Specificity of Certain Methods for the Determinations of Pancreatic Lipase

Vol. 55, No.5 Printed in U.S.A.

GASTROENTEROLOGY

Copyright© 1968 by The Williams & Wilkins Co.

SPECIFICITY OF CERTAIN METHODS FOR THE DETERMINATIONS OF PANCREATIC LIPASE J. A. BARROWMAN, M.B., AND BENGT BoRGSTROM , M .D. Division of Physiological Chemistry, Chemical Center, University of Lund, Lund, Sweden

Pancreatic lipase (glycerol ester hydrolase, EC 3. 1. 1. 3) belongs to the large family of esterases, a group of rather ill-defined enzymes of broad specificity. The unique ability of lipase to attack water-insoluble esters, such as long chain triglycerides in emulsion, offers a ready means of distinguishing it from other esterases. It is possible that lipase has an absolute requirement for water-insoluble substrates, acting at an oil-water interface, although very slight activity against aqueous solutions of short chain triglycerides, such as triacetin, has been demonstrated with preparations of purified porcine lip ase. 1 Reliable and convenient methods for the estimation of lipase in serum, urine, and duodenal contents are of considerable diagnostic importance in investigation of disease of the pancreas and biliary tract. Many methods for this estimation have been proposed but lipase determination has been used less frequently in clinical practice than assay of other pancreatic enzymes; this is particularly true for lipase in serum, which, because of the very low levels of enzyme normally present, requires a very sensitive method. Of all of the methods for lipase measurement, titrimetric assays are probably the most widely used; methods such as that described by Marchis-Mouren et al. 2 use Received M arch 15, 1968. Accepted April 26, 1968. Address requests for reprints to: Bengt Borgstrom, M.D., Division of Physiological Chemistry, Chemical Center, P . 0 . Box 740, Lund 7, Sweden. Dr. Barrowman is t he recipient of a W ellcomeSwedish Travelling R esearch Fellowship. The t echnical assistance of Miss M aj-Lis Sandqvist is gratefully acknowledged. 601

continuous titration to a constant pH, while, in the Cherry and Crandall method, 3 the fatty acid released is titrated at the end of the reaction. Techniques using manometry, 4 phototurbidimetry, 5 • 6 and stalagmometry 7 have also been employed. Another group of methods utilizes chromogenic8-10 and fluorogenic substrates, 11 - 13 while assays using radioactively labeled triglyceride substrates have been described.14 Recently, it has been shown that gel filtration of rat pancreatic juice proteins separates lipase, whose molecular weight is approximately 40,000, from an enzyme of molecular weight approximately 70,000 which attacks water-soluble esters, micellar solutions of monoglycerides, and cholesterol esters.15 These enzymes are also readily separated by ion exchange chromatography. These means of separation have been chosen in this present study to allow examination of the specificity of some recently proposed methods for the determination of lipase, in particular those which use esters of j3-naphthol, p-nitrophenol, fluorescein , and 4-methyl umbelliferone. Materials and Methods Pancreatic juice enzymes. Adult laboratory rats of approximately 350 g were used . Pancreatic fistulas were established and the juice from several animals was collected for 2 or 3 days; it was frozen as it collected and was subsequently lyophilized and stored at below 0 C. One milliliter of juice yielded approximately 25 mg of powder whose protein content was found to be approximately 70% by weight. Gel filtration . Sephndex G-75 (lot 667) and G-100 (lot 6164) were used in columns 1.5 by 85 em. Twenty milligrams of lyophilized pnn-

