: 153274, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32771537

RESUMO

BACKGROUND: Astragali Radix (AR), a common Traditional Chinese Medicine (TCM), is commonly used for treating nephrotic syndrome (NS) in China. At present, the research on the efficacy of AR against NS is relative clearly, but there are fewer researches on the mechanism. PURPOSE: The aim of this study was to evaluate the potential beneficial effects of AR in an adriamycin-induced nephropathy rat model, as well as investigate the possible mechanisms of action and potential lipid biomarkers. METHODS: In this work, a rat model of NS was established by two injections of ADR (3.5 + 1 mg/kg) into the tail vein. The potential metabolites and targets involved in the anti-NS effects of AR were predicted by lipidomics coupled with the network pharmacology approach, and the crucial metabolite and protein were further validated by western blotting and ELISA. RESULTS: The results showed that 22 metabolites such as l-carnitine, LysoPC (20:3), and SM (d18:1/16:0) were associated with renal injury. Moreover, SMPD1, CPT1A and LCAT were predicted as lipids linked targets of AR against NS, whilst glycerophospholipid, sphingolipid and fatty acids metabolism were involved as key pathways of AR against NS. Besides, AR could play a critical role in NS by improving oxidative stress, inhibiting apoptosis and reducing inflammation. Interestingly, our results indicated that key metabolite l-carnitine and target CPT1 were one of the important metabolites and targets for AR to exert anti-NS effects. CONCLUSION: In summary, this study offered a new understanding of the protection mechanism of AR against NS by network pharmacology and lipidomic method.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Síndrome Nefrótica/tratamento farmacológico , Animais , Carnitina/metabolismo , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Ácidos Graxos/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Lipidômica , Lisofosfatidilcolinas/metabolismo , Masculino , Síndrome Nefrótica/induzido quimicamente , Síndrome Nefrótica/metabolismo , Ratos Sprague-Dawley
19.
J Biol Chem ; 295(34): 12167-12180, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641497

RESUMO

Recently, eicosanoid-lysophospholipids were identified as novel metabolites generated from the direct cyclooxygenase- or lipoxygenase-catalyzed oxidation of 2-arachidonoyl-lysophospholipids produced from either phospholipase A1-mediated hydrolysis of diacyl arachidonoyl-phospholipids or through the cytochrome c-catalyzed oxidative hydrolysis of the vinyl ether linkage of arachidonoyl-plasmalogens. Although the metabolic pathways generating eicosanoid-lysophospholipids have been increasingly appreciated, the signaling functions of eicosanoid-lysophospholipids remain largely unknown. Herein, we demonstrate that 2-12(S)-HETE-lysophospholipids as well as nonesterified 12(S)-HETE are potent lipid mediators that activate THP-1 human monocytic cells to generate tumor necrosis factor α (TNFα) and interleukin 8 (IL8). Remarkably, low nanomolar concentrations of 12(S)-HETE-lysophospholipids, but not other oxidized signaling lipids examined activated THP-1 cells resulting in the production of large amounts of TNFα. Moreover, TNFα release induced by 12(S)-HETE-lysophospholipids was inhibited by the TNFα converting enzyme inhibitor TAPI-0 indicating normal processing of TNFα in THP-1 cells stimulated with these agonists. Western blotting analyses revealed that 12(S)-HETE-lysophospholipids activated the phosphorylation of NFκB p65, suggesting activation of the canonical NFκB signaling pathway. Importantly, activation of THP-1 cells to release TNFα was stereoselective with 12(S)-HETE favored over 12(R)-HETE. Furthermore, the EC50 of 2-12(S)-HETE-lysophosphatidylcholine in activating THP-1 cells was 2.1 nm, whereas the EC50 of free 12(S)-HETE was 23 nm Additionally, lipid extracts of activated platelets were separated by RP-HPLC demonstrating the coelution of 12(S)-HETE with fractions initiating TNFα release. Collectively, these results demonstrate the potent signaling properties of 2-12(S)-HETE-lysophospholipids and 12(S)-HETE by their ability to release TNFα and activate NFκB signaling thereby revealing a previously unknown role of 2-12(S)-HETE-lysophospholipids in mediating inflammatory responses.


Assuntos
Lisofosfatidilcolinas/metabolismo , Monócitos/metabolismo , Transdução de Sinais , Animais , Ciclo-Oxigenase 1/metabolismo , Humanos , Camundongos , Monócitos/citologia , Células THP-1 , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
20.
Nutrients ; 12(7)2020 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-32708260

RESUMO

(1) Background: Little is known on impacts of ready-to-use therapeutic food (RUTF) treatment on lipid metabolism in children with severe acute malnutrition (SAM). (2) Methods: We analyzed glycerophospholipid fatty acids (FA) and polar lipids in plasma of 41 Pakistani children with SAM before and after 3 months of RUTF treatment using gas chromatography and flow-injection analysis tandem mass spectrometry, respectively. Statistical analysis was performed using univariate, multivariate tests and evaluated for the impact of age, sex, breastfeeding status, hemoglobin, and anthropometry. (3) Results: Essential fatty acid (EFA) depletion at baseline was corrected by RUTF treatment which increased EFA. In addition, long-chain polyunsaturated fatty acids (LC-PUFA) and the ratio of arachidonic acid (AA)/linoleic acid increased reflecting greater EFA conversion to LC-PUFA, whereas Mead acid/AA decreased. Among phospholipids, lysophosphatidylcholines (lyso.PC) were most impacted by treatment; in particular, saturated lyso.PC decreased. Higher child age and breastfeeding were associated with great decrease in total saturated FA (ΣSFA) and lesser decrease in monounsaturated FA and total phosphatidylcholines (ΣPC). Conclusions: RUTF treatment improves EFA deficiency in SAM, appears to enhance EFA conversion to biologically active LC-PUFA, and reduces lipolysis reflected in decreased ΣSFA and saturated lyso.PC. Child age and breastfeeding modify treatment-induced changes in ΣSFA and ΣPC.


Assuntos
Transtornos da Nutrição Infantil/sangue , Transtornos da Nutrição Infantil/dietoterapia , Fenômenos Fisiológicos da Nutrição Infantil/fisiologia , Fast Foods , Alimentos Especializados , Fenômenos Fisiológicos da Nutrição do Lactente/fisiologia , Metabolismo dos Lipídeos , Lipídeos/sangue , Fatores Etários , Aleitamento Materno , Criança , Transtornos da Nutrição Infantil/metabolismo , Pré-Escolar , Ácidos Graxos Essenciais/sangue , Ácidos Graxos Insaturados , Feminino , Glicerofosfolipídeos/sangue , Glicerofosfolipídeos/metabolismo , Humanos , Lactente , Lisofosfatidilcolinas/sangue , Lisofosfatidilcolinas/metabolismo , Masculino , Paquistão , Índice de Gravidade de Doença
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