Abstract
The present data show a fast and efficient biological sample processing method for the extraction of thiamine (vitamin B1) and its mono-(TMP) and di-(TDP) phosphate esters from hippocampus, thalamus and prefrontal cortex (PFC) and blood sample of the rodents. In addition, using the hippocampus and standards of these three compounds we validated an isocratic fluorescence HPLC procedure for a simultaneous detection of them in a single chromatogram within a total run time of about 12 min. Reproducibility for TDP, TMP and B1 was 2.66%, 4.50% and 7.43% (intraday) and 37.54%, 25.39% and 25.87% (interday), respectively. Recovery assays were between 96.0% and 101.7%. The calibration curves were linear and the concentrations of the three compounds, all in nanomolar range, were determined in the brain areas and in the blood samples. When compared to the current methods in the literature, this new method provides information on essential variables, such as linearity range and limit of detection, reproducibility and stability of thiamine, TMP and TDP in rat brain samples. The present data on sample processing and B1 and its phosphate ester level determinations are the first to be validated using hippocampus samples of rats.
Highlights
Vitamin B1 consists of free thiamine, a pyrimidyl substituted thiazole, and its phosphate esters thiamine monophosphate (TMP), thymidine diphosphate
Trichloroacetic acid (TCA), potassium ferricyanide (K3Fe(CN)6), thiamine hydrochloride, thiamine pyrophosphate (TDP), thiamine monophosphate chloride (TMP) and hydrochloric acid were purchased by Sigma-Aldrich
In a total run time of less than 12 min the thiamine and its phosphorylated forms were eluted from the HPLC system
Summary
(TDP) and thiamine triphosphate [1]. TMP is considered as a substrate to generate the active form (TDP or TPP) by the action of thiamine pyrophosphokinase and to produce B1 by thiamine phosphatase [2] [3] [4] [5]. Decreased levels of B1 and its phosphorylated forms can be induced by inadequate intake and/or absorption of thiamine, associated or not with alcohol abuse [6] [7] [8]. Thiamine deficiency (TD) causes brain dysfunctions known as Wernicke’s Encephalopathy and Wernicke-Korsakoff Syndrome (WKS), respectively considered by some authors as the acute and chronic stages of the same disorder. A plenty of studies have been carried out using these models to understand the neurobiological mechanisms related to the behavioral deficits usually associated with TD.
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