A multiple-site competitive model has been developed to evaluate quantitatively the equilibrium competition of drugs that bind to multiple classes of binding sites on human serum albumin (HSA). The equations, which are based on the multiple-class binding site model, assume that competition exists at individual sites, that the binding parameters for drug or drug competitor pertain to individual sites, and also that the binding parameters for drug or competitor at any given site are independent of drug or competitor bound at other sites. For the drug-competitor pairs, ethacrynic acid (EA) -caproic acid (C6), -lauric acid (C12), and -palmitic acid (C16), the reaction heat of EA binding to HSA was measured in the absence and the presence of fatty acids at the molar ratio of 3:1 with HSA at pH 7.4 and 37°C by isothermal titration microcalorimetry. The calorimetric titration data induced by the presence of fatty acids were directly compaired to the computer simulation curves by the corresponding multiple-site competititve models, which were precedently calculated from binding parameters of EA and fatty acids. In the case of EA-C12 or -C16 competitive binding, EA binding at the first and the second classes of binding sites on HSA were instantaneously inhibited by C12 or C16, resulting that the binding constant of the first class of binding sites of EA were decreased and that the second class of binding sites on HSA entirely disappeared. In the competition between EA and C6, the first class of binding sites of EA was diminished by C6, resulting in the decrease of the binding constants and the number of binding sites in the first class of EA, whereas, the second class of binding sites was unaffected. The multiple-site competitive model assuming site-site competition could be directly comparable to the calorimetric data and be suitable to account for the competitive processes for drugs bound to the multiple-class of binding sites on HSA.
Authors:G. Behbehani, A. Divsalar, A. Saboury, F. Faridbod, and M. Ganjali
Thermodynamics of the interaction between erbium(III) chloride, Er3+, with human serum albumin (HSA), was investigated at pH 7.0 and in phosphate buffer by isothermal titration calorimetry.
Our recently, solvation model was used to reproduce the enthalpies of HSA interaction by Er3+ over a broad range of metal ion concentration. The solvation parameters recovered from our new model, attributed to the structural
change of HSA and its biological activity. The binding parameters for the interaction of Er3+ and HSA indicate that the concentrations of Er3+ have no significant effects on the structure of HSA.
Authors:Sigrid Mennickent, Ricardo Fierro, Mario Vega, Marta Diego, C. Godoy, Cristina Cifuentes, and Arnoldo Miranda
A method using high-performance thin-layer chromatography (HPTLC) for quantitation of sertraline in human serum was developed and validated. It includes a liquid-liquid extraction with diethyl ether-chlorophorm (75:25, v/v) and carbamazepine as internal standard. Chromatographic separation was performed on precoated silica gel F254 HPTLC plates using a mixture of toluene-ethyl acetate-methanol-glacial acetic acid (4.5:1.5:1.0:0.5, v/v) as mobile phase. The method showed good relationship (r = 0.998) (1.00–70.00 ng/band, corresponding to 0.01 and 0.70 ng/μL in serum after extraction process and applying 10 μL to the chromatographic plates). This range includes serum concentration found in literature (0.02–0.50 ng/μL). The % RSD values of intra- and inter-assay were between 0.88–2.75 and 0.76–3.36, respectively. The limit of detection and the limit of quantification were 0.12 and 0.25 ng/band. The accuracy values were between 94.65% and 98.93%. RF for N-desmethylsertraline or norsertraline (major metabolite of sertraline), sertraline, and carbamazepine were 0.23, 0.45, and 0.71, respectively. Ten samples of patient volunteers who were using sertraline as a treatment for major depression were analyzed using the method developed and found sertraline concentrations between 0.05 and 0.32 ng/μL. In conclusion, the method is precise, accurate, selective, and sensitive for quantitative determination of sertraline in human serum, and it could be used for pharmacotherapy adherence evaluation.
Radiotracer experiments have been used to study the retention of Na from 14M HNO3 mineralized serum samples by HAP in both batch and column processes. The specific capacity of HAP depends nonlinearly on
the ratio initial Na-amount/amount of HAP. Pre-irradiation removal of Na by HAP-columns and of Cl by evaporation permits Cu,
Mg an K in human serum to be determined by neutron activation analysis.
