Comparison of three strip-type tests and two laboratory methods for salivary buffering analysis

This study evaluated the correlation between three strip-type, colorimetric tests and two laboratory methods with respect to the analysis of salivary buffering. The strip-type tests were saliva-check buffer, Dentobuff strip and CRT(®) Buffer test. The laboratory methods included Ericsson's laboratory method and a monotone acid/base titration to create a reference scale for the salivary titratable acidity. Additionally, defined buffer solutions were prepared and tested to simulate the carbonate, phosphate and protein buffer systems of saliva. The correlation between the methods was analysed by the Spearman's rank test. Disagreement was detected between buffering capacity values obtained with three strip-type tests that was more pronounced in case of saliva samples with medium and low buffering capacities. All strip-type tests were able to assign the hydrogencarbonate, di-hydrogenphosphate and 0.1% protein buffer solutions to the correct buffer categories. However, at 0.6% total protein concentrations, none of the test systems worked accurately. Improvements are necessary for strip-type tests because of certain disagreement with the Ericsson's laboratory method and dependence on the protein content of saliva.

Influence of high-conductivity buffer composition on field-enhanced sample injection coupled to sweeping in CE

The aim of this work was to clarify the mechanism taking place in field-enhanced sample injection coupled to sweeping and micellar EKC (FESI-Sweep-MEKC), with the utilization of two acidic high-conductivity buffers (HCBs), phosphoric acid or sodium phosphate buffer, in view of maximizing sensitivity enhancements. Using cationic model compounds in acidic media, a chemometric approach and simulations with SIMUL5 were implemented. Experimental design first enabled to identify the significant factors and their potential interactions. Simulation demonstrates the formation of moving boundaries during sample injection, which originate at the initial sample/HCB and HCB/buffer discontinuities and gradually change the compositions of HCB and BGE. With sodium phosphate buffer, the HCB conductivity increased during the injection, leading to a more efficient preconcentration by staking (about 1.6 times) than with phosphoric acid alone, for which conductivity decreased during injection. For the same injection time at constant voltage, however, a lower amount of analytes was injected with sodium phosphate buffer than with phosphoric acid. Consequently sensitivity enhancements were lower for the whole FESI-Sweep-MEKC process. This is why, in order to maximize sensitivity enhancements, it is proposed to work with sodium phosphate buffer as HCB and to use constant current during sample injection.

Preparation and optimization of calcium fluoride particles for dental applications.

Fluorides are used in dental care due to their beneficial effect in tooth enamel de-/remineralization cycles. To achieve a desired constant supply of soluble fluorides in the oral cavity, different approaches have been followed. Here we present results on the preparation of CaF2 particles and their characterization with respect to a potential application as enamel associated fluoride releasing reservoirs. CaF2 particles were synthesized by precipitation from soluble NaF and CaCl2 salt solutions of defined concentrations and their morphology analyzed by scanning electron microscopy. CaF2 particles with defined sizes and shapes could be synthesized by adjusting the concentrations of the precursor salt solutions. Such particles interacted with enamel surfaces when applied at fluoride concentrations correlating to typical dental care products. Fluoride release from the synthesized CaF2 particles was observed to be largely influenced by the concentration of phosphate in the solution. Physiological solutions with phosphate concentration similar to saliva (3.5 mM) reduced the fluoride release from pure CaF2 particles by a factor of 10-20 × as compared to phosphate free buffer solutions. Fluoride release was even lower in human saliva. The fluoride release could be increased by the addition of phosphate in substoichiometric amounts during CaF2 particle synthesis. The presented results demonstrate that the morphology and fluoride release characteristics of CaF2 particles can be tuned and provide evidence of the suitability of synthetic CaF2 particles as enamel associated fluoride reservoirs.

