Assuntos
Cerebrosídeo Sulfatase/metabolismo , Cromatografia Líquida de Alta Pressão , Ensaios Enzimáticos/métodos , Leucócitos/enzimologia , Espectrometria de Massas em Tandem , Adulto , Pré-Escolar , Ensaios Enzimáticos/instrumentação , Feminino , Humanos , Cinética , Leucodistrofia Metacromática/diagnóstico , Leucodistrofia Metacromática/enzimologia , Masculino , Pessoa de Meia-Idade , Padrões de Referência , Especificidade por Substrato , Sulfoglicoesfingolipídeos/análise , Sulfoglicoesfingolipídeos/metabolismo , Sulfoglicoesfingolipídeos/normas , Espectrometria de Massas em Tandem/normasRESUMO
Sphingolipids are a highly diverse category of bioactive compounds. This article describes methods that have been validated for the extraction, liquid chromatographic (LC) separation, identification and quantitation of sphingolipids by electrospray ionization, tandem mass spectrometry (ESI-MS/MS) using triple quadrupole (QQQ, API 3000) and quadrupole-linear-ion trap (API 4000 QTrap, operating in QQQ mode) mass spectrometers. Advantages of the QTrap included: greater sensitivity, similar ionization efficiencies for sphingolipids with ceramide versus dihydroceramide backbones, and the ability to identify the ceramide backbone of sphingomyelins using a pseudo-MS3 protocol. Compounds that can be readily quantified using an internal standard cocktail developed by the LIPID MAPS Consortium are: sphingoid bases and sphingoid base 1-phosphates, more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, and these complex sphingolipids with dihydroceramide backbones. With minor modifications, glucosylceramides and galactosylceramides can be distinguished, and more complex species such as sulfatides can also be quantified, when the internal standards are available. LC ESI-MS/MS can be utilized to quantify a large number of structural and signaling sphingolipids using commercially available internal standards. The application of these methods is illustrated with RAW264.7 cells, a mouse macrophage cell line. These methods should be useful for a wide range of focused (sphingo)lipidomic investigations.
Assuntos
Extratos Celulares/química , Cromatografia Líquida/métodos , Metabolismo dos Lipídeos , Espectrometria de Massas por Ionização por Electrospray/métodos , Esfingolipídeos/análise , Animais , Linhagem Celular , Ceramidas/análise , Ceramidas/normas , Ácidos Graxos/normas , Camundongos , Camundongos Endogâmicos BALB C , Controle de Qualidade , Padrões de Referência , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Esfingolipídeos/isolamento & purificação , Esfingolipídeos/normas , Esfingomielinas/análise , Esfingomielinas/normas , Esfingosina/análogos & derivados , Esfingosina/análise , Esfingosina/normas , Sulfoglicoesfingolipídeos/análise , Sulfoglicoesfingolipídeos/normas , Espectrometria de Massas em TandemRESUMO
3-O-Sulfogalactosylceramides (sulfatides) accumulate in the genetic disease metachromatic leukodystrophy which is due to a defect in the catabolic enzyme, arylsulfatase A. Clinical diagnosis is usually confirmed by in vitro enzymatic deficiency of arylsulfatase A activity. The diagnosis may be complicated because of arylsulfatase A pseudo-deficiencies and another cause of MLD, sphingolipid activator B deficiency. As large quantities of sulfatides can be found in the urine in this disease, sulfatiduria appears as an extremely useful test. As recently enzyme replacement is underway, the quantitative determination, using an internal standard, appears particularly useful as a follow-up. Thus a non-physiological sulfatide was synthesized for this purpose, i.e. 3-O-sulfo-beta-D-C17 galactosylceramide (3-O-Sulfo-D: -Galactosyl-beta1'-->1-N-Heptadecanoyl-D-erythro-Sphingosine). It has been prepared through condensation of an azidosphingosine derivative with a protected D-galactopyranosyltrichloroacetimidate. Reduction of the azide was followed by acylation of a C-17 fatty acid. The key step was achieved by selective sulfation of the desired hydroxyl group on the sugar residue of the galactosylceramide using the stannylene methodology to give a 3'-sulfated beta-galactosyl C-17 ceramide.