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Titolo:
An improved high-performance liquid chromatographic method for the determination of sphingosine-1-phosphate in complex biological materials
Autore:
Ruwisch, L; Schafer-Korting, M; Kleuser, B;
Indirizzi:
Free Univ Berlin, Inst Pharm Pharmakol & Toxikol, D-14195 Berlin, Germany Free Univ Berlin Berlin Germany D-14195 Toxikol, D-14195 Berlin, Germany
Titolo Testata:
NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY
fascicolo: 3, volume: 363, anno: 2001,
pagine: 358 - 363
SICI:
0028-1298(200103)363:3<358:AIHLCM>2.0.ZU;2-O
Fonte:
ISI
Lingua:
ENG
Soggetto:
SPHINGOSINE 1-PHOSPHATE RECEPTOR; PROTEIN-COUPLED RECEPTOR; PROGRAMMED CELL-DEATH; SWISS 3T3 FIBROBLASTS; FOCAL ADHESION KINASE; QUANTITATIVE MEASUREMENT; SPHINGOLIPID METABOLISM; CALCIUM MOBILIZATION; MEDIATED ACTIVATION; SIGNALING PATHWAY;
Keywords:
sphingosine-1-phosphate; dihydrosphingosine-1-phosphate; HPLC; serum; thrombocytes; keratinocytes; 1 alpha,25-dihydroxyvitamin D-3;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
37
Recensione:
Indirizzi per estratti:
Indirizzo: Kleuser, B Free Univ Berlin, Inst Pharm Pharmakol & Toxikol, Konigin LuiseStr 2-4, D-14195 Berlin, Germany Free Univ Berlin Konigin Luise Str 2-4 Berlin Germany D-14195
Citazione:
L. Ruwisch et al., "An improved high-performance liquid chromatographic method for the determination of sphingosine-1-phosphate in complex biological materials", N-S ARCH PH, 363(3), 2001, pp. 358-363

Abstract

Sphingosine-1-phosphate (SPP) has been proposed to act both as an intracellular second messenger and as an extracellular mediator via specific cell surface receptors. Based on the increasing diverse cellular roles methods toquantify endogenous and exogenous SPP are highly required. Here, we report a rapid HPLC method that allows quantification of SPP in the picomolar range even in complex biological systems. A two-step lipid extraction serves to separate SPP from most interfering phospholipids and sphingolipids. Importantly, dihydrosphingosine-1-phosphate (dihydro-SPP), not detectable in all cultured cells and biological samples in considerable amounts, possesses equal extraction properties and therefore is an ideal internal standard. Following extraction SPP and dihydro-SPP are converted to fluorescent isoindol derivatives by ortho-phthaldialdehyde (OPA) and separated by HPLC using a gradient program with methanol and 0.07 M K2HPO4 as eluents. With this procedure we were able to obtain reproducible measurements of SPP over a broad range from 0.5 pM to 0.2 nM. The identity of SPP and dihydro-SPP was confirmed by the use of the ion pair reagent tetraammoniumsulfate, which induced a shift of both peaks but did not alter peak areas. Moreover, enzymatic conversions to sphingosine and sphinganine by bovine intestinal mucose alkaline phosphatase (AP) excluded the existence of overlapping compounds. Levels of SPP were determined in a variety of biological samples like serum, thrombocytes, primary keratinocytes and several cell lines. Furthermore,we were able to detect increases of intracellular SPP levels in human keratinocytes after exposure to 1 alpha ,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3) for which a stimulation of sphingosine kinase activity has been recognized.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 04/04/20 alle ore 15:27:27