Background Hyperbilirubinemia is a condition that affects most infants, but typically self-resolves and is not harmful. However, if bilirubin levels exceed neuroprotective defenses, the compound can cross the blood-brain barrier and have neurotoxic and potentially fatal effects. Treatment of neonatal hyperbilirubinemia with phototherapy is necessary for the prevention of kernicterus. Guidelines for the use of phototherapy in infants born at ≥ 35 weeks’ gestation were published by Bhutani et al. and endorsed by the American Academy of Pediatrics. Consensus-based recommendations for phototherapy treatment and exchange transfusion of premature infants were published in 2012 by Maisels, et al. However, there are no published recommendations for the timing of screening for hyperbilirubinemia in NICU patients. In 2012, the Kapʻiolani Medical Center for Women & Children Neonatology Division implemented internal guidelines for phototherapy with recommendations for the timing of screening serum bilirubin levels, based on the group’s opinion. Five years later, the current study queried whether these guidelines for screening were appropriate. Objective The present study sought to describe current practices of obtaining serum bilirubin levels and the use of phototherapy in the NICU during the first five days of life. It was hypothesized that many bilirubin levels obtained at ≤ 48 hours of life are below published recommended treatment thresholds and are potentially unnecessary. Methods Retrospective chart review was performed on all infants admitted to the NICU at < 24 hours of life, from July 2016-June 2017. Eligible infants were divided into three gestation age groups: ≤ 28, 29-35, and ≥ 36 weeks at birth. Patient demographics, bilirubin levels, and phototherapy treatment were noted. The primary outcome of interest was the percent of serum bilirubin levels obtained during the first 48 hours of life that did not meet phototherapy treatment criteria. Results 931 charts were reviewed. Infants born at ≤ 28, 29-35 and ≥ 36 weeks’ gestation made up 10%, 51% and 39% of the cohort. Overall mortality was 3%, and no exchange transfusions were performed during the study period. At least one serum bilirubin level was obtained for 96% of the patients, but only 55% were treated with phototherapy within the first five days of life. Phototherapy was rarely prescribed on day of life (DOL) 1 (0.7%). By DOL 2, a total of 563 bilirubin levels were obtained, but only 108 infants (19%) were treated with phototherapy. However, one-third of these patients’ bilirubin levels did not meet published criteria for treatment. The timing of phototherapy treatment varied by gestational age. Ninety percent of infants born ≤ 28 weeks’ gestation who received phototherapy were treated starting between DOL 2-3. In contrast, eighty-five percent of infants born ≥ 29 weeks’ gestation who received phototherapy, started on DOL 3-5. Discussion Far more bilirubin levels were obtained than courses of phototherapy prescribed. Given the distinct patterns of phototherapy for infants of varying gestational age, there is ample opportunity to improve resource utilization with targeted recommendations for obtaining screening bilirubin levels in the neonate without early jaundice.

Neonatal jaundice is a physiological condition which has potentially deleterious outcomes. Elevated serum bilirubin levels are well-documented antioxidants and have been shown to disrupt cellular membranes of Gram-positive organisms under specific conditions. To determine whether bilirubin had antimicrobial potential against neonatal sepsis organisms, relevant isolates were identified by clinical audit and assessed for sensitivity. 26 clinical isolates including 15 CoNS, 7 GBS, E. coli, E. faecalis, K. oxytoca and A. haemolyticum were characterised biochemically, genetically, and by MALDI Biotype. GBS isolates showed a significant reduction in growth from 100–82.0%(±6.1%), between 0–400μM bilirubin– supplemented CBA (p=0.005). A physiologically relevant liquid model with 100μM bilirubin was developed to test growth reduction. Results showed slight growth reduction in isolates at specific time points, but species specific. Transcriptomic analysis was performed on three GBS isolates to determine effect of bilirubin exposure on gene expression. 17 genes were differentially expressed between 100μM bilirubin and solvent control; 16 up-regulated and one down-regulated with bilirubin. Most significantly, a 5 gene cluster describing multiple components of the phosphotransferase system and two ABC transporter genes were up-regulated, potentially to remove bilirubin from the cell. Proteomic analysis was completed to study protein expression: 12 proteins were identified by LC-MS from 2-DGE and Progenesis SameSpots analysis. Of these, 6 were up- and 6 down-regulated with bilirubin. Up-regulated proteins included two ABC transporter components, phosphoglycerate kinase, S-ribosylhomocysteinase, and two transcription regulators: GroEL/GntR. Down-regulated was iron ABC transporter, NeuB, ornithine carbamoyltransferase and ssDNA binding proteins controlling transcription and translation. This study concluded that bilirubin may play a protective role during the neonatal period; it can be considered an antimicrobial compound which disrupts Grampositive organisms such as GBS, an important agent in early-onset sepsis. The results from this study could be used to develop novel antimicrobial treatments based on identified molecular targets.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

