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PubMed1983

Potentiometric titration of thiols, cationic surfactants and halides using a solid-state silver-silver sulphide electrode.

A rugged, low resistance silver-silver sulphide solid-state electrode for determining pharmaceuticals as authentic samples or in dosage forms by potentiometric titration is described. Sodium tetraphenylborate, mercury(II) acetate and silver nitrate (0.01) M were employed as titrants in the analysis of cationic surfactants (cetylpyridinium chloride, benzethonium chloride, benzalkonium chloride and chlorhexidine salts), antithyroid drugs (methimazole and propylthiouracil) or sodium halides respectively.

Journal Article↗

PubMed1988

Chloride determination in serum by a flow-injection analysis precipitation pseudo-titration technique using a flow-through all-solid-state silver electrode.

A simply constructed, tubular, all-solid-state, flow-through silver electrode for flow-injection analysis (F.I.A.) is described. Use of a single line manifold accommodating the silver electrode, with a low level of silver ion (5 x 10(-4) M) in the carrier stream, is a useful method to determine chloride in serum, by means of a precipitation pseudo-titration F.I.A. technique. The sampling frequency is about 60/h.

Journal Article↗

PubMed1995

FIA titrations of ephedrine in pharmaceutical formulations with a PVC tetraphenylborate tubular electrode.

A flow injection system for the titration of ephedrine in pharmaceutical products with potentiometric detection was developed. For this purpose a tetraphenylborate tubular electrode was constructed. The electrode was prepared without inner reference solution and with a PVC membrane based on tetrapentylammonium tetraphenylborate as ion exchanger and 2-nitrophenylphenyl ether as mediator solvent. Its operational characteristics were evaluated in a low dispersion manifold and compared with more conventionally shaped electrodes using the same sensor. In the pH range 2.5-11.5, the electrodes showed linear response between 3.8 x 10(-6) and 0.1 M with a slope of -56.4 mV/log[BPh4]. Ephedrine determinations in pharmaceutical products were carried out in a single channel manifold with a mixing chamber incorporated and using the tubular electrode as detector. Recovery rates of 98.6 +/- 2.5% were obtained in the analysis of tables, nasal drops and syrups with a sampling rate of about 60 h-1.

Electrodes↗

PubMedDate not supplied

The initiator titration model: computer simulation of chromosome and minichromosome control.

The initiator titration model was formulated to explain the initiation control of the bacterial chromosome. In particular, features concerning the replication behaviour of minichromosomes, such as their high copy number and Escherichia coli's ability to coinitiate chromosome and many minichromosome origins, were considered during the formulation of the model. The model is based on the initiator protein DnaA and its binding sites, DnaA boxes, in oriC, in the dnaA promoter and at other positions on the chromosome. Another important factor in the model is the eclipse period created by the hemimethylation of a new oriC which makes it refractory to initiation. The model was analysed by computer simulations using a stochastic approach varying the different input parameters, and the resulting computer cells were compared with data on living E. coli cells. Here we present the outcome of a few of these simulations concerning the eclipse period, in silico-shift experiments blocking initiation or elongation of replication, and introduction of minichromosomes into the computer cells. We also discuss the synthesis of DnaA protein in the computer cells. From our simulations, we conclude that, whether true or not, the model can mimic the in vivo initiation control of E. coli.

Bacterial Proteins↗

PubMed1995

Does monitoring end-tidal isoflurane concentration improve titration during general anesthesia?

