Direct detection of endogenous histamine in rat peritoneal mast cells by in-capillary derivatization high-performance capillary electrophoresis.
A simple method for the detection of endogenous histamine in rat peritoneal mast cells was evaluated using on-line mode in-capillary derivatization high-performance capillary electrophoretic (ICD-HPCE) techniques, which were previously developed by our group [S. Oguri et al., J. Chromatogr. A, 787 (1997) 253-260]. The method involves a suspension of peritoneal mast cells (1 x 10(6) cells/ml of saline) collected from a male Wistar rat (eight weeks of age), which are directly introduced into the capillary tube from the anodic end by hydrostatic injection (at 25 cm height, for 2-20 s). When a high-voltage potential (25 kV) is applied to the capillary, which is already filled with the run buffer containing both a lysing reagent (SDS, sodium dodecyl sulfate,) and a derivatizing reagent (OPA, o-phthalaldehyde; NAC, N-acetylcysteine), histamine in the mast cells was detected at high-sensitivity level without further procedures. During ICD-HPCE, the mast cells injected in the capillary were lysed with the lysing reagent, free histamine released from the cell was labeled with the derivatizing reagent, and its derivative was electromigrated, separated and detected with a fluorescence detector (excitation wavelength at 340 nm, emission wavelength at 450 nm) in a fused-silica capillary (75 cm x effective length x 50 microm I.D.). The run buffer used was a 20 mM phosphate-borate buffer (pH 10) containing 20 mM SDS, 2 mM OPA and 2 mM NAC. This method was also examined with regard to the possibility of its use for determination of histamine at the single mast cell level.
Oguri
S
S
Department of Home Economics, Aichi-Gakusen University, Okazaki City, Aichi, Japan. s-oguri@gakusen.ac.jp
Ohta
Y
Y
Suzuki
C
C
D000818:Animals; D002452:Cell Count; D019075:Electrophoresis, Capillary / Q000379:methods*; D006632:Histamine / Q000032:analysis*; D007202:Indicators and Reagents; D008297:Male; D008407:Mast Cells / Q000737:chemistry*; D010529:Peritoneal Cavity / Q000166:cytology*; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate
D007202:Indicators and Reagents; D012967:Sodium Dodecyl Sulfate; D006632:Histamine