<p><strong>Background</strong> Subarachnoid hemorrhage is a condition with high rates of mortality and morbidity. The diagnosis requires an urgent cerebral computed tomography scan and also a lumbar puncture if the scan fails to demonstrate intracranial blood. In Sweden the cerebrospinal fluid (CSF) is analyzed by spectrophotometric scanning for the presence of hemoglobin and bilirubin. The aim of the study was to develop a quantitative diazo reagent based analysis of cerebrospinal fluid bilirubin as a replacement for spectrophotometric scanning.</p><p><strong>Methods</strong> The CSF bilirubin assay on an Architect C8000 chemistry analyzer was compared with spectrophotometry using patient samples.</p><p><strong>Results</strong> The method correlates with spectrophotometry, has a good linearity and precision.</p><p><strong>Conclusions</strong> Quantitative bilirubin measurement offers shorter turnaround times, simplifies the interpretation of the results and reduces work load in comparison with spectrophotometry.</p>

Background Subarachnoid hemorrhage is a condition with high rates of mortality and morbidity. The diagnosis requires an urgent cerebral computed tomography scan and also a lumbar puncture if the scan fails to demonstrate intracranial blood. In Sweden the cerebrospinal fluid (CSF) is analyzed by spectrophotometric scanning for the presence of hemoglobin and bilirubin. The aim of the study was to develop a quantitative diazo reagent based analysis of cerebrospinal fluid bilirubin as a replacement for spectrophotometric scanning. Methods The CSF bilirubin assay on an Architect C8000 chemistry analyzer was compared with spectrophotometry using patient samples. Results The method correlates with spectrophotometry, has a good linearity and precision. Conclusions Quantitative bilirubin measurement offers shorter turnaround times, simplifies the interpretation of the results and reduces work load in comparison with spectrophotometry.