STUDY OBJECTIVE: To assess the value of end-tidal anesthetic gas monitoring with respect to intraoperative hemodynamic stability and recovery times. DESIGN: Randomized blinded study. SETTING: Operating rooms at a university teaching hospital. PATIENTS: 120 ASA I and II patients receiving general anesthesia maintained with isoflurane and nitrous oxide (N2O). INTERVENTIONS: Following a standardized induction technique, patients were assigned to either an end-tidal isoflurane monitored (n = 60) or unmonitored (n = 60) group. During each operation, the anesthesiologist attempted to maintain an adequate "depth of anesthesia" by varying the administered concentration of isoflurane with or without information from an end-tidal isoflurane monitor. Intraoperative hemodynamic stability was assessed by determining the variation from a preincisional "baseline" mean arterial pressure (MAP) value established during a 10 minute interval immediately prior to the surgical incision. Recovery times were recorded from discontinuation of isoflurane and N2O until awakening, orientation, and postanesthesia care unit discharge. MEASUREMENTS AND MAIN RESULTS: Intraoperative hemodynamic stability was assessed in each patient and reported as the average error from the baseline MAP, absolute average error from the baseline MAP, coefficients of variation of heart rate (HR), systolic and diastolic MAP, and end-tidal isoflurane concentrations. Both study groups had similar intraoperative MAP and HR values, average error and coefficients of variation for the hemodynamic variables, as well as similar numbers of episodes of hypertension, hypotension, tachycardia, and bradycardia. Finally, the two groups were comparable with respect to early recovery times and postoperative side effects. CONCLUSIONS: This study suggests that end-tidal isoflurane monitoring does not improve the titration of isoflurane during general anesthesia.

Adult↗

PubMed1993

Titration of the in vivo uptake of 16 alpha-[18F]fluoroestradiol by target tissues in the rat: competition by tamoxifen, and implications for quantitating estrogen receptors in vivo and the use of animal models in receptor-binding radiopharmaceutical development.

We have measured in vivo the uptake of 16 alpha-[18F]estradiol (FES) by target tissues in the immature rat at increasing dose levels (obtained by dilution of [18F]FES with unlabeled estradiol). This was done to examine the binding capacity of target tissues in vivo and to determine whether the uptake in receptor-rich tissues was flow limited, as this has implications concerning the appropriateness of using receptor-rich tissues in experimental animals as models for FES uptake by receptor-poor breast tumors in humans. We also wanted to establish the dose level of the anti-estrogen tamoxifen required to block target tissue uptake of FES. We found that in untreated rats, specific uptake in the uterus saturated at c. 180 pmol/g, in the ovary at c. 54 pmol/g and in the muscle at c. 2 pmol/g. At an intermediate dose of tamoxifen (570 micrograms/kg), uptake saturated at somewhat lower levels, and at a high tamoxifen dose (1710 micrograms/kg), yet lower specific uptake was evident. In the FES titrations at low dose levels of FES, both the uterus and the ovaries, but not the muscle, showed characteristics of flow-limited uptake, i.e. the uptake-to-dose ratio reached a maximum level. This flow limitation suggests that only when receptor levels are sufficiently low will the FES uptake be related to receptor concentration. While receptor-rich tissues such as the rat uterus will show this flow limitation, the receptor concentration in most primary and metastatic human breast tumors is sufficiently low, so that the uptake should parallel receptor content. In in vivo distribution studies, target tissues (or tumors) with low receptor content will be more fully saturated and ligand more readily displaced. Also, uptake by secondary target tissues (i.e. those with a lower content of estrogen receptor, such as muscle, thymus and kidney) may be better models for assessing the effectiveness of new breast tumor imaging agents than uptake by receptor-rich tissues.

Animals↗

PubMed1990

The effect of tissue and cellular pH on the selective biodistribution of porphyrin-type photochemotherapeutic agents: a volumetric titration study.

Volumetric titration of aqueous solutions of haematoporphyrin IX (HP) yields two inflexion points, whereas four pK values can be obtained via mathematical fitting of the experimental data. Assignment of all pK protonation sites is made. A zwitterion is proposed as the neutral species of HP. Evaluation of the pK values of HP in the presence of sodium dodecyl sulphate (SDS) leads to a drastically altered ionic species distribution. On the basis of the distribution diagrams obtained, a pH-sensitive twofold mechanism is proposed for the selective biodistribution of porphyrin-type photochemotherapeutic agents, one involving tissue distribution and the other involving cell membrane penetration by the neutral zwitterion.

