This experiment describes a simple protocol for teaching acid–base titrations using potentiometry, conductivity, and/or photometry to determine end points without an added indicator. The chosen example examines the titratable acidity of a red wine with NaOH. Wines contain anthocyanins, the colors of which change with pH. Importantly, at the equivalence point, anthocyanins maintain their color, and this effect can be captured optically using a desktop scanner. RGB-based color values are obtained from the digitized images using a dedicated application and employed to generate plots of |v| versus titrant volume. The end point is the point at which the slope (d|v|/Δ/dV) of the titration curve is at a maximum; (d|v| is a vector expression of the color change). The photometric titrations were carried out using a plastic 96-well immunology plate and a flat-bed scanner, and RGB values were extracted simultaneously from all 96 wells in less than 5 min from the images using the ImageJ plugin “ReadPlate”. The wine was also titrated using conventional potentiometric and conductometric techniques, and methods were compared using F-test, t-test, and one-way ANOVA. The potentiometric titration yields pH versus titrant volume curves, and the equivalence point is where the slope, or the slope of the derivative curve (dpH/dV), is greatest. The conductometric titration yields conductivity versus titrant volume curves, and the end point is determined as the volume where the slope exhibits a marked change. All titration and derivative curves were plotted and analyzed using spreadsheet software.
Momentum is growing worldwide to use in vitro high-throughput screening (HTS) to evaluate human health effects of chemicals. However, the integration of dosimetry into HTS assays and incorporation of population variability will be essential before its application in a risk assessment context. Previously, we employed in vitro hepatic metabolic clearance and plasma protein binding data with in vitro in vivo extrapolation (IVIVE) modeling to estimate oral equivalent doses, or daily oral chemical doses required to achieve steady-state blood concentrations (Css) equivalent to media concentrations having a defined effect in an in vitro HTS assay. In this study, hepatic clearance rates of selected ToxCast chemicals were measured in vitro for 13 cytochrome P450 and five uridine 5'-diphospho-glucuronysyltransferase isozymes using recombinantly expressed enzymes. The isozyme-specific clearance rates were then incorporated into an IVIVE model that captures known differences in isozyme expression across several life stages and ethnic populations. Comparison of the median Css for a healthy population against the median or the upper 95th percentile for more sensitive populations revealed differences of 1.3- to 4.3-fold or 3.1- to 13.1-fold, respectively. Such values may be used to derive chemical-specific human toxicokinetic adjustment factors. The IVIVE model was also used to estimate subpopulation-specific oral equivalent doses that were directly compared with subpopulation-specific exposure estimates. This study successfully combines isozyme and physiologic differences to quantitate subpopulation pharmacokinetic variability. Incorporation of these values with dosimetry and in vitro bioactivities provides a viable approach that could be employed within a high-throughput risk assessment framework.
The L L-amyloid (AL L) peptide has previously been shown to enhance phenylephrine or endothelin-1 induced constriction of aortic rings in vitro. The characteristics of AL L vasoactivity (dose, fragment length, timing) suggest that the mechanism is distinct from AL L cytotoxicity. To identify which properties of AL L determine its biological activity on vessels, we investigated a number of AL L analogues and fragments, individually and in combination, including those that are known to be associated with Alzheimer's disease (AL L IÀRP ) and hereditary cerebral hemorrhage with amyloidosis^Dutch type (AL L(22Q) IÀRH ). The vasoactivity appears to be related to the conformation adopted by the peptide in solution. The L L-pleated sheet rich AL L IÀRP and AL L(22Q) IÀRH were each less vasoactive than the mainly random coil wild type AL L IÀRH . However, the most vasoactive AL L peptides were combinations which contain mixtures of random coil and L L-sheet structure. The finding that peptides containing low or high levels of L L-pleated conformation are less vasoactive than those containing intermediate amounts of this structural motif allows us to propose the existence of a transitional form between random coil and L L-pleated that is the vasoactive species of AL L. This is the first time that AL L conformational intermediates have been identified and a biological activity associated with them.z 1998 Federation of European Biochemical Societies.
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