I´m having an issue with fecal TAG analysis in mice, and I would appreciate some advice from more experienced people.
I´d like to analyse different lipids in fecal samples.
1) I collect mice feces after 24h period and store them in -80°C
2) To obtain a representative sample I grind all feces from one mouse in mortar and take 100 mg of the powder to analyse.
3) I mix this powder with 400 ul of water and homogenize it some more.
4) I transfer this homogenate into chloroform:methanol 2:1 mixture and perform a kind of modified Folch method to extract lipids from this sample.
5) I end up with chloroform phase containing theoretically all lipids. I evaporate the liquid under the nitrogen stream. I make two such aliquots.
6) First aliquot: Cholesterol assessment. I resuspend lipids in abs. isopropanol and measure the cholesterol content with lachema kit reagent - no problem, I obtain consistent results, which correspond with results from different papers. Extraction obviously works well.
7) Second aliquot: Triglycerides assessment. I resuspend lipids in 300 ul of 3M KOH in 65% EtOH and incubate samples in 70°C for two hours. This is to saponify TAGs, because than I use enzymatic kit to measure content of glycerol (GPO based kit). This procedure works well for samples from liver for example, but in feces I repeatedly measure zero concentration of TAGs, which is surely wrong.
Do you know where the problem is? Could something in feces samples interfere with this enzymatic assay? do you have some experience with TAG assessment in feces of rodents? Thanks in advance for any hints!
People regularly measure some TAGs in fecal samples, I really don´t think the concentration should be zero. I will surely try the positive control, thanks for that suggestion, it´s only logical.
Lipid extraction is ok but then I would solve triglycerides in sodium cholate 0.5%, sonicate for 10 min in ultrasound bath for doing an emulsion) and determine triglycerides with a commercial kit (for glycerol but with lipases).
I think the glycerol must be lost after the saponification step (I guess you washed potassium with water?); why didn't you use a TG assay kit on you isoprop extract?
I am not sure there is anything wrong as fatty acid absorption is very efficient and so there should be very little TG. If you really think you have TG, you can take the Folch extract and run a conventional old-fashioned TLC. I would expect no TG. So I think your answer is not wrong.
To Jean: I used that comercial kit on the isopropanol extract before and obtained no results as well, so my coleague advised me to include saponification. By the way this exact procedure works perfectly with liver samples, so the problem must be in feces I guess.
and thanks everyone for suggestions, of course I thought along the same line - that there needn´t necessarily be TGs in feces samples. But I saw many articles which contained this measurement and they all showed some measurable concentrations of TGs in mice´s feces. Moreover, we used high-fat feeding (33% fat in diet), so I would really expect at least something...
Hi jana,
You might want to explore chemical analyses of the obtained liberated fatty acids.
Your enzymatic assy could be severely interferred by the numerous metabolites present in the fecal excretion as you mentioned earlier.
Dont go for saponification. U can take folch extract, reconstitute in isopropanol containing triton x and estimate triglycride using lipase, glycerol kinase, glycerol 3 p oxidasr, peroxidase system. This rea ction when triglycerjde is nt preseny in a lipoprotein aggregate can be painfuully slow. May take 45 mins or so. So a bit of patiencr will help. Good luck
Chemical analysis of ffa is fine, but is prone to errors. U have got to be extremely careful. If u wanna do chemical estimation, use the cuprizone method, its fantastic and simple
U said u used the kit method on folch ex reconstituted in isoprop. Did the kit contsin lipase?
Hi Jana,
Check that the expected TG mass and the resulting concentration is high enough to be registered in the TG assay. You may have to combine samples or change measuring techniques.
The TG assay is designed to produce the glycerol backbone from a TG and then measures the glycerol content. Of course, not all liquids are compatible with the assay designed for straight serum (organic solvents,etc.) . That you to added the saponification step means that you suspect interference, it should still work but added steps add more issues. For myself, I tested the compatibility of potential delivery liquids with the TG assay liquid with a known standard at different ratios to make sure it was working properly before testing fecal samples. (e.g., 20, 40, 60 ul of test sample in 300 ul of TG assay liquid)
There is a Larry Rudel's methods paper from the 90's that is a good source, at least for cholesterol and CE. They use Folch extractions in to enzymatic assays. It might be a good source of ideas.
