Dear Samir,

The following is an easy protocol for total lipid quantification:

Lipid Quantification Kit (Colorimetric) (see attached file).

Catalog Number

STA-613 100 assays

Introduction

Lipids are a diverse group of molecules that include monoglycerides, diglycerides, trigylcerides, fats,

sterols, and others. Not only do lipids define and preserve cellular membrane integrity, but they are

also involved in cellular processes such as membrane trafficking, signal transduction, apoptosis, and

energy storage. Perturbation in the metabolism of lipids has been linked to many diseases such as

cancer, diabetes, Alzheimer’s disease, and coronary heart disease.

In order to study lipids, they must often be extracted first from tissues or cellular cultures (1) and then

quantified. Some methods for lipid quantification have been described that are quite sensitive, however

they require expensive equipment such as an HPLC machine, light scattering detection technology, or a

latroscan TLC-FID analyser (2,3).

Cell Biolabs’ Lipid Quantification Kit measures the lipid content (unsaturated fatty acids only) of

samples using the sulfo-phospho-vanillin method (4), resulting in a simple colorimetric readout

amenable to multi-well plate detection. First, a crude or purified lipid source is applied to a 96 well

plate. Then concentrated sulfuric acid is added and the samples are heated to solubilize and prime the

total lipid sample, followed by the addition of vanillin in an acid solution. The lipids react with vanillin

in the presence of the acids to form a colorimetric product that is easily detected on a microplate reader.

Each kit provides sufficient reagents to perform 100 assays including standards and unknown samples.

Related Products

1. STA-612: Lipid Extraction Kit (Chloroform Free)

2. STA-241: Human Low Density Lipoprotein

3. STA-242: Human Very Low Density Lipoprotein

4. STA-243: Human High Density Lipoprotein

5. STA-369: OxiSelect™ Human Oxidized LDL ELISA Kit (MDA-LDL Quantitation)

6. STA-384: Total Cholesterol Assay Kit (Colorimetric)

7. STA-391: HDL and LDL/VLDL Cholesterol Assay Kit

8. STA-394: HDL Cholesterol Assay Kit

9. STA-396: Serum Triglyceride Quantification Kit (Colorimetric)

10. STA-398: Free Glycerol Assay Kit (Colorimetric)

11. STA-618: Free Fatty Acid Assay Kit (Colorimetric)

12. STA-600: Phosphatidylcholine Assay Kit

Kit Components

1. Vanillin Reagent (Part No. 261301): One 10 mL amber bottle.

2. Purified Lipid Standard (Part No. 261302): One 100 μL vial at 100 g/dL.

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Materials Not Supplied

1. Concentrated Sulfuric Acid (18M)

2. DMSO or other organic solvent

3. Glass tubes, 15 mL conical tubes, microcentrifuge tubes, or 96 well plates

4. 10 µL to 1000 µL adjustable single channel micropipettes with disposable tips

5. 50 µL to 1000 µL adjustable multichannel micropipette with disposable tips

6. Multichannel micropipette reservoir

Storage

Store the kit at -20ºC.

Preparation of Samples

 Plasma: Collect blood with an anticoagulant such as citrate, EDTA, heparin or oxalate and mix by

inversion. Centrifuge the blood at 1000 x g at 4°C for 10 minutes. Collect plasma supernatant

without disturbing the white buffy layer. Sample should be tested immediately or frozen at -80°C

for storage.

 Serum: Collect blood in a tube with no anticoagulant. Allow the blood to clot at room temperature

for 30 minutes. Centrifuge at 2500 x g for 20 minutes. Remove the yellow serum supernatant

without disturbing the white buffy layer. Samples should be tested immediately or frozen at -80°C

for storage.

 Cultured Cells or Tissue Samples: Prepare lipids using Cell Biolabs’ Lipid Extraction Kit (Cat.

#STA-612) or Folch method (Ref. 1).

Preparation of Purified Lipid Standard

Thaw the Purified Lipid Standard at room temperature and prepare a dilution series of Purified Lipid

Standards in the concentration range of 0 to 2500 mg/dL in dimethylsulfoxide (DMSO) or desired

solvent (Table 1).

Standard

Tubes

100 g/dL Purified Lipid

Standard (μL)

DMSO or other

organic solvent (µL)

Standard

(mg/dL)

1 10 390 2,500

2 200 of Tube #1 200 1,250

3 200 of Tube #2 200 625

4 200 of Tube #3 200 313

5 200 of Tube #4 200 156

6 200 of Tube #5 200 78

7 200 of Tube #6 200 39

8 0 100 0

Table 1. Preparation of Purified Lipid Standard Curve.

