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Measurement of Liver Triglyceride Content    

Hani JouihanHani Jouihan
DOI:   10.21769/BioProtoc.223
Published:   Vol 2, Iss 13, July 05, 2012
Biochemistry > Lipid > Lipid measurement
Mammalia > Murine > Liver > Lipid
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In this protocol

Abstract

This assay is designed to measure relative lipid accumulation of experimental treatments compared to controls. The reagent measures the concentration of glycerol released after lysing the cells and hydrolyzing the triglyceride molecules. The triglyceride concentration can then be determined from the glycerol values.

Materials and Reagents

  1. EtOH
  2. KOH
  3. MgCl2
  4. Triglyceride (GPO Trinder) reagent A (Sigma-Aldrich, catalog number: 337-40A )
    [Please note that this product has been replaced by free glycerol reagent (F6428) from Sigma]
  5. Glycerol Standards (Sigma-Aldrich, catalog number: 339-11 or G7793 )
  6. Ethanolic KOH (see Recipes)

Equipment

  1. Microfuge tube
  2. Glycerol
  3. Microreader
  4. Cuvette

Procedure

  1. Creation of saponified, neutralized liver extract
    1. Moved 100-300 mg of liver to pre-weighed microfuge tube.
    2. Record tube + liver weight.
    3. Add 350 μl ethanolic KOH.
    4. Incubate overnight 55 °C
    5. Vortex early in incubation till the tissue is completely digested.
    6. By morning, tissue should be digested and no oil layer should be visible (if oil layer is present then one must digest longer, and may need more ethanolic KOH).
    7. Bring volume to 1,000 μl with H2O: EtOH (1:1).
    8. Spin in microfuge 5 min, move supernatant to new tube.
    9. Bring volume of supernatant to 1,200 λ with H2O: EtOH (1:1), vortex.
    10. Move 200 μl to new eppendorf tube, add 215 μl 1 M MgCl2, vortex.
    11. 10 min on ice.
    12. Microfuge 5 min, move supernatant to new tube.

  2. Assay of glycerol content
    1. Reconstitute reagent A according to instructions for determination of glycerol content.
    2. Pipette 1 ml of reconstituted reagent A into cuvettes.
    3. Add samples, standards and blank.
      Standards: 10 μl + 20 μl H2O
      Samples: 30 μl
      Blank: 30 μl H2O
    4. Shake.
    5. Incubate 15 min & read at 540 nm.

  3. Calculation of liver TG content
    1. Note that the Sigma glycerol standards are expressed as triglyceride (triolein) equivalents.
    2. Subtract blank from all samples/standards.
    3. Create standard curve by linear regression.
    4. Determine cuvette (triolein equiv) (CTE) glycerol concentration by comparison to standard curve.
    5. Liver TG content (in mg TG / gram liver)
      = CTE (mg/dl)*(10/30)*(415/200)*0.012 (dl)/wt (gr)

Recipes

  1. Ethanolic KOH (2 parts EtOH: 1 part 30% KOH)
  2. 1 M MgCl2
  3. H2O: EtOH (1:1)

Acknowledgments

This protocol was previously used in Norris et al. (2003).

References

  1. Norris, A. W., Chen, L., Fisher, S. J., Szanto, I., Ristow, M., Jozsi, A. C., Hirshman, M. F., Rosen, E. D., Goodyear, L. J., Gonzalez, F. J., Spiegelman, B. M. and Kahn, C. R. (2003). Muscle-specific PPARgamma-deficient mice develop increased adiposity and insulin resistance but respond to thiazolidinediones. J Clin Invest 112(4): 608-618.
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Jouihan, H. (2012). Measurement of Liver Triglyceride Content. Bio-protocol 2(13): e223. DOI: 10.21769/BioProtoc.223.
Q&A

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S Bharathi
S Bharathi
University of Pittsburgh
Hi Hani,

Just a quick clarification. Would RT be ok for incubation, or is 37C better
7/14/2016 1:30:01 PM Reply
Shilpi Islam
Shilpi Islam
Hirosaki University
Dear sir
Thanks a lot for the protocol. I want to measured liver triglyceride, cholesterol and phospholipid for my sheep samples. I have some inquiry. Please inform me. 1) Can you explain me the calculation, specially what is 10/30; 415/200 and 0.012 (dL)?; I don't understand. 2) You suggested that extracted lipid sample would be used for cholesterol, so can I use plasma cholesterol kit for liver cholesterol determination or not? 3) Can I use this extracted liquid for phospholipid or not and plasma kit is considerable or not? 4) Sigma supplied all of require reagents for liver triglyceride?
7/2/2014 1:51:09 AM Reply
Mike Bieraugel
Mike Bieraugel
Allergan
The PI I'm working with prefers the protocols I used on his studies appear in peer reviewed journal articals? Do you know if this one is? If so, can you provide the reference? Do you know if this protocol is also suitable for measurements of total cholesterol with assay kits using cholesterol esterase, cholesterol oxidase, and peroxidase?
Thanks so much!
5/9/2014 11:30:21 AM Reply
Hani Jouihan
Hani Jouihan
Division of Endocrinology, Gerontology and Metabolism, Stanford University School of Medicine, USA