Acid-Base Equilibrium↗

PubMed1994

Systemic heparinization during peripheral vascular surgery: thromboelastographic, activated coagulation time, and heparin titration monitoring.

Fifteen patients (9 male, 6 female) undergoing peripheral vascular surgery were monitored during surgery for evidence of subclinical anticoagulation using the activated coagulation time (ACT), thromboelastography (TEG), and heparin titration monitoring. Assessments were made at 30-minute intervals before and after the occlusion clamp. Mean (+/- SD) ACT values preoperatively were 111 (17) seconds, and 10 minutes after 5,000 IU of heparin, the ACT was 264 (57) seconds (P < 0.001). Intraoperatively, there was a significant decline in ACT values at 30 minutes (ACT 228 [50] sec, P < 0.005) and 60 minutes (200 [46] sec, P < 0.001) postheparin. No significant difference in ACT was observed between samples drawn distally and proximally to the clamp. TEG profiles were abolished in all patients immediately following heparinization. However, in 2 patients nearly complete return of the TEG coagulation profile was observed prior to the termination of the procedure and was associated with ACT values less than 160 seconds. The heparin device was unable to accurately monitor heparin elimination at these low doses. Variability of patient response to heparinization necessitates the use of intraoperative monitoring of anticoagulation during peripheral vascular surgery.

Aged↗

PubMed2005

Mediated spectroelectrochemical titration of proteins for redox potential measurements by a separator-less one-compartment bulk electrolysis method.

One-compartment bulk electrolysis and simultaneous spectroscopic measurements are realized in a conventional spectroscopic cuvette without separator by using a mesh-type working electrode with extremely large surface area and a wire-type counter electrode with very small surface area. Spectrophotometric monitoring revealed complete electrolysis in a first-order kinetics. This technique was applied to mediated titration of cytochrome c and bilirubin oxidase for determining their redox potentials. Kinetics for the solution redox reaction between protein and mediator is described. The subtraction of spectral background due to mediator adsorption is very easy because of high reproducibility. The experiments can be done under completely anaerobic conditions. Low-absorbance protein samples (of low concentrations or small absorption coefficients) and hydrophobic proteins (such as membrane-bound proteins) are acceptable for measurements.

Cytochromes c↗

PubMed2005

Statistical error in isothermal titration calorimetry: variance function estimation from generalized least squares.

The method of generalized least squares (GLS) is used to assess the variance function for isothermal titration calorimetry (ITC) data collected for the 1:1 complexation of Ba(2+) with 18-crown-6 ether. In the GLS method, the least squares (LS) residuals from the data fit are themselves fitted to a variance function, with iterative adjustment of the weighting function in the data analysis to produce consistency. The data are treated in a pooled fashion, providing 321 fitted residuals from 35 data sets in the final analysis. Heteroscedasticity (nonconstant variance) is clearly indicated. Data error terms proportional to q(i) and q(i)/v are well defined statistically, where q(i) is the heat from the ith injection of titrant and v is the injected volume. The statistical significance of the variance function parameters is confirmed through Monte Carlo calculations that mimic the actual data set. For the data in question, which fall mostly in the range of q(i)=100-2000 microcal, the contributions to the data variance from the terms in q(i)(2) typically exceed the background constant term for q(i)>300 microcal and v<10 microl. Conversely, this means that in reactions with q(i) much less than this, heteroscedasticity is not a significant problem. Accordingly, in such cases the standard unweighted fitting procedures provide reliable results for the key parameters, K and DeltaH(degrees) and their statistical errors. These results also support an important earlier finding: in most ITC work on 1:1 binding processes, the optimal number of injections is 7-10, which is a factor of 3 smaller than the current norm. For high-q reactions, where weighting is needed for optimal LS analysis, tips are given for using the weighting option in the commercial software commonly employed to process ITC data.

