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Clinical Chemistry 46: 612-619, 2000;
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(Clinical Chemistry. 2000;46:612-619.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

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

Peter Schadewaldt1,a, Hans-Werner Hammen1, Kamalanathan Loganathan1, Annette Bodner-Leidecker1,2 and Udo Wendel2

1 Deutsches Diabetes Forschungsinstitut an der Heinrich-Heine-Universität, Auf’m Hennekamp 65, D-40225 Düsseldorf, Germany.

2 Kinderklinik, Heinrich-Heine-Universität, Moorenstrasse 5, D-40225 Düsseldorf, Germany.
a Address correspondence to this author at: Deutsches Diabetes Forschungsinstitut, Klinische Biochemie, Auf’m Hennekamp 65, D-40225 Düsseldorf, Germany. Fax 49-211-3382-603; e-mail schadewa{at}uni-duesseldorf.de

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.






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