Preventing Kernicterus: Serum Albumin-Bilirubin Binding

预防核黄疸：血清白蛋白-胆红素结合

• 批准号: 7613740
• 负责人: Eileen S. Krenzel
• 金额: $ 3.73万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2010-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7613740
• 关键词: Preventing; Kernicterus; Serum; Albumin; Bilirubin

DESCRIPTION (provided by applicant): Unconjugated bilirubin (UCB) is the normal, but toxic end product of red blood cell catabolism and circulates in the plasma. However, because of its very low aqueous solubility of <70 nM, it is transported primarily by human serum albumin (HSA). UCB is conjugated in the liver into a water-soluble compound, conjugated bilirubin, for excretion into the bile. In near term and full term infants the enzyme responsible for conjugating bilirubin is not yet fully functional and UCB levels can exceed 175 ?M, though never exceeding the binding capacity of HSA, with more UCB being presumably pushed into the lower affinity binding sites. Since the circulating HSA now contains a high amount of bound UCB, UCB can more easily diffuses through plasma membranes, allowing UCB to accumulate in tissues, causing jaundice. Similarly, these high circulating levels of TSB (>428 ?M) also diffuse across the blood brain barrier where it causes severe neurological deficits in untreated infants. This disease is Kernicterus, or bilirubin-induced encephalopathy, and causes athetoid cerebral palsy, deafness, upgaze abnormalities, and hypoplasia of baby teeth enamel. Since this devastating disease is preventable and reversible with the proper treatment, the American Academy of Pediatrics has developed treatment guidelines. In order to have healthy birth outcomes and to prevent these children from becoming dependent on the healthcare system, it is imperative to efficiently and quickly identify those infants in need of treatment. For this, measures of the total serum bilirubin in the body are performed to determine the potential for bilirubin toxicity. However, this measurement has been deemed a poor determinant of bilirubin toxicity. In order to rectify this, a more complete understanding of the mechanisms of transport of bilirubin throughout the body is needed. We propose to determine the primary and secondary binding sites on HSA for UCB. The exact binding site(s) of UCB have evaded years of research, and these sites and their relationships to fatty acid (FA) and drug binding sires needs to be known at the molecular level. We will use novel applications of 1D and 2D Nuclear Magnetic Resonance (NMR) Spectroscopy, to study aqueous complexes of UCB with HSA and with/without FAs at their physiological concentrations. For direct studies of binding and identification of the primary bilirubin binding site, we will use 13C-labeled bilirubin synthetic analogs together with our methods established for identifying FA sites, namely drug displacement techniques and HSA mutants. Additionally, for clinical applications, it is critical to identify the lower affinity sites and determine in a site specific manner how FA and drug binding forces UCB into the lower affinity sites. These additional studies will utilize unlabeled bilirubin and displacement by 13C-labeled FA. Not only does this proposal identify the primary and secondary bilirubin binding sites on HSA, but will also study how drug binding affects bilirubin binding, for example, predicting whether binding of therapeutic drugs may have an unintended effect of displacing bilirubin into lower affinity sites, and thus increasing the chances for accumulation of UCB in the tissues and brain.

描述(由申请人提供)： 未结合胆红素(UCB)是红细胞分解代谢的正常但有毒的终产物，在血浆中循环。然而，由于其极低的水溶解度&lt；70 nm，它主要由人血清白蛋白(HSA)转运。UCB在肝脏中结合成一种水溶性化合物--结合胆红素，然后排泄到胆汁中。在近足月和足月婴儿中，负责结合胆红素的酶还没有完全发挥作用，UCB水平可以超过175M，但永远不会超过HSA的结合能力，推测更多的UCB被推入低亲和力结合部位。由于循环中的人血清白蛋白现在含有大量结合的脐带血，脐带血可以更容易地通过质膜扩散，使脐带血在组织中积聚，从而引起黄疸。类似地，这些循环中高水平的TSB(&gt；428？M)也会扩散到血脑屏障，在那里它会导致未经治疗的婴儿严重的神经功能障碍。这种疾病是核脑病，或胆红素引起的脑病，导致手足徐动性脑瘫、耳聋、仰视异常和乳牙釉质发育不全。由于这种毁灭性的疾病通过适当的治疗是可以预防和可逆的，美国儿科学会已经 制定了治疗指南。为了有健康的分娩结果，并防止这些儿童依赖医疗保健系统，必须有效和快速地确定那些需要治疗的婴儿。为此，需要测量体内的血清总胆红素，以确定胆红素毒性的可能性。然而，这种测量被认为是胆红素毒性的一个不好的决定因素。为了纠正这一点，需要对胆红素在体内的转运机制有更全面的了解。我们建议确定UCB在HSA上的主要和次要结合部位。UCB的确切结合位点(S)已经逃避了多年的研究，这些结合位点及其与脂肪酸(FA)和药物结合SIRS的关系需要在分子水平上知道。我们将使用一维和二维核磁共振波谱的新应用，研究生理浓度下UCB与HSA和含/不含FAs的水溶液络合物。为了直接研究结合和鉴定初级胆红素结合位点，我们将使用13C标记的胆红素合成类似物和我们建立的鉴定FA位点的方法，即药物置换技术和HSA突变体。此外，对于临床应用，关键是识别低亲和力部位，并以特定部位的方式确定FA和药物结合如何迫使UCB进入低亲和力部位。这些额外的研究将利用未标记的胆红素和13C标记的FA的置换。这一建议不仅确定了人血清白蛋白上的主要和次要胆红素结合部位，还将研究药物结合如何影响胆红素结合，例如预测治疗药物的结合是否会产生意想不到的效果，将胆红素置换到低亲和力部位，从而增加脐带血在组织和大脑中积聚的机会。