Human Endogenous Metabolite

在低剂量 (5 µM) 下，Taurolithocholic acid /牛磺石胆酸 (TLCA) 往往会使 PKC e 亚型的膜相关部分提高 44.1% ± 40.2%[1]。移动 PKC 同工型的激活需要 PKC 的 epsilon 同工型选择性易位至肝细胞膜，这是由 TLCA (10 µM) 诱导的[1]。

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蛋白激酶C（PKC）同工酶家族在肝细胞分泌的调节中起着关键作用。疏水性和胆汁淤积性胆汁酸，牛磺酸胆酸（TLCA），在分离的肝细胞中充当强效的钙离子激动剂。然而，其对PKC亚型的影响尚未阐明。在这里，我们研究了低微摩尔浓度的TLCA对PKC亚型分布和膜相关PKC活性的影响。使用蛋白质印迹和免疫荧光技术在短期培养的分离大鼠肝细胞中确定PKC亚型的分布。放射化学法测定PKC活性。Taurolithocholic acid /TLCA（10微摩尔/升）诱导εPKC选择性易位47.9%+/-20.5%（与对照组相比，P<.02；n=7），但α-、δ-和ζPKC未选择性易位至肝细胞膜，而佛波醇酯佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）（1微摩尔/L）导致所有可移动异构体α-、Δ-和εPKC易位，如免疫印迹所示。免疫荧光研究表明，TLCA（10微摩尔/升）选择性地将ε-PKC转运到分离的大鼠肝细胞联的小管膜，但PMA（1微摩尔/L）主要转运到细胞内和基底外侧膜。TLCA（10μmol/L）和PMA（1μmol/L）均能刺激膜结合PKC活性60.5%+/-45。分别为8%（与对照组相比P<0.05；n=5）和72.4%+/-37.2%（P<0.05；n=5）。较低浓度（5微摩尔/升）的TLCA效果较差。由于εPKC的激活与囊泡介导的分泌细胞膜蛋白靶向和插入的损伤有关，因此推测TLCA通过激活小管膜上的εPKC来降低肝细胞的胆汁分泌能力是有吸引力的。[1]

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分离的大鼠肝细胞中PI3K依赖性PKB（PKB/Akt）活性[2]
磷酸化PKB（Ser-473）的量是PI3K通路激活的敏感读数（27，32），在短期培养中，肝细胞中的Taurolithocholic acid /牛磺酸/TLCA（5μmol/l）显著增强（图7），60分钟后达到对照组的194±46%（与对照组相比p<0.005；与TUDCA相比p<0.05；与TCA相比p<0.01）。相比之下，TUDCA（10μmol/升）仅短暂增加PKB活性，而TCA（10µmol/升，在所选实验条件下没有影响（图7）。因此，TLCA在体外显著影响了分离肝细胞中的PI3K活性，而TUDCA在低微摩尔浓度下给药时仅对PI3K通路产生轻微的短暂影响。

大鼠肝细胞对和灌注大鼠肝脏中的Taurolithocholic acid /牛磺石胆酸 (TLCA) 通过 PI3K 依赖性机制表现出胆汁淤积作用 [2]。
Taurolithocholic acid /牛磺酸胆酸（TLCA）是一种强效的胆汁淤积剂。我们最近的工作表明，TLCA通过蛋白激酶Cepsilon（PKCepsilon）依赖性机制损害肝胆分泌、运输蛋白插入肝细胞顶膜和胆汁流动。磷脂酰肌醇3-激酶（PI3K）的产物刺激PKCε。我们研究了PI3K在离体灌流大鼠肝脏（IPRL）和离体大鼠肝细胞偶联物（IRHC）中对TLCA诱导的胆汁淤积的作用。在IPRL中，TLCA（10微摩尔/升）使胆汁流量受损51%，使辣根过氧化物酶（囊泡分泌的标志物）的胆汁分泌受损46%，Mrp2底物2,4-二硝基苯基-S-谷胱甘肽受损95%，并刺激PI3K依赖性蛋白激酶B（PI3K活性的标志）受损154%，使PKEpsilon膜结合受损23%。在IRHC中，TLCA（2.5微摩尔/升）使荧光胆汁酸（胆酰甘氨酰荧光素）的小管分泌受损50%。选择性PI3K抑制剂渥曼青霉素（100 nmol/升）和抗胆固醇胆汁酸牛磺脱氧胆酸（TUDCA，25微摩尔/升）独立和相加地逆转了TLCA对IPRL中胆汁流量、胞吐、有机阴离子分泌、PI3K依赖性蛋白激酶B活性和PKCε膜结合的影响。渥曼青霉素还逆转了IRHC中受损的胆汁酸分泌。这些数据强烈表明，TLCA通过PI3K和PKCε依赖的机制发挥胆汁淤积作用，这些机制被牛磺脱氧胆酸以PI3K非依赖的方式逆转[2]。

