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]
    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
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]
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]    
  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
  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]
    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]
Table 2: Different assay methods used for Lipase activity