Thesis (M.S.)--University of Wisconsin--Madison, 1966. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph.D.)--Boston University / TryptoPhan and its metabolites have been implicated in various mental disorders including schizopnrenia. This serious disease of unknown etiology has become an increasingly important subject of biochemical investigation. In order to understand more fully the metabolism of tryptopnan in schizophrenia, an investigation of urinary metabolites was undertaken, utilizing radioactivity as a marker. Bats were studied to perfect the techniques and to provide possible indications of the metabolites to be looked for in man. TryptoPhan was administered to humans orally, to duplicate the normal route of intake, and intravenously, to obtain indications of tissue activity without the tryptopnan having been acted on by intestinal bacteria. The techniques employed were a combination of two-dimensional ascending paper chromatography and radioautography. The latter was useful in the visual identification of the urinary metabolites of radioactive C14-labelled tryptophan. Through the use of extraction procedures with various solvents, selective separation and identification were made possible. Concentration of the extracts provided for the detection of substances present in only minute quantities [TRUNCATED]

Behavioral and biochemical correlates of the carbohydrate and tryptophan induced increase in brain serotonin were investigated in a series of 4 experiments. Experiment 1 was conducted to establish the nadir of brain tryptophan during the dark phase of the light cycle, Following a 16 hour fast, brains were removed, at 1600, 1800, 2000, 2.400, and 0400, hrs for tryptophan determination. Analyses indicated no differences in brain tryptophan throughout the dark period. The time course and peak concentrations of the carbohydrate and tryptophan induced increase in brain tryptophan and serotonin were determined in Experiment 2. Rats were fasted from 0030 to 1730 and then offered a control diet and injected with saline or 50 mg/kg tryptophan, or offered a high carbohydrate, protein-free meal and injected with saline. One hour after treatment and hourly for the next 3 hours, brains were obtained for analysis of tryptophan and serotonin. Tryptophan injected rats exhibited a peak in brain tryptophan at 1 hour post injection and a fall in tryptophan to control levels by 2 hours. Carbohydrate fed animals exhibited an increase in brain tryptophan at all times observed. Elevated brain serotonin was found in both tryptophan and carbohydrate treated animals. Experiment 3 was conducted to establish a behavioral correlate of brain serotonin. Behaviors investigated included: latency to step-down and explore a novel chamber and acquisition and extinction of a passive avoidance response. Animals were fed ad libitum, and 1 hour (1700) prior to behavioral testing injected with saline or 50 mg/kg tryptophan. Animals did not differ on measures of passive avoidance acquisition or extinction. However, tryptophan injected animals were found to exhibit a longer latency to step-down and explore a novel chamber than controls. In Experiment 4, plasma corticosterone, latency to step-down, rearing, urination, and defecation in a novel chamber were assessed. Animals were fasted from 2400 to 1700 and injected and fed as in Experiment 2. One and 2 hours following treatment, behaviors were observed. Thereafter, brains were removed for determination of tryptophan and serotonin and blood obtained for plasma corticosterone analysis. In tryptophan administered rats, brain tryptophan was observed to peak at 1 hour post injection and to remain higher than controls at 2 hours post injection. Carbohydrate fed rats were found to exhibit higher levels of brain tryptophan than control animals at both times assayed. Brain serotonin was found to peak in tryptophan treated rats at 1 hour post injection and to remain elevated at 2 hours. No changes in brain serotonin were revealed in carbohydrate fed animals. No group differences were observed for any of the behavioral measures taken. However, increased plasma corticosterone was found in rats fed the high carbohydrate meal. These data revealed that injection of tryptophan resulted in an increased latency to step-down and explore a novel chamber when animals were fed ab libitum, whereas carbohydrate ingestion resulted in an increase in plasma corticosterone with no effect on behavior. Confirmation that serotonin mediated these biochemical and behavioral changes awaits further research. / Land and Food Systems, Faculty of / Graduate