Analysis of Variance↗

PubMed2005

Beta-D-glucosidase reaction kinetics from isothermal titration microcalorimetry.

The cellobiase activities of nine thermal stable mutants of Thermobifida fusca BglC were assayed by isothermal titration microcalorimetry (ITC). The mutations were previously generated using random mutagenesis and identified by high-temperature screening as imparting improved thermal stability to the beta-D-glucosidase enzyme. Analysis of the substrate-saturation curves obtained by ITC for the wild-type enzyme and the nine thermally stabilized mutants revealed that the wild type and all the mutants were subject to binding of a second substrate molecule. Furthermore, the "inhibited" enzyme-substrate complexes were shown to retain catalytic activity. In the case of three of the BglC mutants (N178I, N317Y/L444F, and N317Y/L444F/A433V), binding of a second substrate molecule resulted in improved cellobiose turnover rates at lower substrate concentrations. No correlation between denaturation temperatures of the mutants and activity on cellobiose at 25 degrees C was evident. However, one particular mutant, BglC S319C, was significantly improved in both thermal tolerance and cellobiase activity with respect to those of the wild-type BglC. The triple mutant, N317Y/L444F/A433V, had a 5 degrees C increase in denaturation temperature while maintaining activity levels similar to that of the wild type at higher substrate concentrations. ITC provided a highly sensitive and nondestructive means to continuously monitor the reaction of BglC with cellobiose, resulting in abundant data sets that could be rigorously analyzed by fitting to known enzyme kinetics models. One distinct advantage of using data from the ITC was the empirical validation of the pseudo steady state assumption, a necessary condition for obtaining solutions to the proposed mechanisms.

Actinomycetales↗

PubMed2005

Energetics of carbohydrate binding to Momordica charantia (bitter gourd) lectin: an isothermal titration calorimetric study.

Physico-chemical and carbohydrate binding studies have been carried out on the Momordica charantia (bitter gourd) seed lectin (MCL). The lectin activity is maximal in the pH range 7.4-11.0, but decreases steeply below pH 7.0. The lectin activity is mostly unaffected in the temperature range 4-50 degrees C, but a sharp decrease is seen between 50 and 60 degrees C, which could be correlated to changes in the structure of the protein as seen by circular dichroism and fluorescence spectroscopy. Isothermal titration calorimetric studies show that the tetrameric MCL binds two sugar molecules and the binding constants (Kb), determined at 288.15 K, for various saccharides were found to vary between 7.3 x 10(3) and 1.52 x 10(4)M(-1). The binding reactions for all the saccharides investigated were essentially enthalpy driven, with the binding enthalpies (DeltaHb) at 288.15 K being in the range of -50.99 and -43.39 kJ mol(-1), whereas the contribution to the binding reaction from the entropy of binding was negative, with values of binding entropy (DeltaSb) ranging between -99.2 and -72.0 J mol(-1)K(-1) at 288.15 K. Changes in heat capacity (DeltaCp) for the binding of disaccharides, lactose and lactulose, were significantly larger in magnitude than those obtained for the monosaccharides, methyl-beta-D-galactopyranoside, and methyl-alpha-D-galactopyranoside, and could be correlated reasonably well with the surface areas of these ligands. Enthalpy-entropy compensation was observed for all the sugars studied, suggesting that water structure plays an important role in the overall binding reaction. CD spectroscopy indicates that carbohydrate binding does not lead to significant changes in the secondary and tertiary structures of MCL, suggesting that the carbohydrate binding sites on this lectin are mostly preformed.

Binding Sites↗

PubMed2004

Oral appliance titration and nasal resistance in nonapneic subjects.

