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1.
Biochim Biophys Acta ; 1771(7): 853-63, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17531529

RESUMO

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used for a detailed analysis of cellular phospholipid and cholesterol efflux in free cholesterol (FC) loaded human primary fibroblasts and human monocyte-derived macrophages (HMDM) loaded with enzymatically modified LDL (E-LDL). Although both cell models differed significantly in their cellular lipid composition, a higher apoA-I specific efflux was found for monounsaturated phosphatidylcholine (PC) species together with a decreased contribution of polyunsaturated PC species in both cell types. Moreover, medium chain sphingomyelin (SPM) species SPM 14:0 and SPM 16:1 were translocated preferentially to apoA-I in both cell types. In contrast to fibroblasts, HMDM displayed a considerable proportion of cholesteryl esters (CE) in basal and apoA-I specific efflux media, most likely due to secretion of CE associated to apoE. Analysis of HDL(3) mediated lipid efflux from HMDM using D(9)-choline and (13)C(3)-FC stable isotope labeling revealed significantly different D(9)-PC and D(9)-SPM species pattern for apoA-I and HDL(3) specific efflux media, which indicates a contribution of distinct cellular phospholipid pools to apoA-I and HDL(3) mediated efflux. Together with a partial loading of fibroblasts and HMDM with HDL(3)-derived CE species, these data add further evidence for retroendocytosis of HDL. In summary, analysis of apoA-I/ABCA1 and HDL(3) mediated lipid efflux by ESI-MS/MS demonstrated a preferential efflux of monounsaturated PC and medium chain SPM to apoA-I. Moreover, this is the first study, which provides evidence for distinct cellular phospholipid pools used for lipid transfer to apoA-I and HDL(3) from the analysis of phospholipid species pattern in HMDM.


Assuntos
Apolipoproteína A-I/farmacologia , Fibroblastos/efeitos dos fármacos , Lipoproteínas HDL3/farmacologia , Monócitos/efeitos dos fármacos , Fosfatidilcolinas/metabolismo , Esfingomielinas/metabolismo , Transporte Biológico/efeitos dos fármacos , Isótopos de Carbono , Células Cultivadas , Colesterol/metabolismo , Meios de Cultura , Deutério , Fibroblastos/química , Fibroblastos/metabolismo , Humanos , Marcação por Isótopo , Monócitos/química , Monócitos/metabolismo , Fosfatidilcolinas/química , Espectrometria de Massas por Ionização por Electrospray , Esfingomielinas/química
2.
Biochim Biophys Acta ; 1761(1): 121-8, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16458590

RESUMO

Analysis of free cholesterol (FC) is not well suited for electrospray ionization (ESI); however, cholesteryl ester (CE) form ammonium adducts in positive ion mode and generate a fragment ion of m/z 369 upon collision-induced fragmentation. In order to allow parallel analysis of FC and CE using ESI tandem mass spectrometry (ESI-MS/MS), we developed an acetyl chloride derivatization method to convert FC to cholesteryl acetate (CE 2:0). Derivatization conditions were chosen to provide a quantitative conversion of FC to CE 2:0 without transesterification of naturally occurring CE species. FC and CE were analyzed by direct flow injection analysis using a fragment of m/z 369 in a combination of selected reaction monitoring (SRM) and precursor ion scan for FC and CE, respectively. Quantification was achieved using deuterated D(7)-FC and CE 17:0/CE 22:0 as internal standards as well as calibration lines generated by addition of FC and naturally occurring CE species to the respective sample matrix. The developed assay showed a precision and detection limit sufficient for routine analysis. A run time of 1.3 min and automated data analysis allow high throughput analysis. Loading of human skin fibroblast and monocyte derived macrophages with stable isotope labeled FC showed a potential application of this method in metabolism studies. Together with existing mass spectrometry methodologies for lipid analysis, the present methodology will provide a useful tool for clinical and biochemical studies and expands the lipid spectrum that can be analyzed from one lipid sample on a single instrumental platform.


Assuntos
Ésteres do Colesterol/análise , Colesterol/análise , Cromatografia em Camada Fina , Espectrometria de Massas por Ionização por Electrospray/métodos
3.
Biochim Biophys Acta ; 1740(2): 155-61, 2005 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-15949682

RESUMO

Vitamin A and its naturally occurring derivatives 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (ATRA) exert a variety of biological effects including immunomodulation, growth, differentiation, and apoptosis of normal and neoblastic cells. In order to directly study the effects of these retinoids on macrophage gene expression and lipid metabolism, primary human monocytes and in vitro differentiated macrophages were stimulated with beta-carotene, 9-cis RA, and ATRA and global gene expression profiles were analyzed by Affymetrix DNA-microarrays and differentially regulated genes were verified by quantitative TaqMan RT-PCR. Among others, we have identified a strong up-regulation of a cluster of genes involved in cholesterol metabolism including apolipoproteins (apoC-I, apoC-II, apoC-IV, apoE), the scavenger receptor CD36, steroid-27-hydroxylase (CYP27A1), liver X receptor alpha (LXRalpha), and ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1). Since the CYP27A1 gene displayed the strongest up-regulation on the mRNA level, we cloned various deletion constructs of the promoter region and analyzed the response to retinoids in macrophages. Thereby, a novel retinoic acid-responsive element could be located within 191 bp of the proximal CYP27A1 promoter. To further assess the functional consequences of retinoid receptor action, we carried out phospholipid and cholesterol efflux assays. We observed a strong induction of apoA-I-dependent lipid efflux in stimulated macrophages, implicating an important role for retinoids in cellular functions of macrophages.


Assuntos
Metabolismo dos Lipídeos , Macrófagos/metabolismo , Retinoides/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apolipoproteína A-I/farmacologia , Apolipoproteínas/genética , Antígenos CD36 , Colesterol/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Fosfolipídeos/metabolismo , Receptores Imunológicos/genética , Receptores do Ácido Retinoico/agonistas , Receptores Depuradores , Retinoides/farmacologia , beta Caroteno/farmacologia
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