RESUMO
Oxidative derivatives of cholesterol such as 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 25-hydroxycholesterol, and (25S),26-hydroxycholesterol are endogenous ligands for the liver X receptors (LXRα and LXRß). The LXRs are nuclear hormone receptors known as "intracellular cholesterol sensors" because of their ability to bind to and be activated by oxysterols at circulating concentrations. Oxysterols are expressed in a tissue-specific manner and are generally at least 104 to 106-fold less abundant than cholesterol. Thus, the extraction and measurement of oxysterols from plasma and tissues are facilitated by the removal of bulk sterols by solid phase extraction prior to quantitative analysis by mass spectrometry. In this chapter we describe step by step methods for extracting and quantitating oxysterols from biological samples using electrospray ionization LC/MS/MS.
Assuntos
Cromatografia Líquida , Oxisteróis/sangue , Espectrometria de Massas em Tandem , Animais , Humanos , Ligantes , Camundongos , Oxisteróis/isolamento & purificação , Oxisteróis/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Extração em Fase SólidaRESUMO
Four new oxysterols 1â»4 along with previously known oxygenated sterols 5â»14 were isolated from the sponge Inflatella sp., collected from the Sea of Okhotsk. Structures of 1â»4 were elucidated by the detailed NMR spectroscopic and mass-spectrometric analyses as well as by comparison of the corresponding experimental data with those reported in literature. The influence of compounds 1â»14 on the viability of neuronal Neuro2a cells treated by 6-hydroxydopamine and reactive oxygen species (ROS) formation in these cells was investigated.
Assuntos
Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Oxisteróis/química , Oxisteróis/farmacologia , Transtornos Parkinsonianos/tratamento farmacológico , Poríferos/química , Animais , Antiparkinsonianos/química , Antiparkinsonianos/isolamento & purificação , Antiparkinsonianos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Oxidopamina , Oxisteróis/isolamento & purificação , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Oxysterols play important roles in development and diseases, but can be highly challenging to analyze. To ensure satisfactory measurements, oxysterols must typically be separated with chromatography prior to detection. Here, we will devote attention to the chromatography of oxysterols, focusing on gas chromatography and liquid chromatography. We will present the role of stationary phases, mobile phases, and dimensions and geometries of particles/columns. We discuss how these parameters may affect the chromatography, regarding factors such as speed and resolution. Finally, we present some less explored avenues for separation of oxysterols.
Assuntos
Cromatografia Gasosa/métodos , Cromatografia Líquida/métodos , Oxisteróis/isolamento & purificaçãoRESUMO
Oxysterols (OHC) are biologically active cholesterol metabolites circulating in plasma that may be formed enzymatically (e.g. 24S-OHC, 25-OHC and 27-OHC) or by autoxidative mechanisms (e.g. 7-ketocholesterol, 7ß-OHC and 25-OHC). Oxysterols are more soluble than cholesterol and are reported to exert inflammatory, cytoprotective and apoptotic effects according to concentration and species. Esterified oxysterols have been analysed in people with dementia and cardiovascular diseases although there is no consistent relationship between oxysterol esters and disease. However, oxysterol esters are held in lipoprotein core and may not relate to the concentration and activity of plasma free oxysterols. Methodological limitations have challenged the analysis of free oxysterols to date. We have developed a fast, sensitive and specific quantitative LC-MS/MS, multiple reaction monitoring (MRM) method to target five oxysterols in human plasma with analyte recoveries between 72% and 82% and sensitivities between 5 and 135â¯pg/ml. A novel method was used to investigate the hypothesis that simvastatin may reduce the concentrations of specific plasma free oxysterols in hypercholesterolaemia. Twenty healthy male volunteers were recruited (aged 41-63 years); ten were asymptomatic with high plasma cholesterol >â¯6.5â¯mM and ten were healthy with normal plasma cholesterol (<â¯6.5â¯mM). Simvastatin (40â¯mg/day) was prescribed to those with hypercholesterolaemia. Plasma samples were taken from both groups at baseline and after three months. Simvastatin reduced plasma cholesterol by ~35% (pâ¯<â¯0.05) at the end of three months. Oxysterols generated by autoxidation (but not enzymatically) were elevated up to 45 fold in hypercholesterolaemic midlife men. Plasma oxysterols were restored to those of healthy controls after simvastatin intervention suggesting that autoxidation is either prevented by simvastatin directly or that autoxidation is less prevalent when plasma cholesterol concentrations are within the normal range.
Assuntos
Colesterol/metabolismo , Hipercolesterolemia/tratamento farmacológico , Oxisteróis/sangue , Sinvastatina/administração & dosagem , Adulto , Cromatografia Líquida , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/patologia , Masculino , Pessoa de Meia-Idade , Oxisteróis/isolamento & purificação , Espectrometria de Massas em TandemRESUMO
We have developed a workflow to extract, separate, and semi-quantify bioactive oxysterols from mouse colon tissues and fecal matters using solid- and liquid-phase extractions, enzymatic and chemical modifications, and stable-isotope dilution LC/MS/MS. The method was applied to a dextran sodium sulphate (DSS)-induced mouse colitis model, which revealed that one particular dihydroxycholesterol (diOHC), 7α,25-diOHC, was significantly elevated in both colon tissue and fecal matters of mice with colitis compared to that in naïve mice. The extent of 7α,25-diOHC elevation was positively correlated with colitis severity.