通过免疫印迹技术测定分离的大鼠肝细胞中PKB/Akt的活性。简而言之，在接种后4小时（见上文），细胞与Taurolithocholic acid /牛磺胆酸/TLCA（5μmol/l；浓度>5μmol/l时，TLCA在短期培养中对分离的肝细胞造成可见损伤）、TUDCA（10μmol/l）、TCA（10µmol/l）或仅使用载体Me2SO（对照，0.1%，v/v）孵育5、15、30和60分钟。然后将培养皿放在冰上，刮取细胞并立即冷冻（-80°C）。将休克冷冻细胞在冰冷的裂解缓冲液（1ml/100mg）中均质化，并如上所述进行处理[2]。

Bile acid secretion by IRHC was assessed by measuring the hepatocellular uptake and secretion of 1 μmol/liter cholylglycylamido fluorescein (CGamF) into the canalicular space as previously described. CGamF was synthesized according to Schteingart et al. and was kindly provided by Dr. Alan Hofmann. Four hours after isolation, hepatocytes (on coverslips) were briefly transferred to HEPES buffer. Then, cells were pretreated for 15 min at 37 °C with (i) Me2SO (0.1%, v/v), (ii) 100 nmol/liter wortmannin and Me2SO, (iii) Me2SO for 5 min, and 2.5 μm Taurolithocholic acid /TLCA (in Me2SO, 0.1%, v/v) for 10 min, (iv) 100 nmol/liter wortmannin and Me2SO for 5 min, and 100 nmol/liter wortmannin and 2.5 μm Taurolithocholic acid /TLCA (in Me2SO, 0.1%, v/v) for 10 min, (v) Me2SO for 5 min and 5 μmol/liter Taurolithocholic acid /TLCA (in Me2SO, 0.1%, v/v) for 10 min, and (vi) 100 nmol/liter wortmannin and Me2SO for 5 min, and 100 nmol/liter wortmannin and 5 μmol/liter Taurolithocholic acid /TLCA for 10 min. Cells were then transferred for 5 min to HEPES buffer containing 1 μmol/liter fluorescent CGamF at 37 °C to allow adequate loading of the fluorescent bile acid and transferred back for 10 min to their previous dishes (i-vi). Hepatocyte secretion was stopped by placing coverslips in ice-cold HEPES buffer on ice, and cells were viewed immediately on a Zeiss LSM 510 microscope (Thornwood, NY). Laser settings were optimized for a dynamic range to avoid saturation of the fluorescence. The same settings were used for all conditions. Cells were analyzed on the confocal laser scanning microscope by one investigator (C. J. Soroka) who was blinded to the experimental conditions. Couplets were selected based upon the presence of a well defined canalicular space as determined under bright field optics. Images were then acquired with rapid scanning to avoid quenching of the fluorescence. Quantitation of uptake (uptake = (F° cell + F° can)/μm2) and secretion (% secretion = [F° can/(F° cell + F° can)] × 100) of CGamF was performed as previously published, except that NIH Image software was used.[2]

[1]. Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes. Hepatology. 1999 Feb;29(2):477-82.