Initial mandibular position might be a key factor leading to rapid therapeutic outcome in oral appliance therapy for patients with obstructive sleep apnea (OSA). The purpose of this study was to investigate the effects of an adjustable oral appliance on nasal resistance in 7 nonapneic patients. Upright and supine nasal resistance was measured for each subject with an adjustable oral appliance in place in 3 mandibular positions: most retruded (MAX0), maximum protrusion (MAX100), and 67% of MAX100 (MAX67). A significant decrease in upright and supine nasal resistance was observed between MAX0 and MAX67 (P < .01), and between MAX0 and MAX100 (P < .01), but not between MAX67 and MAX100. In addition, there was a positive correlation between the nasal resistance at MAX0 and the nasal resistance change from MAX0 to MAX67 (P < .01), indicating that when the mandible was advanced from MAX0 to MAX67, the more nasal resistance at MAX0, the greater the reduction in nasal resistance at MAX67. These findings suggest that MAX67 might be indicated as the initial mandibular position and that gradual anterior titration of mandibular position beyond MAX67 would give OSA patients rapid therapeutic effects by reducing the nasal resistance. Moreover, OSA patients with augmented nasal resistance at MAX0 might have a greater reduction in nasal resistance in response to MAX67 in oral appliance therapy.

Adult↗

PubMed2006

Effect of individualizing starting doses of a statin according to baseline LDL-cholesterol levels on achieving cholesterol targets: the achieve cholesterol targets fast with atorvastatin stratified titration (ACTFAST) study.

AIMS: To investigate whether selecting the starting dose of atorvastatin according to baseline and target (<2.6 mmol/L) LDL-cholesterol (LDL-C) values would allow high-risk subjects to achieve target LDL-C concentration within 12 weeks, with the initial dose or a single uptitration. METHODS AND RESULTS: Twelve-week, prospective, open-label trial that enrolled 2117 high-risk subjects (statin-free [SF] or statin-treated [ST]). Subjects with LDL-C >2.6 mmol/L (100mg/dL) but <or=5.7 mmol/L (220 mg/dL) were assigned a starting dose of atorvastatin (10, 20, 40 or 80 mg/day) based on LDL-C and status of statin use at baseline, with a single uptitration at 6 weeks, if required. There was no washout for ST subjects. At study end, 80% of SF (82%, 82%, 83% and 72% with 10, 20, 40 and 80 mg, respectively) and 59% of ST (60%, 61% and 51% with 20, 40 and 80 mg, respectively) subjects reached LDL-C target. In the ST group, an additional 21-41% reduction in LDL-C was observed over the statin used at baseline. Atorvastatin was well tolerated. CONCLUSION: This study confirms that individualizing the starting dose of atorvastatin according to baseline and target LDL-C values (i.e. the required LDL-C reduction), allows a large majority of high-risk subjects to achieve target safely, within 12 weeks, with the initial dose or with a single titration.

Aged↗

PubMed2004

Dissociation constants of phenothiazine drugs incorporated in phosphatidylcholine bilayer of small unilamellar vesicles as determined by carbon-13 nuclear magnetic resonance spectrometric titration.

The dissociation constants (pKms) of the phenothiazine drugs promazine, chlorpromazine, and triflupromazine, incorporated in the phosphatidylcholine (PC) bilayer of small unilamellar vesicles (SUV), were investigated by a 13C nuclear magnetic resonance (NMR) titration method employing their N-13CH3 (ionizable group) labelled derivatives. Use of the labelled drugs enabled direct observations of the ionization equilibrium of the N-dimethyl group. A second derivative spectrophotometric study proved that 95-98% of the phenothiazine species in the sample solutions (200 microM phenothiazine in the presence of 27 mM PC SUV) were incorporated into the PC bilayer, which simplified the calculation of pKm values by allowing that the phenothiazines in the aqueous phase could be neglected. The pKm values were calculated from the chemical shift dependence of the N-dimethyl 13C NMR signal on the pH value of sample solutions. The pKm values obtained were smaller than those measured in aqueous solutions by about one unit. The existence of cholesterol (30 mol%) in the PC bilayer showed little effect on the pKm values, suggesting that cholesterol in the bilayer does not largely affect the interfacial region where the N-dimethyl group of the incorporated phenothiazines is located. The results offered clear evidence for the pKm decrease and provided their precise values.

