Gut: first published as 10.1136/gut.28.3.330 on 1 March 1987. Downloaded from Guit, 1987, 28, 330-335

Raised serum concentrations of pancreatic enzymes in cigarette smokers

M A DUBICK, C N CONTEAS, H T BILLY, A P N MAJUMDAR, AND M C GEOKAS From the Enzymology Research Laboratory, Department ofMedicine, Veterans Administration Medical Center, Martinez, CA, and Departments ofMedicine and Biological Chemistry, University ofCalifornia, Davis, CA, USA

SUMMARY Circulating concentrations of digestive enzymes, certain lysosomal hydrolases and protease inhibitors were measured in 19 heavy smokers and 13 non-smokers before (basal) and at 15, 30, and 60 minutes after a single intravenous injection of secretin (75 CU). In smokers, basal serum amylase and immunoreactive pancreatic elastase 2 (IRE2) concentrations were about 100% and 25% higher respectively, than in the non-smokers, whereas, no differences were observed in basal immunoreactive cationic trypsinogen (IRCT) concentrations and in acid phosphatase and f- glucuronidase activities between the two groups. Furthermore, a single injection of secretin to cigarette smokers significantly increased serum amylase, IRCT and IRE2 by 155%, 200%, and 100%, respectively when compared with their corresponding basal levels. No such increment was observed in the non-smokers. In addition, there were no significant differences in serum trypsin or elastase inhibitory capacity or immunoreactive ac,-protease inhibitor and c(2- levels between smokers and non-smokers. The levels and inhibitory capacity of these protease inhibitors was also not affected by secretin injection. These data suggest that cigarette smoking enhances the http://gut.bmj.com/ responsiveness of the exocrine pancreas to a physiological stimulus such as secretin, with resultant substantial increase in the concentrations of pancreatic hydrolases in blood.

Cigarette smoking is a well recognised risk factor in injection in cigarette smokers. We have recently the aetiology of pulmonary emphysema,'- coronary shown that exposure of isolated rat pancreatic acini on October 1, 2021 by guest. Protected copyright. artery disease and aortic aneurysms, '7 and has been to nicotine greatly stimulates the secretion of pre- linked to the development of pancreatitis' and formed exportable hydrolases and newly synthesised pancreatic carcinoma. Although the mechanism(s) .'7 18 This observation indicates that nicotine that relate smoking to these diseases are not well exerts a direct effect on the exocrine pancreatic cell. understood, enzymes with elastase-like activity have In addition to nicotine, tobacco smoke also con- been implicated in the pathogenesis of emphysema tains other cytotoxic agents which may affect and aortic aneurysms."zL" exocrine pancreatic function. For example, recent Accumulating evidence suggests that nicotine data from this laboratory demonstrate that acetalde- affects exocrine pancreatic secretion. Several hyde, the primary metabolite of ethanol and a investigators'- have shown that cigarette smoking constituent of tobacco smoke'` inhibits cholecysto- in man or infusion of nicotine in experimental kinin induced amylase release from isolated pan- animals inhibits both basal and secretin mediated creatic acini and the binding of cholecystokinin to stimulation of pancreatic fluid and bicarbonate secre- acinar membranes." tion with no apparent change in output. In The present study was undertaken to further contrast, Balldin, et al" have observed raised serum investigate alterations in the responsiveness of the concentrations of amylase, cationic trypsinogen and exocrine pancreas to secretin stimulation and its pancreatic secretory trypsin inhibitor after secretin effect on the protease antiprotease balance in heavy Addres%s for corresplondenece Dr Michael I)Lic;k Depi.rtment of Medicinec cigarette smokers. The circulating levels of digestive (I5IF). VA MediedlCenter, Martinei. (A 94553, USA enzymes, certain lysosomal hydrolases and protease Received fOr publitcation 3 July I986. inhibitors were measured in serum from non- 330 Gut: first published as 10.1136/gut.28.3.330 on 1 March 1987. Downloaded from Circulating enzymes in smokers 33 smokers and heavy smokers of different ages before Hydrolases and after a single iv injection of secretin. Further- [3-glucuronidase and acid phosphatase activity was more, efforts were also made to relate these findings determined colorimetrically according to Sigma to the history of alcohol consumption in the subjects. Technical Bulletins 325 and 104, respectively. 13- glucuronidase activity was expressed a microgram Methods phenolphthalein liberated/h/ml. Acid phosphatase activity was expressed as [tmoles p-nitrophenol SUBJECTS liberated/h/ml. Nineteen smokers (16 men, three women) were recruited from the patients and staff of the VA Gamma-glutamyl transpeptidase Medical Center (Martinez, CA). Except for one Gamma-glutamyl transpeptidase activity was subject, all had smoked for a minimum of eight years assayed by the colorimetric procedure outlined in with a smoking history of 65-7±81 pack years Sigma Technical Bulletin 545. (mean±SEM). The majority smoked at least two packs of cigarettes/day. The subjects ranged in age RADIOIMMUNOASSAYS from 30-68 years (52.2±2.5) and 13 of them con- Serum immunoreactive cationic trypsin(ogen) sumed significant amounts of alcohol regularly. (IRCT) and immunoreactive elastase 2 (IRE2) con- Although coffee consumption was not controlled for centrations were determined by radioimmunoassay this study, Andriulli et al'' found that coffee intake as previously described' except that 0 12% normal did not affect serum trypsin concentrations after rabbit immunoglobulin was removed from the assay secretin injection. Nine young, healthy, volunteer buffer. After a four day incubation at 40C, the students (four men, five women), ranging in age from antigen- complex was precipitated with 25-35 years, served as controls with no history of goat-antirabbit immunoglobulin bound to agarose. A smoking or alcohol abuse. In addition, four male dilution of 1:1 000 000 of specific antiserum to both non-smokers, ranging in age from 53-76 years served enzymes was used. Three appropriate dilutions were as controls to the more aged smokers. Subjects were analysed in duplicate and results were expressed as fasted overnight before testing and the smokers were ng/ml of cationic trypsin(ogen) or elastase 2 by allowed to smoke up to the test period. comparison with purified human cationic trypsin or elastase 2 as standards.

