Serum Lipase Determination with an Olive Oil Substrate Using a Three-Hour Incubation Period

Serum Lipase Determination with an Olive Oil Substrate Using a Three-Hour Incubation Period Roderick P. MacDonald and Royal 0. Lefave The effect of various emulsifying agents, ph, buffers, and accelerators on the Cherry- Crandall procedure for determining serum lipase was studied. A method is proposed which uses a 3-hour incubation period. Olive oil emulsified with 7% acacia is the substrate. This is buffered with tris buffer, ph 8.0; magnesium acetate and EDTA are added as accelerators. Other conditions which result in an increased rate of hydrolysis are discussed. 1T1E DETERMINATION of serum lipase levels is generally recognized to be a valuable aid in the diagnosis of acute pancreatitis (1-3). Following an attack of acute pancreatitis, lipase remains elevated in blood after the amylase level has returned to normal. The Cherry-Crandall (4) procedure for determination of serum lipase has been in general use for many years, and is the basis of the method in Standard Methods of Clinical Chemistry (5). The inherent disadvantage of this technic is the required 16- to 24-hour incubation period (6). Methods have been proposed requiring a shorter time of incubation. Bunch and Emerson (7), using what is essentially the Nothman, Pratt, and Benotti (8) procedure, found adequate amounts of hydrolysis after 4 hours incubation-provided the enzyme level was elevated. Tietz (9), using olive oil with barbiturate buffer at ph 8.0, was able to obtain sufficient hydrolysis for satisfactory determination after 6 hours incubation. Olive oil has generally been used as the substrate. Other substrates proposed include tributyrin (10), alpha naphthyl laurate (ii), beta naphthyllaurate (12,13), phenyllaurate (14), and Tween 20 (15). From the Department of Pathology, Harper Hospita1, Detroit, Mich. Supported in part by the Mrs. Bruce Lockwood Fund. Received for publication Oct. 6, 1961. 609

510 MACDONALD & LEFAVE Clinical Chemistry Bunch and Emerson followed 8 patients with pancreatitis and found elevated lipase levels only when using an olive oil substrate. No elevation in tributyrinase occurred. Henry et at. (16) confirmed this finding and reported that the lipolytic enzyme (or enzymes) occurring in sera from patients with acute pancreatitis differs from the lipase in pancreatic extracts. They proposed the term pancreatitis lipase for the enzyme which rises during acute pancreatitis and requires olive oil as a substrate. The rate of hydrolysis of olive oil by lipase is slow, and various accelerators for the reaction have been proposed. These include bile salts (17,18), calcium ions (17), taurocholate with eserine (13), cholesterol (19), lima-bean trypsin inhibitor (20), and others (21,22). We have investigated several modifications of the Cherry-Crandall method in an attempt to decrease the time of incubation. We used only olive oil as a substrate, since its specificity is indisputable. We performed approximately 1800 determinations using various emulsifying agents, buffers, and accelerators, and variations in ph in order to find conditions which would result in the highest rate of hydrolytic activity. We were able to obtain a satisfactory amount of hydrolysis after 3 hours incubation using 7% acacia as an emulsifying agent, and adding EDTA and Mg as activators. All modifications studied were compared to the 16-hour technic of Henry in Standard Methods of Clinical Chemistry (5). We will refer to this technic as the standard method. In all experiments, units of lipase are expressed as the milliliters of 0.05 N NaOH required to neutralize the fatty acids produced by hydrolysis under the conditions of the test. Experimental Table 1 gives typical results of 4-hour and 16-hour incubation periods using the standard method. This is in agreement with Henry (16), who found that values at 4 hours averaged about 50 per cent of those at 16 hours. Table 1. LIPASE VuEs AT 4- AND 16-Houa INCUBATION (STANDARD METHOD) 4hr. l6hr. 0.29 0.42 0.23 0.36 0.26 0.57 1.55 2.95

