Analysis of concentration and (13)C enrichment of D-galactose in human plasma.
BACKGROUND
A stable-isotope dilution method for the sensitive determination of D-galactose in human plasma was established.


METHODS
D-[(13)C]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-(12)C-, 1-(13)C-, and U-(13)C(6)-labeled D-galactose, respectively. The D-galactose concentration was quantified on the basis of the (13)C-labeled internal standard.


RESULTS
The method was linear (range examined, 0.1-5 micromol/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 micromol/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) micromol/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.
Schadewaldt
P
P
Deutsches Diabetes Forschungsinstitut an der Heinrich-Heine-Universität, Auf'm Hennekamp 65, D-40225 Düsseldorf, Germany. schadewa@uni-duesseldorf.de
Hammen
H W
HW
Loganathan
K
K
Bodner-Leidecker
A
A
Wendel
U
U
D000293:Adolescent; D000328:Adult; D001786:Blood Glucose / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes; D005215:Fasting; D005260:Female; D005690:Galactose / Q000097:blood* / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D005949:Glucose Oxidase; D006801:Humans; D007201:Indicator Dilution Techniques; D008297:Male; D008875:Middle Aged
D001786:Blood Glucose; D002247:Carbon Isotopes; D005949:Glucose Oxidase; D005690:Galactose