An improved method for assaying phosphatidylcholine in mouse tissue.
BACKGROUND
To measure levels of phosphatidylcholine (PtdCh) in various mouse tissues, we developed a rapid and precise method using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) and an immobilized enzyme column. To generate an example data set, the effect of methoxamine (an alpha1-adrenergic agonist) on the PtdCh levels was examined by this method in the artery and the submandibular gland of the mouse in vivo.


METHODS
Under our modifications of the method of Zapata et al. [J. Neurosci. 18 (1998) 3597], the mixture of lipophilic choline metabolites (PtdCh, lyso-PtdCh, and sphingomyelin) extracted by chloroform from the tissue homogenate was dried without prior separation and hydrolyzed with free choline by a 1-N perchloric acid solution containing ethylhomocholine (an internal standard for choline assay) at 90 degrees C for 1 h. Subsequently, the hydrolyzed mixture was injected directly into the HPLC system for PtdCh assay.


RESULTS
The present method permitted PtdCh assay within 5 min in one chromatographic run. Recovery of an authentic PtdCh sample was 99% (n = 10). The within-run coefficients of variation for choline derived from PtdCh in the same tissue samples were 0.6% (n = 10) and 1.3% (n = 30). Under the present method, the lowest and highest PtdCh values in tissue samples were about 2 micromol/g (eye ball) and 29 micromol/g (spinal cord), respectively. Methoxamine significantly decreased PtdCh levels and increased free choline levels in mouse artery and submandibular gland.


CONCLUSIONS
Under the present sample processing procedure, the choline values originating from lyso-PtdCh and sphingomyelin were much less than those originating from PtdCh hydrolysis. Thus, it was possible to inject the hydrolyzed mixture directly into the HPLC system for PtdCh assay. Since the present method provides simple, rapid, and highly reliable PtdCh determination, it is suitable for routine assay of PtdCh in a large number of samples.
Murai
Shigeo
S
Department of Pharmacology, School of Dentistry, Iwate Medical University, Morioka, 020-8505, Japan.
Saito
Hiroko
H
Shirato
Rhuichi
R
Tamura
Haruki
H
Yamada
Arisa
A
Kato
Hirohisa
H
D000316:Adrenergic alpha-Agonists / Q000493:pharmacokinetics; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D004563:Electrochemistry / Q000379:methods*; D004800:Enzymes, Immobilized; D008729:Methoxamine / Q000493:pharmacokinetics; D051379:Mice; D010713:Phosphatidylcholines / Q000032:analysis*; D015203:Reproducibility of Results; D013363:Submandibular Gland / Q000378:metabolism; D014018:Tissue Distribution
D000316:Adrenergic alpha-Agonists; D004800:Enzymes, Immobilized; D010713:Phosphatidylcholines; D008729:Methoxamine