Simultaneous, quantitative analysis of UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, UDP-glucose and UDP-galactose in human peripheral blood cells, muscle biopsies and cultured mesangial cells by capillary zone electrophoresis.
The aim of this study was to develop and evaluate a capillary zone electrophoretic (CE) procedure for the accurate quantification of the UDP-hexosamines as well as for the corresponding UDP-hexoses in samples from various biological origins. Testing different buffer conditions, voltages, capillary dimensions and temperatures, optimal results were achieved with a 90 mM borate buffer, pH 9.0, at 18 degrees C and 15.5 kV in an uncoated fused-silica capillary of 50 cm x 50 microm and a detection wavelength of lambda = 262 nm. The total procedure, i.e., including variations of the sample preparation, showed coefficients of variation for the peak areas between 4. 1% and 10.4% in mesangial cells (n = 7) and between 7.8 and 10.3% (n = 6) in leukocytes for the components of interest. To improve precision, an internal standard was used for calibration. The limit of detection for all compounds is an absolute amount of 180 fmol, sufficient for the precise analysis of UDP-sugars in a limited amount of biological samples, such as human leukocytes (obtained from a 10 mL blood sample), muscle biopsies (< or = 100 mg), and mesangial kidney cells (ca. 2.5 x 10(5) cells). This reproducible, quantitative analysis of all four UDP-sugars from various biomedically relevant origins by CZE is a definite improvement over the generally used high performance liquid chromatography (HPLC) procedures. The CZE method allows the study of the flux through the hexosamine pathway in diabetes mellitus and other diseases in a simple, quantitative and accurate way.
Lehmann
R
R
Klinisch-chemisches Zentrallabor der Universitätskliniken, Abteilung Innere Medizin IV, Tübingen, Germany. rainer.lehmann@med.uni-tuebingen.de
Huber
M
M
Beck
A
A
Schindera
T
T
Rinkler
T
T
Houdali
B
B
Weigert
C
C
Häring
H U
HU
Voelter
W
W
Schleicher
E D
ED
D001773:Blood Cells / Q000737:chemistry; D002478:Cells, Cultured; D019075:Electrophoresis, Capillary / Q000379:methods*; D005920:Glomerular Mesangium / Q000737:chemistry; D006801:Humans; D018482:Muscle, Skeletal / Q000737:chemistry; D014531:Uridine Diphosphate Galactose / Q000032:analysis* / Q000737:chemistry; D014532:Uridine Diphosphate Glucose / Q000032:analysis* / Q000737:chemistry; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000032:analysis* / Q000737:chemistry; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000032:analysis* / Q000737:chemistry
D014531:Uridine Diphosphate Galactose; D014537:Uridine Diphosphate N-Acetylglucosamine; D014536:Uridine Diphosphate N-Acetylgalactosamine; D014532:Uridine Diphosphate Glucose