| Title: | Measurement of choline and choline metabolite concentrations using high-pressure liquid chromatography and gas chromatography-mass spectrometry |
| Author(s): | Pomfret EA; daCosta KA; Schurman LL; Zeisel SH; |
| Address: | "Department of Pathology, Boston University School of Medicine, Massachusetts 02118" |
| DOI: | 10.1016/0003-2697(89)90091-2 |
| ISSN/ISBN: | 0003-2697 (Print) 0003-2697 (Linking) |
| Abstract: | "We have developed a reproducible and sensitive procedure for the isolation and measurement of choline, phosphocholine, glycerophosphocholine, phosphatidylcholine, lysophosphatidylcholine and acetylcholine in a single 100-mg sample of biological tissue. Tissues were spiked with 14C-methyl- and 2H-methyl- or 15N-choline labeled internal standards for each compound. They were extracted with chloroform/methanol/water and the aqueous and organic phases were dried. The organic phase was resuspended in chloroform/methanol (1/1, v/v) and an aliquot was applied to a silica-gel thin-layer chromatography plate. The plate was developed in chloroform/methanol/water (65/30/4, v/v). Segments which cochromatographed with external standards of phosphatidylcholine and lysophosphatidylcholine were stained, scraped, and hydrolyzed in 6 M methanolic-HCl at 80 degrees C for 60 min, liberating free choline. The aqueous phase was resuspended in methanol/water and injected onto a silica HPLC column. Choline and its metabolites were eluted using a binary nonlinear gradient of acetonitrile/ethanol/acetic acid/1 M ammonium acetate/water/0.1 M sodium phosphate (800/68/2/3/127/10, v/v changing to 400/68/44/88/400/10, v/v). Peaks were detected with an on-line radiometric detector, collected, and dried under vacuum. Each choline ester was digested in 6 M HCl at 80 degrees C to form choline. Choline was then converted to the propionyl ester and demethylated with sodium benzenethiolate. This volatile derivative was then isolated using gas chromatography and measured with a mass selective detector. Deuterated internal standards were used to correct for variations in recovery. Choline, glycerophosphocholine, phosphocholine, phosphatidylcholine, lysophosphatidylcholine, and acetylcholine were measured in rat liver, heart, muscle, kidney, plasma, red blood cells, and brain and in human plasma.(ABSTRACT TRUNCATED AT 250 WORDS)" |
| Keywords: | "Adolescent Adult Animals Choline/*analogs & derivatives/*analysis Chromatography, High Pressure Liquid/methods Gas Chromatography-Mass Spectrometry Humans Male Rats Rats, Inbred Strains;" |
| Notes: | "MedlinePomfret, E A daCosta, K A Schurman, L L Zeisel, S H eng HD16727/HD/NICHD NIH HHS/ RR-00533/RR/NCRR NIH HHS/ Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S. 1989/07/01 Anal Biochem. 1989 Jul; 180(1):85-90. doi: 10.1016/0003-2697(89)90091-2" |
