Analysis of concentration and C-13 enrichment of D-galactose in human plasma

Background: A stable-isotope dilution method for the sensitive determinatio n of D-galactose in human plasma was established. Methods: D-[C-13]Galactose was added to plasma, and the concentration was m easured after D-glucose was removed from the plasma by treatment with D-glu cose oxidase and the sample was purified by ion-exchange chromatography. Fo r gas chromatographic-mass spectrometric analysis, aldononitrile pentaaceta te derivatives were prepared. Monitoring of the [MH-60](+) ion intensities at m/z 328, 329, and 334 in the positive chemical ionization mode allowed t he assessment of 1-C-12-, 1-C-13-, and U-C-13(6)-labeled D-galactose, respe ctively. The D-galactose concentration was quantified on the basis of the C -13-labeled internal standard. Results: The method was linear (range examined, 0.1-5 mu mol/L) and of good repeatability in the low and high concentration ranges (within- and betwee n-run CVs <15%). The limit of quantification for plasma D-galactose was <0. 02 mu mol/L. Measurements in plasma of postabsorptive subjects yielded D-ga lactose concentrations (mean +/- SD) of 0.12 +/- 0.03 (n = 16), 0.11 +/- 0. 04 (n = 15), 1.44 +/- 0.54 (n = 10), and 0.17 +/- 0.07 (n = 5) mu mol/L in healthy adults, diabetic patients, patients with classical galactosemia, an d obligate heterozygous parents thereof, respectively. These data were cons iderably lower (3- to 18-fold) than the values of a conventional enzymatic assay. The procedure was also applied successfully in a stable-isotope turn over study to evaluate endogenous D-galactose formation. Conclusions: The present findings establish that detection of D-galactose f rom endogenous sources is feasible in human plasma and show that erroneousl y high results may be obtained by enzymatic methods. (C) 2000 American Asso ciation for Clinical Chemistry.