Also, be aware that not all fats are the same and not all TG are equally absorbed. For example, a tristearate is absorbed much more poorly compared to a triolein, This makes oleate absorption close to 95% (like a lot of FA) while stearate is closer to 70%.. You may want to compare the fat content in your mouse diet compared to those you are emulating. They may have more left in the feces than yours. Unless you're inducing steatorrhea.
To Apurva: Yes, the kit contained lipases. It works well for plasma samples, we also use it to asses TGs in liver, but only with the previous saponification step - in this step it does not interfere with the assay.
I shall try to measure FFAs as you suggested and also repeat the extraction with some tripalmitoylglycerol added to see, whether the extraction and saponification process kills these arteficial TGs. I shall poste the result :-) And Eric thanks a lot for your comprehensive answer! I will consider all those stuff and read Rudel´s paper as well, I´m also struggling with cholesterol assessment, so it seems to be really valuable!
Fecal TG levels on a normal chow diet are very low compared to liver. The saponification method you are using is not particularly sensitive. Take a greater amount of fecal material for extraction in Folch, perhaps a couple of grams if you have it. Evaporate down under nitrogen or argon. Add some PBS and then sonicate the mixture for about a minute on ice. Sonication creates micro-emulsions with residual phospholipids. Then simply use a commercially available test kit as if you were doing plasma analysis. The method works nicely. Indeed, you can use the emulsified aliquot for any lipid assays. One more point: there are a couple of commercial kits which correct for free glycerol if you think this may be a significant confounder. Two manufacturers I recall are Wako (Japan) and Randox. Best of luck...
i suggest you analysis the fatty acid contents with GC,but not the glycerol.
I agree with John. Liver is much more TG rich and a much different makeup so it is hard to compare with feces. Also, the Wako kit he suggests I've used with success. The KOH is probably making your enzymatic assay impossible. No need for it with the functional lipases in the kit. Very nice suggestions to try by John (do the things he says in glass and avoid plastics as the hydrophobic molecules will stick to it and the plastic will even extract as part of the Folch!). GC-FAME that others are suggesting is a different approach and doesn't get at your TG question, especially with the gut flora involved. Just one point: if you back-calculate based on what you expect in results (absorbance value) - you can figure out how much starting material you need.to begin with
As a person who has had to smell feces in organic solvents, baking, in acid, etc. I empathize with your dilemma
GCMS-fame analysis seems the best way for your problem. Metanolic KOH hydrolysis is a bit more efficient of Ethanolic one. Alternatively you can test an acid hydrolysis. See the Cesareo’s work for the detail. "Fecal sample preparation methods for gas chromatography analysis of fatty acids of ruminants fed different amounts of
rumen protected conjugated linoleic acids (CLA)"
Just by curiosity i wonder how you distinguish between DAG, 2-MAG and TAG with the kit based analyses? In faeces, it is my understanding that DAGs and 2-MAG are in higher or similar concentrations asTAG, due to the very effecient hydrolysis by the pancreatic lipase.Is that incorrect? Most (if not all) kits are based on release of the fatty acids with a lipase, followed by a reaction of the released glycerol with glycerol dehydrogenase. These must also give a signal for DAG and MAG? As far as I know, TLC-FID, HPLC-ELSD, LC-MS or preparative TLC followed by GC-FID is the only ways to analyze TAG in samples with high concentrations of the hydrolytic products.
Best wishes
Since in Feces lipids are not coming in their native form, and somehow they modified during their passage and technically termed as "Fecal Sterol'. So I doubt you will able to detect fecal TGs with conventional enzymatic assay kit used for liver TGs estimation. Thanks
Hi Jana,
I plan to collect mouse feces as well. How much (wet weight) could you get during 24h per mouse?
I'd appreciate for that.
Best,
Yingyun