Vol. 8, No. 5, 1962 SERUM LIPASE 511 Emulsifying Agents Table 2 shows typical results obtained when various emulsifying agents were substituted in the standard method. Tween 20, Triton X-100, and Tergitol were discarded because the emulsion separated on standing. Acacia did not permit separation, and we confirmed the find- Table 2. EFFECT Op VARYING EMULSIFYING AGENTS IN LIPASE STANDARD METHOD Emulsifying agent Increa8e in rate of activity Amount of separation Tween 20 - High Triton X-100 - High Tergitol - Some Acacia, 5% - None Aeacia, 7% 1+ None Aerosol + acacia 2+ None Aerosol O.T. 3+ Slight ing of Tietz (9) that a 7% concentration was superior to a 5%. Olive oil emulsions prepared with Aerosol OT gave highest rates of hydrolysis, but tended to separate on standing. A mixture of both aerosol and acacia had only a slight advantage over the acacia alone. During our studies we tried several brands of mineral oil. We found the type recommended by Tietz (Fisher, Best, U.S.P.) to be best-producing more stable emulsions, giving more reproducible results, and definitely showing higher rates of hydrolysis. Preservatives Sodium benzoate is commonly used as a preservative for the olive oil substrate. We also used propylparasept to determine if either compound lowered the hydrolytic rate. Freshly prepared substrate was used simultaneously with substrates containing the 2 preservatives. No differences were noted in the 3 series. Buffers and ph Buffer systems studied were M/15 phosphate buffers at ph 7.0 (5), 7.4, and 8.2; 1 M NH4C1/N114011 (19) at ph 8.5; and 0.2 M Tris buffer at ph 8.0 (9). Representative results are shown in Table 3. Tris buffer at ph 8.0 gave best results, although the other buffers above ph 8.0 gave rates of hydrolysis higher than the phosphate buffers of ph 7.0 and 7.4. In a single instance when the Tris buffer was incorrectly prepared, and was actually ph 7.8, lower results were obtained.

512 MACDONALD & LEFAVE Clinical Chemistry Table 3. EFFECT OF VARYING BUFFER AND ph IN LIPASE STANDARD METHOD AFTER 16 Howis INCUBATION USING DIFFERENT SPECIMENS NH4CZINHCOH P04 buffer buffer Trw buffer ph 7.0 ph 7.4 ph 8.2 ph 8.5 ph 8.0 0.77 0.81 0.89 0.92 0.77 1.07 0.89 1.07 1.12 1.33 0.40 1.06 0.56 1.03 0.45 0.95 Accelerators We studied the effect of possible accelerators on a substrate mixture containing olive oil, 7% acacia, sodium benzoate, and Tris buffer, ph 8.0-although occasionally we also varied these constituents. Representative results obtained from adding 0.2 ml. of 0.01 M calcium acetate and 0.5 ml. of 0.01 M MgSO4 and using acacia plus aerosol as emulsifying agents are shown in Table 4. These concentrations of Ca++ and Mg+ + did not appear either to increase or decrease the rate of enzymatic hydrolysis of the substrate. Other salt concentrations had a similar effect although much higher levels had an inhibitory effect (greatest from 1 ml. of 0.01 M calcium acetate, less from 0.5 ml.). The sodium salt of ethylene diamine tetra-acetic acid was added to Table 4. EmCT os CA AND M& SALTS ON LIPASE Ac,rivlTY* 4-hr. incubation 0.2 ml. 0.01 H 0.8 ml. 0.01 H 16-hr. incubo.t4ont No salt added Ca(02H80,), MgSOe 1.04 0.51 0.47 0.28 0.99 0.60 1.07 0.48 1.15 0.44 0.95 0.43 2.04 1.21 0.39 0.17 0.81 0.56 Substrate: Olive oil, Trw buffer, acacia and aerosol, at ph 8. IStandard Method.

