Diazotization of kynurenine by acidified nitrite secreted from indoleamine 2,3-dioxygenase-expressing myeloid dendritic cells.
J Immunol Methods
; 332(1-2): 162-9, 2008 Mar 20.
Article
en En
| MEDLINE
| ID: mdl-18158157
ABSTRACT
Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan metabolism along the kynurenine (Kyn) pathway regulates T-cell responses in some dendritic cells (DC) such as plasmacytoid DC. A Kyn assay using HPLC showed that samples were frequently deproteinized with trichloroacetic acid (TCA). In the present study, bone marrow-derived myeloid DC (BMDC) were differentiated from mouse bone marrow cells with GM-CSF. CpG oligodeoxynucleotides (CpG) induced the expression of IDO protein with NO production in BMDC cultured for 24 h. The concentrations of Kyn in the culture supernatants were not increased by stimulation with CpG but rather decreased by based on the Kyn assay after deproteinization with TCA. The level of Kyn exogenously added into the cell-free culture supernatant of BMDC stimulated with CpG was severely decreased by deproteinization with TCA but not methanol, and the decrease was prevented when BMDC was stimulated with CpG in the presence of a NOS inhibitor. Under acidic conditions, Kyn reacted with nitrite produced by BMDC, and generated a new compound that was not detected by Ehrlich reagent reacting with the aromatic amino residue of Kyn. An analysis by mass spectrometry showed the new compound to be a diazotization form of Kyn. In conclusion, the deproteinization of samples by acidic treatment should be avoided for the Kyn assay when NO is produced.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Compuestos Azo
/
Células Dendríticas
/
Indolamina-Pirrol 2,3,-Dioxigenasa
/
Quinurenina
/
Nitritos
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
Idioma:
En
Revista:
J Immunol Methods
Año:
2008
Tipo del documento:
Article
País de afiliación:
Japón
Pais de publicación:
HOLANDA
/
HOLLAND
/
NETHERLANDS
/
NL
/
PAISES BAJOS
/
THE NETHERLANDS