Carbon Isotopes↗

PubMed2006

Isothermal titration calorimetric study defines the substrate binding residues of calreticulin.

Earlier we established using modeling studies the residues in calreticulin (CRT) important for sugar-binding (M. Kapoor, H. Srinivas, K. Eaazhisai, E. Gemma, L. Ellgaard, S. Oscarson, A. Helenius, A. Surolia, Interactions of substrate with calreticulin, an endoplasmic reticulum chaperone, J. Biol. Chem. 278 (8) (2003) 6194-6200). Here, we discuss the relative roles of Trp-319, Asp-317, and Asp-160 for sugar-binding by using site-directed mutagenesis and isothermal titration calorimetry (ITC). Residues corresponding to Asp-160 and Asp-317 in CNX play important role towards sugar-binding. From the present study we demonstrate that the residue Asp-160 is not involved in sugar-binding, while Asp-317 plays a crucial role. Further, it is also validated that cation-pi interactions of the sugar with Trp-319 dictate sugar-binding in CRT. This study not only defines further the binding site of CRT but also highlights its subtle differences with that of calnexin.

Amino Acids↗

PubMed2005

Gamma-irradiated RecD overproducers become permanent recB-/C- phenocopies for extrachromosomal DNA processing due to prolonged titration of RecBCD enzyme on damaged Escherichia coli chromosome.

The RecBCD enzyme of Escherichia coli consists of three subunits RecB, RecC and RecD. RecBCD enzyme activities are regulated by its interaction with recombination hotspot Chi. Biochemical and genetic evidence suggest that interaction with Chi affects RecD subunit, and that RecD polypeptide overproduction antagonizes this interaction, suggesting that intact RecD replaces a Chi-modified one. We used bacteria with fragmented chromosomes due to double-strand breaks inflicted by UV and gamma-irradiation to explore in which way increased concentrations of RecBCD's individual subunits affect DNA metabolism. We confirmed that RecD overproduction alters RecBCD-dependent DNA repair and degradation in E. coli. Also, we found that RecB and RecC overproduction did not affect these processes. To determine the basis for the effects of RecD polypeptide overproduction, we monitored activities of RecBCD enzyme on gamma-damaged chromosomal DNA and, in parallel, on lambda and T4 2 phage DNA duplexes provided at intervals. We found that gamma-irradiated wild-type bacteria became transient, and RecD overproducers permanent recB(-)/C(-) phenocopies for processing phage DNA that is provided in parallel. Since this inability of irradiated bacteria to process extrachromosomal DNA substrates coincided in both cases with ongoing degradation of chromosomal DNA, which lasted much longer in RecD overproducers, we were led to conclude that the RecB(-)/C(-) phenotype is acquired as a consequence of RecBCD enzyme titration on damaged chromosomal DNA. This conclusion was corroborated by our observation that no inhibition of RecBCD activity occurs in gamma-irradiated RecBCD overproducers. Together, these results strongly indicate that RecD overproduction prevents dissociation of RecBCD enzyme from DNA substrate and thus increases its processivity.

Bacteriophage T4↗

PubMed2004

Direct spectroelectrochemical titration of glutathione.

Previous potentiometric attempts to determine the formal potential (E'(0)) of key intracellular redox buffer glutathione resulted in contradictory values. We have developed a spectroelectrochemical method using direct reduction on metal oxide electrodes. Disulfide absorbance at 258 nm was used to titrate glutathione in the thin layer cell reversibly. At conditions close to physiological ([GSH] = 0.001-0.005 mol/l, pH = 7.34; I = 0.1 mol/l; T = 298.15 K), we have measured glutathione E(0)' = -0.22 +/- 0.02 V (NHE), corroborating the results of equilibrium measurements.

Electrochemistry↗