Vol. 8, No. 5, 1962 SERUM LIPASE 513 Table 5. COMPARISON OF LIPASE VALUES OBTAINED USING 3H0UR PROPOSED METHOD AND 16-HouR STANDARD METHOD 16-hr. incubation* 3-hr. incubationt 3.50 2.16 3.50 1.98 3.60 2.03 3.60 2.10 5.76 3.86 3.70 1.07 11.32 9.14 5.02 2.82 1.57 1.20 0.84 0.77 0.83 0.69 5Standard method. fusing EDTA and Mg. an incubation mixture containing olive oil, acacia, sodium benzoate, Tris buffer, ph 8.0, and various concentrations of Mg+ +. The optimum Mg salt concentration was 0.5 ml. of 0.01 M Mg(C2H302)2. The rate of hydrolysis was only slightly better than when the same amount of MgSO4 was substituted. EDTA concentrations used were 0.1, 0.2, 0.4,0.5 ml. of 0.O1M EDTA (0.372%) and 0.1 ml. of 0.5% EDTA. The optimum level of the EDTA is 0.1 ml. of a 0.5% solution. Substrate mixtures prepared with either the EDTA or Mg + alone did not result in the increased hydrolytic rate observed when both were included. Typical results after 3 hours incubation using the optimum EDTA and Mg + are shown in Table 5. Table 6 gives typical results from the substitution of a calcium salt for the magnesium salt. There is no in- Table 6. EFFECT OF CA + EDTA ON LIPASE ACTIvITY* 4-hr. incubation 16-hr. incubation No salt added 0.3 ml. 0.01 H CaCI, + EDTA 1.18 0.51 0.78 0.40 0.99 0.57 1.17 0.51 0.70 0.31 1.46 0.80 0.46 0.21 0.50 0.28 58nbstrate: Tris buffer, ph 8.0; acacia 7%; 0.1 ml. 0.01 M DTA.

514 MACDONALD & LEFAVE Clinical Chemistry crease in the hydrolytic rate above that of the standard method when Ca + and EDTA are added to the incubation mixture. Tietz found that 3 ml. of emulsion resulted in less separation during the 6-hour incubation period than lesser amounts of emulsion. We also found this to be true in our procedure. We tried adding 0.5, 1.5, 2.4, and 2.5 ml. of water to the 3 ml. of emulsion, and obtained higher results with the latter two amounts. A comparison of results obtained on the same specimen in 1 through 6 hours incubation using the Standard, Tietz, and our procedure is shown in Fig. 1. The break in these curves between 3and 4 hours was consistently found. This break is accentuated as the rate of hydrolysis increases. 3.0 Mg + EDTA 2.5 Tletz Method w 2.0 Fig. 1. Lipase activity; Std. Method comparison of three methods I. 5 at hourly intervals for 6 hours. I.o 0.5 0 0 I 2 3 4 5 6 INCUBATION TIME (HOURS) Method On the basis of our experimental results, the following procedure is proposed. Reagents Olive Oil Emulsion Transfer 100 ml. of distilled water to a Waring Blendor, add 7 gm. of acacia (Gum Arabic, TLS.P.) and 0.2 gm. of sodium benzoate. Blend until dissolved. Slowly add 100 ml. of olive oil (Fisher, Best, TJ.S.P., Catalog No. 0-111) and mix for 10 mm. at high speed. A hand homogenizer may be used to prepare this emulsion if the mixture is passed through it at least 10 times. Store this reagent in a refrigerator. Avoid freezing or exposure to high temperatures.