[2]. Taurolithocholic acid exerts cholestatic effects via phosphatidylinositol 3-kinase-dependent mechanisms in perfused rat livers and rat hepatocyte couplets. J Biol Chem. 2003 May 16;278(20):17810-8.

Taurolithocholic acid is the bile acid taurine conjugate of lithocholic acid. It has a role as a human metabolite. It is a monocarboxylic acid amide and a bile acid taurine conjugate. It is functionally related to a lithocholic acid. It is a conjugate acid of a taurolithocholate.
Taurolithocholic acid has been reported in Homo sapiens, Bos taurus, and Aeromonas veronii with data available.
A bile salt formed in the liver from lithocholic acid conjugation with taurine, usually as the sodium salt. It solubilizes fats for absorption and is itself absorbed. It is a cholagogue and choleretic. Sodium taurolithocholate is a bile acid. It is functionally related to a taurolithocholic acid.
See also: Sodium taurolithocholate (annotation moved to).

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In the present study, co-administration of a PI3K inhibitor not only reversed TLCA-induced impairment of bile secretion but also cellular damage as determined by lactate dehydrogenase release (Table I). The improvement in bile flow alone could not account for this effect since TUDCA also improved secretion in TLCA-treated livers but failed to abolish the cell damage induced by TLCA in IPRL. Future studies will be necessary to elucidate the role of PI3K in TLCA-induced acute liver cell damage.
The present data suggest that PI3K represents a potential target of future anticholestatic treatment strategies. It should be mentioned, however, that PI3K may activate a survival pathway in rat hepatocytes treated with the hydrophobic bile acid, taurochenodeoxycholic acid (TCDCA) which protects liver cells from TCDCA-induced damage in vitro as well as in vivo (Rust C, unpublished observation). Interestingly, the taurochenodeoxycholic acid-induced survival pathway did not involve PKB activation in vitro. Thus, different bile acids may exert differential effects on PI3K- and PKB-mediated processes in liver cells. It remains to be clarified whether involvement of different PI3K isoforms or action in different subcellular compartments may contribute to these diverse effects of bile acids on PI3K and PKB.
In summary, the present study demonstrates that TLCA-induced impairment of bile flow, hepatobiliary exocytosis, secretion of bile acids, and other organic anions as well as liver cell damage is mediated by PI3K- and putatively PKCε-dependent mechanisms. TUDCA reversed the inhibitory effects of TLCA on bile secretion by a PI3K-independent mechanism.[2]

C26H45NO5S

483.7

483.302

C, 64.56; H, 9.38; N, 2.90; O, 16.54; S, 6.63

516-90-5

6042-32-6; 516-90-5

439763

Typically exists as solid at room temperature

1.169g/cm3

81-82ºC

1.54

6.347

115.57

3

5

7

33

825

9

CC(CCC(=O)NCCS(=O)(=O)O)C1CCC2C1(CCC3C2CCC4C3(CCC(C4)O)C)C

QBYUNVOYXHFVKC-GBURMNQMSA-N

InChI=1S/C26H45NO5S/c1-17(4-9-24(29)27-14-15-33(30,31)32)21-7-8-22-20-6-5-18-16-19(28)10-12-25(18,2)23(20)11-13-26(21,22)3/h17-23,28H,4-16H2,1-3H3,(H,27,29)(H,30,31,32)/t17-,18-,19-,20+,21-,22+,23+,25+,26-/m1/s1

2-[[(4R)-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonic acid

Lithocholic acid; taurine conjugate; TAUROLITHOCHOLIC ACID; 516-90-5; 2-[[(4R)-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonic acid; CHEBI:36259; DTXSID20965925; N-(3alpha-hydroxy-5beta-cholan-24-oyl)-taurine; ST 24:1;O2;T; Acid, Taurolithocholic; Taurolithocholic acid; Lithocholyltaurine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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