RESUMEN
A reversed-phase high-performance liquid chromatography method (HPLC) with coulometric electrochemical detection has been developed and validated for the simultaneous analysis of pyridoxamine-5'-phosphate (PMP), pyridoxamine (PM), pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxine (PN) and 4-pyridoxic acid (4-PA) in human plasma and serum. The isocratic separation was achieved on C(18) column (250mmx4.6mm, I.D., 5microm) with a mobile phase consisted of 35mM sodium phosphate containing 2.5mM heptanesulfonic acid, adjusted to pH 3.2 with 85% orthophosphoric acid and 12% methanol (v/v). Within-run and between-run precisions expressed by the relative standard deviations were less than 2.7% and 7.7% for all the analysed vitamins and 4-PA, respectively. The limits of detection (LOD) and quantification (LOQ) were: 0.8 and 2.6nM, 1.1 and 3.8nM, 1.5 and 4.5nM, 1.3 and 4.2nM, 1.1 and 3.7nM, 2.1 and 6.3nM for PMP, PM, PLP, PL, PN and 4-PA, respectively. The recoveries ranged from 90.4% to 98.4%. Stability of vitamins was checked under a variety of storage conditions. The developed application demonstrated acceptable sensitivity, precision, accuracy, stability, and linearity over the physiological concentration range. The major advantage of the proposed method is its great simplicity.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Técnicas Electroquímicas/métodos , Vitamina B 6/sangre , Adulto , Estabilidad de Medicamentos , Femenino , Humanos , Modelos Lineales , Masculino , Ácido Piridóxico/sangre , Ácido Piridóxico/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Vitamina B 6/metabolismoRESUMEN
The increased plasma level of homocysteine have been shown to be the sensitive marker for the folate, vitamin B6 and cobalamins deficiency and an independent risk factor for the cardiovascular disease, neutral tube defects and a potential causal risk factor for neuropsychiatric disorders. The blood and plasma homocysteine levels except for genetic defects are influenced by age, gender, efficiency of detoxication systems, one or more unhealthy lifestyle factors, such as high alcohol consumption, low nutritional intake of vitamins, high coffee consumption, acquired disorders and lack of physical exercise. Many studies confirm that active tobacco smoking and environmental tobacco smoke (ETS) have been significantly associated with hiperhomocysteinemia. In metabolic pathway of homocysteine the important role played folic acid, as a donor of methyl group in re-methylated reaction to methionine and vitamin B6. It acts as the cofactor in transsuphuration reactions of homocysteine to cystathionine and cysteine. Hence, the aim of this work was to compare the plasma folate and vitamin B6 concentrations in smokers and passive smokers with a hiperhomocysteinemia (> 15 micromol/L). It was observed that the plasma folate levels in active (n = 30) and passive smokers (n = 29) groups decrease statistically significant (P < 0.001) in comparison to non-smokers (n = 37). The calculated Spaermann's correlations coefficient of total plasma homocysteine level and plasma folate concentrations in the non-smoker group indicated a weak, statistically insignificant correlation (r = -0.103, P = 0.542). However, the above relationship in passive and active smokers were statistically significant (r = -0.495, P 0 0.01; r = -0.672, P < 0.001, respectively). The decrease of vitamin in B6 plasma was observed in all active smokers group (P < 0.01) and men smokers comparing to non-smokers (P < 0.001). There was no observed significant correlations between hiperhomocysteinemia and vitamin B6 in all studied groups. The results indicated that hiperhomocysteinemia have strong negative impact on folate levels in active and passive smokers. The tobacco smoke exposure have negative influence on the status of vitamin B6. The lack of significant correlation between increased homocysteine levels and vitamin B6 status confirmed hypothesis that hiperhomocysteinemia is not depended on vitamin B6 concentrations in plasma.