Vol. 8, No. 5, 1962 SERUM LIPASE 55 Shake well before using. Discard reagent if separation of the emulsion is noticeable after shaking. N NaOH Indicator Solution Dissolve 1.0 gm. of thymolphthalein and 0.5 gm. of phenolphthalein in 95% ethanol and dilute to 100 ml. Tris buffer, ph 8.0 Mix 25 ml. of 0.2 M tris (hydroxymethyl) aminomethane (2.43 gm./100 ml.) with 25 ml. of 0.1 N HC1. Dilute to 100 ml. with distilled water. This reagent should be checked on a ph meter, and if necessary adjusted to the correct ph. Ethanol, 95 per cent EDTA solution, 0.5 per cent Dissolve 0.5 gm. of the disodium salt of ethylenediarnine tetra-acetic acid in 100 ml. of distilled water. EDTA solution, dilute Dilute 4 ml. of 0.5 per cent EDTA solution to 100 ml. with distilled water. Magnesium acetate, 0.01 M Dissolve 2.145 gm. of Mg(C2H302)2. 41120 in water and dilute to 100 ml. Procedure Transfer 2.5 ml. of dilute EDTA solution (equivalent to 0.1 ml. of 0.5% EDTA solution plus 2.4 ml. of water) to each of 2 test tubes. Add 0.5 ml. of 0.01 M magnesium acetate, 1.0 ml. of tris buffer, ph 8.0, and 3.0 ml. of olive oil emulsion. Into 1 of these tubes pipet 1.0 ml. of serum. Label this tube test and the other tube blank. Vigorously shake both tubes (a vortex mixer is very convenient). Transfer another 1.0 ml. sample of serum to a 50-mi. Erlenmeyer flask labeled blank. Store this flask in the refrigerator. Incubate the test and blank test tubes in a 37#{176} water bath for 3 hours. Transfer the contents of the blank tube into the similarly labeled Erlenmeyer flask. Transfer the contents of the test tube into a clean 50-ml. Erlenmeyer flask. Add 3.0 ml. of 95% ethanol to both flasks by first pipetting the alcohol into the test tubes, then shake and transfer this solution to the appropriate flask. Add 4 drops of indicator solution to each flask. Using a 5.0-ml. microburet, titrate the mixtures to a definite blue color with 0.05 N NaOH. The color intensity of both flasks should be matched. Subtract the blank titration value from the test value. This difference represents the units of lipase, expressed as milliliters of 0.05 N NaOH required to neutralize the fatty acids produced by hydrolysis under the conditions of the test.

516 MACDONALD & LEFAVE Clinical Chemistry Normal Values Figure 2 shows the units of lipase found in the sera of a series of 98 subjects. None of these was known to have symptoms which would suggest the possibility of an abnormal lipase value. This graph shows skewing toward the lower values, therefore the data were converted to Cl) I: t10 e Fig. 2. Lipase levels determined in 98 normal sub- 6 jects, using proposed 3-hour method. 2 -i 0 203.40 5O0 70.8OO 1.00 UNITS LIPASE logarithms and the distribution treated as lognormal (23). The geometric mean was 0.25 U. and the 95 per cent limits 0.06 to 1.02 U. The chi-square formula for testing fit was applied to the lognormal distribution and this distribution was found to be appropriate. Discussion The purpose of this work was to study various modifications of the Cherry-Crandall technic which would permit shortening the period of incubation. We limited our investigation to an olive oil substrate since it is generally acceptable (16, 24, 25). We recognized that while it would be necessary to use normal sera for the hundreds of analyses in our preliminary work, data supporting any new procedure would have to be obtained using abnormal sera. The superiority of acacia, tris buffer at ph 8.0, and 3.0 ml. of emulsion confirm the work of Tietz. Many other emulsifying agents have been proposed (13, 19, 20, 26, 27). Wills (19) found that anionic detergents inhibit, cationic detergents (under certain conditions) increase, and nonionic detergents have no effect on the rate of reaction. Minard (26) used Tween 20, 60, and 80 to emulsify corn oil, with pork pancreatin as a source of enzyme, and found inhibition of the reaction -the greatest in Tween 80 and least in Tween 20. The inhibition could be reversed by bile salts. The probable role of bile salts is as an emulsifier rather than as an accelerator. The Tweens are monoesters of