You can check papers from Dr. Ronald Kahn @ Harvard, you should be able to see this protocol referenced in one of his older papers. Alternatively, your PI can pay $$$ to buy commercially available kits.
Don't know if assay will work with the other conditions mentioned.
Best

5/12/2014 8:51:53 AM

Jessica Han
Jessica Han
North Carolina A&T State University
Thank you very much for the protocol. I have couple questions need help with
1) How much supernatant do you expect at step 12? We use the protocol for high fat mice liver sample, and only got 30-50 ul supernatant. Is it normal? Should we digest longer at the steps 3-6?

2)Can the supernatant at step 12 be stored at -20?

Thanks a lot!

3/6/2014 2:44:22 PM Reply
Hani Jouihan
Hani Jouihan
Division of Endocrinology, Gerontology and Metabolism, Stanford University School of Medicine, USA

Hi Jessica
I can't recall exactly how much sup to expect from step 12. However, since you are using a HF fed mice, you might want to use couple of different volumes of ethanolic KOH, and while you at it, try to incubate longer. The more effort you put upfront in optimizing the assay, the better off you will be in the long term.
Yes, sups can be stored at -20C. If you anticipate repeated measurements of the same sample, prepare aliquots to prevent freez-thaw issues.
Hope this helps

3/7/2014 8:20:42 AM

Jessica Han
Jessica Han
North Carolina A&T State University

Thanks very much! I will do different extraction volume of ethanolic KOH. Another question is what is the centrifuge speed at step 8 and 12?

3/7/2014 8:44:30 AM

Jessica Han
Jessica Han
North Carolina A&T State University

At step 9, after bring up to 1200 ul with 1:1 H2O:Ethanol, can this solution be stored at -20 for later process?

Thanks again.

3/7/2014 8:52:46 AM

Bronson Bronson A Haynes
Bronson Bronson A Haynes
Eastern Virginia Medical School
What is the purpose of the magnesium chloride? Is it essential to have this step?
11/16/2013 8:57:11 AM Reply
Hani Jouihan
Hani Jouihan
Division of Endocrinology, Gerontology and Metabolism, Stanford University School of Medicine, USA

Hi Bronson
the purpose of using MgCl2 is to serve as a precipitating agent for lipids.
For more details, I recommend googling for "Triglyceride Assay and magnesium chloride" you will find a lot of detailed info about this topic.

11/18/2013 6:07:23 AM

Bronson Bronson A Haynes
Bronson Bronson A Haynes
Eastern Virginia Medical School
Hello Hani!

Are you able to measure liver triglyceride content using a triglyceride kit that is used for serum/plasma? If not, please provide an explanation.
8/13/2013 5:34:07 PM Reply
Hani Jouihan
Hani Jouihan
Division of Endocrinology, Gerontology and Metabolism, Stanford University School of Medicine, USA

Hi Bronson
A short answer is; No, I have not used serum TG kit to measure the liver TG.
However, since TG should be the same regardless of the origin, I won't be surprised to see that you can use the serum kit with liver sample. The only trick here would be the way you are going to extract the TG from liver. So, if you decide to process the liver to extract the TG, make a serial dilution of this extract and test them all at once. This is important since liver might have way more TG than normally found in serum and the kit dynamic detection range might not detect very hi TG levels. Therefore, by diluting your extracts (1:2, 1:5, 1:10...ect) you might get it to work.
Good luck

8/14/2013 6:22:02 AM

Wil Wil
Wil Wil
University of Toronto
Hi Hani,

Thanks for the protocol.

I was wondering if you have tried this protocol to measure plasma triglyceride levels as well? If not, would you expect it to also work?

Thanks
8/2/2013 11:35:39 AM Reply
Hani Jouihan
Hani Jouihan
Division of Endocrinology, Gerontology and Metabolism, Stanford University School of Medicine, USA

Hi Wil
I would assume it should work, especially that you have already separated the plasma, so no blood-cells would interfere with the reading. However, I never tested plasma.
If you decided to try the protocol with plasma, make sure to include a positive control (from liver, for example) and used several different volumes of plasma so that your values are withing the linear range of the standard curve.
Good luck

8/2/2013 2:13:27 PM

Park Eunjung
Park Eunjung
Gyeong sang national university
Hi, Hani

you said that triglyceride reagent A (please note that this product has been replaced by free glycerol reagent). What is "replace" mean?

I thought that "replace" is changed A(TG reagent) to B(free glycerol reagent).
but, I saw that datasheet of two reagents in sigma homphage.

