An investigation of the relationship between antemortem and postmortem drug concentrations in blood

In the field of postmortem toxicology, principles from pharmacology and toxicology are combined in order to determine if exogenous substances contributed to ones death. In order to make this determination postmortem and (whenever available) antemortem blood samples may be analyzed. This project focused on evaluating the relationship between postmortem and antemortem blood drug levels, in order to better define an interpretive framework for postmortem toxicology. To do this, it was imperative to evaluate the differences in antemortem and postmortem drug concentrations, determine the role microbial activity and evaluate drug stability. Microbial studies determined that the bacteria Escherichia coli and Pseudomonas aeruginosa could use the carbon structures of drugs as a source of food. This would suggest prior to sample collection, microbial activity could potentially affect drug levels. This process however would stop before toxicologic evaluation, as at autopsy blood samples are stored in tubes containing the antimicrobial agent sodium fluoride. Analysis of preserved blood determined that under the current storage conditions sodium fluoride effectively inhibited microbial growth. Nonetheless, in many instances inconsistent drug concentrations were identified. When comparing antemortem to postmortem results, diphenhydramine, morphine, codeine and methadone, all showed significantly increased postmortem drug levels. In many instances, increased postmortem concentrations correlated with extended postmortem intervals. Other drugs, such as alprazolam, were likely to have concentration discrepancies when short antemortem to death intervals were coupled with extended postmortem intervals. While still others, such as midazolam followed the expected pattern of metabolism and elimination, which often resulted in decreased postmortem concentrations. The importance of drug stability was displayed when reviewing the clonazepam/ 7-aminoclonazepam data, as the parent drug commonly converted to its metabolite even when stored in the presence of a preservative. In instances of decreasing postmortem drug concentrations the effect of refrigerated storage could not be ruled out. A stability experiment, which contained codeine, produced data that indicated concentrations could continue to decline under the current storage conditions. The cumulative data gathered for this experiment was used to identify concentration trends, which subsequently aided in the development of interpretive considerations for the specific analytes examined in the study.

Conformational dynamics associated with ligand binding to vertebrate hexa-coordinate hemoglobins

Neuroglobin (Ngb) and cytoglobin (Cygb) are two new additions to the globin family, exhibiting heme iron hexa-coordination, a disulfide bond and large internal cavities. These proteins are implicated in cytoprotection under hypoxic-ischemic conditions, but the molecular basis of their cytoprotective function is unclear. Herein, a photothermal and spectroscopic study of the interactions of diatomic ligands with Ngb, Cygb, myoglobin and hemoglobin is presented. The impact of the disulfide bond in Ngb and Cygb and role of conserved residues in Ngb His64, Val68, Cys55, Cys120 and Tyr44 on conformational dynamics associated with ligand binding/dissociation were investigated. Transient absorption and photoacoustic calorimetry studies indicate that CO photo-dissociation from Ngb leads to a volume expansion (13.4±0.9 mL mol-1), whereas a smaller volume change was determined for Ngb with reduced Cys (ΔV=4.6±0.3 mL mol-1). Furthermore, Val68 side chain regulates ligand migration between the distal pocket and internal hydrophobic cavities since Val68Phe geminate quantum yield is ∼2.7 times larger than that of WT Ngb. His64Gln and Tyr44Phe mutations alter the thermodynamic parameters associated with CO photo-release indicating that electrostatic/hydrogen binding network that includes heme propionate groups, Lys 67, His64, and Tyr 44 in Ngb modulates the energetics of CO photo-dissociation. In Cygb, CO escape from the protein matrix is fast (< 40 ns) with a ΔH of 18±2 kcal mol-1 in Cygbred, whereas disulfide bridge formation promotes a biphasic ligand escape associated with an overall enthalpy change of 9±4 kcal mol-1. Therefore, the disulfide bond modulates conformational dynamics in Ngb and Cygb. I propose that in Cygb with reduced Cys the photo-dissociated ligand escapes through the hydrophobic tunnel as occurs in Ngb, whereas the CO preferentially migrates through the His64 gate in Cygbox. To characterize Cygb surface 1,8-ANS interactions with Cygb were investigated employing fluorescence spectroscopy, ITC and docking simulations. Two 1,8-ANS binding sites were identified. One binding site is located close to the extended N-terminus of Cygb and was also identified as a binding site for oleate. Furthermore, guanidinium hydrochloride-induced unfolding studies of Cygb reveal that the disulfide bond does not impact Cygb stability, whereas binding of cyanide slightly increases the protein stability.

Optimizing the Synthesis of AZN

Azulenyl nitrone (AZN) is a bright green compound that can be used to stain different compounds, including plastics. When these stained plastics are irradiated, as they commonly are in the sterilization of medical devices, AZN changes color from green to red, constituting a permanent change. This would make obsolete the current methods of radioactive labeling and maintain the integrity of medical equipment. Although a method of synthesis is already in place, the aim was to improve the yield significantly and find a more efficient and cost-effective procedure. Last year, the procedure used resulted in 18 to 20% of AZN synthesized at the most favorable conditions. With that in mind, this year modifications were done in the hopes of improving the yield. The solvent was changed to a mixture of isopropanol and triethylamine, a stronger base, and a catalytic amount of N-tertbutyl hydroxylamine hydrochloride was used (around 4 equivalents). The reaction time was also increased to 7 days, rather than 2. After several trials, the samples were run through column chromatography and the average yield was 70%, a much more promising result than that obtained last year. There is still research to be done to improve the technicalities of the procedure, including altering the amounts of N-tertbutyl hydroxylamine hydrochloride to try and obtain similar data with fewer amounts. This portion of the research will be done in the second half of the year. In the meantime, however, a novel and more efficient method of synthesis has been established for the production of AZN that can be potentially commercialized.

Optimizing the Synthesis of AZN

Azulenyl nitrone (AZN) is a bright green compound that can be used to stain different compounds, including plastics. When these stained plastics are irradiated, as they commonly are in the sterilization of medical devices, AZN changes color from green to red, constituting a permanent change. This would make obsolete the current methods of radioactive labeling and maintain the integrity of medical equipment. Although a method of synthesis is already in place, the aim was to improve the yield significantly and find a more efficient and cost-effective procedure. Last year, the procedure used resulted in 18 to 20% of AZN synthesized at the most favorable conditions. With that in mind, this year modifications were done in the hopes of improving the yield. The solvent was changed to a mixture of isopropanol and triethylamine, a stronger base, and a catalytic amount of N-tertbutyl hydroxylamine hydrochloride was used (around 4 equivalents). The reaction time was also increased to 7 days, rather than 2. After several trials, the samples were run through column chromatography and the average yield was 70%, a much more promising result than that obtained last year. There is still research to be done to improve the technicalities of the procedure, including altering the amounts of N-tertbutyl hydroxylamine hydrochloride to try and obtain similar data with fewer amounts. This portion of the research will be done in the second half of the year. In the meantime, however, a novel and more efficient method of synthesis has been established for the production of AZN that can be potentially commercialized.