ABSTRACT
Introducción: Los métodos utilizados para la medición de Vitamina D tienen varios aspectos metodológicos sin definir: método de referencia, trazabilidad con estándar internacional, especificidad, etc.; situación que genera grandes controversias entre los resultados obtenidos. Objetivo: Comparar los resultados de la medición de Vitamina D con dos inmunoensayos de diferente especificidad y estandarización, y su impacto en la interpretación clínica de los mismos en individuos no tratados y tratados con Vitamina D2 o D3. Materiales y Métodos: Para la comparación de los métodos se consideraron dos grupos: G1 (95 muestras de suero de pacientes algunos de los cuales recibían Vitamina D2, otros Vitamina D3 y otros sin medicación) y G2 (40 muestras de suero luego de excluir a los 55 pacientes que recibían Vitamina D2). Resultados: Al analizar las medias de los resultados obtenidos por cada método, se hallaron diferencias altamente significativas (p<0.0001) en G1 y diferencias significativas (p< 0.0038) en G2. Cuando se compararon los resultados de EQLIA vs RIE, no se halló correlación (r:-0,00671; p=0,9483) en G1 pero sí en G2. (r=0.898, p<0.0000). Si bien en G1 se halló concordancia entre los resultados de los métodos se observa gran dispersión de los mismos (media 20,8 ng/ml). En cambio en el G2 se observa una mejor concordancia entre ellos (media 6.4 ng/ml). Conclusiones: Esta comparación de métodos nos permitió identificar las dos situaciones críticas que debemos considerar cuando nos solicitan la medición de Vitamina D: primero la condición del paciente en cuanto a si ha sido o no suplementado con Vitamina D y en ese caso si se le administró D2 o D3 y segundo: el método a emplear para su determinación. Las discrepancias entre los resultados de vitamina D obtenidos por diferentes metodologías impactarán en la utilidad clínica del informe emitido por el laboratorio y consecuentemente en la interpretación y decisión médica.
Introduction: The methods used for vitamin D measurement have several methodological aspects still not defined, such as reference method, traceability of international standard, specificity, etc, which generates great discrepancies between the results obtained. Objective: To compare the results of vitamin D measurements obtained with two different immunoassays of different specificity and standardization, and their impact on clinical interpretation in patients treated and not treated with vitamin D2 or D3. Materials and Methods: Two groups are considered for comparison of the methods: G1 (95 serum samples from patients who were receiving vitamin D2, vitamin D3 or no medication) and G2 (40 serum samples after excluding the 55 patients who received vitamin D2). Results: There are great differences between the samples measured by both assays. We found highly significant differences between means in G1 (p <0.0001) and significant differences in G2 (p < 0.0038). No correlation was observed between EQLIA vs. RIA in the G1 (r = -0.00671, p = 0.9483) but good correlation in G2. (r = 0.898, p < 0.0000). Although ECLIA was in agreement with RIA, in G1 the results of the methods shows a wide dispersion around the mean of the difference 20.8 ng / ml. In contrast, in G2 there is a better match between results (mean of the difference 6.4 ng/ml). Conclusions: This comparison of methods allowed us to identify two critical situations to consider when measuring vitamin D: first, whether the patient has been supplemented or not with Vitamin D, and in that case, if he/she has been given D2 or D3, and secondly, the method used for measurement. Discrepancies between the results of vitamin D measurements obtained by different methodologies will impact on the clinical usefulness of the report issued by the laboratory, and consequently in the interpretation and medical decision.
ABSTRACT
Correolide is a novel immunosuppressant that inhibits the voltage-gated potassium channel K(v)1.3 [Felix et al. (1999) Biochemistry 38, 4922-4930]. [(3)H]Dihydrocorreolide (diTC) binds with high affinity to membranes expressing homotetrameric K(v)1.3 channels, and high affinity diTC binding can be conferred to the diTC-insensitive channel, K(v)3.2, after substitution of three nonconserved residues in S(5) and S(6) with the corresponding amino acids present in K(v)1.3 [Hanner et al. (1999) J. Biol. Chem. 274, 25237-25244]. Site-directed mutagenesis along S(5) and S(6) of K(v)1.3 was employed to identify those residues that contribute to high affinity binding of diTC. Binding of monoiodotyrosine-HgTX(1)A19Y/Y37F ([(125)I]HgTX(1)A19Y/Y37F) in the external vestibule of the channel was used to characterize each mutant for both tetrameric channel formation and levels of channel expression. Substitutions at Leu(346) and Leu(353) in S(5), and Ala(413), Val(417), Ala(421), Pro(423), and Val(424) in S(6), cause the most dramatic effect on diTC binding to K(v)1.3. Some of the critical residues in S(6) appear to be present in a region of the protein that alters its conformation during channel gating. Molecular modeling of the S(5)-S(6) region of K(v)1.3 using the X-ray coordinates of the KcsA channel, and other experimental constraints, yield a template that can be used to dock diTC in the channel. DiTC appears to bind in the water-filled cavity below the selectivity filter to a hydrophobic pocket contributed by the side chains of specific residues. High affinity binding is predicted to be determined by the complementary shape between the bowl-shape of the cavity and the shape of the ligand. The conformational change that occurs in this region of the protein during channel gating may explain the state-dependent interaction of diTC with K(v)1.3.