|dc.description.abstract||Bilirubin is an endogenous molecule that is formed from the degradation of haem when senescent red blood cells have been transported to the liver, kidneys and spleen. Since its discovery, it has been thought of as a waste product that required excretion upon degradation due to accumulation leading to detrimental health conditions such as neonatal hyperbilirubinaemia and kernicterus. As such, the primary research focus on bilirubin has been on developing treatment protocols for reducing elevated levels of bilirubin, as well as optimising and developing more sensitive, robust and streamlined methods of measuring serum and urine bilirubin levels. However, there has since been a change in focus whereby recent research has demonstrated the antioxidative activity of bilirubin and has led to the postulation that it also has a physiological role as a protective cellular antioxidant, producing bilirubin oxidation by-products as a result of scavenging reactive oxygen and nitrogen species.
There is a need for identifying and clarifying this developing area of research that also suggests that bilirubin oxidation end products could be associated with a variety of health conditions. This study commenced with a scoping review to distinguish the different types of bilirubin oxidation end products in the literature, for the purpose of conducting further detailed literature reviews, identifying gaps and informing the direction of experimental work for the thesis. The scoping review identified two pyrrole-containing oxidative by-products of bilirubin as biotripyrrins, biopyrrins, or bilirubin oxidative metabolites (BOMs) and bilirubin oxidation products (BOXes). A third pyrrole-containing compound was identified that has been associated with mental illness and oxidative stress, with speculation on its origin. Due to the structural similarities shared with this compound and BOMs and BOXes, it was considered important for further literature review. Two broad themes emerged from the scoping review as a focus for the subsequent reviews on the three groups of pyrrole compounds: (i) elucidation, characterisation and chemical synthesis of bilirubin oxidation end products, (ii) and proposed clinical application or pathophysiology for the bilirubin oxidative end products, including both human and animal studies.
It was found that BOMs were serendipitously discovered when researchers were developing an enzyme-linked immunosorbent assay (ELISA) to detect total and unconjugated bilirubin. This led to the characterisation of these bilirubin metabolites that were hypothesised to result from conditions of oxidative stress. A number of clinical studies reported elevated levels of these metabolites for a wide variety of unrelated health conditions, particularly for cardiovascular and mental health conditions.
The findings from the literature review conducted on BOXes identified that these oxidised forms of bilirubin have been speculated as a causative agent of vasospasm in patients suffering from subarachnoid haemorrhage (SAH). There has been a focus on identifying the causes of vasospasm in patients with SAH due to the condition being described as clinically frustrating and challenging to manage. Initial reports on BOXes identified these compounds in the cerebral spinal fluid (CSF) of SAH patients and researchers were able to characterise compounds related to bilirubin albeit at poor yields. A few reports that have investigated these compounds in in vitro and in vivo models corroborated the earlier findings however the low yields acquired from initial methods of synthesis reflected the lack of reports on the mode of action of BOXes. Recent publications have provided insight into the syntheses of these compounds at relatively high yields.
The final literature review was conducted on urine pyrroles that have been the subject of considerable controversy, primarily relating to their chemical structure, as well as their role in mental health, which is not recognised or accepted by mainstream medicine. Urine pyrroles were originally identified in the 1960’s in patients with schizophrenia due to the purple colour observed when their urine samples reacted positively to Ehrlich’s reagent hence the name ‘Mauve factor’. Considerable efforts in identifying the compounds through qualitative and semiquantitative methods showed that the compounds were highly unstable hindering the characterisation and methods of syntheses. Due to their unstable nature, misidentifications were frequently reported in the literature, and together with their undetermined pathophysiological origin, compounded the controversy. Despite the unclear nature of the exact biochemical mechanisms of urine pyrroles, contemporary treatment and diagnosis of ‘pyrrole disorder’ or ‘pyroluria’ is gaining popularity, highlighting the importance of identifying the exact chemical structures as well as investigating the pathophysiology behind this elusive condition.
The literature reviews provided the impetus for the direction of the experimental work. A number of intermediate compounds were derived from a series of photooxidation reactions using 2,4-dimethyl-3-ethyl-pyrrole. This provided insight into the solvent conditions required for these intermediates to be isolated, with preliminary results showing close structural similarities to compounds shown in reports originally investigating urine pyrroles and more recently in the characterisation of BOXes. Further, two intermediate compounds that previously have not been reported in the literature were synthesised through the photooxidation reactions. Both compounds followed similar methods of preparation and subsequent work-up with the distinction being the time of irradiation under ultraviolet light when in the presence of hydrogen peroxide (H2O2). One compound was irradiated for a period of over 96 hours before undergoing organic solvent work-up after being quenched with sodium hydrogen (NaOH) whereas the other novel compound was irradiated for a period of 48-72 hours. It was found that differing times in irradiation could provide insight into the dynamic system of these sensitive photooxidation reactions. Despite rigorous efforts in work-up and purification, the exploratory experiments in this study have shown that the compounds will readily interconvert into different compounds. This in turn directed the proceeding review and experimental work investigating the reactivity of the chemical trap para-dimethylaminobenzaldehyde, colloquially known as Ehrlich’s reagent, since a suitable chemical trapping agent would likely be needed for these highly reactive compounds.
The literature reviews revealed that specifically for BOM research, there were a lack of reports pertaining the baseline reference ranges when utilising the ELISA biopyrrin kits. Establishing reference ranges may allow for accurate comparisons with established clinical markers that could validate BOMs as a robust clinical tool. The findings from the literature review on BOXes literature review showed that a continuation of research to establish whether these compounds are the cause or result of vasospasm in patients with SAH could allow for future therapeutic development. The long history on urine pyrroles and its withstanding controversy shows a need for further research specifically determining whether zinc and vitamin B6 are able to form complexes with urines pyrroles. Finally, further experimental work will help clarify and understand the mechanisms relating to pyrrole-containing molecules for the investigation of other chemical trapping agents. Overall, this is the first study to identify and provide an in-depth review of bilirubin oxidation end products and their relationship to human health. The results collated from the scoping review, literature reviews and experimental work may provide a useful starting point to continue the early work on urine pyrroles from the 1970’s and support the developing research that is currently emerging for BOM and BOX compounds. Bilirubin oxidation end products may emerge as the missing link in the pathophysiology of a number of chronic health conditions.||