Vol. 8, No. 5, 1962 SERUM LIPASE 517 polyethylene sorbitan with oleic, stearic, and lauric acids, and in themselves may be a substrate for pancreatic lipase (15). We investigated some possible emulsifying agents. Although Aerosol 0.T. gave somewhat higher values, acacia had to be added to prevent separation. With aerosol better results were obtained in a 4-hour incubation period than in a 16-hour period-using as a comparison equivalent times for the standard method. Separation would account for these poorer results after the longer incubation time. The slight advantages of the combined aerosol and acacia did not recommend this technic. Tietz reported the highest hydrolytic rate at ph 8.0 (9). He recommended a barbiturate buffer, but noted that the tris buffer had a slightly better buffer capacity and a slightly higher rate of hydrolysis. We found that all of the higher p11 buffers gave better results, but selected the tris buffer because of these advantages. Tietz pointed out that since serum is a strong buffer, it is the ph of the mixture after addition of the serum that is important. In the procedure we are proposing, the ph of the mixture after addition of serum varies from 7.82 to 8.01. The use of tris as a buffer has been discussed by Gomori (28). Tietz studied the activity of other serum esterases. Using methyl butyrate as a substrate, he found an optimum p11 of 7.0. Therefore, the higher ph (8.0) not only gives a higher hydrolytic rate for lipase, hut at least partially eliminates the effect of esterases proper. Tietz stated that the optimal ph for determination of lipase in serum from patients with acute pancreatitis and from normal subjects is the same. Our studies would support this, since there is an increased rate of activity in normal sera at ph 8.0. There is conflicting evidence as to whether olive oil lipase is normally found in human serum. Comfort and Osterberg (29) believe it is present, while Gomori (13) states there are only negligible levels of lipase in normal human serum. It seems probable that pancreatic lipase is actually a group of related enzymes (30). The predominant enzyme being measured would depend on the conditions of the test procedure. Serum lipase may be either pancreatic or extrapancreatic in origin, and recurrence of serum lipase may exist 2-3 weeks after total pancreatectomy (8). Willst#{228}tter (17) suggested that Ca ions activate lipase, acting by removal of the fatty acids formed during hydrolysis by formation of un-ionized or insoluble soaps, thereby preventing a decrease in hydrolytic rate by a reverse reaction. Others have also reported this accelerating effect of Ca + (24, 26). Henry (16) reported inhibition of the reaction by addition of 5 ml. of 2% calcium acetate. We found no effect from lower concentrations of Ca++, but inhibition from higher

518 MACDONALD & LEFAVE Clinical Chemistry concentrations. Nonetheless, the formation of insoluble calcium soaps from the fatty acids released by hydrolysis seems to be a reasonable assumption. Mg ion has been suggested as a lipase activator (31) but has not to our knowledge been used in the assay of the enzyme. Our results after adding it to the incubation mixture were disappointing. However, inclusion of EDTA in the mixture gave considerably better results. The combination of Mg + and EDTA in the incubation mixture resulted in higher rates of hydrolysis than any other technic which we used. The use of EDTA as an accelerator was suggested by the work of Matsuo and Greenberg (32), who used it to neutralize the inhibitory effect of heavy metals on homoserine deaminase. We postulate that the action of the EDTA is to bind the Ca already present in the serum. This seems supported by the finding that the optimum level of EDTA for increased rate of hydrolysis is also approximately the amount of EDTA required for chelating the Ca ions ordinarily found in serum. The EDTA would prevent the formation of calcium soaps and not permit Ca+ + to act as an inhibitor. Since EDTA preferentially chelates Ca before the added Mg would still exist in an ionized form, and be available to act as an enzyme accelerator. The formation of magnesium soaps is a possibility, but we are unable to explain the actual role of the magnesium ion in the reaction. The EDTA or Mg added individually did not increase the hydrolytic rate, so that both were contributing to the reaction. We tried adding an excess of EDTA after the incubation period with the idea of removing the ions participating in soap formation, but these experiments did not give any higher titration values. The use of magnesium sulfate instead of magnesium acetate did not appreciably alter the hydrolytic rate. The break in the time curves shown in Fig. 1 was a consistent finding. Mattson and Beck (33) have shown that hydrolysis of triglycerides proceeds in a series of stepwise reactions from triglyceride to 1,2-diglyceride to 2-monoglyceride. This break may represent the end of one of these steps and the beginning of the next. We did not study optimum temperature, but Henry has shown this to be 40#{176}.However, it is usually more convenient to incubate at 37#{176}. In one series, when a water bath was erroneously set at 350, lower values were obtained. The use of thymolphthalein as an indicator for titrating the liberated fatty acids to ph 10.5 was recommended by Henry (8). However, this gives a rather indistinct end-point. Addition of 0.5 gm. of phenolphthalein to the 1% thymolphthalein solution resulted in a sharper

