Lipase measurement

The serum lipase measured with this assay is often increased in patients with nonpancreatic disorders, particularly gastrointestinal and hepatobiliary... 

The specific activities of human digestive lipases measured from the in vivo and in vitro lipolysis of test meals. Gastroenterology 119, 949-960. 

Carriere, F., Renou, C., Lopez, V., De Caro, J., Ferrato, F., Lengsfeld, H., De Caro, A., Laugier, R., and Verger, R. (2000) The Specific Activities of Human Digestive Lipases Measured from the In Vivo and In Vitro Lipolysis of Test Meals, Gastroenterology 119, 946-960. 

Sample Collection and Enzyme Stability. Serum samples are collected with chemically clean, sterile glassware. Blood is allowed to clot at room temperature, the clot is gently separated from the test tube with an applicator stick, and the blood is centrifuged for 10 minutes at 1,000 g. If the red cells are known to contain the enzymes whose activity is being measured, as in the case of LD, even slightly hemolyzed serums must be discarded. When acid phosphatase is to be measured, the serum should be placed immediately in ice and processed as soon as possible, or it should be acidified by the addition of a small amount of sodium citrate. Anticoagulants such as EDTA, fluoride and oxalate inhibit some serum enzymes. However, heparin activates serum lipoprotein lipase. 

Numerous workers have found that measurements of serum lipase activity are useful in the diagnosis of pancreatitis (83, 84, 85). Despite this, serum lipase determinations are not usually performed in clinical laboratories, probably due to inherent problems associated with the conventional methods, based on an emulsified lipid substrate. The methods are also not very suitable for manual batch analysis nor for automation due to laborious post incubation procedures. 

Cherry and Crandall in 1932 (86) used olive oil as substrate with gum acacia as the emufsTfier. This method has served as the basis for a number of modifications that increased the stability of the emulsion, decreased incubation time and gave better precision. When a serum sample is incubated with a stabilized olive oil emulsion, lipase acts at the interface of substrate and water to hydrolyze olive oil into fatty acid plus diglycerides, and to a small extent to monoglycerides and glycerol. The bile salt sodium deoxycholate activates the reaction. These methods measure the liberated fatty acids by titration with a standardized NaOH solution. An indicator such as phenolphatalein, thymolphthalein or methyl red or a pH meter are used to detect the end point. 

Lippi et. al (87) and Dirstine (88) circumvented titration by converting the liberated fatty acids into copper salts, which after extraction in chloroform are reacted with diethyldithio-carbamate to form a colored complex which is measured photometrically. While the end point appears to be more sensitive than the pH end point determination, the advantages are outweighed by the additional steps of solvent extraction, centrifugation and incomplete extraction when low concentrations of copper salts are present. Other substrates used for the measurement of lipase activity have been tributyrin ( ), phenyl laurate (90), p-nit ro-pheny1-stearate and 3-naphthyl laurate (91). It has been shown that these substrates are hydrolyzed by esterases and thus lack specificity for lipase. Studies on patients with pancreatitis indicate olive oil emulsion is definitely superior to water soluble esters as substrates for measuring serum lipase activity. 

Seligson s group (95) has published a similar turbidimetric procedure but used nephelometry to measure continuously the effect of lipase on the light scattering of an olive oil emulsion. The instrumentation and approach is the same as that described above for the nephelometric determination of amylase. The method according to the authors is fast and precise with good specificity and sensitivity. The short time required for analysis makes it suitable for emergency use. The technical simplicity permits this method to be easily automated, and it appears to be the lipase method of choice. 

Yang, J. S. and Biggs, H. G. Rapid, reliable method for measuring serum lipase activity. Clin. Chem. (1971),... 

In our previous work [63], we studied the hydrolysis kinetics of lipase from Mucor javanicus in a modified Lewis cell (Fig. 4). Initial hydrolysis reaction rates (uri) were measured in the presence of lipase in the aqueous phase (borate buffer). Initial substrate (trilinolein) concentration (TLj) in the organic phase (octane) was between 0.05 and 8 mM. The presence of the interface with octane enhances hydrolysis [37]. Lineweaver-Burk plots of the kinetics curve (1/Uj.] = f( /TL)) gave straight lines, demonstrating that the hydrolysis reaction shows the expected kinetic behavior (Michaelis-Menten). Excess substrate results in reaction inhibition. Apparent parameters of the Michaelis equation were determined from the curve l/urj = f /TL) and substrate inhibition was determined from the curve 1/Uj.] =f(TL) ... 

