HEME SYNTHESIS--EARLY DETECTION OF HEMOGLOBINOPATHIES

血红素合成——血红蛋白病的早期检测

• 批准号: 2017134
• 负责人: JACQUELINE M HIBBERT
• 金额: $ 7.25万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2017134
• 关键词: blood disorder diagnosis; body composition; body weight; diagnosis design /evaluation; early diagnosis; electrofocusing; erythrocytes; glycine; heme; hemoglobin; hemoglobin F; hemoglobinopathy; hemoprotein biosynthesis; hemoprotein metabolism; high performance liquid chromatography; human subject; mass spectrometry; protein degradation; stable isotope

Many attempts have been made to obtain fundamental information about blood diseases by estimating the life span of the red blood cell. While a number of different clinical methods have been used, most are unreliable and none is based on the direct measurement of hemoglobin synthesis rate. The primary objective of this proposal is to develop an in vivo method to enable measurement of the rate of fractional hemoglobin synthesis and red blood cell life span, in hematologic diseases. The method has been adapted from work described in the 1940's when hemoglobin was labeled with 15N- glycine in vivo and the heme was isolated for analysis. The amino acid glycine is required for the synthesis of the protoporphyrin of hemoglobin. Progress in mass spectrometry analysis and the availability of 15N labels now make such techniques feasible. Furthermore, it will be simple and more affordable than the original method described. The secondary objective is to apply this measurement to sickle cell disease, which provides a model of changed red blood cell metabolism. Intermittent oral doses of 15N- glycine, 400mg over 12 hours will label heme and globin. The heme is ultimately lost in the stool as bilirubin and therefore this label will not be recycled to heme synthesis. Blood will be collected at times zero (pre dose), 12 and 24 hours, and the heme and globin will be isolated from the red blood cell hemolysates. The intracellular free glycine pool will be measured to represent the precursor pool enrichment for heme synthesis. The glycine will be isolated with high performance liquid chromatography, from a trichloroacetic acid extract of the red blood cell. Enrichments of 15N will be measured by mass spectrometry so that fractional hemoglobin turnover rate and red blood cell survival time can be calculated. This method will simultaneously measure rates of in vivo synthesis and degradation of fetal and adult or sickle hemoglobin. The advantages of this proposed method over the clinical use of chromium 51 (51 Cr) to tag the red cells, are the accuracy and directness of the measurement. Accuracy of the 51 Cr method is affected by leakage of chromium from the red cell after the cells are removed from the subject, tagged in vitro and then reintroduced to the subject. The tagged cells may also be metabolically perturbed by this procedure. Finally, the proposed method presents no radiation hazard and enables the dynamics of red cell metabolism to be measured, as well as the estimates of red cell survival time. In addition the procedure measures whole body protein turnover using the 15N-glycine, urine collection and mass spectrometry. This can be correlated with the fractional hemoglobin metabolism, to determine how change in the metabolism of this one protein, is reflected in the whole body. This method to measure the dynamics of hemoglobin metabolism will have wide application as a tool for in vivo characterization of all conditions resulting from altered hemoglobin synthesis. It may also eventually prove useful in measuring platelet survival and turnover rates as well as other blood constituents affected by disease.

已经进行了许多尝试以获取有关血液的基本信息 通过估计红细胞的寿命来疾病。而一个数字 已经使用了不同的临床方法，大多数是不可靠的，没有 基于血红蛋白合成率的直接测量。这 该建议的主要目的是开发一种体内方法 启用分数血红蛋白合成速率和红色 血细胞寿命，在血液学疾病中。该方法已改编 根据1940年代描述的工作，当时血红蛋白被标记为15n- 甘氨酸在体内和血红素被分离进行分析。氨基酸 甘氨酸是血红蛋白原磷脂的合成所必需的。 质谱分析的进展和15N标签的可用性 现在使这样的技术可行。此外，它将更简单且越来越多 负担得起的是所描述的原始方法。第二个目标是 将此测量应用于镰状细胞疾病，该疾病提供了模型 红色血细胞代谢变化。 15n-的间歇性口服剂量 甘氨酸，400毫克超过12小时的甘氨酸将标记血红素和球蛋白。血红素是 最终像胆红素一样在粪便中迷失，因此该标签将 不回收为血红素合成。血液将在零时收集 （预剂量），12和24小时，血红素和球蛋白将与 红细胞流血。细胞内游离甘氨酸池将 测量以表示血红素合成的前体池富集。 甘氨酸将通过高性能液相色谱分离， 来自红细胞的三氯乙酸提取物。丰富 15N将通过质谱法测量，以便分数血红蛋白 可以计算营业率和红细胞存活时间。这 方法将同时测量体内合成的速率和 胎儿和成人或镰状血红蛋白的降解。优势 这项提出的方法是在51铬（51 cr）的临床用途上进行标记的方法 红细胞是测量的准确性和直接性。 51 Cr方法的准确性受铬泄漏的影响 将细胞从受试者中取出后的红细胞，在体外标记，并 然后重新引入对象。标记的单元也可能是 该过程的代谢扰动。最后，提出的方法 提出没有辐射危险，并实现红细胞的动态 要测量的代谢以及红细胞存活的估计值 时间。此外，该过程使用 15N-甘氨酸，尿液收集和质谱法。这可以 与分数血红蛋白代谢相关，以确定 这种一种蛋白质的代谢变化反映在整体上 身体。这种测量血红蛋白代谢动力学的方法将 具有广泛的应用作为体内表征的工具 血红蛋白合成改变导致的条件。它也可能 最终被证明可用于测量血小板存活和周转率 以及其他受疾病影响的血液成分。