Capillary-based analytical methodologies were developed to meet the need for metabolite determination in two major areas. The first area is the determination of free bilirubin in sera for the management of jaundiced neonates under critical conditions. Three sensitive detection techniques were investigated, Quantum dots (QD) mediated fluorescence, Chemiluminescence (CL) and Microelectrode detection. Four different types of QDs were synthesized for the direct bilirubin determination. The CAH-capped CdTe QDs were selected as it shows the best performance compared to organic dyes and other QDs. Its optimized preparation conditions are: refluxing solution containing Cd/Te/CAH (1:0.5:2.4 w/w) for 4 hours at 100 °C. From Transmission Electron Microscope characterization, nano-size QDs with an uniform size distribution, high luminescence and good stability were obtained. The optimized detection conditions were: incubation of bilirubin with CAH-capped CdTe QDs (5 10-6 mol/L) in water at pH=5.6 and 20 oC for 8 min prior to spectrofluorometric determination (λex=473 nm and λem=580 nm). A linear working range from 0.043-0.86 μg/mL with 0.9943 correlation coefficient and 2 ng/mL detection limit (LOD, S/N=3) were achieved. Results from nFIA-CL indicate a quick response within seconds though a poorer LOD (S/N=3) of 15 μg/mL for the direct bilirubin determination. The third technique investigated used an enzyme microelectrode and it was found to be able to couple with capillary electrophoresis (CE) in frontal analysis (FA) for the determination of free bilirubin in serum samples. Making use of the micron size of the carbon-fiber electrode, a new MCNTs (Multi-wall Carbon Nanotubes) modified CFMEs (Carbon fiber microelectrodes) was fabricated within a microchip-CE device with three guided channels to enable electrodes alignment. Method to immobilize bilirubin oxidase (BOD) onto the CFMEs surface by the carbodiimide chemistry achieved the highest detection sensitivity. Under optimized conditions (sample introduced by hydrodynamic injection at △H (20 cm), and a running/detection buffer (10 mM phosphate) at pH 7.4, working potential for amperometric detection at +0.8 V), a linear working range between 1-40 μg/mL and a detection limit (S/N=3) at 0.15 μg/mL for free bilirubin was achieved. The second area for metabolite determination was developing a new analytical method for the management of APL (acute promyelocytic leukemia) patients under arsenic treatment, a drug required continued monitoring. The analytical requirements include a high detection sensitivity and the capability to provide timely results for multiple drug residues. Using a 20 mM phosphate as the running buffer and 0.05mM CTAH (Cetyl-trimethyl-ammonium hydroxide) as an additive for EOF reversal, co-EOF (co-electroosmotic flow) stacking was established to enhance up to 200 times of the detection limit for arsenite. Satisfactory baseline separation for arsenite, arsenate, MMA (Methylarsonic acid) and DMA (Dimethylarsinic acid) was achieved with linear working ranges (correlation coefficients > 0.999) from 1-50 μg/mL for arsenate and DMA, 0.5-50 μg/mL for MMA as well as 0.1-50 μg/mL for arsenite. Detection limits (S/N=3, n=3) achievable for arsenate, arsenite, MMA and DMA were found to be 0.41 μg/mL, 0.01 μg/mL, 0.04 μg/mL and 0.32 μg/mL respectively at levels meeting the requirement for APL patient urine monitoring. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Felaktig provtagning kan orsaka hemolys som kan påverka mätresultatet av S-bilirubin koncentrationen vid användning av diazometoder. Bilirubin är ett gulfärgat pigment som erhålls när hemolglobin bryts ner. Vätebindningar som finns i bilirubinstrukturen stabiliserar molekylen och avgör om den är löslig eller inte löslig i vatten. Bilirubin konjugeras i levern och blir vattenlösligt, därefter utsöndras bilirubin via gallan och tarmen. Hyperbilirubinemi uppstår vid ökad nedbrytning av erytrocyter eller vid leverskada. Ökad bilirubinkoncentration i blodet sker ofta hos nyfödda och diffunderar till kroppens celler och orsakar icterus. Studien gjordes med syftet att studera hur olika grad av hemolys påverkar analys av S-bilirubin-koncentrationen. S-bilirubinkoncentration analyserades kolormetrisk i Olympus AU2700. Diazoreaktionen sker mellan bilirubin och diazotiserad sulfanilsyra som bildar en färgad produkt, azobilirubin. Azobilirubin analyseras genom absorbansmätning vid 540 nm. Absorbansen är proportionell mot totala bilirubinkoncentrationen. En hemolysat-spädningslösning gjordes och användes som en spädningsslösning till 16 stycken olika serumprover för att få olika hemolysgrader 0 g/L, 0,5 g/L, 1 g/L, 3 g/L respektive 5 g/L. Resultatet visade att hemolys sänkte S-bilirubinkoncentrationen vid mätning i instrumentet som använder diazimetoder. Vid högre grad av hemolys som tillsattes till S-bilirubinkoncentrationen desto lägre blev S-bilirubinkoncentrationen som analyserades i proverna. Alla prover som hade låg S-bilirubinkoncentration hade större avvikelser jämfört med de prover som hade hög S-bilirubinkoncentration. Vid tillsats av hemolys med 0,5 g/L till prover med S-bilirubinkoncentrationen under 20 µmol/L låg avvikelsen mellan 12,00 % och 16,00 % medan vid tillsats av samma hemolysgrad till S-bilirubinkoncentrationen över 20 µmol/L låg avvikelsen mellan 0,67 % och 4,00 %. Slutsatsen är att patientprover som har S-bilirubinkoncentrationer lägre än 20 µmol/L och med hemolys inte kan mätas, prover från patienter med hyperbilirubinemi och hemolysgrad lägre än 1 g/L kan analyseras i de fall de är svåra att ta om t.ex på nyfödda.

Murine cytochrome P450 2A5 (CYP2A5) is an interesting enzyme for its unique regulation and its involvement in liver injury caused by various well-known pathological conditions or hepatotoxins. It has been reported that CYP2A5 is upregulated following exposure to chemical hepatotoxins and during pathophysiological conditions in which the levels of most Cytochrome P450s are either unchanged or down-regulated. Recently bilirubin has been identified as the first endogenous substrate for CYP2A5 and it has been suggested that CYP2A5 plays a major role in bilirubin clearance as an alternative mechanism to BR conjugation by UGT1A1. This study investigated the mechanisms of gene regulation and cytoprotective role of CYP2A5 in response to bilirubin treatment in liver. Our results demonstrate that bilirubin induces CYP2A5 expression at the mRNA and protein levels by increasing CYP2A5 transcription via a mechanism that involves Nrf2 activation. Furthermore, our results suggest that induced CYP2A5 plays a cytoprotective role against bilirubin toxicity by directly lowering the cellular levels of bilirubin and by inhibiting caspase-3 activation.

Thesis (Ph. D.)--University of Wyoming, 2005. / Title from PDF title page (viewed on Nov. 14, 2007). Includes bibliographical references.