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1 LOCUS for Homocysteine and Related Vitamins, University of Bergen, N-5021 Bergen, Norway.
aAddress correspondence to this author at: LOCUS for Homocysteine and Related Vitamins, Department of Pharmacology, University of Bergen, N-5021 Bergen, Norway. Fax 47-55-973115; e-mail per.ueland{at}ikb.uib.no.
Background: The quaternary ammonium compounds, choline and betaine, and dimethylglycine (DMG) reside along a metabolic pathway linked to the synthesis of neurotransmitters and membrane phospholipids and to homocysteine remethylation and, therefore, folate status. Lack of a convenient, high-throughput method for the determination of these compounds has prevented population-based studies of their possible associations with lifestyle, nutrition, and chronic diseases.
Methods: Serum or plasma samples were deproteinized by mixing with three volumes of acetonitrile that contained d9-choline and d9-betaine as internal standards. We used a normal-phase silica column for the separation of choline (retention time, 2.8 min), betaine (1.3 min), DMG (1.15 min), and internal standards, which were detected as positive ions by tandem mass spectroscopy in the multiple-reaction monitoring mode, using the molecular transitions m/z 104
60 (choline), m/z 113
69 (d9-choline), m/z 118
59 (betaine), m/z 127
68 (d9-betaine), and m/z 104
58 (DMG).
Results: For all three metabolites, the assay was linear in the range 0.4400 µmol/L, and the lower limit of the detection (signal-to-noise ratio = 5) was
0.3 µmol/L. The within- and between-day imprecision (CVs) was 2.17.2% and 3.58.8%, respectively. The analytical recovery was 87105%. The fasting plasma concentrations (median, 25th75th percentiles) were 8.0 (7.09.3) µmol/L for choline, 31.7 (27.041.1) µmol/L for betaine, and 1.66 (1.302.02) µmol/L for DMG in 60 healthy blood donors. In individuals who had eaten a light breakfast, plasma concentrations of all three metabolites were significantly (2530%) higher than in fasting individuals.
Conclusion: This is the first method for the combined measurement of choline, betaine, and DMG in human plasma or serum. The assay is characterized by simple sample preparation, no derivatization, high throughput, imprecision (CV) <10%, detection limits below the values seen in volunteers, and the high specificity provided by tandem mass spectroscopy.
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