Do Triglyceride reagent A and free glycerol reagent have to mix??
4/17/2013 6:55:50 PM Reply
Hani Jouihan
Hani Jouihan
Division of Endocrinology, Gerontology and Metabolism, Stanford University School of Medicine, USA

Park, seeing the data sheets does NOT mean that the reagents are commercially available.
If you happen to still have reagent A, use it. If not, then you get the alternative, which happens to be the "Free Glycerol Reagent (F6428)".
Check the dictionary to see what "replace" means
I did not mention that you need to mix TG reagent A and Free glycerol reagent

4/18/2013 5:39:11 AM

Park Eunjung
Park Eunjung
Gyeong sang national university
Thank you for your protocol.

Triglyceride Reagent A (sigma#337-40A) was discontinued, and another TG reagent is sigma#T2449.
Accoding to your protocol, reagent is used 1ml per sample.
But, new TG reagent (sigma#T2449) accoding to instructions in sigma homephage can 10ml sufficient for 50 tests.
How much volum new TG reagent per sample into cuvettes?
1ml or 200ul ??
4/15/2013 12:55:28 AM Reply
Hani Jouihan
Hani Jouihan
Division of Endocrinology, Gerontology and Metabolism, Stanford University School of Medicine, USA

Hi Park
Since I did not use Sigma's new replacement product, I can't precisely tell you what to use.
However, you can optimize the assay according to your needs by keeping the ratio of sample: reagents at a constant. this way, you can reduce the total assay volume (let's say from 1000ul to 200 ul) by dividing all reagents and sample volumes by 5. this will keep the actual ratios among reactants constant, despite the reduction in assay total volume.
Like any newly introduce change in experiments, it is advisable that you test few different times before you reach a reproducible result. As long as you always have your standard included in each run, you should be OK in terms of extrapolating your experimental values.

4/17/2013 8:08:55 AM

Ryan Walker
Ryan Walker
USC
Hi,
Nice protocol. I have a few questions.

1) our spectrophotometer does not have a 540nm filter. We do have 535 and the tech seemed to think that this was "close enough". any thoughts on that?

2) what is your procedure for creating the glycerol standard titrations to generate the regression line from the data?

3) is there a way to add steps to obtain a protein fraction from this protocol?

4) we are planning to use the protocol on both muscle and triglyceride (which the literature says is fine). have you had success with both?

5) we may not have 100mg of tissue for all samples. if we have 50mg, for example, must we adjust the volume of all the reagents or does this not matter b/c the calculations are based on total weight of tissue?

Thanks a lot!
3/20/2013 4:23:28 PM Reply
Hani Jouihan
Hani Jouihan
Division of Endocrinology, Gerontology and Metabolism, Stanford University School of Medicine, USA

1) our spectrophotometer does not have a 540nm filter. We do have 535 and the tech seemed to think that this was "close enough". any thoughts on that?
? This is good enough.


2) what is your procedure for creating the glycerol standard titrations to generate the regression line from the data?

? This is to be determined empirically. You might want to look up some of the available commercial kits to see what concentrations of standard they have use. I would usually have a bulk idea on the expected TG amounts, so I will tailored my standard concentrations to for my use.

3) is there a way to add steps to obtain a protein fraction from this protocol?

? You can not use the same piece of liver to get TG and protein. The chemical treatment used to extract TG will not be suitable to do protein determination. Alternatively, you can dedicate another piece from the same tissue to do protein analysis.


4) we are planning to use the protocol on both muscle and triglyceride (which the literature says is fine). have you had success with both?

? What do you mean by “Muscle and TG”? I think you meant muscle and liver. In any case, this procedure should work on any tissue that you suspect to store TG.


5) we may not have 100mg of tissue for all samples. if we have 50mg, for example, must we adjust the volume of all the reagents or does this not matter b/c the calculations are based on total weight of tissue?

? This you have to adjust the volumes and calculations. I did not have this issue because liver and muscle usually provide ample material. If you are dealing with a mouse model that store excessive amounts of TG, you can use less tissue material.

3/27/2013 7:36:17 AM

We wish to know whether there is an universal equation for calculating TG concentration in the liver (Tg (mg)/Liver (gm) from the CTE (mg/dL) value obtained in a TG assay? Could you explain to us your equation: CTE (mg/dL)*(10/30)*(415/200)*0.012(dL)/wt(gr)that is listed in the protocol "Measurement of Liver Triglyceride Content" ?

2/23/2012 9:04:53 AM Reply
Hani Jouihan
Hani Jouihan
Division of Endocrinology, Gerontology and Metabolism, Stanford University School of Medicine, USA

There is no universal equation for calculating TG concentration. You can check protocols of commercially available TG kits and you will see several methods/equation for calculation.

In the equation, the (10/30) is the ratio of standard used (10ul) to the total volume of test sample which is 30ul.
The (415/200): the 41corresponds to the volume (415ul) from where the tested sample came out (step 10: Move 200 µl to new epp tube, add 215 µl 1M MgCl2, vortex)
The 200 is for the 200ul that we took out of step 9
Finally the 0.012 corresponds to the the final volume obtained in step 9 (1200ul)
Hope this helps

2/24/2012 7:30:32 AM

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