| Assay |
Substrate used |
Product analyzed |
Principle |
Remarks |
Reference |
| Turbidimetry assay |
Tween20 in the presence of CaCl2 |
Released fatty acids |
Optical density increase at 500 nm due to precipitation in the form of calcium salts |
Simple and quantitative but Tweens are not specific substrates for lipases |
[89] |
| Interfacial tensiometry |
Lipid monolayer spread on surface of aqueous phase |
Fatty acids
|
Monitoring of surface pressure change due to dissolution of lipids using electro microbalance and Teflon barrier |
Highly sensitive reliable measurements,
|
[114]
[115]
|
| |
Oil water interface |
Fatty acids |
Tensiometers for oil drop method |
Low amounts of lipids used, but requires very sophisticated equipments |
[116] |
| Atomic force microscopy
|
Lipid bilayer supported on Mica |
Fatty acids |
Lipid dissolution forms holes in the bilayer an the increase in area of holes with time is monitored using real time images |
Nano scale assay and hence requires very sophisticated instruments |
[91] |
| Infrared spectroscopy |
Triacyl glycerols
(TAG) |
Free fatty acids and Fatty acid esters |
In the Fourier transform IR spectrum Fatty acid esters peak at 1751 cm-1 and FFAs at 1715 cm-1 and hence can be quantitated on the basis of molar extinction coefficients |
Expensive and sophisticated equipments required |
[117] |
| Titrimetry |
Stirred emulsion of TAG, tributyrin, Olive oil emulsified with gum Arabic |
Fatty acids |
pH stat method –Neutralization of released FFAs using titrated NaOH |
Most common procedure, sensitive to within 1 μmol fatty acid released per min, disadvantage if FFAs are not fully ionized |
[87]
[88]
|
| Spectrophotometry (Colorimetry) |
Lipid at lipid water interface Olive oil emulsion
Olive oil emulsion in presence of copper |
Safranin
Free fatty acids converted to |
Absorbance change of Safranin due to change in net negative charge at the lipid water interface
Formation of a copper soap. The copper complex is estimated spectrophotometrically at 440 nm |
Lipase activities as low as 50 mU can be detected
Sensitivity and efficacy improved for specific |
[83]
[84] |
| |
reagent
|
copper soaps.
Rhodamine G-FFA complex |
The complex develops a pink color. Absorbance read at 513 nm |
purposes by many researchers
Reproducibility is difficult |
[85]
|
| |
Para-nitrophenly esters |
Para nitro phenol |
Yellow coloured product which is measured at 410 nm |
Convenient and quick method, used commonly. These esters are liable to spontaneous hydrolysis and also by non-specific estetrases |
[86] |
| Flourimetry |
TAG with alkyl groups substituted with fluorescent group (Pyrenic acylglycerol derivatives) |
Free fatty acids
|
Shift in fluorescence wavelength after hydrolysis
|
Rapid assay but expensive substrate, Chemically modified Tag is poorly hydrolyzed
|
[118]
[88]
|
| |
Triaclyglycerol in the presence of fatty acid binding protein conjugated to an acrylodan fluorophore |
Free fatty acids
|
Fluorescence emission wavelength changes from 432 nm to 505 nm upon binding
|
Detection of concentration as low as 1 nM. Kit commercially available
|
[119]
|
| |
Phosphatidylch online containing naturally fluorescent parinaric acid |
Parinaric acid |
Detection of parinaric acid. Excitation and emission wavelengths of parinaric acid -324 nm and 420 nm |
Low quantities can be detected |
[120] |
| |
|
|
|
|
|
| Chromatography |
Lipids, TAGs |
Fatty acids |
Thin layer chromatography and quantititative analysis of FFAs by densitometric or auto radiographic methods when TAGs are labeled |
Detection of as small as a few pmoles of fatty acids. Time consuming and not continuous |
[121] |
| Electron microscopy |
Lipids |
Fatty acids |
Electron microscopy detection of fatty acids |
---------------- |
[90] |
| Immunodetection |
----------------- |
Lipase |
ELISA using monoclonal antibodies specific to antigens on Lipase |
Detection of both active and inactive form |
[122] |
