酮体的生成和利用

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1、 酮体的生成和利用酮体的生成和利用【实验目的实验目的】 了解酮体的生成部位及掌握测定酮体生成与利用的方法。 【实验原理实验原理】 在肝脏线粒体中，脂肪酸经 -氧化生成的过量乙酰辅酶 A 缩合成酮体。酮体包括乙酰 乙酸、-羟丁酸和丙酮三种化合物。肝脏不能利用酮体，只有在肝外组织，尤其是心脏和 骨骼肌中，酮体可以转变为乙酰辅酶 A 而被氧化利用。 本实验以丁酸为基质，与肝匀浆一起保温，然后测定肝匀浆液中酮体的生成量。另外， 在肝脏和肌肉组织共存的情况下，再测定酮体的生成量。在这两种不同条件下，由酮体含 量的差别我们可以理解以上的理论。本实验主要测定的是丙酮的含量。 酮体测定的原理：在碱性溶液中碘可

2、将丙酮氧化成为碘仿。以硫代硫酸钠滴定剩余的 碘，可以计算所消耗的碘，由此也就可以计算出酮体（以丙酮为代表）的含量。反应式如 下：CH3COCH3十 3I2十 4NaOH CHI3十 CH3COONa 十 3NaI 十 3H2O I2十 2Na2S2O3 Na2S4O6十 2NaI【实验材料实验材料】1. 实验器材实验器材 试管；移液管；锥形瓶；滴定管及架。 2. 实验试剂实验试剂(1) 0.1% 淀粉液。 (2) 0.9% NaCl 溶液。 (3) 15% 三氯乙酸。 (4) 10 NaOH 溶液。 (5) 10 HCl 溶液。 (6) 0.5mol/L 丁酸溶液：取 5ml 丁酸溶于 100

3、ml 0.5mol/L NaOH 中。 (7) 0.1mol/L 碘液：I2 12.5g 和 KI 25g 加水溶解，稀释至刻度 1L，用 0.1mol/L Na2S2O3标定。(8) 0.02mol/L Na2S2O3: 24.82g Na2S2O35H2O 和 400mg 无水 Na2CO3溶于 1L 刚煮沸的 水中，配成 0.1mol/L 溶液，用 0.1mol/L KIO3标定。临用时将标定 Na2S2O3溶液 稀释成 0.02mol/L。 【实验操作实验操作】1. .标本的制备：标本的制备： 将兔致死，取出肝脏，用 0.9% NaCl 洗去污血，放滤纸上，吸去表面的水分，称取肝 组织

4、 5g 置研钵中，加少许 0.9% NaCl 至总体积为 10ml，制成肝组织匀浆。另外再取后腿 肌肉 5g，按上述方法和比例，制成肌组织匀浆。2. .保温和沉淀蛋白质：保温和沉淀蛋白质： 取试管 3 只，编号，按下表操作：管号 试剂 ABC肝组织匀浆2.0 ml2.0 ml预先煮沸的 肝组织匀浆2.0 mlpH 7.6 的 磷酸盐缓冲液4.0 ml4.0 ml4.0 ml正丁酸2.0 ml2.0 ml2.0 ml43水浴保温 60 分钟肌组织匀浆4.0 ml预先煮沸的 肌组织匀浆4.0 ml4.0 ml43水浴保温 60 分钟15%三氯醋酸3.0 ml3.0 ml3.0 ml摇匀后，用滤纸过

5、滤，将滤液分别收集在 3 支试管中,为无蛋白滤液。3. .酮体的测定酮体的测定 取锥形瓶 3 只，按下述编号顺序操作：编 号 试 剂123无蛋白滤液5.0 ml5.0 ml5.0 ml0.1mol/L I2-KI3.0 ml3.0 ml3.0 ml10% NaOH3.0 ml3.0 ml3.0 ml摇匀，静置 10 分钟，向各管中加入 10% HCl 3ml,加 1%淀粉液 1 滴呈兰色，分别用 0.02mol/L Na2S2O3滴定至溶液呈亮绿色为止。 【实验结果与计算实验结果与计算】 肝脏生成的酮体量（mmol/g）=(CA)Na2S2O3的摩尔数1/6 肌肉利用的酮体量（mmol/g）=