Characterization of the stereoselective biotransformation of ketamine to norketamine via determination of their enantiomers in equine plasma by capillary electrophoresis

A robust CE method for the simultaneous determination of the enantiomers of ketamine and norketamine in equine plasma is described. It is based upon liquid-liquid extraction of ketamine and norketamine at alkaline pH from 1 mL plasma followed by analysis of the reconstituted extract by CE in the presence of a pH 2.5 Tris-phosphate buffer containing 10 mg/mL highly sulfated beta-CD as chiral selector. Enantiomer plasma levels between 0.04 and 2.5 microg/mL are shown to provide linear calibration graphs. Intraday and interday precisions evaluated from peak area ratios (n = 5) at the lowest calibrator concentration are < 8 and < 14%, respectively. The LOD for all enantiomers is 0.01 microg/mL. After i.v. bolus administration of 2.2 mg/kg racemic ketamine, the assay is demonstrated to provide reliable data for plasma samples of ponies under isoflurane anesthesia, of ponies premedicated with xylazine, and of one horse that received romifidine, L-methadone, guaifenisine, and isoflurane. In animals not premedicated with xylazine, the ketamine N-demethylation is demonstrated to be enantioselective. The concentrations of the two ketamine enantiomers in plasma are equal whereas S-norketamine is found in a larger amount than R-norketamine. In the group receiving xylazine, data obtained do not reveal this stereoselectivity.

Rapid determination of gemcitabine in plasma and serum using reversed-phase HPLC

Gemcitabine (2'2'-difluorodeoxycytidine) is a pyrimidine analog used in the treatment of a variety of solid tumors. After intravenous (i.v.) administration, it is rapidly inactivated to 2'-deoxy-2',2'-difluorouridine (dFdU). A sensitive analytical method for the quantitation of gemcitabine is required for the assessment of alternative dosage and treatment schemes. A rapid and robust RP-HPLC assay for analysis of gemcitabine in human and animal plasma and serum was developed and validated using 2'-deoxyuridine (dU) and 5-fluoro-2'-deoxyuridine (5FdU) as internal standards. It is based on protein precipitation, the use of an Atlantis dC18 column of 100 mm length (inner diameter, 4.6 mm; particle size, 3 microm) and isocratic elution using a 10 mM phosphate buffer, pH 3.0, followed by isocratic elution with the same buffer containing 3% of ACN. For gemcitabine, RSD values for intraday and interday precision were < 4.4 and 5.3%, respectively, the LOQ was 20 ng/mL, and the assay was linear in the range of 0.020-20 microg/mL with an accuracy of > or =89%. The recovery for gemcitabine, dU and 5FdU was 86-98%. The assay was applied to determine gemcitabine levels in plasma samples of patients collected during and shortly after conventional infusion of 25-30 mg/kg body mass (levels: 2.0-18.9 microg/mL) and rats that received lower doses (1.5 mg/kg) via i.v., subcutaneous and oral drug administration (levels: 0.20-2.60 microg/mL). It could also be applied to estimate dFdU levels in human plasma.

Electrophoretically mediated microanalysis for characterization of the enantioselective CYP3A4 catalyzed N-demethylation of ketamine

Execution of an enzymatic reaction performed in a capillary with subsequent electrophoretic analysis of the formed products is referred to as electrophoretically mediated microanalysis (EMMA). An EMMA method was developed to investigate the stereoselectivity of the CYP3A4-mediated N-demethylation of ketamine. Ketamine was incubated in a 50 μm id bare fused-silica capillary together with human CYP3A4 Supersomes using a 100 mM phosphate buffer (pH 7.4) at 37°C. A plug containing racemic ketamine and the NADPH regenerating system including all required cofactors for the enzymatic reaction was injected, followed by a plug of the metabolizing enzyme CYP3A4 (500 nM). These two plugs were bracketed by plugs of incubation buffer to ensure proper conditions for the enzymatic reaction. The rest of the capillary was filled with a pH 2.5 running buffer comprising 50 mM Tris, phosphoric acid, and 2% w/v of highly sulfated γ-cyclodextrin. Mixing of reaction plugs was enhanced via application of -10 kV for 10 s. After an incubation of 8 min at 37°C without power application (zero-potential amplification), the capillary was cooled to 25°C within 3 min followed by application of -10 kV for the separation and detection of the formed enantiomers of norketamine. Norketamine formation rates were fitted to the Michaelis-Menten model and the elucidated values for V(max) and K(m) were found to be comparable to those obtained from the off-line assay of a previous study.

