DETERMINATION OF GLYCEROL
Acylglycerols must be first hydrolyzed and, if possible, under conditions that do not cleave the phosphate ester of phospholipids and the glycosyl residue of glycosyl diglycerides, these compounds may be also present in the extract. Thus, under these conditions, even crude tissue extracts may be analyzed without prior purification.
1- Micromethod: determination with enzyme reactions
Small glass test tubes
Water bath or heating block
Spectrofluorimeter with quartz cuvettes
Tetraethylammonium hydroxide (TEAH) (20% aqueous solution) (FW 147.3) - Ethanol - 0.1 M HCl - Hexane - Hydrazine hydrate (85% aqueous solution, FW 50) - 0.4 M Glycine (FW 75) - 1 M MgCl2 - 2% NAD in water - 3% ATP in 5% NaHCO3 - 0.1M phosphate buffer pH 7.4 containing 1mM EDTA - 0.1M EDTA pH 10 - Glycerokinase (EC 18.104.22.168) diluted to 5 units/ml with phosphate buffer - a-Glycerophosphate dehydrogenase (EC 22.214.171.124) diluted to 20 units/ml with phosphate buffer.
Selective hydrolysis of acylglycerols
Aliquots of up to 50 nmoles (about 40 µg) of lipids are transferred to small glass tubes and evaporated. Add 0.1 ml of 50 mM TEAH in ethanol (dilute 1/30 the initial solution) and heat at 60°C during 30 min. Add 0.1 ml of 0.1 M HCl and mix. Extract fatty acids with 2 ml hexane. After mixing and centrifugation, eliminate the hexane layer (it can be used for fatty acid analysis), the lower phase is used for glycerol determination.
Determination of glycerol
For 10 samples mix the following solutions:
Hydrazine: 5 ml, Glycine: 5 ml, EDTA: 0.1 ml, MgCl2: 0.025 ml, NAD: 0.25 ml, ATP: 0.25 ml.
To each tube, containing 0.2 ml of hydrolyzate (with 1 to 50 nmoles of glycerol), glycerol standard or water, add 0.7 ml of the working solution, mix and measure the initial fluorescence (F1) (excitation: 360 nm, emission: 415 nm).
To each tube, add 0.050 ml of each enzyme suspension, mix and measure the final fluorescence (F2) after 30 min.
Fluorescence values are converted to nmoles of glycerol by comparison with values for standard glycerol amounts.
It is easily verified that phospholipids yield very small amounts of glycerophosphate but no glycerol.
The method can be used to determine the glycerol concentration in plasma after a simple deproteinisation at 100°C (after dilution with 3 volumes of water).
A flow-injection analytical system based on the lipase generation of glycerol from triacylglycerols and electrochemical detection was proposed in continuous operation (Wu LC et al., Anal Biochem 2005, 346, 234). It was possible to analyze up to 15 samples per hour.
2 - Macromethod: colorimetric determination
When samples contain up to 1 mg acylglycerols, a simple colorimetric procedure may be used based on the formation of formaldehyde during the periodate oxidation of glycerol.
Periodate reagent: dissolve 65 mg NaIO4 in 90 ml of water, add 10 ml acetic acid, mix and add 7.7 g ammonium acetate, mix.
Acetylacetone reagent: add 2.5 ml of acetylacetone to 247.5 ml of isopropanol, mix and store in the dark.
Lipid samples must be hydrolyzed as previously described.
Add 1 ml of periodate reagent to the hydrolysate and keep 5 min at room temperature.
Add 2.5 ml acetylacetone reagent, mix and warm 20 min at 50°C
Cool and read absorbance at 410 nm against a reagent blank.
Calibrate with known amounts of glycerol (2 to 50 µg/tube).
Comments: Note that glucose and other hexoses give the same color reaction. If glycoside-containing acylglycerols are present, a hydrolysis step with 2 M HCl at 100°C during 1 h is necessary to cleave glycosidic bond before glycerol analysis.
Instead of the colorimetric determination of the formaldehyde appearing during the oxidation, it is possible to quantify the formic acid also produced by potentiometric titration using an acid-base reaction (Naviglio D et al., Food Chem 2007, 102, 399).