6、(CB)Na2S2O3的摩尔数1/6A: 滴定样品 1 消耗的 Na2S2O3 ml 数。 B: 滴定样品 2 消耗的 Na2S2O3 ml 数。 C: 滴定样品 3 消耗的 Na2S2O3 ml 数。【思考题思考题】为什么只有在肝外组织，酮体才可以被氧化利用？Experiment 11 Production and Degradation of Ketone Bodies【Purpose】Understand the production organs and master the method used for production and degradation of ketone bo

7、dies measurement.【Principle】Within the mitochondria of liver, the excess acetyl-CoA produced during fatty acid - oxidation is converted to acetoacetate, -hydroxybutyrate, and acetone, this group of molecules is called the ketone bodies. Liver can not use ketone bodies as an energy source. Only in se

8、veral tissues out of liver, most notably cardiac and skeletal muscle, ketone bodies are converted to acetyl-CoA, the acetyl-CoA is then oxidated to generate energy . We use butyric acid as initial stuff in this experiment, the butyric acid is heated with the liver plasm, and then measure the content

9、 of ketone bodies in liver plasm. Moreover, measure the content of ketone bodies under the condition of coexistence of liver plasm and skeletal muscle in reaction system. We can comprehend the above theories from the difference of the ketone bodies content under the two different conditions. We dete

10、rmine the content of acetone in this experiment. The ketone bodies measurement principle is shown below: In alkaline aqua the iodine can oxidize acetone to become iodoform , titrate the remainder iodine in the reaction system with hyposulphite, we can calculate the consumption of iodine according to

11、 the result of hyposulphite titration, we can also calculate the content of ketone bodies (take acetone as to represent) according to the titration result.The equation of Reaction is as follows: CH3COCH3十 3I2十 4NaOH CHI3十 CH3COONa 十 3NaI 十 3H2O I2十 2Na2S2O3 Na2S4O6十 2NaI【Materials】1. Apparatus: Tube

12、s, Pipets, Flasks Burettes and burette support. 2. Reagents: (1) 0.1% aqua of starch. (2) 0.9% aqua of NaCl. (3) 15% trichlorine acetic acid. (4) 10% aqua of NaOH. (5) 10% aqua of HCl.(6) 0.5mol/L butyric acid: Dissolve 5ml butyric acid in 100ml 0.5mol/L NaOH. 70.1mol/L aqua of iodine: Dissolve I2 1

13、2.5g and KI 25g in distilled water, dilute the solution to 1L, demarcate the solution with 0.1mol/L Na2S2O3. 80.02 mol/L Na2S2O3: Dissolve 24.82 g Na2S2O3 5 H2O and 400 mg anhydrous Na2CO3 in 1L fresh boiled water to get 0.1 mol/L solution, demarcate the solution with 0.1 mol/L KIO3. Dilute the solu

14、tion to 0.02 mol/L just before using.【Procedures】1. Preparation of the specimen: Execute the rabbit, take out the liver, scour off the blood with 0.9% NaCl, put the liver on filter paper to suck away the surface humidity, weigh 5 g of the liver organize, place it into the mortar, add a few 0.9% NaCl

15、 to total volume 10 ml. Then take 5 g muscle of rear leg, make it into the liver organization plasm according to above- mentioned method and comparisons. 2. Heat preservation and precipitation of the protein: Take three tubes, number the tubes and operate as the table followed：Tube No. ReagentABCThe

16、 liver organization plasm 2.0 ml2.0 mlThe liver organizationn plasm that boiled in advance 2.0 mlPhosphoric acid salt buffer liquid of pH7.64.0 ml4.0 ml4.0 mlorthobutyric acid2.0 ml2.0 ml2.0 mlHeat preservation at 43 water bath for 60 minutes.The muscle organization plasm4.0 mlThe muscle organization plasm that boiled in advance4.0 ml4.0 mlHeat preservation at 43 water bath for 60 minutes.15%