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creatic juice were applied in most cases and the elution was performed at 4 C with a 0.15 M sodium chloride solution. Flow rates were approximately 10 ml per hr and fractions of 2.5 ml were collected. I on exchange chmmatography. Whatman microgranular DEAE-cellulose (DE 52) was used in columns 2.5 by 28 em. Three hundred fifty milligrams of rat pancreatic juice proteins were applied and the cationic proteins were eluted with a 0.005 M Tris-HCl buffer, pH 8.0. Anionic proteins eluted with a gradient from 0 to 1 M sodium chloride in this buffer. Substrates and reagents. The conjugated bile salts were synthesized according to Hofmann.'• Their purity when checked by thin layer chromatography was found to be better than 97%. Mono-olein (Myverol, Eastman Kodak Company) and triolein (glycerol trioleate, British Drug Houses) were purified on alumina columns. The products were checked for purity on thin layer chromatograms. {?-Napthy! acetate and laurate were obtained from Dajac laboratories (Leominster, Mass.) . Free {?-naphthol was removed by several recrystallizations of the esters from ethanol. The following esters were synthesized from the alcohol and appropriate acyl chloride and t heir purity was checked by thin layer chromatography and also, in some cases, by melting point determination: {?-naphthyl oleate, p-nitrophenyl acetate and laurate, fluorescein diacetate, dicaproate, and dioleate, and 4-methyl umbelliferone caproate and caprate. I ncubation systems. Those systems using emulsified triglyceride and micellar monoglyceride have already been described.'" {?-Naphthol esters . The procedures used were similar to those described by N achlas and Blackburn17 which employ a substrate mixture containing 5% acetone in water. Aliquots of 25 fLliters from fractions from the chromatography columns were generally taken for each incubation. When the effect of the addition of bile salts on the hydrolysis of {?-naphthyl laurate and oleate was studied, the appropriate amounts of bile salt were added to bring their concentration in the incubation mixture to the desired level. In other respects the procedure was identical with the above. All estimations of {?-napht hol ester hydrolysis were performed at_ 25 C and the incubation period was 30 mm. p-Nitmphenol esters. Hydrolysis of p-nitrophenyl acetate was measured in a system containing 1 ml of a saturated aqueous solution of this ester and 1 ml of sodium phosphate buffer, pH 6.3, 0.15 M in sodium, containing a

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50-p.liter aliquot of a fraction from the chromatographic separation. In some experiments, bile salts were added t o the incnbation mixture. All incubations were performed at 37 C for 20 min and the p-nitrophenol released was estimated by reading the solutions in a spectrophotometer at 400 mfL against a blank prepared as above but containing no enzyme. In the case of p-nitrophenyl laurate, the following substrate mixture was prepared: 25 ml of 0.05 M Tris-HCl buffer (pH 8.2), 25 mg of p-nitrophenyl lam·ate, and 50 mg of decane. This mixture was subjected to ultrasonic vibration at full effect for 10 min with a Branson sonicator (Branson I nstruments, Inc., Stamford, Conn.). Incubations were perfo rmed with 1 ml of this emulsion, freshly prepared, added to 1 ml of water containing 50 p.liters of the fractions . The incubations were performed at 37 C for 5 min after which 3 ml of acetone were added and t he solutions were read at 400 mfL against a blank prepared without enzyme. In some experiments with p-nit rophenyl laurate, bile salts were added to the incubation mixture. Fluorescein esters. Stock solutions of these esters were prepared with methyl Cellosolve. From these, solutions of 5 x w-• M were prepared in Tris-HCl buffer, pH 8.0, after the method of Kramer and Guilbault.12 Two milliliter aliquots were incubated at 25 C for 2 min together with the enzyme source, and free fluorescein was measured in a Jobin-Yvon spectrofluorimeter with an exciting wavelength of 470 mfL and an emission wavelength of 510 mfL . Suitable controls were included to correct for the spontaneous hydrolysis of fluorescein diacetate . The aliquots from the fractions used in this study were usually 25 or 50 fLliters. Esters of 4--methyl umbelliferone. Stock solutions of these esters (1 X 10-• M) in 6% methyl Cellosolve in water were prepared according to Jacks and Kircher.'• The incubation mixture consisted of 3 ml of 0.05 M Tris-HCI buffer, pH 7.4, containing 6 fLmoles of substrate. Fifty- or 100-fLliter aliquots from column fractions were added and the reaction was measured for 60 sec in a Jobin-Yvon spectrafluorimeter with an exciting wavelength of 370 mfL and an emission wavelength of 450 mfL. In one experiment, a saturated aqueous solution of 4-methyl umbellife rone caproate was substituted for the stock solution described above.