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Assay Protocol

Note: Sulfuric acid is highly corrosive and can damage certain types of plastics. Avoid using plastics

that are sensitive to sulfuric acid such as polystyrene, and test plastics prior to attempting this assay by

adding 100 L of sulfuric acid and heating to 90ºC for 10 minutes. Sulfuric acid should be handled

with care. Gloves, a lab coat, and protective eyewear should be worn during handling. Sulfuric acid

should be stored in glassware only and be pipetted in a fume hood.

1. Add 15 μL of samples or standards into microcentrifuge tubes or a 96-well plate.

Note: For samples in DMSO, skip steps 2 and 3 and proceed to step 4.

2. Incubate samples and standards uncovered at 90°C for 30 minutes to completely evaporate organic

solvents.

Note: This step is optional for aqueous, non-organic based samples, which will not evaporate

during heating.

3. Transfer samples to 4°C for 5 minutes.

4. Add 150 μL of 18M sulfuric acid.

5. Incubate samples at 90°C for 10 minutes.

6. Transfer samples to 4°C for 5 minutes.

7. Transfer 100 µL of each standard and unknown sample into a clean 96-well plate.

8. Read samples at OD 540 nm to determine background.

9. Add 100 L of Vanillin Reagent and mix carefully.

Note: The Vanillin Reagent tends to precipitate and may require incubation at 37°C for 15-30

minutes to go into solution.

10. Incubate samples at 37°C for 15 minutes.

11. Read samples at OD 540 nm to determine signal.

12. Subtract background from signal.

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Example of Results

The following figures demonstrate typical Lipid Quantification Kit (Colorimetric) results. One should

use the data below for reference only. This data should not be used to interpret actual results.

Figure 1: Lipid Quantification Kit Standard Curve.

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Figure 2: Detection of lipids from Human Serum or Human Plasma. Fifteen microliters of

undiluted Human Serum, Human Plasma, or Negative Control buffer were analyzed using the Lipid

Quantification Kit (Colorimetric).

References

1. Folch J, Lees M, and Slone Stanley GH (1956) J. Biol. Chem. 226, 497-509.

2. Lutzke BS and Brauler JM. (1990) J. Lipid Res., 31, 212-2130

3. Fraser AJ, Tocher DR, and Sargent JR (1985) J. Exp. Marine Biol. And Ecol. 88, 91-99.

4. Chabrol E and Charonnat R (1937) Presse Med. 45, 1713-1714.

Hoping this will be helpful,

Rafik

The method you could follow will depend much on the type of sample. Is it serum or a cell/tissue/organelle sample?

Totally agree with Begoña. It also depends on whether you want to use the lipids for further analysis, how much polar lipids your sample contains, the available equipment at your lab and so on.

https://www.researchgate.net/publication/222271833_Comparison_of_different_methods_for_total_lipid_quantification_in_meat_and_meat_products

Folch is one of the most used method, specially in animal tissue lipids extraction. It is based on a mixture of solvents (chloroform and methanol). It satisfactorily extracts most polar lipids, and also non-polar one (although there could be problem with very fatty tissues). You can find a description in the link above

Article Comparison of different methods for total lipid quantificati...

Totally agree with Begoña and Jorge. In any case for animal tissues, specially of marine organisms , the method described by Bligh & Dyer (1959) involving a three solvent system (chloroform, methanol & water) is quite easy to peform providing  the possibility of using reduced tissue samples.

Totally agree with Begoña and Jorge. In any case for animal tissues, specially of marine organisms , the method described by Bligh & Dyer (1959) involving a three solvent system (chloroform, methanol & water) is quite easy to peform providing  the possibility of using reduced tissue samples.

Agreed with those who say it depends on sample type and amount. If you want to get a rough estimate, you can perform a Bligh&Dyer extraction and weigh the extracted lipids after solvent evaporation. Doesn't get any cheaper than that

 (add some buffer to your tissue or scraped cells from a culture plate or whatever for a total volume of X ml, add X ml Chloroform and 2X ml Methanol, shake like hell, let it sit one hour, add X ml buffer and X ml chloroform, shake again and let sit, then centrifuge a bit (optional) if you need to separate phases, take the chloroform phase (usually the bottom one), add 2X ml chloroform to the remaining liquid, shake and let sit, collect the bottom phase of that one and add it to the previously collected chloroform phase, dry the chloroform and weight the pellet (= total lipids)). Of course, you need to weigh the glass tube in which you collect the chrloroform phases first :)

As regards to the critics to the B & D Method appearing in the manuscript referred above just a quick comment: the method is originally tested with fresh cod (highly hydrated meat, salt rich)  and final proportions of choloroform:methanol:water should be about (2:2:1.8) for best partition. I did not check those figures but they won´t differ much. Else, it requires much less tissue than the quantity referred by authors: 5 gr, I suppose fresh (raw) with a variety of hydration levels ( from fresh pork to dry cured ham) for the amount of solvent added.  Solvents added in the Folch method (20 to 1) have evidently worked better.