CLINICAL CHEMISTRY Originl Article Lipse nd Pncretic Amylse Activities in Tissues nd in Ptients with Hypermylsemi FRED APPLE, PH.D, PETER BENSON, M.D., LYNNE PREESE, MT, M.B.A., STEVEN EASTEP, M.D., LAURA BILODEAU, M.D., AND GREG HEILER, M.D. Lipse, pncretic mylse, nd totl mylse ctivities were mesured in nondisesed nd disesed humn pncretic tissues nd in six different loctions of the humn digestive system. In ddition, it ws determined whether serum lipse nd pncretic mylse tests could replce the totl mylse test to improved dignostic efficiency in the evlution of cute pncretitis in hypermylsemic ptients. Nondisesed pncretic tissue contined 4.5 times more lipse ctivity thn totl mylse ctivity. Disesed pncretic tissue contined less ctivity for both lipse nd totl mylse compred to norml tissue. The totl mylse ctivity of the pncres ws comprised solely of pncretic mylse. Tissue obtined from six different ntomic loctions in the digestive system contined 35 to 45 times less lipse nd totl mylse ctivity compred to the pncres. Totl mylse ctivity of the digestive system tissues were comprised of 25% pncretic nd 75% slivry isomylses. Lipse, pncretic mylse, nd totl mylse levels lso were determined in seril serum smples from 17 consecutive hypermylsemi ptients dmitted with possible cute pncretitis. The serum lipse level remined higher thn norml longer thn either the totl mylse nd pncretic mylse levels. In ptients with hypermylsemi of pncretic origin, poor correltion ws observed t dmission between serum pncretic mylse nd serum lipse. Not ll ptients with elevted lipse hd n elevted pncretic mylse level nd vice vers. However, in every ptient pncretic disese would hve been detected by the elevtion of either lipse or pncretic mylse levels. Dignostic efficiency for pncretic disese using serum pncretic mylse, lipse, nd totl mylse tests ws 94.1%, 76.5%, nd 64.7%, respectively. These dt suggest tht lipse nd pncretic mylse tests re specific for the pncres nd might be considered replcements for totl mylse s the stt or routine lbortory test for the dignosis of pncretic tissue injury. (Key words: Pncretic injury; Lipse; Pncretic mylse; Serum enzyme ssys) Am J Clin Pthol 1991; 96:610-614 Severl orgns contribute to mylse ctivities in serum, nd vriety of extrpncretic disorders contribute to n increse in totl serum mylse ctivity. 3 In norml individuls, totl serum mylse ctivity results from mixture of pncretic isomylse (found lmost exclusively in the pncres) nd slivry isomylse (found in vrious orgns). Overll pncretic isomylse constitutes bout 40% of the totl mylse ctivity. 4 The cinr cells of the pncres lso re the primry source of serum lipse. Thus, serum lipse ctivity should increse the specificity for the dignosis of cute pncretitis. The dignosis of cute pncretitis is not uncommonly From the Deprtment of Lbortory Medicine nd Pthology, mde Hennepin County Medicl Center, Minnepolis, in the presence of normomylsemi. In recent Minnesot. The dignosis of cute pncretitis hs lwys been chllenge to clinicins. During the pst 10 yers, techniques such s ultrsonogrphy, lprotomy, endoscopic retrogrde cholngiopncretogrphy, nd computed xil tomogrphy, s well s clinicl findings hve ided in the evlution of pncretitis.' However, the dignosis often hs relied on lbortory mesurement of serum totl mylse ctivity. 2 With the incresing incidence of pncretitis during the pst severl yers, efforts hve been concentrted on improving the specificity nd simplicity of enzyme ssys for the dignosis of cute pncretitis. Received November 5, 1990; received revised mnuscript nd ccepted for publiction My 17, 1991. Supported in prt by Boehringer Mnnheim Dignostics nd Robert Gnz, M.D., for the collection of tissue specimens. Address reprint requests to Dr. Apple: Clinicl Lbortories #812, Hennepin County Medicl Center, 701 Prk Avenue South, Minnepolis, Minnesot 55415. study by Clvien nd ssocites, 5 68% of 65 normomylsemic ptients with cute pncretitis were found to hve elevted lipse levels. Until recently, the clinicl efficcy of pncretic mylse nd lipse ssys hve been limited by the lck of specific nd sensitive utomted ssys. 2,6 Improvements in technology for both the lipse nd pncretic isomylse ssys now fford lbortory workers 610
