Abstract
1) Determination of total thiamin: Blood was deproteinized and the supernatant was adjusted to pH 4.5 with CH_3COONa. Then diastase was added to the supernatant and incubated at 37℃ for 8〜10 hours. For HPLC, the mobile phase (0.2M NaH_2PO_4) was eluted at a flow-rate of 0.3ml/min. A sample was loaded on the sample loop and then injected into the column. Potassuim ferricyanide (0.05%) in sodium hydroxide (15%) solution applied at 0.3ml/min by a proportioning pump was mixed with the column effluent to convert thiamin into a fluorophore. The intensity of the fluorophore was measured with a spectrofluorophotometer and recorded graphically. Using this method, thiamin concentrations in the blood of humans and animals were determined. Average thiamin concentrations in the blood were 1.54×10^<-7>mol/l, 8.09×10^<-7>mol/l and 8.57×10^<-7>mol/l in humans, rats and rabbits, respectively. This method is simple and highly reproducible, and its sensitivity is sufficient for determination of the thiamin content of as low as 20μl blood samples. 2) Determination of thiamin and its phosphate esters: Blood was deproteinized and centrifuged. Aliquots of the samples are applied to a μBondapak C_<18> column attached to a high-performance liquid chromatograph. The mobile phase (0.2M NaH_2PO_4 in 0.3% acetonitril) was eluted at a flow-rate of 1.0ml/min. Addition of potassium ferricyanide/sodium hydroxide solution to the column effluent with a proportioning pump converted thiamin phosphate into fluorophores, the intensities of which were measured with a spectro-fluorophotometer. In human blood, thiamin pyrophosphate was present in the highest concentration, followed by thiamin triphosphate. Thiamin monophosphate and thiamin were present in small amounts. In rat blood, thiamin pyrophosphate was found in the largest amount, followed by thamin monophosphate. Thiamin triphosphate and thiamin were present in small amounts. Reliability and sensitivity of these methods were satisfactory.
