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Developmental study for diagnostic techniques of congenital hepatobiliary disorders by fetal bile acids

胎儿胆汁酸诊断先天性肝胆疾病技术的进展研究

基本信息

批准号：
01571182
负责人：
IKEGAWA Shigeo
金额：
$ 1.41万
依托单位：
Higashi Nippon Gakuen University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

项目摘要

To develop a diagnostic technique for congenital hepatobiliary disorders, qualitative and quantitative analysis of the conjugated 1beta- and 6alpha-hydroxy bile acids including common bile acids has been performed by high-performance liquid chromatography with chemiluminescence detection. Analysis of urine samples from newborns, normal women and women with pregnancy showed a large defderence in bile acid composition and conjugation mode, suggesting the distinctiveness of the bile acid metabolism during fetal and neonetal periods.Anti-tauro 1beta-hydroxycholic acid antisera have been prepared by immunizing rabbits with bovine serum albumin conjugates, in which the glycine and beta-alanine derivatives of 1 -hydroxycholic acid were coupled at the C-24 position. The antisera had high affinity and specificity for conjugated 1beta-hydroxycholic acid ; cross-reactivities for glyco 1beta-hydroxycholic acid were 100% and those for glycine and taurine conjugates ranged from 43.1 to 0.23%. Concentrations of conjugated 1beta-hydroxycholic acid were determined by radioimmunoassay with the more specific antiserum to be significantly large amounts in newborns at 0 - 20 days after birth, ranging from 0.29 to 18.51 mug/ml, when compared to normal women (<0.12 mug/ml) and women with late pregnancy (<1.13 mug/ml).To facilitate mass screening for congenenital biliary atresia, radioimmunoassay of conjugated 1 -hydroxycholic acid in dried blood spotted on filter paper was developed. The assay method was sufficiently sensitive and specific to determine the bile acid in one disc (3mm i. d., 4mul of whole blood). Preliminary tests suggested that the efficiency of this screening is acceptable, in which the levels of the patients (2.35 ng/disc) were significantly higher values than those of normal newborns (0.97 ng/disc).

为了建立先天性肝胆疾病的诊断技术，采用高效液相色谱化学发光检测对包括普通胆汁酸在内的共轭1 β -和6 α -羟基胆汁酸进行了定性和定量分析。新生儿、正常妇女和妊娠妇女的尿液样本分析显示，胆汁酸组成和结合模式有很大差异，提示胎儿和新生儿时期胆汁酸代谢的独特性。用牛血清白蛋白偶联物免疫家兔制备了抗牛黄- 1 -羟基胆酸抗血清，其中1 -羟基胆酸的甘氨酸和β -丙氨酸衍生物偶联在C-24位。该抗血清对共轭1 β -羟基胆酸具有较高的亲和力和特异性；葡萄糖- 1 -羟基胆酸的交叉反应活性为100%，甘氨酸和牛磺酸偶联物的交叉反应活性为43.1 ~ 0.23%。用放射免疫法测定新生儿出生后0 - 20天的共轭1 β -羟基胆酸浓度，与正常妇女（<0.12马克杯/毫升）和妊娠晚期妇女（<1.13马克杯/毫升）相比，特异性更强的抗血清含量明显较高，为0.29 ~ 18.51马克杯/毫升。为了便于对先天性胆道闭锁进行大规模筛查，建立了滤纸沾点干血中共轭1 -羟基胆酸的放射免疫测定方法。该方法具有足够的灵敏度和特异性，可测定一个圆盘（直径3mm，全血4ml）中的胆汁酸。初步试验表明，这种筛查的效率是可以接受的，其中患者的水平（2.35 ng/椎间盘）明显高于正常新生儿的水平（0.97 ng/椎间盘）。