APPLE ET AL. 611 Lipse nd Pncretic Amylse Activity in Hypermylsemi the bility to provide clinicins with improved dignostic enzymology testing for the evlution of ptients with cute or chronic bdominl distress with possible pncretitis, replcing the nonspecific serum totl mylse 7-9 ssy. The present study hd two primry gols. First, we quntitted the ctivity of lipse, pncretic mylse, nd totl mylse in nondisesed nd disesed humn pncreses nd in humn digestive system tissues to determine tissue specificity for ech enzyme. Second, we determined the clinicl dignostic efficiency of pncretic mylse nd lipse ssys in serum from ptients dmitted through the emergency deprtment with the possible dignosis of cute pncretitis. Tissues METHODS Pncreses were hrvested within 9 hours fter deth from two mle subjects (A nd B) who died s result of hed trum in motor vehicle ccidents. Neither hd clinicl or gross indiction of pncretitis. Pncreses lso were hrvested within 7 hours fter deth from femle subject (C) nd mle subject (D) who died s result of trum in motorcycle ccident nd from drug overdose, respectively. Both of the ltter subjects hd known histories of lcoholism nd chronic pncretitis. Ech of the ltter intct pncreses t hrvest hd gross indictions of ptchy ft necrosis nd hemorrhge. Pncres frgments (100-150 mg) were obtined from six ntomic loctions from the hed (loction 6) to til (loction 1) of the pncres (Tble 1). Visible res of necrosis were voided during tissue smpling. In ddition, tissue specimens (5 mg) lso were obtined t biopsy from six different n- Antomic Loction TABLE 1. LIPASE AND AMYLASE ACTIVITIES IN NORMAL AND DISEASED PANCREATIC TISSUE FROM FOUR SUBJECTS Nondisesed Lipse, U/g Disesed A B C D Nondisesed Amylse, U/g Disesed A B C D 1 1624 1700 540 950 320 380 180 265 2 1772 1730 2320 1850 388 382 432 295 3 1492 1555 1120 820 304 320 344 422 4 2124 2010 1535 1295 524 595 132 206 5 1904 2040 1938 1685 428 480 112 226 6 1900 1985 824 1108 444 425 90 252 Men 1802 1836 1380 1301 401 434 215 277 SD 224 201 678 438 82 97 140 77 Loction I denotes the til nd loction 6 denotes the hed of the pncres; results re expressed s U/g wet weight of tissue; mylse ctivity is 100% pncretic mylse. SD stndrd devition. tomic loctions of the digestive system, s well s from six different pncreses. Tissues were trimmed to remove excess connective tissue nd immeditely frozen t 70 C nd thwed overnight t 4 C before the nlyses. All tissues were homogenized in 1- to 5-mL ice-cold phosphte buffer (0.2 mol/ L potssium phosphte, ph 7.0) nd centrifuged to remove cellulr debris. The superntnts were nlyzed for totl mylse, pncretic mylse, nd lipse ctivities, s described below (in the Ptients section). Results re expressed s U/g wet weight tissue or s U/mg totl protein. Agrose gel electrophoresis ws used to determine qulittively the distribution of tissue isomylses. 10 Ptients Serum ws obtined t dmission s well s pproximtely 12, 24, nd 48 hours fter dmission in 17 consecutive hypermylsemic ptients dmitted through the emergency deprtment with the possible clinicl dignosis of cute pncretitis. Serum ws frozen t - 20 C until time of ssy. For enzyme clernces, men enzyme vlues were clculted t ech time point (Fig. 2). The dignosis of pncretitis nd dmission to the hospitl initilly ws bsed on history of bdominl pin tht ws consistent with cute pncretitis. The enzyme vlues obtined in the emergency deprtment were not used to estblish the dignosis but were used for clinicl confidence. The etiology of the pncretitis in most cses (9 of 14) ws lcohol buse. The etiologies of the remining five cses were unknown. The finl dignoses in 35% of cses were confirmed by lprotomy, computed tomogrphy, ultrsonogrphy, or t utopsy. Serum totl mylse nd lipse (with colipse cofctor) ctivity were determined on the utomted Kodk Ektchem 400 nlyzer (Estmn Kodk, Rochester, NY) ccording to the mnufcturer's protocols. Reference rnges were 37-117 U/L nd 12-198 U/L, respectively. Pncretic isomylse ws determined on Cobs-Bio (Roche Dignostic Systems, Montclir, NJ) using the Boehringer Mnnheim (Indinpolis, IN) enzymtic colorimetric test, pncretic lph mylse p-nitrophenylmlthoheptoside. 