Mackay, Gillian Moira
Kynurenines in neurological disorders.
PhD thesis, University of Glasgow.
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The kynurenine pathway is thought to be involved in neurological disorders but its precise role and the mechanisms involved have yet to be established. Tryptophan can be metabolised via this pathway to produce the neurotoxic N-methyl-D-aspartate (NMDA) receptor agonist, quinolinic acid (QUIN), and the direct generators of reactive oxygen species, 3-hydroxykynurenine (3HKYN) and 3-hydroxyanthranilic acid (3HANA), as well as the neuroprotective NMDA receptor antagonist, kynurenic acid (KYNA). High performance liquid chromatography (HPLC) methods were successfully developed and validated for measuring tryptophan, kynurenine, KYNA, 3HANA and anthranilic acid (ANA) in blood samples, using absorbance and fluorescence detection. The method for determining 3HKYN using electrochemical detection was more problematic and was only used for tryptophan loaded samples and their respective baseline samples. Using HPLC, the concentrations of tryptophan, kynurenine, KYNA, 3HKYN and 3HANA were measured in the blood of Huntington's disease (HD) patients and patients with chronic brain injury, where the injury had occurred at least one year previously. QUIN was also determined for these patients using gas chromatography-mass spectrometry (GC-MS). In addition, the dynamics of the kynurenine pathway were investigated following oral tryptophan depletion and loading. In contrast to these chronic conditions, patients with acute stroke were also studied. The concentrations of tryptophan, kynurenine, KYNA, ANA and 3HANA were determined in the blood of the stroke patients, examining any changes in these concentrations during the two weeks after the stroke. The extent of inflammation and oxidative stress were also assessed for all patients, by measuring the levels of neopterin and the lipid peroxidation products, malondialdehyde and 4-hydroxynonenal. Patients with late stage HD showed abnormal tryptophan metabolism via the kynurenine pathway, together with increased inflammation and oxidative stress. Increased levels of kynurenine together with increased kynurenine: tryptophan (K:T) ratios, indicating greater indoleamine 2,3-dioxygenase (IDO) activity, were observed in blood samples from HD patients in comparison with healthy control subjects. In conjunction with this increased IDO activity, there was a decrease in the ratios of KYNA: kynurenine, suggesting decreased kynurenine aminotransferase (KAT) activity. Inflammation, which may be stimulating IDO activity, could also be decreasing KAT activity, suggested by negativecorrelations between the KYNA: kynurenine ratios and the inflammatory marker, neopterin. The inactivity of KAT suggests a small deficiency in KYNA over a long period of time which could cause a reduction in NMDA receptor antagonism, resulting in slow progressive excitotoxicity contributing to the neurodegeneration in HD. Low KYNA: kynurenine ratios were observed in baseline and tryptophan depleted samples, but after tryptophan loading, HD patients showed similar ratios compared with control subjects. This suggests that loading may be beneficial for HD patients, as more of the neuroprotectant, KYNA can potentially be produced. However, the results suggest that concentrations of the neurotoxin, QUIN, may also be increasing after tryptophan loading. Low concentrations of 3HKYN and 3HANA, with no change in QUIN levels, were also observed in the blood of HD patients. 3HANA levels continued to be decreased for the HD patients after loading. This may suggest degradation of 3HKYN and 3HANA by autoxidation producing reactive oxygen species which could contribute to the high levels of oxidative stress found in these patients. Tryptophan loading in healthy control subjects showed significant increases in the inflammatory marker, neopterin, and in the lipid peroxidation products. These results should be considered when tryptophan loading is used in psychiatric practice and in diets high in tryptophan, such as the Atkins diet. Patients with severe chronic brain injury showed similar alterations in kynurenine pathway metabolism as HD patients, at baseline and throughout the loading and depletion protocols. Although the brain injury had occurred at least one year previously, these patients showed persistent inflammation and oxidative stress, demonstrated by their increased levels of neopterin and lipid peroxidation products compared with healthy controls. In baseline blood samples, there were increased K:T ratios indicating greater IDO activity in the patients. Patients with chronic brain injury showed decreased concentrations of the neuroprotectant, KYNA, as well as low KAT activity, indicated by the decreased KYNA: kynurenine ratios. After tryptophan loading, K:T ratios decreased and the KYNA: kynurenine ratios increased in patients in comparison with controls, suggesting a reversal in the activities of the enzymes IDO and KAT. Similar levels of the inflammatory marker, neopterin, were observed in patients and controls after tryptophan loading. This suggests that these changes in IDO and KAT activities may be related to inflammation. As for the HD patients, patients with chronic brain injury showed lower levels of 3HKYN and 3HANA in their blood, with no change in QUIN levels. These metabolites may be undergoing autoxidation, producing reactive oxygen species which contribute to the ongoing oxidative stress in these patients.The kynurenine pathway was activated following an acute stroke, as indicated by the increased K:T ratios, suggesting greater IDO activity. Stroke patients also had raised levels of neopterin and lipid peroxidation products, indicating inflammation and oxidative stress. There were no changes in the blood concentrations of kynurenines, neopterin or lipid peroxidation products during the fourteen days after a stroke. Stroke patients had reduced levels of 3HANA in their blood, as observed for the HD and chronic brain injury patients. There were negative correlations between the concentration of 3HANA and the volume of the brain lesion, measured by computed tomography (CT) scan, demonstrating the importance of the decreased concentrations of 3HANA. In addition, there were increased levels of ANA in the blood of the stroke patients and the ratios of 3HANA: ANA also correlated with brain lesion volume. Another measurement which correlated with lesion volume was lipid peroxidation, suggesting that oxidative stress contributes to the extent of the brain damage after a stroke. This may suggest that the role of 3HANA in stroke is related to its autoxidation and the generation of reactive oxygen species. Increased concentrations of KYNA were observed in patients who died within three weeks of having a stroke. These high levels of KYNA may have been produced following excitotoxicity and the generation of free radicals, and may cause excessive NMDA receptor blockade or reduced mitochondrial adenosine triphosphate (ATP) synthesis, thus contributing to cell death. The kynurenine pathway was activated and showed abnormal metabolism in all the patient groups, suggesting a potential role for these metabolites in neuronal dysfunction in HD, chronic brain injury and acute stroke. Further work is required to elucidate the role of tryptophan metabolites and whether they may have a direct contribution to neuronal damage in neurological disorders.
||tryptophan, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, quinolinic acid, tryptophan loading, tryptophan depletion, Huntington's disease, brain injury, stroke, neopterin, lipid peroxidation, HPLC
||Q Science > Q Science (General)
||College of Medical Veterinary and Life Sciences > Institute of Neuroscience and Psychology
||Stone, Prof Trevor W
|Date of Award:
Dr Gillian Moira Mackay
||Copyright of this thesis is held by the author.
||25 Mar 2008
||10 Dec 2012 13:14
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