تعیین سطح پلاسمایی تیامین در بیماران همودیالیزی: یک روش دقیق کروماتوگرافی با کارکرد عالی

    Background and Aim: Renal replacement therapies are usually associated with changes in trace elements and metabolism of vitamins. Multivitamins are administered routinely to dialysis patients in dialysis centers. Thiamin plasma level is a good indicator of nutritional state of the patient. In this study, a sensitive high-performance liquid chromatography HPLC method for determination of the concentration of thiamin in human plasma has developed by which thiamin level is detected in plasma of hemodialysis patients. The aim of the study was determination of thiamin blood levels in chronic hemodylysis patients in order to evaluate their need for thiamin supplements.Materials and Methods: In this experimental study, the plasma of nine Iranian patients on hemodialysis were analyzed for their thiamin levels and compared to the thiamin levels in a group of healthy Iranian subjects. The procedure was based on pre-column oxidation of thiamin to thiochrome followed by fluorescence detection. Plasma (1ml) was deproteinized with trichloroacetic acid and thiamin was extracted with diethyl ether. Then, cyanogens bromide (0.3 M) was added to convert thiamin to thiochrome. Samples (25µl) were applied to a Novapak C8, 4µm (4.6 × 250 mm) column. The mobile phase consisted of methanol: phosphate buffer (30mM) in the ratio of 45:55 and 0.05% sodium lauryl sulfate. Data were analyzed using SPSS V13. Statistical analysis was done by t-test and One- way Anova test.Results: A precise and reproducible HPLC method was developed for determination of thiamin in plasma. The minimum detection limit was 0.2 ng/ml and percentage recovery was 85%. Inter and Intra day assay variabilities were determined for 1,5 and 15 ng/ml thiamin spikes in plasma and coefficient of variations were in the range 0.2-2.94%. The average plasma thiamin level in 10 healthy Iranian subjects was 3.07±0.95 ng/ml and in Iranian patients was 4.72±1.12 ng/ml and 4.29±0.67 ng/ml before and after hemodialysis, respectively. Our study has shown that the mean plasma thiamin level in Iranian patients on hemodialysis has no significant difference with its level in healthy Iranian subjects. The thiamin level also remained unchanged before and after the hemodialysis.Conclusion: The HPLC method used for determination of thiamin in plasma was sensitive, accurate, reproducible and suitable for kinetic studies of thiamin. According to our findings the thiamin level in patients undergoing hemodialysis has no significant difference with healthy subjects and it seems that dietary vitamin is sufficient for the normal functions of the body, and taking viamin supplementation is not necessary for these patients.