STUDY ON PHYSIOLOGICAL SIGNIFICANCE OF BRAIN BILE ACID

胆汁酸在肝脏中由胆固醇合成。在肠腔中，它们帮助脂肪分解和脂肪吸收，然后在回肠和近端结肠吸收后返回肝脏。由于其有效的肝脏摄取，胆汁酸在外周血中的浓度低。使用LC/ESI-MS，我们最近发现三个未结合的胆汁酸在大鼠脑细胞质组分。鹅去氧胆酸（CDCA）仅在用高浓度盐酸胍提取后检测到，表明其与蛋白质非共价结合。本研究以3 β-羟基-5-胆烯酸（3 β-hydroxy-5-cholenoic acid，3 β，7 α-dihydroxy-5-cholenoic acid，7 α-hydroxy-3-oxo-4-cholenoic acid，3 β，7 α-dihydroxy-5-cholenoic acid）为底物，在体外培养条件下，研究了3 β-羟基-5-胆烯酸（3 β-hydroxy-5-cholenoic acid，3 β，7 α-dihydroxy-5-cholenoic acid，7 α-hydroxy-3-oxo-4-cholenoic acid，3 β，7 α-hydroxy-3-oxo-4-cholenoic acid，3 β，7 α-hydroxy-3-oxo-4-cholenoic acid，3 β，7 α-hydroxy-3-oxo-4-cholenoic acid，3 β，7 α-hydroxy-hydroxy-5-cholenoic acid<18>结果 ...更多信息 表明3 β-羟基-5-胆烯酸在微粒体制备中依赖于NADPH转化为3 β，7 α-二羟基-5-胆烯酸。3 β，7 α-二羟基-5-胆烯酸在微粒体制备中依赖于NAD ^+生成7 α-羟基-3-氧代-4-胆烯酸，最后在大鼠脑胞浆酶作用下依赖于NADPH将Δ 4 - 3-酮转化为CDCA。结果表明，大鼠脑组织中存在由3 β-羟基-5-胆烯酸转化为CDCA的酶活性。将大鼠脑细胞质部分施加到凝胶上，并通过使用高浓度盐酸胍提取蛋白质。双向凝胶电泳分离蛋白质混合物，并与对照蛋白质图谱进行比较。几种蛋白质特异性富集的亲和提取。采用MALDI-TOFMS分析胶内酶解后的肽段混合物，并采用肽质量指纹法进行蛋白质鉴定。共鉴定出9种蛋白质，其中包括微管蛋白α和β、14 - 3 - 3蛋白A，它们可能与阿尔茨海默病有关。少

Bile acids are synthesized in the liver from cholesterol. In the intestinal lumen, they assist lipolysis and the absorption of fats, and then return to the liver upon absorption in the ileum and proximal colon. Because of their efficient hepatic uptake, bile acids have low concentrations in the peripheral blood. Using LC/ESI-MS, we have recently found three unconjugated bile acids in the rat brain cytoplasmic fraction. Chenodeoxycholic acid (CDCA) was detected only upon extraction with high concentrations of guanidine hydrochloride, indicating that it is bound non-covalently to protein. In the present study, we demonstrated the enzyme activity from 3β-hydroxy-5-cholenoic acid to CDCA in rat brain tissue, and we identified the candidates of CDCA-binding proteins.To distinguish from endogenous contaminants, ^<18>O-hydroxy-5-cholenoic acid, 3β,7α-dihydroxy-5-cholenoic acid, and 7α-hydroxy-3-oxo-4-cholenoic acid were prepared and used as substrates for the in vitro incubation. The results … More suggested that 3β-hydroxy-5-cholenoic acid was NADPH-dependently converted to 3β,7α-dihydroxy-5-cholenoic acid in microsomal preparation. And 7α-hydroxy-3-oxo-4-cholenoic acid was NAD^+-dependently produced from 3β,7α-dihydroxy-5-cholenoic acid in microsomal preparation, and finally, Δ4-3-keto form was NADPH-dependently converted to CDCA by rat brain cytosolic enzyme. These findings indicated the presence of the enzyme activity from 3β-hydroxy-5-cholenoic acid to CDCA in rat brain tissue.Affinity gel was prepared by immobilization of CDCA. The rat brain cytoplasmic fraction was applied to the gel, and proteins were extracted by using high concentrations of guanidine hydrochloride. The protein mixture was separated by two-dimensional gel electrophoresis, and the protein map was compared with the control protein map. Several proteins were specifically enriched by the affinity extraction. The peptide fragment mixtures obtained from in-gel tryptic digestion was analyzed by MALDI-TOFMS, and the protein identification was performed by peptide mass fingerprinting method., Nine proteins were identified, and they have included tubulin α and β, 14-3-3 protein ζ, which may relate to Alzheimer's disease. Less