TITLE: UC Davis - Tissue TG & TC Protocol
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
The lipid extraction method is based on work by Folch 1957. Tissue is homogenized and put into a 2:1 chloroform and methanol mix. The mix is separated into two phases by adding a salt solution. The upper phase contains any non-lipid substances and the lower phase contains the chloroform with the lipids. The upper phase is removed and a sample is taken from the bottom phase and evaporated. The sample is reconstituted with 2-propanol with a volume to bring it to the desired concentration to assay.</div><div class = "text-block"><span>
Triglycerides are enzymatically hydrolyzed by lipase to free fatty acids and glycerol. The glycerol is phosphorylated by adenosine triphosphate (ATP) with glycerol kinase (GK) to produce glycerol-3- phosphate and adenosine diphosphate. Glycerol-3-phosphate is oxidized by dihydroxyacetone phosphate (DAP) by glycerolphosphate oxidase producing hydrogen peroxide (H</span><span style = "vertical-align:sub;">²</span><span style = "vertical-align:sub;vertical-align:sub;">O</span><span style = "vertical-align:sub;">²</span><span>). In a Trinder5 type color reaction catalyzed by peroxidase, the H</span><span style = "vertical-align:sub;">²</span><span style = "vertical-align:sub;vertical-align:sub;">O</span><span style = "vertical-align:sub;">²</span><span> reacts with 4-aminoantipyrine (4-AAP) and 3,5-dichloro-2- hydroxybenzene sulfonate (DHBS) to produce a red colored dye. The absorbance of this dye is proportional to the concentration of triglycerides present in the sample.
</span></div><div class = "text-block">Cholesterol esters are enzymatically hydrolysed by cholesterol esterase to cholesterol and free fatty acids. Free cholesterol, including that originally present, is then oxidized by cholesterol oxidase to cholest-4-en-3- one and hydrogen peroxide. The hydrogen peroxide combines with HBA and 4-aminoantipyrine to form
a chromophore (quinoneimine dye) which may be quantitated at 500-550nm.</div></div>

[STEPS]
?. Weigh out tissue (minimum of 50 mg) accurately and record, then transfer to a mortar and pestle along with 2 g of sodium sulfate (Fisher S415-212). Grind the tissue with the mortar and pestle until completely homogenous with salt.
?. Transfer homogenized mix to a 16×125 mm screw-cap glass tube (Fisher 14-959-35A) using a funnel (Fisher 03-865).
?. Add 4ml of ACS grade methanol (Fisher A412-4) with a volumetric pipet (Fisher 13-650-2E) then 8ml of ACS grade chloroform (Fisher C298-4) with a volumetric pipet (Fisher 13-650-2J). Screw the cap on (Fisher 14-959-36A), vortex, and store overnight at 4°C. Repeat steps 2-5 for all samples.
?. Add 2.4ml of a 0.7% sodium chloride (Fisher S271-3) solution to each tube and DO NOT VORTEX! The liquid will separate into 2 phases with the salt solution and methanol on top and the chloroform on the bottom. Store for at least 24 hours at 4°C.
?. Aspirate off the supernatant and any particulate matter floating on top of the chloroform layer. Be careful not to aspirate too much of the chloroform.
?. Take a 5ml sample of the chloroform using a volumetric pipet (Fisher 13-650-2F) and then transfer into a 16×125mm open top glass test tube (Fisher 14-961-30).
?. Put all the test tubes containing the chloroform sample into the Evap-O-Rac (Cole-Parmer EW-01610- 25) and evaporate with 100% nitrogen gas until completely dry. Takes about 4 hours.
?. Add ACS grade 2-propanol (Fisher A417-4) to each tube (volume will depend on amount of sample and concentration of TG or TC in tissue, 0.25 ml for liver and 0.15 ml for muscle is a good estimate), vortex at high speed for 1 minute with parafilm over the top of the tube, then use a pipet to transfer the 2-propanol to a microfuge tube.
?. There will be bits of tissue floating in the 2-propanol so centrifuge the microfuge tube for 1 min at 10000×g, then transfer the clean supernatant to another threaded microfuge tube (Fisher 02-681-339) and cap (Fisher 02-681-358).
?. Reconstitute powdered TG or TC reagent with only 25 ml of distilled water to make a 2X solution.
?. Add150 μl of PBS to each well.IMPORTANT: Make sure not to add any bubbles to the wells when dispensing reagents, this will interfere with reading in the platereader.
?. Add 3 μl of calibrator and sample to each well. Read at 540 nm.IMPORTANT: Add PBS to wells first and then add samples rather than adding samples to blank wells and adding PBS after. The 2-propanol will evaporate quickly if the 3μl is added to a blank well.
?. Add 150 μl of 2X reagent to each well. Incubate at 37°C for 5 minutes. Read at 540 nm.
?. Subtract blank readings from final readings.The assay will be linear so the unknown samples can be
calculated as (Sample Absorbance ÷ Calibrator Absorbance) × Calibrator Concentration.
?. Convert the assay TG or TC value in mg/dl to mg/ml then multiply by the total ml of 2-propanol used to get the total mg in the 5 ml chloroform sample that was evaporated. Multiply by the ratio of the total chloroform used and the chloroform sample taken (8/5) to get the total mg extracted from the chloroform. Divide by the tissue weight to get mg of TG or TC per g of tissue.