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Lipid Extraction from Mouse Feces
小鼠排泄物中的脂质提取   

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Abstract

The analysis of feces composition is important for the study of energy metabolism, which comprises various measurements of energy intake, energy expenditure, and energy wasting. The current protocol describes how to measure energy-dense lipids in mouse feces using a modification of the method proposed by Folch et al. (1957).

Keywords: Energy metabolism(能量代谢), Lipid excretion(脂质排泄), Feces(粪便)

Materials and Reagents

  1. Mouse feces
  2. Normal saline (about 5-10 ml per mouse, depending on the amount of feces) (e.g., Sigma-Aldrich)
  3. Chloroform in methanol (2:1 by volume) (about 5-10 ml per mouse)

Equipment

  1. Forceps suitable for picking up mouse feces
  2. Sieve, coarse enough to hold back mouse feces
  3. Optional: Flour sifter
  4. 50-ml and 15-ml conical polypropylene tubes (one of each per mouse)
  5. 15-ml glass tubes (one per mouse)
  6. Tube stands
  7. Analytical balance
  8. Chemical fume hood
  9. Potter-Elvehjem tissue grinder and drill
  10. Centrifuge with rotor to hold 15-ml tubes (e.g., Beckman Coulter GS-6 with GH-3.8 swing bucket rotor or similar)
  11. 22G 1½ hypodermic needles (two per mouse)
  12. 5-ml syringes (one per mouse)

Procedure

  1. Collect mouse feces
    1. Replace bedding in all of the mouse cages. It is best to use bedding material that consists of small particles; you may want to ask your local animal facility for available choices.
    2. Feed the mice (one per cage) ad libitum for a number of days (e.g., 3-4 days).
    3. Place the mice in new cages.
    4. Run the bedding in the old cages over a coarse sieve that holds back feces and other large particles.
    5. Optionally, run the feces and other particles over the flour sifter.
    6. Using the forceps, pick up all the feces from one mouse and place it in a 50-ml conical tube that is labeled with the mouse number. Repeat for all mice.
    7. Obtain the total feces weight per mouse.
    8. Weigh exactly 1,000 mg of feces per mouse and powderize it using the tissue grinder.

  2. Prepare glass tubes
    1. Label each glass tube with a corresponding mouse number.
    2. Weigh the tube on an analytical balance; make a note of the tube number and weight.

  3. Extract the lipids
    1. Add 5 ml of normal saline to 1,000 mg of powderized feces in a 15-ml tube and vortex.
    2. Add 5 ml of chloroform: methanol mix and vortex.
    3. Centrifuge the suspension at 1,000 x g (about 2,000 rpm with above-mentioned rotor) for 10 min at room temperature. Afterwards you will see two liquid phases that are separated by a solid phase (Figure 1); there may be another, small solid phase at the bottom. The lower liquid phase contains the extracted lipids in chloroform: methanol.
    4. Holding the plastic tube above a glass tube, carefully insert a 22G½ needle through the tube wall into the lower of the two liquid phases; then, remove the needle. Caveat: Potential hazard: When inserting needles into the plastic tubes, make sure to apply gently, constant pressure. Twisting the needle may facilitate the insertion. Do not keep your fingers at the opposite wall of the tube!
    5. Attach an air-filled syringe to another 22G½ needle; carefully insert the needle at the top of the lower of the two liquid phases, just below the solid phase (Video 1).

      Video 1. Removal of lipid phase from plastic tube. The video shows how to insert a needle into the polypropylene tube and drain the lipid phase from the tube.

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player

    6. Hold the tube above the glass tube that is labelled with the mouse’s number and carefully inject the air; the lower liquid phase will be drained from the plastic tube and be collected into the glass tube.

  4. Evaporation
    1. Place the glass tubes in a stand under a fume hood.
    2. Leave the tubes alone for 3-4 days until all liquid is evaporated.
    3. Weigh the tubes on the analytical balance.
    4. Subtract the empty tube weights from the new tube weights to obtain the lipid mass per 1,000 mg of feces.
    5. Multiply the lipid mass by the total weight of feces in grams, and divide it by the number of days that the feces were collected for to obtain the mouse’s average fecal lipid excretion per day.