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The regulation of tryptophan-2,3-dioxygenase (TDO) (EC 1.13.1.12) and, to a lesser extent, pineal indoleamines, both in vitro and in vivo, is examined in this study. Rat liver TDO is a cytosolic enzyme which plays a crucial role in the regulation of circulating tryptophan (TRP) levels. Stimulation of this enzyme by heme enhances the catabolism of TRP, making less TRP available for uptake into the brain and other tissues, and for protein synthesis. At pH 7, the enzyme has an approximate Km of 100μM, is subject to substrate inhibition immediately beyond Sopt([S] at Vmax), and response of the enzyme is cooperative in both uninhibited and inhibited regions. Hill analysis of the uninhibited region reveals a biphasic plot and two classes of binding sites. Negative cooperativity is brought about through deprotonation of the enzyme. Substrate iphibition also occurs at both acidic and basic pH values with concomitant shifts in Sopt. The results obtained indicate that substrate inhibition could be an additional mechanism whereby the flux through the TRP-kynurenine pathway is regulated. TDO is subject to a diurnal rhythm, with peak activity during the pre-dark period and the loweSt activity towards the end of the dark period. It is possible that the enzyme controls the synthesis of the neurotransmitter serotonin (5-HT), and that the circadian rhythm in TDO activity is due to the endogenous rhythm of melatonin (aMT) production by the pineal gland. In the present study, aMT displaces TRP from bovine serum albumin (BSA) in vitro, and it is therefore possible for the indoleamine to regulate the availability of TRP for uptake into the brain for conversion to its derivatives. Chronic intraperitoneal administration of aMT affects physiological hepatic parameters in rats, such as TDO activity and stromal fatty acid composition, whilst no observable effect is demonstrable with respect to protein synthesis, nucleic acid metabolism, membrane fatty acid composition and pineal indole biosynthesis. On the other hand, chronic treatment of rats with antidepressants, the tricyclic desmethylimipramine (DMI) and the selective serotonin reuptake inhibitor (SSRI), fluoxetine, reveals significant negative alterations in TDO concentrations and pineal indole amine synthesis. Combining aMT with any of these two drugs normalises the activity of the hepatic enzyme. DMI is found to be an effective inhibitor of TDO in the micromolar range in vitro, and also affects total enzyme concentrations in vivo. Fluoxetine has no effect on TDO in vitro, but in vivo also reduces total enzyme levels in the liver. However, the SSRI does not affect conjugation between apo- and holoenzyme. Instead, it decreases extant holoenzyme levels. Indoleamine synthesis by the pineal gland, in organ culture, is altered by both antidepressants, although in different ways. DMI increases N-acetylserotonin levels and reduces the output of methoxyindole acetic acid and meth6xytryptophol. Fluoxetine treatment markedly reduces aMT concentrations and also brings about high levels of the 5-HT catabolites, 5-hydroxytryptophol and 5-hydroxyindole acetic acid. Insulin also lowers aMT synthesis significantly in pineal organ cultures, via a mechamsm that involves inhibition of the enzyme, N-acetyl transferase, that regulates aMT synthesis. The effects of insulin on pineal indole metabolism are due to the observation that a carbohydrate rich diet which induces insulin release elevates plasma TRP and brain 5-HT, but has no effect on pineal TRP and indole amine synthesis. It could thus be possible for insulin to have an effect on the pineal, since the latter is outside the blood brain barrier. The finilings of this study support the biogenic amine deficiency hypothesis, implicating some of the major biogenic amines such as noradrenaline (NA), 5-HT and aMT in depression. There is believed to be a deficiency of NA and 5-HT at their respective synapses in the depressed state. The drug DMI could act, firstly, by inhibiting TDO and thus increasing plasma TRP levels, and could, secondly, stimulate NA release and inhibit NA reuptake at the pineal membrane. The combined effect would be to enhance aMT synthesis, with eventual remission. Fluoxetine, on the other hand, appears to utilize a slightly different mode of action to DMI, which seems to focus on the preservation of 5-HT. The fact that aMT counteracts the effects of both antidepressants, and restores the activity of TDO to that of the controls, is also consistent with the observation that the therapeutic action of drugs such as these coincides willi the restoration of normal plasma levels of the neurohormone in depressives. In view of the biogenic amine deficiency hypothesis of depression and the contentious claim that TDO is the major peripheral determinant of brain TRP, brain 5-HT and ultimately aMT, the regulation of TDO is investigated and discussed. The study concludes that TDO activity is regulated by a number of endogenous compounds which are mainly derivatives of TRP, such as aMT and oxidized nicotinamide adenine dinucleotide and exogenous substances, of which DMI and fluoxetine are but two. In addition, modulation of IDO activity in depression appears to be an important aspect of antidepressant action. aMT, the product of the pineal gland, also has the potential to increase plasma TRP and hence forebrain TRP levels, and ultimately 5-HT concentrations, firstly by displacing TRP from serum albumin and secondly by inhibiting TDO.

