Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings

Abstract

The aim of this study was to design a convenient, specific, sensitive, and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs were used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent because more than half of the fatty acids from Parinari oil are known to contain 9,11,13,15-octadecatetraenoic acid (parinaric acid) in its esterified form. The presence of detergents (sodium taurodeoxycholate, CHAPS, Sulfobetaine SB12, Tween ® 20, Brij ® 35, Dobanol ® , n-dodecylglucoside) above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase in the fluorescence intensity is linear with time and proportional to the amount of lipase added. This new method, performed under non-oxidative conditions, was applied successfully to detecting low lipase levels in crude protein extracts from plant seeds and could be scaled down to microtiterplate measurements. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8). Lipase activity can also be assayed in acidic media (pH 5) using human gastric lipase. This simple and continuous assay is compatible with a high sample throughput and might be applied to detecting true lipase activities in various biological samples. -Beisson, F., N. Ferté, J. Nari, G. Noat, V. Arondel, and R. Verger. Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings.

Development of a High-Throughput Assay for Measuring Phospholipase A Activity Using Synthetic 1,2-α-Eleostearoyl-sn-glycero-3-phosphocholine Coated on Microtiter Plates

“…The detection sensitivity of this coated method is, however, around 50 times lower compared to that obtained with the fluorescent assays. Beisson et al 24 set up a lipase assay using parinaric acid-containing triacylglycerols, purified from Parinari glaberrimum seed oil, and quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions. Unlike the fluorescence method, which is solely based on the micellar solubilization of the free fatty acid released, the UV spectrophotometric method is based on the coated phospholipid-containing α-eleostearic acid and it can be performed without detergent, which would be convenient when assaying phospholipases sensitive to detergents.…”

“…Following hydrolysis, the binding between albumin and the free parinaric acid released corresponded to an increase in the total fluorescence intensity. The drawback of this method is, on the one hand, the susceptibility of parinaric acid to oxidation by atmospheric oxygen and, on the other hand, the need for the selected detergent to solubilize the released parinaric acid into mixed micelles …”

“…The drawback of this method is, on the one hand, the susceptibility of parinaric acid to oxidation by atmospheric oxygen and, on the other hand, the need for the selected detergent to solubilize the released parinaric acid into mixed micelles. 24 Here we have developed a specific and continuous assay using the ultraviolet (UV) spectrophotometric properties of natural α-eleostearic acid, incorporated into PC at positions sn-1 and sn-2, to measure PLA activity. α-Eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) was purified from Aleurites fordii seed oil (tung oil).…”

Oral lipolysis and its association with diet and the perception and digestion of lipids: A systematic literature review

“…In summary, a literature review (Beisson et al, 2001) described the screening assays for quantifying lipase activity on the principles and practical aspects of this methodological consideration. It was concluded that the pH-stat method, which enabled screening over an extended range of pH values due to the inclusion of several pH indicators, is suitable for pure lipase assays, and a method based on natural fluorescent substrates, as described in Beisson et al 1999, is more suitable for crude biological sample assays (Beisson et al, 1999).…”

A simple, rapid, and sensitive fluorescence-based method to assess triacylglycerol hydrolase activity

“…Attempts have been made to measure ATGL activity using fluorogenic substrates (30) such as dibutyrilfluoresceine (31), parinaric acid (32,33), umbelliferone (34,35), resorufin (24), pyrenic compounds, and BODIPY-C12 (4,(36)(37)(38). These assays have some disadvantages, namely the compounds are spontaneously hydrolyzed at pH 8.8, are susceptible to oxidation by atmospheric oxygen (39), and are poorly hydrolyzed by lipases (38,40). Nitrobenzoxadiazole (NBD) is a better fluorophore because it is small, does not interact with colored compounds, and is insensitive to oxidation (41,42).…”