      Figure 1. Two samples of dissolved feces after centrifugation. The figure shows the three layers after centrifugation of feces suspended in chloroform:methanol. Phase ‘1’ is the aqueous phase. Phase ‘2’ contains insolubles. Phase ‘3’ contains the extracted lipids.

Notes

In the authors’ hands, 12-week-old male C57BL/6 mice fed with standard chow (LabDiet Formulab 5008) excrete 5.8 ± 0.5 [SD] grams of feces and 8.6 ± 4 mg of fecal lipids per day (lipid content in feces, 6 ‰). On a high-fat diet (Harlan Teklad TD 93075), they excrete 2.5 ± 0.4 grams of feces and 10.0 ± 1.4 mg of fecal lipids per day (lipid content in feces, 19 ‰).

Acknowledgments

This protocol (Kraus et al., 2014) is based on a method reported by Folch et al. (1957). The work was by supported grants from the Deutsche Forschungsgemeinschaft (KR 3475/1-1) and American Heart Association (09POST2250499) to D.K., grants from the NIH (KO8 DK090149, R01 DK100385, BNORC P30 DK046200 and NORCH P30 DK040561) to Q.Y., and grants from the NIH (R37 DK43051, P30 DK57521) as well as a grant from the JPB foundation to B.B.K.

References

  1. Folch, J., Lees, M. and Sloane Stanley, G. H. (1957). A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226(1): 497-509.
  2. Kraus, D., Yang, Q., Kong, D., Banks, A. S., Zhang, L., Rodgers, J. T., Pirinen, E., Pulinilkunnil, T. C., Gong, F., Wang, Y. C., Cen, Y., Sauve, A. A., Asara, J. M., Peroni, O. D., Monia, B. P., Bhanot, S., Alhonen, L., Puigserver, P. and Kahn, B. B. (2014). Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity. Nature 508(7495): 258-262.

简介

粪便组成的分析对于能量代谢的研究是重要的,其包括能量摄取,能量消耗和能量浪费的各种测量。 目前的方案描述了如何使用Folch等人(1957)提出的方法的修改来测量小鼠粪便中的能量密度脂质。

关键字:能量代谢, 脂质排泄, 粪便

材料和试剂

  1. 鼠粪
  2. 生理盐水(约5-10ml /小鼠,取决于粪便的量)(例如,Sigma-Aldrich)
  3. 氯仿在甲醇(2:1体积)(约5-10ml /小鼠)中

设备

  1. 镊子适合捡鼠标
  2. 筛子,足够粗,可以抓住鼠标粪便
  3. 可选:面粉筛
  4. 50毫升和15毫升锥形聚丙烯管(每只小鼠一只)
  5. 15ml玻璃管(每只小鼠一只)
  6. 管架
  7. 分析天平
  8. 化学通风橱
  9. Potter-Elvehjem组织研磨机和钻机
  10. 用转子离心以保持15ml管(例如,具有GH-3.8摇臂转子或类似物的Beckman Coulter GS-6)
  11. 22G1½皮下注射针(每只小鼠两只)
  12. 5 ml注射器(每只小鼠一只)

程序

  1. 收集小鼠粪便
    1. 更换所有鼠标笼子的床上用品。 最好是使用床上用品 由小颗粒组成的材料; 你可能想问你 当地动物设施可供选择。
    2. 每天喂食小鼠(每笼一只),随意喂食数天(例如,3-4天)。
    3. 将鼠标放在新的笼子里。
    4. 在老笼子里的床上用一个粗筛子,把粪便和其他大颗粒运走。
    5. 或者,将粪便和其他颗粒运到面粉筛上
    6. 使用镊子,拿起所有的粪便从一个老鼠,并把它   在用小鼠标号标记的50-ml锥形管中。 重复 对所有小鼠
    7. 获得每只鼠的总粪便重量。
    8. 每只小鼠称量正好1,000mg的粪便,并使用组织研磨机将其粉碎。