Results

As shown previously, activities of rat pancreatic juice enzymes against micellar

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monoglyceride and emulsified triglyceride are readily separable by gel filtration and ion exchange chromatography. As the former enzyme attacks such typical esterase p-nitrophenyl acetate, and the latter attacks emulsified long chain triglycerides, the trivial names esterase and lipase are used below for convenience. In all separations by gel filtration or ion exchange chromatography, the patterns of activity of these two enzymes were determined and t he patterns against the test substrates were compared with them. Esters of (3 -naphthol. The patterns of activity against (3-naphthyl lam·ate and oleate were found to correspond closely with t hose against emulsified triglyceride, while (3-naphthyl acetate hydrolysis paralleled activity against micellar monoglyceride. There was little or no cross specificity. Concentrations of 3 mM sodium taurodeoxycholate in t he incubation mixture greatly enhanced t he activity of esterase toward (3-naphthyl acetate while the same concentration of bile salt in the {3-naphthyl lam·ate or oleate incubations resulted in a marked change in specificity of the substrate which was now very strongly hydrolyzed by esterase in addition to lipase. ViTith concentrations of 0.8 and 0.4 mM of this bile salt in the incubation mixture, actiYity by both enzymes against the substrates was also found. Similar resu lts were obtained with concentrations of 0.5, 1.0, and 3.0 mM sodium taurocholate in the incubation mixtures. Esters of p-nitrophenol. Activity against p-nitrophenyl acetate was found to be confin ed to t he fractions containing esterase. As with (3-naphthyl acetate, the hydrolysis of p-nitrophenyl acetate was greatly enhanced by t he presence of 3 mM sodium taurodeoxycholate in the incubation mixture. Hydrolysis of emulsion s of p-nitrophenyl laurate was confined to fractions containing lipase but t he addition of sodium taurodeoxycholate to a concentration of 3 mM resulted in a similar change to that observed with (3-naphthyl laurate in the presence of bile salt, i.e., the appearance of strong hydrolytic activity by esterase; the activity of lipase in this system was reduced.

Esters of fluorescein. Three esters of fluorescein were synthesized, viz., the eliacetate, dicaproate, and dioleate. The first two are crystalline at 25 C while the oleate is an oil. The hydrolytic activity of unseparated rat pancreatic juice was tested against several emulsions of the dioleate; negligible hydrolysis was found. The other two esters were strongly hydrolyzed by rat pancreatic juice. Figures 1 and 2 show t he separation of pancreatic juice proteins by gel filtrat ion and the location of optimal activity against these esters. In the system used, the activity of esterase toward fluorescein diacetate appeared to be greater than toward dicaproate. In the case of fluorescein dicaproate, a second peak of activity was found (fig. 2). The elution volume of this peak corresponds to that for proteins of an approximate molecular weight of 25,000 as judged by calibration of the columns with standard proteins. A small secondary peak of activity against emulsified triolein is present in this area, but activity against fluorescein dicaproate is absent in the area showing maximal activity toward emulsified triglyceride. With ion exchange chromatography on DEAE-cellulose at pH 8.0, lipase is cationic and es-

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Fra . 1. Chromatography of lyophilized rat pancrea tic juice on Sephadex G-75. Activity against micellar monoglyceride, emulsified triglyceride, and fluorescein diacetate. The uni ts of acti vit~-- for the hydrolysis of the glycerides are microequivalents of fatty acid released per fraction per minute. Fluorescein diaceta te hydrolysis is shown as the rate of liberation of fre e fluores~ cein per minute, in arbitrary units, by a 25-,u liter aliquot of each fraction.