The goals of treatment for acute pancreatitis include (1) resolution of nausea, vomiting, abdominal pain, and fever (2) ability to tolerate oral intake (3) normalization of serum amylase, lipase, and white blood cell count and (4) resolution of abscess, pseudocyst, or fluid collection as measured by CT scan. 

J5. Jaume, J. C., Mendel, C. M Frost, P. H., Greenspan, F. S and Laughton, C. W., Extremely low doses of heparin release lipase activity into plasma and can thereby cause artifactual elevations in serum free thyroxine concentration as measured by equilibrium dialysis. Thyroid 6, 79-84 (1996). 

Hydrolysis of Copolyamide-esters (CPAEs) by Lipase (jj,). CPAEs were synthesized by the amide-ester interchange reaction between polyamide and polyester. The length of the polyamide blocks was measured after hydrolysis of ester bonds in CPAE by alkali at 30 C. The infrared spectra after hydrolyzing ester bonds on CPAEs showed that the ester bonds were almost completely removed. The molecular weight distribution of polyamide blocks was examined by GPC (Table II). The following samples were used CPAE-1 (reaction time for synthesis, 1 hr) and CPAE-2 (reaction time, U hr) composed of nylon 6 and PCL at a 50/50 molar ratio, CPAE-3 (reaction time, 1 hr) and CPAE-U (reaction time,... 

In a typical experiment, a small volume of an insoluble surface-active material (dissolved in a water-insoluble solvent such as benzene) is placed atop a clean water surface. As the solvent evaporates away, a film remains and the moving barrier can be adjusted so that the surface film exerts pressure on the mica float. A calibrated torsion balance is used to measure the force that the film exerts on the float. That force divided by the length of the float is the force per unit length or the surface pressure. For studies of lipolysis kinetics , a Langmuir trough can be constructed so that one can measure lipase action under first-order and zero-order conditions. 

Fig. 5. Time course of the lipase-catalyzed hydrolysis of the (R)- and (5)-ester 1 measured with a UV/ Vis plate reader, (a) WT lipase from P. aeruginosa, (b) improved mutant in the first-generation epPCR 16).

One of the first fluorescence-based ee assays uses umbelliferone (14) as the built-in fluorophore and works for several different types of enzymatic reactions 70,86). In an initial investigation, the system was used to monitor the hydrolytic kinetic resolution of chiral acetates (e.g., rac-11) (Fig. 8). It is based on a sequence of two coupled enzymatic steps that converts a pair of enantiomeric alcohols formed by the asymmetric hydrolysis under study (e.g., R - and (5)-12) into a fluorescent product (e.g., 14). In the first step, (R)- and (5)-ll are subjected separately to hydrolysis in reactions catalyzed by a mutant enzyme (lipase or esterase). The goal of the assay is to measure the enantioselectivity of this kinetic resolution. The relative amount of R)- and ( S)-12 produced after a given reaction time is a measure of the enantioselectivity and can be ascertained rapidly, but not directly. 

The use of MALDI-MS for the measurement of low molecular mass compounds is widely accepted now [61], but quantification remains problematic. The main problem is the inhomogeneous distribution of the analytes within the matrix [62]. This leads to different amounts of ions and therefore to different signal intensities at various locations of a sample spot. The simplest and most effective way to overcome this problem is the use of an appropriate internal standard [63]. The use of deuterated compounds with a high molecular similarity to the analyte as internal standards leads to a linear correlation between relative signal intensities and relative amount of the compound to be quantified (Fig. 4b) [64]. Using this approach it is possible to quantitate substrates and products of enzyme catalyzed reactions. Two examples were shown recently by Kang and coworkers [64, 65]. The first was a lipase catalyzed reaction which produces 2-methoxy-N-[(lR)-l-phenylethyl]-acetamide (MET) using rac-a-... 

Fig. 4. a rac-Phenylethylamine (PEA) and 2-methoxy-J T-[(li )-l-phenylethyl]-acetamide (MET) are substrate and product in a lipase catalyzed transesterification, b Linear relationship between relative signal intensities in MALDI-MS and the relative concentrations of PEA/d5-labeled PEA. c Comparison of decrease in PEA during enzymatic conversion as measured by quantitative MALDI-MS and gas chromatography (GC)... 

Table 2.1 Enantiomeric ratios (E) obtained in hydrolysis of butanoates la-15a and transesterifrcation of alcohols lb-9b and 13b-15b using 2-chloroethyl butanoate as acyl donor and lipase B from Candida antarctica (CALB) as catalyst. For significance of and R2 see Figure 2.9. F-values were determined from chromatographically measured ee and ee at several degrees of conversion and using the computer program E K calculator version 2.03. Note that the stereopreference of CALB changes from (R) to (S) when R2=F, ClandBr.

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