9 In this ssy, the ctivity of humn slivry isomylse is inhibited by monoclonl ntibody tht does not ffect pncretic isomylse. The ctivity of pncretic isomylse is then mesured t 37 C using p-nitrophenyl-d-mltopeptoside substrte. The reference rnge ws 0-115 U/L. (It should be noted tht under the current ssy conditions, totl mylse nd pncretic mylse levels re mesured by different ssys; therefore, 1 U/L of totl mylse does not equl 1 U/L of pncretic isomylse.) Dignostic efficiency, sensitivity nd predictive vlues of positive test were clculted. Vol. 96 No. 5
612 CLINICAL CHEMISTRY Originl Article U/G LIPASE, 2200-2000 - 1600-1800- 1400-3000 3 2000 LU < Q. 1000 3 D A, NORMAL 1 i 300 400 500 AMYLASE, U/G B, DISEASED D D G 1 600 i 1 1 1 1 1 1 1 0 100 200 300 400 500 AMYLASE, U/G FIG. 1. Reltionship between mylse nd lipse ctivity (U/g) in tissues obtined from nondisesed (norml) nd disesed pncreses. RESULTS Tble 1 shows the lipse nd mylse ctivities from six distinct ntomic loctions in nondisesed nd disesed pncretic tissue from four subjects. From til to hed in nondisesed tissue (subjects A nd B), lipse ctivity (1819 U/g) ws 4.5 times greter thn mylse ctivity (418 U/g) per grm of wet weight tissue. In tissue obtined from subjects (C nd D) with history of chronic pncretitis, there ws substntilly less ctivity for both lipse (1340 U/g) nd mylse (246 U/g), representing 26% nd 41% depletions. Fourfold differences in mylse nd lipse ctivities existed within ech disesed pncres. Figure 1 shows tht lthough there ws n excellent correltion (r = 0.90) between tissue lipse nd mylse in nondisesed pncreses, there ws very poor correltion (r = 0.17) in enzyme ctivity in the disesed tissue. Tble 2 shows the totl mylse, pncretic mylse, nd lipse ctivities is six different loctions in the digestive system compred to the pncres. The pncres contined 35 to 45 times more ctivity per milligrm of totl protein for totl mylse, lipse, nd pncretic mylse thn ny of the six different digestive system loctions. The totl mylse ctivity of the pncres consisted solely of pncretic mylse, nd the digestive system totl mylse ctivity ws comprised of less thn 50% pncretic mylse t ll six sites. The serum enzyme clernce profiles for totl mylse, pncretic isomylse, nd lipse from the 17 consecutive emergency deprtment dmissions with possible dignosis of cute pncretitis re shown in Figure 2. Although there ws good correltion of totl mylse with pncretic isomylse for ll smples (r = 0.81), the correltion improved (r = 0.95) when smples with norml lipse were excluded. A poor correltion existed in dmission specimens between serum pncretic isomylse nd lipse ctivities, s shown in Figure 3. Tble 3 shows the sensitivity, dignostic efficiency, nd predictive vlue of positive test for ll three ssys bsed on the dmission serum specimen. The dignostic sensitivity nd efficiency with this smll group of ptients for cute pncretitis ws best using serum pncretic mylse, followed by lipse nd totl mylse. DISCUSSION The dignosis of pncretitis often is difficult to mke becuse this condition must be differentited from other bdominl disorders with similr clinicl fetures.' Until TABLE 2. TOTAL AMYLASE, PANCREATIC AMYLASE, AND LIPASE ACTrVITY IN THE PANCREAS AND IN SIX DIFFERENT ANATOMIC LOCATIONS OF THE HUMAN DIGESTIVE SYSTEM Loction Totl Amylse Pncretic Amylse Lipse Pncres (n = 6) Duodenl bulb (n = 4) Pyloric ntrum (n = 11) Angulr notch (n = 1) Corpus (n = 15) Gstroesophgel junction (n = 1) Esophgus(n = 3) 100% pncretic 0.60 (0.73) 0.07(0.13) 0.010 0.08(0.15) 0.94(1.5) 9.0 (7.0) 0.28 (0.45) 0.02 (0.03) 0.01 (0.02) 0.02 (0.3) 7.8 (3.2) 0.13(0.22) 0.03 (0.04) 0.010 0.21 (0.36) 0.01 (0.01) ' Results re expressed s men (stndrd devition) of ctivities, U/mg totl protein. AJ.C.P. November 1991