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terase is anionic. No activity against either fluorescein diacetate or dicaproate was found in the cationic proteins but a strong activity against both esters was found in the anionic proteins, the pattern of activity corresponding well with that shown against micellar monoglyceride. Esters of 4-methyl umb elliferone

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MU) . 4-MU caproate and caprate were tested. Figure 3 shows the pattern of activity toward both esters in fractions obtained by gel filtration of 20 mg of lyophilized pancreatic juice. 4-MU caproate is clearly hydrolyzed by esterase only, while activities corresponding with both esterase and lipase are found for 4-MU caprate. In the one experiment in which a saturated aqueous solution of 4-MU caproate replaced the solution of the ester in 6% methyl Cellosolve, the results obtained were closely similar to those obtained with the latter stock solution. Table 1 summarizes these results and shows the activities of these two enzymes against a number of other substrates. Discu ssion

In this study, the specificity of certain substrates for lipase measurement has been evaluated. In examining the activity of other enzymes interfering with the assay, it is important to consider the source of

the material to be assayed. It should therefore be emphasized that the results described in this study apply to rat pancreatic juice and probably to duodenal contents. In assays of serum lipase, the substrate specificities will require redefinition, especially with respect to the activity of esterases present. The need for a short, simple, and sensitive method for the determination of lipase has prompted the development of a number of methods which use chromogenic and fluorogenic substrates, some of which have been examined in this study. Most of the 10

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TABLE 1. Specificity of pancreatic juice enzymes• esters discussed here have a very limited water solubility. To achieve solution or Substrate Lipase Esterase dispersion of these substances, it is neces- - - - - - - - - - - - - - - - - sary to use small amounts of such solvents Emulsified triolein* ............... +++ as acetone or methyl Cellosolve in aqueous Micellar triolein*.... . ........ . ++ systems. Such systems present a question Emulsified mono-olein (in hepas to the physical state of the substrate. tane)*.. . .......... . ++ The results of this study show that the Emulsified mono-olein (in heptane)-bile salt*. long chain fatty acid esters of ,8-naphthol, ++ ++ i.e., the laurate and oleate, and p-nitro- Micellar mono-olein* . . . . . +++ (+) (+) phenyl laurate, in a heterogeneous system Micellar lecithin* .... are hydrolyzed by lipase. There was little Emulsified cholesterol oleate*. or no detectable activity by the esterase Cholesterol oleate in bile salt dispersian* . . .. . . . . .. . + + on these substrates in the absence of bile /1-Naphthyl acetate*... . . . . . ....... +++ salts. Of the esters of fluorescein which /1-Naphthyllaurate* ........... . ... +++ were tested the diacetate and dicaproate /1-Naphthyl oleate.. . . +++ were both strongly hydrolyzed by esterase /1-Naphthyllaurate-bile salt. + ++ and neither appeared to be split by pan- /1-Naphthyl oleate-bile salt... + ++ creatic lipase. Both the caproate and cap- p-Nitrophenyl acetate ... +++ rate esters of 4-methyl umbelliferone were p-Nitrophenyllaurate .. +++ (+) ++ strongly attacked by esterase while 4- p-Nitrophenyllaurate-bile salt .. +++ methyl umbelliferone caprate appeared to Fluorescein diacetate ... ++ be split by lipase also. The presence of Fluorescein dicaproate. . . .. .. . .. . bile salts, sodium taurocholate and tauro- Fluorescein dioleate. 4-Methyl umbelliferone caproate. +++ deoxycholate, in the systems used here 4-Methyl umbelliferone caprate. ++ +++ seemed to alter the specificity of the substrates ,8-naphthyl laurate and oleate and •Activities: -,none detected;(+), slight or p-nitrophenyl laurate for lipase, resulting doubtful; +, small; ++, moderate; +++, in hydrolysis of these substances by es- strong. The substrates marked with an asterisk terase. The activity of esterase against (*)have been examined in a previous study.'s p-nitrophenyl acetate and ,8-naphthyl acetate was greatly enhanced by the presence lam·ate by the presence of bile salts is observed at levels much below the critical of bile salt in the incubation mixture. The esters of ,B-naphthol have been micellar concentration. Some preliminary widely tested and used as substrates for studies suggest that the solubility of long lipase and esterase. Many of the methods chain esters of ,B-naphthol in bile salt measuring serum lipase employ the addi- micelles is low. Another role of bile salts tion of bile salts as activators of lipase in these systems may be a direct effect on and inhibitors of esterase in order to en- the enzyme or enzyme-substrate complex. hance the specificity of the method (for It has been postulated that sodium tauroexample, Kramer et aJ.l 8 ). The bile salts cholate has such an effect on the enzymeused in these methods are generally sod- substrate complex of porcine lipase and ium cholate or taurocholate. Their role in against ,B-naphthyl acetate and p-nitrothese systems is probably complex: as phenyl acetate is greatly enhanced by the detergents, they are likely to influence the bile salts tested and it is possible that the physical state of the substrate either by patterns of activity obtained against ,Bstabilizing the emulsion or by creating a naphthyllaurate and p-nitrophenyl lam·ate micellar phase in which the substance may are the result of enhancement of a minimal in part be dissolved. The addition of bile activity of esterase against these substrates salt to the substrate mixture of ,B-naphthyl in the absence of bile salt. An emulsion of p-nitrophenyl laurate lam·ate results in considerable decrease in the turbidity of the solution. However, appears to be hydrolyzed by lipase, as the alteration in specificity of ,8-naphthyl suggested by Desnuelle and Savary 20 ; the