Vol. 8, No. 5, 1962 SERUM LIPASE 519 blue end-point at the equivalence point of about ph 10.5, determined by simultaneously checking the ph with a meter. Henry (16) found the blank titration practically constant for a particular olive-oil emulsion and that a blank need not be run for each serum. Tietz recommended an individual blank titration with each serum sample. We found the blank titration value to be fairly constant on all sera except those which were icteric or which contained some other colored substances. If all sera are clear, one blank determination will be satisfactory for the entire series. In all cases in which abnormal values were found using the 16-hour standard method we also found abnormal values in our 3-hour method. These 3-hour values are reasonably elevated for convenient titration, and this technic represents an actual increase in the hydrolytic rate of the enzyme and not a portion of the 16-hour value obtained by the standard method. References 1. Comfort, M. W., Am. J. Digest. Di.s..5. Nutrition 3, 817 (1936). 2. Comfort, M. W., and Osterberg, A. E., Proc. Staff Meet. Mapo Clinic 15, 427 (1940). 3. Ingelfinger, F. 0., New Engl. J. Med. 235, 653 (1946). 4. Cherry, Q. S., and Crandall, L. A., Jr., Am. J. Phpsiol. 100, 266 (1932). 5. Henry, R. J., In Standard Methods of Clinical Chemi8try, New York, Academic Press, Vol. 2, p. 86, 1958. 6. Machella, T. E., A. M. A. Arch. mt. Med. 96, 322 (1955). 7. Bunch, L. D., and Emerson, B. L., Gun. Chein. 2, 75 (1956). 8. Nothman, M. M., Pratt, T. D., and Benotti, J., J. Lab. Clin. Med. 33, 833 (1948). 9. Tietz, N. W., Borden, T., and Stepleton, J. D., Am. J. Gun. Path. 31, 148 (1959). 10. Goldstein, N. P., Epstein, J., and Roe, J. H., J. Lab. din. Med. 33, 1047 (1948). 11. Katz, S., Am. J. Gastroent. 27, 479 (1957). 12. Seligman, A. M., and Nachlas, M. M., J. dun. IflVe8t. 29, 31 (1950). 13. Gomori, G., Am. J. GUn. Path. 27, 170 (1957). 14. Saifer, A., and Perle, G., GUn. Chem. 7, 178 (1961). 15. Archibald, H. M., J. Biol. Chein. 165,443 (1946). 16. Henry, B. J., Sobel, C., and Berkman, S., dun. Cheon. 3, 77 (1957). 17. Willst#{228}tter,R., Waldschmidt-Leitz, E., and Memmen, F., Z. f. physiol. chem. 125, 93 (1923). 18. Loevenliart, A. S., and Souder, C. 0., J. Biol. Chem. 2, 415 (1906-07). 19. Wills, E. D., Biochein. J. 60,529 (1955). 20. Tauber, H., Proc. Soc. Expt. Biol. Med. 90, 375 (1955). 21. Weinstein, S. S., and Wynne, A. M., J. Biol. chem. 112, 649 (1935-36). 22. Mattson, F. H., and Beck, L. W., J. Biol. Chem. 214, 115 (1955). 23. Henry, H. J., Aim. J. CUn. Path. 34, 326 (1960). 24. King, E. J., Gun. Chem. 3, 507 (1957). 25. Ravin, H. A., and Seligman, A. M., Ar#{244}h.Biochem. (5Biophy8. 42, 337 (1953). 26. Minard, F. W.,,J. Bioi. Chem. 200, 657 (1953). 27. Fiore, J. V., and Nord, F. F., Arch. Biochem. 23, 473 (1949). 28. Gomori, G., Proc. Soc. Expt. Biol. Med. 62, 63 (1946). 29. Comfort, M. W., and Osterberg, A. E., J. Lab. Clin. Med. 20, 271 (1934). 30. Frazer, A. C., Power, W. F., and Sammons, H. G., Internatl. Cong. Biochein., Absir. of GommunioatioRI, p. 594, Cambridge, 1949. 31. Smorodintsev, I. A., Cited by A. Ia. Pleshchitser (Gor kii), Clin. dhein. 4, 429 (1958). 32. Matsuo, Y., and Greenberg, D. M., J. Biol. dhem. 234, 507 (1959). 33. Mattson, F. H., and Beck, L. W., J. Biol. Clse,n. 219, 735 (1956).