Simultaneous Kinetic Determinations of Lipase, Chymotrypsin, Trypsin, Elastase, and Amylase on the Same Microtiter Plate

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Abstract
Micromethods are described to determine in 10 min the activity of the five most common pancreatic zymogens: amylase, lipase, trypsin, chymotrypsin, and elastase. Progress of the reactions is monitored at 405 nm, allowing the kinetic determination of the five enzymes on a single 96-well microtiter plate. Amylase activity is measured by the release of p-nitrophenol from a chemically defined substrate. Linearity of the assay is from 10 to 360 U/L of amylase, and activities as low as 0.4 U/L can be easily measured by extending the period of incubation up to 24 h. Chymotrypsin, trypsin, and elastase activities are monitored by the release of p-nitroanilide from specific substrates, and activities are from 25 to 6,500, 15 to 260, and 20 to 600 U/L, respectively. Finally, lipase is determined by the clearing of a commercially available stabilized emulsion of triolein. The lipase determination can be performed from 90 to 3,600 U/L. When microplate methods were compared with conventional procedures, a perfect correspondence was found between the two types of procedure. Factors necessary to convert microplate results to those of conventional assays are provided. These microassays make possible the rapid and simultaneous determination of the three main types of pancreatic hydrolases (a glycohydrolase, three proteases, and a lipase) with < 5 microliters of pancreatic juice by kinetic analysis. They could be easily adopted as routine assays in most research laboratories.

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