Stability and kinetics of nucleic acid triplexes with chimaeric DNA/RNA third strands

A series of chimaeric DNA/RNA triplex-forming oligonucleotides (TFOs) with identical base-sequence but varying sequential composition of the sugar residues were prepared. The structural, kinetic and thermodynamic properties of triplex formation with their corresponding double-helical DNA target were investigated by spectroscopic methods. Kinetic and thermodynamic data were obtained from analysis of non-equilibrium UV-melting- and annealing curves in the range of pH 5.1 to 6.7 in a 10 mM citrate/phosphate buffer containing 0.1M NaCl and 1 mM EDTA. It was found that already single substitutions of ribo- for deoxyribonucleotides in the TFOs greatly affect stability and kinetics of triplex formation in a strongly sequence dependent manner. Within the sequence context investigated, triplex stability was found to increase when deoxyribonucleotides were present at the 5'-side and ribonucleotides in the center of the TFO. Especially the substitution of thymidines for uridines in the TFO was found to accelerate both, the association and dissociation process, in a strongly position-dependent way. Differential structural information on triplexes and TFO single-strands was obtained from CD-spectroscopy and gel mobility experiments. Only minor changes were observed in the CD spectra of the triplexes at all pH values investigated, and the electrophoretic mobility was nearly identical in all cases, indicating a high degree of structural similarity. In contrast, the single-stranded TFOs showed high structural variability as determined in the same way. The results are discussed in the context of the design of TFOs for therapeutic or biochemical applications.

Formation of caries-like lesions in vitro on the root surfaces of human teeth in solutions simulating plaque fluid

Lesion formation on root surfaces of human posterior teeth was studied in acetate/lactate buffers with a background electrolyte composition based on plaque fluid analyses. Lesion depth after 28 days at 37 degrees C was measured in relation to: the presence or absence of cementum; the concentration of undissociated buffer; the presence or absence of magnesium ions at plaque fluid concentration. Each factor was evaluated at several values of -log(ion activity product for hydroxyapatite): pI(HA). Solutions were formulated to minimize variation in pH, which varied by < or =0.03 for a given comparison (individual pI(HA)) and by 0.42-0.82 over the range of pI(HA) within experiments. Lesions on surfaces from which cementum had been ground were significantly deeper than on intact surfaces, but this is considered to be due to subsurface mechanical damage and not to a solubility difference. Neither the concentration of undissociated buffer nor the presence of magnesium ions significantly affected lesion depth. Lesion depth was strongly influenced by the correlated variations in pI(HA) and pH. At pI(HA) 54 and 55, only extremely shallow lesions formed. From pI(HA) 56, lesion depth increased with increasing pI(HA). The results confirm that the solubility of the mineral of root tissues is higher than that of hydroxyapatite, but indicate that it is probably lower than suggested by Hoppenbrouwers et al. [Arch Oral Biol 1987;32:319-322]. For calcium concentrations of 3-12 mM, the critical pH for root tissue mineral was calculated as 5.22-5.66 assuming solubility equivalent to pI(HA) 54 and 5.08-5.51 assuming pI(HA) 55.

A minimally destructive technique for removing the smear layer from dentine surfaces

OBJECTIVES: To develop a minimally destructive technique for removing the smear layer produced by cutting and polishing specimens of dentine prepared for use in experimental studies, e.g. on occlusion of dentinal tubules by oral health products. The aim was to avoid the damage caused by conventional techniques utilising short exposures to solutions with very low pH. METHODS: Two acetate buffers, pH 5.5, containing different concentrations of calcium and phosphate, with -log(ion activity product with respect to hydroxyapatite) (pI(HA)) of 55 or 56, were tested on slices of dentine using scanning electron microscopy (SEM). RESULTS: A solution which, from previous work, was slightly undersaturated with respect to dentine mineral, with a pI(HA) of 56, was found to remove smear layers produced by cutting and/or polishing after 15 min. However, to reliably remove debris occluding the tubules an exposure time of 2h, followed by brief ultrasonication, was necessary. After 2h treatment with this buffer, only a small amount of demineralization of the surface was detectable by SEM, while calcium and phosphorus were detectable by X-ray dispersive spectroscopy. CONCLUSION: It is possible to remove smear layers, and to open dentinal tubules, by a reasonably short exposure to an acidic buffer which is undersaturated with respect to dentine mineral.

