Analysis of Concentration and 13C Enrichment of d-Galactose in Human Plasma

Abstract Background: A stable-isotope dilution method for the sensitive determination of d-galactose in human plasma was established. Methods: d-[13C]Galactose was added to plasma, and the concentration was measured after d-glucose was removed from the plasma by treatment with d-glucose oxidase and the sample was purified by ion-exchange chromatography. For gas chromatographic–mass spectrometric analysis, aldononitrile pentaacetate derivatives were prepared. Monitoring of the [MH-60]+ ion intensities at m/z 328, 329, and 334 in the positive chemical ionization mode allowed the assessment of 1-12C-, 1-13C-, and U-13C6-labeled d-galactose, respectively. The d-galactose concentration was quantified on the basis of the 13C-labeled internal standard. Results: The method was linear (range examined, 0.1–5 μmol/L) and of good repeatability in the low and high concentration ranges (within- and between-run CVs <15%). The limit of quantification for plasma d-galactose was <0.02 μmol/L. Measurements in plasma of postabsorptive subjects yielded d-galactose 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) μmol/L in healthy adults, diabetic patients, patients with classical galactosemia, and obligate heterozygous parents thereof, respectively. These data were considerably lower (3- to 18-fold) than the values of a conventional enzymatic assay. The procedure was also applied successfully in a stable-isotope turnover study to evaluate endogenous d-galactose formation. Conclusions: The present findings establish that detection of d-galactose from endogenous sources is feasible in human plasma and show that erroneously high results may be obtained by enzymatic methods.