Authors:Renuka Munshi, Namrata Gawde, Salman Dalal, and Deepali Ganachari
A simple and reliable high-performance thin-layer chromatographic (HPTLC) method has been developed and validated for the determination of topiramate in human serum which will help in therapeutic drug monitoring that aids in the clinical management of patient therapy. This method uses dipping method of derivatization to make the non-chromophore topiramate visible for detection. Pre-coated silica gel F254 HPTLC plates were used to carry out chromatographic separation using a mixture of toluene–ethanol (9:1) as the mobile phase. Derivatization was carried out using dipping method. Betamethasone was used as internal standard. Densitometric detection was carried out at 417 nm. The method was validated for linearity, precision, selectivity, limit of detection and limit of quantification and accuracy. Linear calibration curves in the range of 0.25 to 40 μg band−1 gave a correlation coefficient of 0.9963. The intra-day (n = 6) and inter-day (n = 18) precision, expressed as the relative standard deviation, were in the range of 2.89% to 3.41% and from 2.97% to 4.20%. The limit of detection and the limit of quantification were found to be 0.36 μg band−1 and 1.10 μg band−1. The accuracy was calculated as percentage recovery and was found to be 97.41% and 110.7%. The method was specific and showed no matrix interference. Compared to other methods employed in the determination of topiramate from blood, this method is cost-effective, simple, and reliable to carry out therapeutic drug monitoring of topiramate.
Authors:N. L. Mehany, M. T. El Kolaly, S. M. Ayyoub, and S. E. M. Hassan
Development, optimization and validation of immunoradiometric assay (IRMA) using solid phase-cellulose particles for the measurement of TSH in human serum or plasma are described. The preparation of 125I anti-TSH monoclonal antibody (MoAb) was carried out by two different methods (Chloramine-T and Iodogen). It was found that Ch-T method gave approximately the same results as the iodogen method. The activation of cellulose particles using 1,1-carbonyl diimidazole (CDI) and coupling of these solid phase particles with sheep anti-TSH were carried out. Optimization and validation of the assay were undertaken. The reproducibility as measured by the intra- and inter-assay variations is acceptable. The recovery and dilution tests indicated accurate calibration and appropriate matrix. No significant position effect was recognized. The different modes of sampling tested did not affect significantly the results of the present study. The present technique agreed well with the currently used commercial kit (DPC, IRMA). The cellulose particles of the present system for estimation of TSH retain their characteristics during storage for 6 months at 4 °C. In conclusion, this technique proved to be sensitive, specific, precise and accurate for routine laboratory use.
Authors:Akifumi Eguchi, Takeshi Enomoto, Norimichi Suzuki, Miho Okuno, and Chisato Mori
from all donors. This study was approved by the Ethics Committee of Chiba University, Japan. Humanserum samples ( n = 26) were collected from Chiba Prefecture, Japan [ 16 ]. Whole blood samples were collected by a certified physician, and serum
Authors:R. Cornelis, J. Versieck, L. Mees, J. Hoste, and F. Barbier
Vanadium in serum has been investigated by the aid of neutron activation analysis (8 min irradiation at 8·1013 n·cm−2·s−1 in the reactor FR-II of the Kernforschungszentrum in Karlsruhe). The lyophilized samples were dry-ashed before irradiation
and the52V activity extracted after irradiation. The values for V in the sera of 22 healthy males ranged from 0.029–0.939 ng V·ml−1. There is a real assumption that some of the high figures are due to persons being contaminated with V. The 18 healthy females
yielded a mean value of 0.033±0.012 ng V·ml−1 for 17 of them and one additional value of 0.139 ng V·ml−1. These V-data are the lowest ever reported in the literature. The analyses of two packed blood cell samples yielded 0.031
and 0.020 ng·g−1, indicating that about 68% of the total V in blood is present in serum. There was no correlation between the V-content and
age, nor between the V-content and the cholesterol, triglycerides or the lipoprotein fractions in serum.
Authors:M. Al-Janabi, S. Khachadoorian, S. Moussa, and Z. Yousif
Higher than 90% of113mIn radioactivity was bound to microaggregates. The liver uptake in mice was (80%) with low lung uptake (1.3%). With respect to99mTc-microaggregated albumin, the radiochemical yield was higher than 95%. The liver uptake in mice was about (80%) with low lung uptake (1.7%). The stability of the microaggregates was followed for two months.
Gel filtration and neutron activation analysis methods were combined in work aimed at the development of methods for the determination
of metals firmly bound to protein in human blood serum. The values obtained are semiquantative. A purely instrumental technique
afforded data on Cu, Fe, Zn, Al and Mn but values for the latter two only were determined since Cu, Fe and Zn are already
well known. The concentrations for Al and Mn were found to be 300 ppb and 0.4 ppb, respectively. Despite the high resolution
of Ge(Li) detectors, no other metals were found. For the determination of any others, radiochemical separations are necessary
and were used to obtain data on the following elements: As 3 ppb, Ag 3 ppb, Au 0.003 ppb, Cr 1 ppb, Sb 1 ppb, Co, Cd, Mo and
Sn could not be detected and upper limits were estimated to be 0.1 ppb, 2 ppb, 2 ppb, and 1 ppb, respectively.