APPLE ET AL. 613 Lipse nd Pncretic Amylse Activity in Hypermylsemi Dy After Admission to Hospitl o o 0 200 400 600 800 1000 1200 LIPASE U/L FlG. 2. Enzyme clernces fter the onset of bdominl pin in ptients dmitted for pncretitis for serum lipse, pncretic mylse, nd totl mylse ctivities; ULN = upper limit of norml reference rnge. Ech time point represents the men vlue of ech enzyme for ll 17 ptients. FlG. 3. Reltionship between dmission serum pncretic mylse nd lipse ctivities in ll ptients dmitted with possible dignosis of cute pncretitis (r = 0.16). vilble, improved serum lipse ssy (with colipse cofctor) nd pncretic mylse ssy (which uses inhibiting monoclonl ntibodies ginst slivry mylse) hve been shown cliniclly to be interchngeble tests for the dignosis of pncretitis. 5 ' 7 ' 9121415 Although it is not thoroughly representtive popultion for studying dignostic efficiency, the current study demonstrted tht serum pncretic mylse nd serum lipse were the most cliniclly sensitive nd dignosticlly efficient ssys in ptients treted in the emergency deprtment for possible cute pncretitis. This correlted with severl other lrger popultion studies. Lott nd co-workers 7 showed tht the clinicl sensitivity nd specificity of lipse ssy (100% nd 62%, respectively) ws much better thn totl mylse ssy (96% nd 34%, respectively) in 175 ptients dmitted for cute pncretitis. Clvien nd co-workers 5 showed tht cute pncretitis nd normmylsemi were not n uncommon combintion; however, the serum lipse level ws elevted in 68% of 65 normmylsemic cses. For the emergency dignosis of cute pncretitis in more thn 250 cses of vried clinicl findings, lipse nd pncretic mylse were considerbly more sensitive nd specific, with few to no flse-positive results when compred to totl mylse. 14 Finlly, Vn Lente nd ssocites 15 hve shown tht lipse nd pncretic mylse re interchngeble serum mrkers, with high clinicl sensitivity nd specificity for cute pncretitis. These findings demonstrte tht the mesurement of totl mylse is not sefficient s the new nd improved serum lipse- nd pncretic-specific mylse ssys. Multiple fctors contribute to the bsence of hypermylsemi in the presence of hyperlipsemi. These include n erlier return to norml for serum mylse during hospitliztion (Fig. 2) nd the inbility of disesed pncreses to relese mylse (Tble 1). In the present investigtion, we describe two dditionl mechnisms. First, lipse ctivity ws four times greter thn mylse ctivity in the pncres (Tble 1). Second, pncretic tissue obtined from chronic pncretitis subjects demonstrted substntil decline in both mylse nd lipse ctivity, with mylse ctivity showing greter decrese compred to lipse (41 % versus 26%; Tble 1, Fig. 1). These findings recently, the serum totl mylse test hs been the dignostic lbortory test of choice in this setting. However, severl studies (s well s the tissue nd serum results described in the current study) show tht serum lipse nd serum pncretic (specific) mylse ssys re more sensitive nd specific for the dignosis of pncretic injury 2,4,5,7,11-15 The rpid, simple-to-perform, nd redily TABLE 3. SENSITIVITY, EFFICIENCY, AND PREDICATIVE VALUES OF SERUM PANCREATIC ENZYME ASSAYS Sensitivity (%) Efficiency (%) Positive predictive vlue (%) Totl Amylse 64.3 64.7 90.0 Lipse 87.5 76.5 85.7 Pncretic Amylse 92.9 94.1 100 Vol. 96 No. 5