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mechanism which results in the alteration in specificity in the presence of bile salts may be similar to that operating in the case of the long chain esters of ,8-naphthol. Of the fluorogenic substrates, all, with the exception of fluorescein dioleate, were hydrolyzed by esterase. In addition, activity against 4-methyl umbelliferone caprate was found in fractions of pancreatic juice containing lipase. Sephadex chromatography revealed two peaks of activity against fluorescein dicaproate; the first corresponded to esterase while the second to a protein of approximately 25,000 molecular weight. It is possible that this latter enzyme is chymotrypsin which has been shown to hydrolyze the fluorescein esters. 21 Although unseparated lyophilized rat pancreatic juice exhibits no tryptic activity, fractions obtained by gel filtration have been shown to contain free trypsin (R. G. H. Morgan, H. Filipek-Wender, and the authors, unpublished data), and it is possible that this may activate chymotrypsinogen during the chromatography. Some broad similarities exist between the fluorogenic substrates and the esters of ,8-naphthol, both with respect to their chemical structure and the means by which they are dispersed in the incubation system. The observation that 4-methyl umbelliferone caprate is split by both esterase and lipase is interesting and a rough parallel may be drawn between the fatty acyl esters of ,8-naphthol and of 4-methyl umbelliferone, insofar as, in the ,8-naphthol series, esters with fatty acids of approximately 10 carbon atoms are hydrolyzed by both lipase and esterase; the specificity for lipase increases with esters of longer chain fatty acids. For example, Ravin and Seligman22 have found such an increase in specificity between ,8-naphthyllaurate and ,8-naphthyl myristate. It is possible that there exists among the higher fatty acid esters of 4methyl umbelliferone a substance with a high specificity for lipase. At present, assays employing the digestion of emulsified long chain triglycerides with titration of released fatty acids are probably the best documented and reliable methods for lipase measurement, although chromogenic and fluorogenic ester systems

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offer the prospect of development of more convenient and highly sensitive methods. Summary

Gel filtration and ion exc~ange chromatography of rat pancreatic juice afford a suitable means for separating lipase (glycerol ester hydrolase) and a larger molecular weight enzyme (carboxylic ester hydrolase) also present in pancreatic juice which attacks both water-soluble and micellar dispersed esters (also long chain glycerides). The specificity of these two enzymes for a series of substrates proposed for lipase determination has been studied. Methods employing emulsified long chain triglycerides as substrates are still the best documented and the most specific for assay of pancreatic lipase. Different methods based on chromogenic and fluorogenic systems have been proposed and may offer prospects of more convenient and sensitive methods. In some of these assay systems, those employing certain diesters of fluorescein, the substrates are not split by lipase under any conditions, while in others using esters of ,8-naphthol and 4-methyl umbelliferone some substrates are split by lipase, some by esterase, and some by both enzymes, depending on factors such as the chain length of the fatty acids and the presence or absence of bile salts in the system. Dispersions of the chromogenic ,8-naphthyl laurate and oleate and p-nitrophenyl laurate are split principally by lipase; the addition of bile salt, even in very low concentrations to the incubation systems, however, alters their specificity in favor of the esterase. REFERENCES 1. Sarda, L., and P. D esnuelle. 1958. Action de Ia

lipase pancn\atique sur les esters en emulsion. Biochim. Biophys. Acta 30: 513-521. 2. Marchis-Mouren, G., L. Sarda, and P. D esnuelle. 1959. Purification of hog pancreatic lipase. Arch. Biochem. 83 : 309-319. 3. Cherry, I. S., and L. A. Crandall, Jr. 1932. The specificity of pancreatic lipase : its appearance in the blood after pancreatir injury. Amer. J. Physiol. 100: 266--273. 4. Rona, P., and A. Lasnitzki. 1924. Eine Methode

