STUDY ON PHYSIOLOGICAL SIGNIFICANCE OF BRAIN BILE ACID

胆汁酸在肝脏中由胆固醇合成。在肠腔内，它们帮助脂肪分解和吸收，然后在回肠和近端结肠吸收后返回肝脏。由于胆汁酸在肝脏的有效摄取，其在外周血中的浓度较低。利用LC/ESI-MS，我们最近在大鼠脑细胞质部分中发现了三种未结合的胆汁酸。鹅去氧胆酸（CDCA）仅在高浓度盐酸胍提取时检测到，表明其与蛋白质非共价结合。在本研究中，我们证实了3β-羟基-5-胆烯酸在大鼠脑组织中转化为CDCA的酶活性，并鉴定了CDCA结合蛋白的候选物。为了区分内源性污染物，制备了^<18> o-羟基-5-胆烯酸、3β、7α-二羟基-5-胆烯酸和7α-羟基-3-氧-4-胆烯酸作为底物进行体外培养。结果表明，3β-羟基-5-胆烯酸在微粒体制备过程中以nadph依赖性转化为3β，7α-二羟基-5-胆烯酸。7α-羟基-3-氧-4-胆烯酸在微粒体制备中由3β、7α-二羟基-5-胆烯酸产生NAD^+依赖性的7α-羟基-3-氧-4-胆烯酸，最后通过大鼠脑细胞质酶以nadph依赖性的Δ4-3-keto形式转化为CDCA。这些结果表明在大鼠脑组织中存在从3β-羟基-5-胆烯酸到CDCA的酶活性。通过固定化CDCA制备亲和凝胶。将大鼠脑细胞质部分应用于凝胶中，用高浓度盐酸胍提取蛋白质。通过双向凝胶电泳分离蛋白混合物，并与对照蛋白图谱进行比较。有几种蛋白通过亲和萃取得到特异性富集。采用MALDI-TOFMS对凝胶内胰消化获得的肽片段混合物进行分析，采用肽质量指纹图谱法对蛋白质进行鉴定。发现9种蛋白，其中包括微管蛋白α和β， 14-3-3蛋白ζ，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