  2. 准备玻璃管
    1. 用相应的鼠标标记每个玻璃管。
    2. 在分析天平上称量管; 记录管的编号和重量。

  3. 提取脂质
    1. 在15ml管中加入5ml生理盐水至1000mg粉末化的粪便中并涡旋
    2. 加入5ml氯仿:甲醇混合物并涡旋
    3. 将悬浮液以1,000×g离心(约2,000rpm, 上述转子)在室温下10分钟。 后来你 将看到由固相分离的两个液相(图1) 1); 在底部可能存在另一个小的固相。 较低 液相含有提取的脂质在氯仿:甲醇中
    4. 将塑料管保持在玻璃管上方,小心插入22G½   针穿过管壁进入两个液相的下部; 然后,取下针。 警告:潜在危险:插入时 针头插入塑料管,确保轻轻应用,恒定 压力。 扭转针可便于插入。 不要保留 你的手指在管的对面墙!
    5. 附加 空气填充注射器到另一个22G½针; 小心插入针 在两个液相的下部的顶部,刚好在固体下面 阶段(视频1)。

      视频1.从塑料中去除脂质相 。视频显示如何将针插入聚丙烯管中  并从管中排出脂质相。
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      获取Adobe Flash Player

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                                                                                                                                                                                                                                            <! - [if!IE]> - > <! - <![endif] - >                                                                                                                                                                                                                                                          

      要播放视频,您需要安装较新版本的Adobe Flash Player。

      获取Adobe Flash Player

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    6. 将管子保持在带有标记的玻璃管上方 小鼠的数量并仔细注入空气; 下层液相会   从塑料管中排出并收集到玻璃管中。

  4. 蒸发
    1. 将玻璃管放在通风橱下的支架中。
    2. 将管子单独放置3-4天,直到所有液体蒸发
    3. 称量分析天平上的试管。
    4. 从新管重量中减去空管重量,以获得每1,000mg粪便的脂质质量
    5. 将脂质质量乘以粪便的总重量(克) 除以粪便被收集的天数 获得小鼠每天的平均粪便脂质排泄

      图1。 离心后溶解的粪便的两个样品。 图显示 三层离心后粪便悬浮 氯仿:甲醇。 相'1'是水相。 阶段'2'包含 不溶物。 相'3'包含提取的脂质。

笔记

在作者的手中,用标准食物(LabDiet Formulab 5008)喂养的12周龄雄性C57BL/6小鼠每天排泄5.8±0.5 [SD]克粪便和8.6±4mg粪便脂质(粪便中的脂质含量 ,6‰。 在高脂肪饮食(Harlan Teklad TD 93075)中,他们每天排泄2.5±0.4克粪便和10.0±1.4毫克粪便脂质(粪便中的脂质含量,19‰)。

致谢

该方案(Kraus等人,2014)基于Folch等人(1957)报道的方法。这项工作得到德国心脏病协会(KR 3475/1-1)和美国心脏协会(09POST2250499)DK的资助,NIH的捐赠(KO8 DK090149,R01 DK100385,BNORC P30 DK046200和NORCH P30 DK040561)到QY,和来自NIH(R37 DK43051,P30 DK57521)的拨款以及来自JPB基金会给BBK的拨款

参考文献

  1. Folch,J.,Lees,M。和Sloane Stanley,G.H。(1957)。 从动物组织中分离和纯化总脂质的简单方法 J Biol Chem 226(1):497-509。
  2. Kraus,D.,Yang,Q.,Kong,D.,Banks,AS,Zhang,L.,Rodgers,JT,Pirinen,E.,Pulinilkunnil,TC,Gong,F.,Wang,YC,Cen, ,Sauve,AA,Asara,JM,Peroni,OD,Monia,BP,Bhanot,S.,Alhonen,L.,Puigserver,P.and Kahn,BB(2014)。 烟酰胺N-甲基转移酶基因敲低可防止饮食诱导的肥胖。 /em> 508(7495):258-262。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kraus, D., Yang, Q. and Kahn, B. B. (2015). Lipid Extraction from Mouse Feces. Bio-protocol 5(1): e1375. DOI: 10.21769/BioProtoc.1375.
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