Stable-isotope dilution analysis of galactose metabolites in human erythrocytes

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-C-13-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 mumol/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 mumol/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 postabsorptive patients under treatment, Q188R-heterozygous parents, and healthy subjects, galactose1-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) mumol/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 C-13-label into erythrocyte galactose metabolites in a D-[1-C-13] galactose in vivo turnover study. Copyright (C) 2003 John Wiley Sons, Ltd.