了解酮體的生成部位及掌握測定酮體生成與利用的方法。

【實(shí)驗(yàn)原理】

在肝臟線粒體中，脂肪酸經(jīng)β-氧化生成的過量乙酰輔酶A縮合成酮體。酮體包括乙酰乙酸、β-羥丁酸和丙酮三種化合物。肝臟不能利用酮體，只有在肝外組織，尤其是心臟和骨骼肌中，酮體可以轉(zhuǎn)變?yōu)橐阴］o酶A而被氧化利用。

本實(shí)驗(yàn)以丁酸為基質(zhì)，與肝勻漿一起保溫，然后測定肝勻漿液中酮體的生成量。另外，在肝臟和肌肉組織共存的情況下，再測定酮體的生成量。在這兩種不同條件下，由酮體含量的差別我們可以理解以上的理論。本實(shí)驗(yàn)主要測定的是丙酮的含量。

酮體測定的原理：在堿性溶液中碘可將丙酮氧化成為碘仿。以硫代硫酸鈉滴定剩余的碘，可以計(jì)算所消耗的碘，由此也就可以計(jì)算出酮體（以丙酮為代表）的含量。反應(yīng)式如下：

 

CH3COCH3十3I2十4NaOH      CHI3十CH3COONa十3NaI十3H2O

I2十2Na2S2O3      Na2S4O6十2NaI

 

【實(shí)驗(yàn)材料】

1. 實(shí)驗(yàn)器材

試管；移液管；錐形瓶；滴定管及架。

2. 實(shí)驗(yàn)試劑

(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100ml 0.5mol/L NaOH中。

(7)      0.1mol/L碘液：I2 12.5g和KI 25g加水溶解，稀釋至刻度1L，用0.1mol/L Na2S2O3標(biāo)定。

(8)      0.02mol/L Na2S2O3: 24.82g Na2S2O3·5H2O和400mg無水Na2CO3溶于1L剛煮沸的水中，配成0.1mol/L溶液，用0.1mol/L KIO3標(biāo)定。臨用時(shí)將標(biāo)定Na2S2O3溶液稀釋成0.02mol/L。

【實(shí)驗(yàn)操作】

1.標(biāo)本的制備：

將兔致死，取出肝臟，用0.9% NaCl洗去污血，放濾紙上，吸去表面的水分，稱取肝組織5g置研缽中，加少許0.9% NaCl至總體積為10ml，制成肝組織勻漿。另外再取后腿肌肉 5g，按上述方法和比例，制成肌組織勻漿。

2.保溫和沉淀蛋白質(zhì)：

取試管3只，編號(hào)，按下表操作：

 

 

 

 

43℃水浴保溫60分鐘

43℃水浴保溫60分鐘

搖勻后，用濾紙過濾，將濾液分別收集在3支試管中,為無蛋白濾液。

3.酮體的測定

取錐形瓶3只，按下述編號(hào)順序操作：

搖勻，靜置10分鐘，向各管中加入10% HCl 3ml,加1%淀粉液1滴呈蘭色，分別用0.02mol/L Na2S2O3滴定至溶液呈亮綠色為止。

【實(shí)驗(yàn)結(jié)果與計(jì)算】

肝臟生成的酮體量（mmol/g）=(C－A)×Na2S2O3的摩爾數(shù)×1/6

肌肉利用的酮體量（mmol/g）=(C－B)×Na2S2O3的摩爾數(shù)×1/6

A: 滴定樣品1消耗的Na2S2O3 ml數(shù)。

B: 滴定樣品2消耗的Na2S2O3 ml數(shù)。

C: 滴定樣品3消耗的Na2S2O3 ml數(shù)。

【思考題】

為什么只有在肝外組織，酮體才可以被氧化利用？

 

Experiment 11  Production and Degradation of Ketone Bodies

 

【Purpose】

Understand the production organs and master the method used for production and degradation of ketone bodies measurement.

【Principle】

Within the mitochondria of liver, the excess acetyl-CoA produced during fatty acid β-oxidation is converted to acetoacetate, β-hydroxybutyrate, and acetone, this group of molecules is called the ketone bodies. Liver can not use ketone bodies as an energy source. Only in several 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 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 determine 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 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:

Tubes, 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.

⑺         0.1mol/L aqua of iodine: Dissolve I2 12.5g and KI 25g in distilled water, dilute the solution to 1L, demarcate the solution with 0.1mol/L Na2S2O3.

⑻         0.02 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 solution 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 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：

 

Heat preservation at 43℃ water bath for 60 minutes.

Heat preservation at 43℃ water bath for 60 minutes.

 

Filter with the filter paper after shaking evenly, collect the filtrate respectively in 3 tubes, then we get the filtrate without protein.

3. Determination of the ketone bodies

Take 3 flasks, operate as below-mentioned serial number in proper order: 

Place Statically for 10 minutes after shaking evenly, add 10% HCl 3ml to each tube, and add one drop of 1% starch liquid to each tube, then we can see the solution presents the color of orchid, titrate with 0.02mol/L Na2S2O3 until the solution presents the color of bright green respectively.

【Result and calculation】

Ketone bodies produced in liver( mmol/g)=(C－A)×Moore content of the Na2S2O3 ×1/6

Ketone bodies consumed in muscle ( mmol/g)=(C－B)×Moore content of the Na2S2O3 ×1/6

A: volume of the Na2S2O3 (ml) consumed during titration of sample 1 

B: volume of the Na2S2O3 (ml) consumed during titration of sample 2

C: volume of the Na2S2O3 (ml) consumed during titration of sample３

【Advisement after experiment】

Ketone bodies are oxidated to generate energy only in several tissues out of liver. Why?