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lipase using fatty acyl esters of 4-methyl zur Bestimmung der Lipase in Korperfii.issumb elliferone. Anal. Bioch em. 21: 279-285. igkeiten und un Gewebe. Biochem. Z. 152: 14. Chino, H., and L. I. Gilbert. 1965. Determina504- 522. 5. Grossberg, A., P. Guth, S. Komarov, and tion of lipase activity by fiorisil column chromatography and liquid scintillation H. Shay. 1953. A phototurbidimetric method spectrometry. Anal Biochem. 10: 395-400. for determination of lipase in canine pancreatic juice. R ev. Canad. Bioi. 12: 495-508. 15. Morgan, R. G. H., J. Barrowman, H. FilipekWender, and B. Borgstrom. 1967. The lipo6. Borgstrom, B. 1957. Determination of panlytic enzymes of rat pancreatic juice. Biocreatic li pase in human small intestinal conchim. Biophys. Acta 146: 314-316. tent. Scand. J . Clin . Lab. Invest. 9: 226-228. 7. Ron a, P., and L. Michaelis, 1911. Uber Ester 16. Hofmann, A. F. 1964. The function of bile und Fettspaltung im Elute und im Serum. salts in fat absorption. M.D. Thesis, University of Lund, Sweden. Biochem. Z . 31: 345-354. 8. Nachlas, M. M., and A. M . Seligman. 1949. 17. Nachlas, M. M ., and R. Blackburn. 1958. The colorimetric determination of urinary Evidence for th e specificity of esterase and lipase by t he use of three chromogenic lipase. J . Bioi. Chern. 230: 1051-1061. 18. Kramer, S. P., M. Bartalos, J. N. Karpa, substrates. J. Bioi. Chern. 181: 343-355. J. S. Mindel, A. Chang, and A. M. Selig9. Huggins, C., and J. Lapides. 1947. Chromoman. 1964. D evelopment of a clinically genic substrates: acyl esters of p-nitrophenol useful colorimetric method for serum lias substrates for the colorimetric deterpase. J . Surg. R es. 4: 23-35. mination of esterase. J. Biol. Chem. 170 : 467-482. 19. Fritz, P. J., and P . Melius. 1963. Mechanism of activation of hog pancreatic lipase by 10. Raderecht, H. J. 1959. Zur Bestimmung der sodium taurocholate. Canad. J. Biochem: Serumlipase. 1. Bestimmung mit Phenyllaurat als Substrat. Clin. Chim. Acta 4: 41: 719-730 . 221-226. 20. Desnuelle, P., and P . Savary. 1963. Specificities 11. Yagi, K, Y. Yamamoto, and J. Okuda. 1961. of lipases. J . Lipid R es. 4: 369-384. Hydrolysis of fatty acid esters of ribofl avin 21. Guilbault, G. G., and D. N. Kramer. 1964. F luorimetric determination of lipase, acylby pancreatic lipase. Nature (London) 191: 174-175. ase, alpha- and gamma-chymotrypsin and inhibitors of th ese enzymes. Anal. Chern. 12. Kram er, D. N ., and G. G. Guilbault. 1963. A 36 : 409-412. substrate for the fiuorimetric determination of lipase activity. Anal. Chem. 35 : 22. Ravin, H. A., and A. M. Seligman. 1953. 588-589. D eterminants for the specificity of action 13. Jacks, T. J., and H . W. Kircher. 1967. Fluoriof pancreatic lipase . Arch. Biochem. 42: 337-354. metric assay for t he hydrolytic activity of