614 CLINICAL CHEMISTRY Article would explin why cute pncretitis, normomylsemi, nd hyperiipsemi re not n uncommon finding in clinicl prctice. Regrding the specificity of elevted serum lipse nd pncretic mylse, ourfindingsshowed tht only the pncres contined substntil ctivities of either enzyme when compred to severl other ntomic loctions in the digestive system (Tble 2). To our knowledge, this is the first report to mesure tissue lipse ctivities in the pncres nd digestive system. Our finding of smll quntity nd mixture of pncretic nd slivry isomylses in nonpncretic tissue correlted with the findings of Whitten nd ssocites. 3 From chrt review in the current study, it ppers tht elevtions of either pncretic mylse or lipse represent direct pncretic injury or secondry relese from the pncres due to other pthologic conditions. Combined mesurement of pncretic mylse with lipse or totl mylse with lipse did not increse the sensitivity or dignostic efficiency (89%, 85%, nd 86%, respectively). Specificity clcultions were not performed in the current study becuse of the high prevlence of disese (pncretitis, 14 of 17 ptients) in the smll ptient popultion studied. From ourfindingswe conclude tht (1) lipse nd pncretic mylse levels re specific for the pncres; (2) for the dignosis of cute pncretitis, clinicl sensitivity nd specificity re improved by mesuring lipse nd pncretic mylse levels; nd (3) ourfindings,together with those of others, provide sufficient informtion to consider replcement of the mesurement of totl serum mylse ctivity s n indictor of cute pncretitis. However, the elimintion of the mesurement of serum totl mylse in clinicl lbortories will depend on the generl vilbility of instrumenttion for the mesurement of serum lipse (colipse) nd serum pncretic mylse levels. When serum lipse nd pncretic mylse ctivities re found to be elevted, it should suggest some form of pncretic tissue injury. REFERENCES 1. Mooss AR. Dignostic tests nd procedures in cute pncretitis. N Engl J Med 1984; 311:639-643. 2. Kolrs JC, Ellis CJ, Levitt MD. Comprison of serum mylse pncretic isomylse nd lipse in ptients with hypermylsemi. DigDisSci 1984;29:289-293. 3. Whitten RO, Chndler WL, Thoms MG, et l. Survey of mylse ctivity nd isomylses in utopsy tissue. Clin Chem 1988; 34: 1552-1555. 4. Pnteghini M, Pgni F. Dignostic vlue of mesuring pncretic lipse nd the P 3 isoform of the pncretic mylse isoenzyme in serum of hospitlized hypermylsemic ptients. Clin Chem 1989;35:417-421. 5. Clvien PA, Robert J, Meyer P, et l. Acute pncretitis nd normomylsemi. Ann Surg 1989; 210:614-620. 6. Koehler DF, Eckfeldt JH, Levitt MD. Dignostic vlue of routine isomylse ssy of hypermylsemic serum. Gstroenterology 1982; 82:887-890. 7. Lott JA, Ptel ST, Swhney AK, et l. Assys of serum lipse: Anlyticl nd clinicl considertions. Clin Chem 1986; 32:1290-1302. 8. Rosenblum JL. Direct, rpid ssy of pncretic isomylse ctivity by use of monoclonl ntibodies with low ffinity for mcromylsemic complexes. Clin Chem 1988; 34:2463-2468. 9. Tietz NW, Burling A, Gerhrdt W, et l. Multicenter evlution of specific pncretic isomylse ssy bsed on double monoclonl-ntibody technique. Clin Chem 1988; 34:2096-2101. 10. Gillrd BK. Quntittive gel-electrophoretic determintion of serum mylse isoenzyme distributions. Clin Chem 1979; 25:1919-1923. 11. Werner M, Steinberg WM, Puley C. Strtegic use of individul nd combined enzyme indictors for cute pncretitis nlyzed by receiver-opertor chrcteristics. Clin Chem 1989; 35:967-971. 12. Kzmierczk SC, VnLente F. Incidence nd source of hypermylsemi fter crdic surgery. Clin Chem 1988; 34:916-919. 13. Hfkenscheid JC, Hessels M, Wetzels JF. Clernce of pncretic nd slivry mylse in norml subjects. Clin Chem 1985; 31: 162-163. 14. d'eril GM, Bosoni T, Lesi C. Pncretic mylse in serum for differentil dignosis of cute pncretitis nd cute bdominl diseses. Clin Chem 1989; 35:2142-2143. 15. VnLente F, Kzmierczk SC. Immunologiclly-derived pncretic mylse, pncretic lipse, nd totl mylse compred s predictors of pncretic inflmmtion. Clin Chem 1989; 35:1542.