For pain research, the orofacial region is unique in a number of ways. The region has complex local anatomy, including substantial sensory innervation from neural pathways, and muscles of facial expression that convey important information concerning pain intensity and associated psychological traits. Although chronic orofacial pain conditions appear prevalent, useful documentation on pain intensity ratings using well established instruments is sparse. In particular, two conditions, atypical facial pain and atypical odontalgia, are poorly understood in aetiology so that definitive treatment modalities are severely limited. The region's local biofluid, saliva, has been used to diagnose various local and systemic disease states, and to quantitate drug concentrations. However, recent studies indicate that saliva also contains some of the same peptides, e.g. bradykinin, that are involved in pain mechanisms. It may be that pharmacological-pharmacokinetic studies of these peptides could shed more information on thesignificance of their presence in saliva. This thesis consists of four major sections. Section 1 comprises of three clinical studies investigating orofacial pain. Section 2 deals with clinical laboratory studies of saliva. Section 3 is concerned with the development of chromatographic methods to assay bradykinin and its pharmacokinetics in saliva. Section 4 uses chromatography for the identification of novel salivary peptides. This thesis, then, presents clinical studies of orofacial pain and pharmacological investigations of saliva as the local biofluid.

A new general method is developed for the optimization of HPLC ternary or pseudo-quaternary mobile phases which are represented by the trilinear coordinate system. This method can predict the global optimum of the mobile phase composition. The global optimum composition along each edge of the triangle and the corresponding selectivity factor of the worst-separated peak pair(s) are used in this method. This method is named the weighted pattern comparison optimization method (WPCO) and is applicable for both known and unknown samples. The WPCO method is simpler than those currently in use. The WPCO method was tested by using 68 literature data sets whose separation response surfaces are different. Results of the WPCO method agree with the results obtained by the minimum α plot method and by the grid search method, and do so with substantially fewer experimental measurements. Compared with the 5% (in eluent composition) step size grid-search procedure, the WPCO method using the same step size reduces the experimental work by 75%. For further reducing the experimental work, the original WPCO method is simplified. In an ordinary HPLC separation, the separation factor and resolution are approximately proportional to the logarithm of the selectivity factor. Based on this, the separation factor replaces the logarithm of selectivity factor in the original WPCO method. This further reduces the experimental work and avoids the error introduced in the measurement of the column dead volume. The simplified WPCO method has been tested in the normal-phase and reversed-phase chromatography separation cases. The simplified WPCO method has been tested by using 27 literature data sets whose separation response surfaces are different. Results of the simplified and original WPCO methods are nearly identical when the capacity factors of the solutes of the worst-separated peak pairs are greater than 5. When the capacity factors are less than 5, the simplified WPCO method is satisfactory in less complex, less critical applications. Two new metabolites of nefopam have been separated from greyhound urine. In the separation process, flash chromatography is used for cleaning up and preseparating the samples in a single step. Compared with other techniques, experimental work is reduced. The structure of one of the newly discovered metabolites is determined using MS and NMR. The most probable structure of the other metabolite is determined using MS. The main metabolic pathways at different doses in greyhounds are studied. / Graduation date: 1991

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 114-126). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 101-107). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 114-126). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 101-107). Also available on the Internet.

Parenteral nutrition (PN) is a method of feeding those incapable of absorbing nutrients from the gastrointestinal tract. All required nutrients are combined in one "big bag". Consequently, many chemical interactions are possible between components. Ascorbic acid (AA) is ubiquitous to both animal and plant kingdoms. Although its biochemistry is not fully understood, dietary deficiency is detrimental to well being, with the most extreme condition being scurvy. AA is water-soluble and frequent intake is therefore required to maintain nutritional status. AA is possibly the most reactive additive in PN mixtures, readily reacting with dissolved oxygen, initially producing dehydroascorbic acid (OHAA). OHAA retains the biological activity of AA. It was the purpose of this study to further knowledge regarding stability of AA and OHAA in PN mixtures, informing pharmaceutical practice to improve safety and efficacy of PN. A stability-indicating HPLC method was optimised for the study of AA and OHAA in PN mixtures. A study of the kinetics of OHAA degradation was undertaken to provide data that could be used to predict OHAA stability. Results obtained indicated a first order reaction. In direct contrast to AA degradation, trace elements did not catalyse OHAA degradation. A further product of AA degradation is oxalic acid (OA) which is potentially toxic. A HPLC method for the determination of OA in PN mixtures was developed and validated, although minimum quantification limits were relatively high (~10J.Lg/ml).The method was used to assess OA appearance in stored PN mixtures, with results indicating that concentrations remained below 10J.Lg/ml even after 35 days storage. The final aspect of this research was to investigate the most likely components of a PN mixture which may "protect" AA from oxidation. a-tocopherol photo-oxidises and therefore may compete with AA for oxygen. As light catalyses the reaction it is possible oxygen reacts more rapidly with a-tocopherol compared with AA. Results indicated 0.- Tocopherol did not oxidise in preference to AA and therefore offered no "protection". Cysteine is a reducing agent included in some amino acid preparations. The average dissolved oxygen content of standard adult PN mixtures was determined, from which the amount of cysteine required to react with dissolved oxygen was calculated. AA instability in PN mixtures was compared with and without cysteine. Results indicated that adding cysteine to PN mixtures 24 hours before addition of AA, resulted in retention of >95% AA. Results obtained from this study have furthered knowledge of the AA degradation profile, its kinetics and the potential influence of other components in PN mixtures. In particular potential strategies for minimising AA degradation are identified therefore ensuring patients receive quantities approaching those prescribed.

(Uncorrected OCR) Abstract Porphobilinogen (PBG) synthase condenses two molecules of aminolaevulinic acid (ALA) to form PBG in heme biosynthesis. The enzyme activity is sensitive to inhibition by heavy metals such as lead. It can act as a biological indicator of chronic lead POis~r\g to identify the risk group, especially in children, so that early treatment can be given to prevent possible permanent damages. A reversed-phase ion-pair HPLC analytical method for the assay of the PBG synthase activity based on detection of PBG production has been validated. A Hypersil CN column (150 x 4.6 mm; 5 urn) was employed together with a mixture of acetonitrile-40 mM phosphate buffer at pH 2.4 with 5 mM 1-heptanesulphonic acid (8:92, v/v). UV detection was performed at 240 nm. PBG was eluted as a spectrally pure peak resolved from its impurities in the methanol-inhibited enzyme reaction. The method was sensitive with a limit of quantitation of 2 ~M. The within-run and between-run precisions were 8.2% and 13.8% respectively. The recovery was 93.4 �7.1% (n=6). The preliminary reference range of the PBG synthase activities in the local pediatric population were from 21.5 to 26.3 ~mol/L RBC/min. Bland and Altman statistical analysis showed that the HPLC assay and the colorimetric assay could not be used interchangeably. The HPLC assay was an alternative way to assess the PBG synthase activities in the human erythrocyte samples. IV / abstract / toc / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy