title: Stable-isotope dilution analysis of galactose metabolites in human erythrocytes.
abstract: An established gas chromatography/mass spectrometry (GC/MS) method, devised for stable-isotope dilution analysis of plasma galactose, was developed to allow determination of erythrocyte (red blood cell, RBC) concentrations of galactose-1-phosphate and other primary metabolites relevant in galactosaemia. Galactose-1-phosphate was enzymatically converted to galactose, and the aldononitrile pentaacetate derivative was separated by gas chromatography and determined by mass spectrometry using chemical ionisation and selected ion monitoring of the [MH-60](+) ion. U-(13)C-Labelled standard was used for quantification. Comparative measurements were conducted using established fluorimetric and radiometric enzymatic methods. The GC/MS analysis for galactose-1-phosphate was linear (range examined 0-600 micromol/L(RBC), packed cells), of acceptable repeatability at low and high concentrations (within and between run CVs <15%), with a limit of quantification of 0.01 micromol/L(RBC). With samples from patients with classical galactosaemia there was a linear correlation with conventional enzymatic assays (r(2) > 0.927). In erythrocytes from post-absorptive patients under treatment, Q188R-heterozygous parents, and healthy subjects, galactose-1-phosphate concentrations (mean +/- SD) were found to be 142 +/- 38 (n = 41), 1.4 +/- 0.2 (n = 8), and 1.9 +/- 0.5 (n = 33) micromol/L(RBC), respectively. In comparison, free galactose concentrations were 3.8 +/- 1.7, 0.49 +/- 0.19, and 0.43 +/- 0.20 mol/L(RBC), respectively. The procedure allowed simultaneous galactitol analysis and proved to be useful to trace incorporation of (13)C-label into erythrocyte galactose metabolites in a D-[1-(13)C]galactose in vivo turnover study.
authors.affiliation: Klinik für Allgemeine Pädiatrie, Universitätsklinikum, Heinrich-Heine-Universität Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf, Germany. schadewa@uni-duesseldorf.de
authors.forename: Peter
authors.initials: P
authors.lastname: Schadewaldt
authors.forename: Loganathan
authors.initials: L
authors.lastname: Kamalanathan
authors.forename: Hans-Werner
authors.initials: HW
authors.lastname: Hammen
authors.forename: Udo
authors.initials: U
authors.lastname: Wendel
mesh_terms: D001774:Blood Chemical Analysis / Q000379:methods*; D004912:Erythrocytes / Q000378:metabolism*; D005260:Female; D004376:Galactitol / Q000097:blood* / Q000378:metabolism; D005690:Galactose / Q000097:blood* / Q000378:metabolism; D005693:Galactosemias / Q000097:blood* / Q000175:diagnosis; D005695:Galactosephosphates / Q000097:blood* / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D008297:Male; D008657:Metabolic Clearance Rate; D011865:Radioisotope Dilution Technique; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D005695:Galactosephosphates; D004376:Galactitol; C029973:galactose-1-phosphate; D005690:Galactose