Determination of thiamin and its phosphate esters in cultured neurons and astrocytes using an ion-pair reversed-phase high-performance liquid chromatographic method.
A sensitive method, based on fluorescence detection, for the determination of thiamin derivatives after precolumn derivatization is described. The separation is achieved on a PRP-1 column using ion-pair reversed-phase HPLC. This method is especially well adapted to the detection of thiamin triphosphate in complex mixtures such as tissue extracts. The detection limit for TTP is 50 fmol. The contents of thiamin derivatives were determined in primary cultures of rat cerebellar granule neurons and cerebral astrocytes. The amount of TTP is about five times higher in neurons than in astrocytes. Thus in rat brain TTP seems to be essentially associated with neurons and the intracellular concentration is estimated to be about 0.2 microM. Our results suggest the existence, in nerve cells, of specific regulatory mechanisms not related to the blood-brain barrier and responsible for the maintenance of thiamin homeostasis in brain.
Bettendorff
L
L
Laboratory of General and Comparative Biochemistry, University of Liège, Belgium.
Peeters
M
M
Jouan
C
C
Wins
P
P
Schoffeniels
E
E
D000818:Animals; D001923:Brain Chemistry; D002478:Cells, Cultured / Q000737:chemistry; D002531:Cerebellum / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005904:Glial Fibrillary Acidic Protein / Q000276:immunology; D007150:Immunohistochemistry; D009474:Neurons / Q000737:chemistry*; D010084:Oxidation-Reduction; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013831:Thiamine / Q000032:analysis* / Q000737:chemistry; D013833:Thiamine Monophosphate / Q000032:analysis / Q000737:chemistry; D013835:Thiamine Pyrophosphate / Q000032:analysis / Q000737:chemistry; D013838:Thiamine Triphosphate / Q000032:analysis* / Q000737:chemistry
D005904:Glial Fibrillary Acidic Protein; D013838:Thiamine Triphosphate; D013833:Thiamine Monophosphate; D013835:Thiamine Pyrophosphate; D013831:Thiamine