Screening and prediction of erosive potential

The literature on the erosive potential of drinks and other products is summarised, and aspects of the conduct of screening tests as well as possible correlations of the erosive potential with various solution parameters are discussed. The solution parameters that have been suggested as important include pH, acid concentration (with respect to buffer capacity and concentration of undissociated acid), degree of saturation, calcium and phosphate concentrations, and inhibitors of erosion. Based on the available data, it is concluded that the dominant factor in erosion is pH. The effect of buffer capacity seems to be pH dependent. The degree of saturation probably has a non-linear relationship with erosion. While calcium at elevated concentrations is known to reduce erosion effectively, it is not known whether it is important at naturally occurring concentrations. Fluoride at naturally occurring concentrations is inversely correlated with erosive potential, but phosphate is probably not. Natural plant gums, notably pectin, do not inhibit erosion, so they are unlikely to interfere with the prediction of erosive potential. The non-linearity of some solution factors and interactions with pH need to be taken into account when developing multivariate models for predicting the erosive potential of different solutions. Finally, the erosive potential of solutions towards enamel and dentine might differ.

Extrinsic causes of erosion. Diet. Chemical factors

pH value, calcium, and phosphate and to a lesser extent fluoride content of a drink or foodstuff are important factors explaining erosive attack. They determine the degree of saturation with respect to tooth minerals, which is the driving force for dissolution. Solutions oversaturated with respect to dental hard tissue will not dissolve it. Addition of calcium (and phosphate) salts to erosive drinks showed protection of surface softening. Today, several Ca-enriched soft drinks are on the market or products with naturally high content in Ca and P are available (such as yoghurt), which do not soften the dental hard tissue. The greater the buffering capacity of the drink or food, the longer it will take for the saliva to neutralize the acid. The buffer capacity of a solution has a distinct effect on the erosive attack when the solution remains adjacent to the tooth surface and is not replaced by saliva. A higher buffer capacity of a drink or foodstuff will enhance the processes of dissolution because more ions from the tooth mineral are needed to render the acid inactive for further demineralization. Further, the amount of drink in the mouth in relation to the amount of saliva present will modify the process of dissolution. There is no clear-cut critical pH for erosion as there is for caries. Even at a low pH, it is possible that other factors are strong enough to prevent erosion.

Erosion in relation to nutrition and the environment.

When considering the erosive potential of a food or drink, a number of factors must be taken into account. pH is arguably the single most important parameter in determining the rate of erosive tissue dissolution. There is no clear-cut critical pH for erosion as there is for caries. At low pH, it is possible that other factors are sufficiently protective to prevent erosion, but equally erosion can progress in acid of a relatively high pH in the absence of mitigating factors. Calcium and phosphate concentration, in combination with pH, determine the degree of saturation with respect to tooth minerals. Solutions supersaturated with respect to enamel or dentine will not cause them to dissolve, meaning that given sufficient common ion concentrations erosion will not proceed, even if the pH is low. Interestingly, the addition of calcium is more effective than phosphate at reducing erosion in acid solutions. Today, several calcium-enriched soft drinks are on the market, and acidic products with high concentrations of calcium and phosphorus are available (such as yoghurt), which do not soften the dental hard tissues. The greater the buffering capacity of the drink or food, the longer it will take for the saliva to neutralize the acid. A higher buffer capacity of a drink or foodstuff will enhance the processes of dissolution because more release of ions from the tooth mineral is required to render the acid inactive for further demineralization. Temperature is also a significant physical factor; for a given acidic solution, erosion proceeds more rapidly the higher the temperature of that solution. In recent years, a number of interesting potentially erosion-reducing drink and food additives have been investigated.