The main goals in electroanalytical sensing are towards improved sensitivity and selectivity, or specificity, of an analyte. There are several approaches to achieving these goals with the main approach being modification of an electrode surface with synthetic or natural catalysts (enzymes), polymers and also utilisation of nanostructured materials. At present, there is a strong movement towards hybrid sensing which couple different properties of two or more surface modification approaches. In this thesis, a range of these surface modifications were explored for analysis and detection of two main analytes: the amino acid, tryptophan (Trp); and, the neurotransmitter, dopamine (DA). Specifically, this thesis aimed to utilise these methods to enhance the sensitivity and selectivity for Trp over an interferent, the indoleamine, melatonin (Mel); and, DA over the vitamin, ascorbic acid (AA). For Trp detection, immobilisation of an enzyme, Tryptophanase (Trpase) resulted in poor selectivity for the analyte. However, enhanced sensitivity and selectivity was achieved through pH manipulation of the electrolyte medium at a Nafion®-modified electrode surface for both Trp and Mel. At pH 3.0, the Mel and Trp anodic peak potentials were sufficiently resolved allowing for an LOD of 1.60 and 1.62 nM,respectively, and permitting the accurate analysis of Trp in a dietary supplement containing Mel. Multi-walled carbon nanotubes (MWCNTs) suspended in Nafion® exhibited further increases in the signal responses of these analytes at pH 3.0 and 7.4 with minimal change in the resolution of the anodic peaks. A lower sensitivity was, therefore, observed at the Nafion® and MWCNT modified electrode compared to the Nafion®-modified electrode at pH 3.0 with LODs of 0.59 and 0.80 nM exhibited for Trp and Mel, respectively. Enhanced selectivity for Trp in the presence of Mel can be achieved with MWCNTs in the presence of metallotetrasulphonated phthalocyanines (MTSPcs) particularly at pH 3.0, owing to cation exchange effects. However, the lack of sensitivity towards Trp, and even Mel, at this CoTSPc and MWCNT modified electrode remains a drawback. For DA, detection at the MWCNT and Nafion® surface resulted in improved sensitivity over that of both the bare electrode (613.0 nM) and the Nafion® modified electrode (1045.1 nM) with a calculated LOD of 133.9 nM at this layer. Furthermore, improvements in the selectivity of DA were achieved at the Nafion® and MWCNT modified electrode as exclusion of AA (150 μM) was achieved. At the MWCNT and CoTSPc surface, AA was excluded up to 130 μM with sensitivity for DA extending as low as 14.3 nM, far greater than observed for Trp and Mel. These concentrations are well within physiological concentration ranges and represent the most significant solution yet in terms of AA exclusion and enhanced sensitivity for DA. An examination of the surface layering by impedance spectroscopy and atomic force microscopy indicates that the success of the hybrid sensor utilising CoTSPc and MWCNTs lay in improved dispersion of MWCNTs and improved electron transfer kinetics, facilitated by the net charge of the materials present. This thesis, thus, showed the utility of a judicious selection of synthetic and biological catalysts, polymers and carbon nanomaterials towards a hybrid approach to the electrochemical sensing of Trp, Mel, DA and AA with focus on sensitivity and selectivity of these analytes.