Original article
An improved method for assaying phosphatidylcholine in mouse tissue

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Abstract

Introduction: 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 α1-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 °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 μmol/g (eye ball) and 29 μmol/g (spinal cord), respectively. Methoxamine significantly decreased PtdCh levels and increased free choline levels in mouse artery and submandibular gland. Discussion: 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.

Introduction

Phosphatidylcholine (PtdCh), a main choline-containing phospholipid, is a principal structural and functional membrane component in mammalian tissues (Exton, 1994). Furthermore, intracellular PtdCh has been suggested as a modulator of cell growth and death Jackowski, 1994, Yen et al., 1999. Thus, many investigators have studied changes in PtdCh levels in biological samples; this has employed a variety of detection methods, such as liquid scintillation spectrometry (Lee, Fellenz-Maloney, Liscovitch, & Blusztajn, 1993), 31P NMR spectrometry (Adinehzadeh, Reo, Jarnot, Taylor, & Mattie, 1999), gas chromatography–mass spectrometry Jope & Jenden, 1979, Pomfret et al., 1989, Stott et al., 2000, and electrochemical detection (ECD) Klein et al., 1993, Klein et al., 1992, Zapata et al., 1998.

In the high-performance liquid chromatography (HPLC)-with-ECD method, lipophilic choline-containing phospholipids (PtdCh, lyso-PtdCh, and sphingomyelin) are extracted from tissue using an organic solvent. Subsequently, PtdCh is isolated from other phospholipids by thin-layer chromatography (TLC) and is acidically hydrolyzed to liberate choline. TLC is a useful procedure for separation of phospholipids Klein et al., 1993, Pomfret et al., 1989. However, the isolation, scraping, and reextraction steps of PtdCh on TLC are laborious, and these steps lower the measurement accuracy. Recently, Zapata et al. (1998) described a simple method for the measurement of PtdCh levels using HPLC-ECD and an enzyme column that immobilizes acetylcholine esterase and choline oxidase. This immobilized enzyme column can efficiently convert acetylcholine and choline to hydrogen, which is detected by an electrochemical detector with a platinum electrode. Since the method does not include the isolation procedure for PtdCh using TLC, it simplifies sample processing and may improve measurement accuracy. However, their method introduces other problems that may be come sources of error, such as (1) the precise pipetting of organic solvent and (2) neutralization and dilution of sample solutions because of the use of a strong acid (6 N HCl) for hydrolysis of PtdCh. Furthermore, their HPLC-ECD method does not use an internal standard necessary for the accurate measurement of choline derived from the hydrolysis of PtdCh.

More recently, we reported a very simple, rapid, and precise HPLC with ECD method for assaying the levels of choline, phosphocholine, and glycerophosphocholine, which are compounds relevant to the biosynthesis and metabolism of PtdCh (Murai, Saito, Shirato, & Kawaguchi, 2001). The advantages of this method include a minimal sample preparation procedure and a high accuracy due to the direct injection of the extraction mixtures and use of an internal standard. Here, we report an improved HPLC with ECD method for the measurement of PtdCh levels, which provides simplicity and accuracy equal to our previous method (Murai et al., 2001). The present method was applied to measuring the steady state levels of PtdCh in various tissues of the mouse. In addition, the validity of the present method was demonstrated by analyzing the effect of methoxamine, an α1-adrenergic agonist, on the PtdCh levels in mouse artery and submandibular gland in in vivo conditions because it was reported that the adrenergic agonists promoted hydrolysis of PtdCh in the artery in ex vivo conditions Gu et al., 1992, Nally et al., 1992.

Section snippets

Chromatography

The HPLC system consisted of a solvent delivery pump (model L-6000, Hitachi, Japan), an autosampler (model L-7250, Hitachi), an analytical column (EICOMpak AC-GEL, 150 mm, 6 mm ID, 10 μm, EICOM, Japan), an immobilized enzyme column (EICOM AC-Enzympak), a catecholamine-trap column (EICOM), a guard column (EICOM Prepak), and a Shimadzu data processor (C-R6A, Shimadzu, Japan). The catecholamine trap column was connected immediately behind the immobilized enzyme column. An electrochemical detector

Results

Fig. 2 shows typical chromatograms of the authentic standard (A) brain sample without (B) and with (C) heating in a 1-N perchloric acid solution. Since there were no other peaks except for EHC (an internal standard) and large choline, injection of the sample was possible every 5 min.

Under the present hydrolytic condition, the recovery rates of authentic PtdCh and lyso-PtdCh were almost 100%. However, the recovery of authentic sphingomyelin was only 0.9%. Under the present chromatographic

Discussion

The main lipophilic choline-containing phospholipids extracted from biological tissues by organic solvents are PtdCh, lyso-PtdCh, and sphingomyelin. In the HPLC-ECD method, the levels of all these compounds are estimated indirectly from the levels of Ch produced by their acidic hydrolysis. Thus, conventional HPLC-ECD methods for PtdCh assay involve isolation of PtdCh by TLC before the acidic hydrolysis of extraction samples (Klein et al., 1993), despite the fact that TLC is laborious and

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