BLOOD SAMPLES AND SECRETIN INJECIION http://gut.bmj.com/ Venous blood was obtained before and 15, 30, and 60 OTHER ASSAYS minutes after a single rapid injection (iv over 1 min) The concentrations of immunoreactive a,-protease of 75 CU of secretin (KabiVitrum CB Laboratories, inhibitor (a,-PI) and a2-macroglobulin (a2-M) in Stockholm, Sweden). Blood was allowed to coagu- serum were determined by the rocket immuno- late on ice for at least two hours and serum was electrophoresis procedure of Laurell and McKay.'4 isolated by centrifugation (2000 g, 15 min). Serum Trypsin inhibitory capacity was determined by

was stored at -80°C until assayed. incubation of a known amount of purified bovine on October 1, 2021 by guest. Protected copyright. trypsin with serum and measuring the inhibition of ENZYMES AND SUBSTRATES trypsin hydrolysis of TAME.2S Inhibitory capacity Bovine trypsin (EC 3.4.4.4) and porcine pancreatic was assessed from the difference in slope in the elastase (EC 3.4.21.11) were obtained from absence (water blank) and presence of serum. Worthington Biochemical Corp, Freehold, NJ. Elastase inhibitory capacity was measured similarly Porcine pancreatic amylase was a product of by inhibition of the hydrolysis of SLAPNA after Calbiochem-Behring (LaJolla, CA). Tosyl-arginine- incubation of purified pancreatic elastase with methyl ester (TAME) and succinyl-tri-alanine-p- serum.26 nitroanilde (SLAPNA) were from Sigma Chemical Serum concentrations were determined Co (St Louis, MO). Agarose was purchased from by the bromocresol green dye-binding assay (Sigma Biorad Laboratories (Richmond, CA). All reagents Chemical Co). was assayed by the used for biochemical measurements were of analyti- method of Walberg et al17 and results were expressed cal grade or the purest commercially available. as milligrams haematin/100 ml.

ENZYME ASSAYS STATISTICAL ANAL YSIS Amylase Results were analysed by Student's t test for com- Amylase concentrations in serum were determined parison of the means between the smokers and by the method of Jung22 using procion yellow coupled non-smokers and basal and stimulated values within to starch as substrate. groups. Gut: first published as 10.1136/gut.28.3.330 on 1 March 1987. Downloaded from 332 332 ~~~~~~~~~~~~~Dubick,Cont'eas, Billy, Ma]'umdar, and Geokas Results 12LV Amylase c It was observed that both the basal and secretin stimulated levels of amylase and immunoreactive 101 cationic trypsin(ogen) and elastase 2 were similar in 81 both the young and aged non-smokers. Conse- quently, all non-smoking subj'ects were combined mg/mi 6- into a single group. In smokers basal serum amylase and immunoreac- 41- tive elastase 2 concentrations were found to be 100% and 24% higher, respectively, than in the non- 21 smokers (Figure). Serum IRCT concentrations be- tween the two groups were, however, the same (Figure). These observations remained unchanged when the smokers were subdivided according to age 1501- Cationic trypsin(ogen) b (>50 years old) or their drinking habits (those who consumed alcohol regularly). 100 A of secretin increased serum single inj'ection mg/mi amylase concentrations an additional 2.6-fold in all a aa a smokers, and 3-2-fold in those who smoked and drank alcohol (smoking+drinking group) (Figure). Serum enzyme concentrations in the smokers group 0' a ~ -11 were higher at all time periods in comparison with c cc controls. Therefore, only the maximum stimulated 200 ELastase 2 levels (at 15 mmn) are reported. Serum amylase concentrations in the non-smoking controls were unaffected by the hormone (Figure). In addition, iSOL serum IRCT and IRE2 levels were 3.4 and 2.1-fold m.g/mI higher, respectively, after secretin inj'ection, than b a.b b

100 http://gut.bmj.com/ their corresponding basal levels (Figure). Again the a greatest stimulation in circulating levels of pancreatic proteases was observed in the 'smoking+drinking' so0V group. In general data from individual smokers showed that the secretin stimulated increase of U'-I' amylase was accompanied by raised serum cationic Basal Stimulated trypsin(ogen) and elastase 2. None ot these changes Figure Concentrations ofamylase, cationic trypsin(ogen) were observed in the non-smoker control subjects and elastase 2 in serum. Data expressed as mean ± SE of on October 1, 2021 by guest. Protected copyright. (Figure). We wish to emphasise that whereas 1O-I9samples. Open bar, non-smokers; closed bar, all trypsinogen and elastase 2 are pancreas specific, the smokers; hatched bar, smokers overSO0years old; stippled origin of the increased concentrations of amylase bar, smokers and drinkers. Means with different letters are cannot be ascertained because assay for amylase significantly different (p<0OO5). isoenzymes was not performed. macroglobulin (u2,-M) tn any of the categories of As shown in Table 1. the basal levels of immuno- smokers were the same or slightly higher in compari- reactive (xi-protease inhibitor ((xi-PI) and u2- son with the controls. Similar results were observed

'Table I Serumt p)rotease-itlllibitor levels atid activity1

.Smokers Non-smooker% All .SmokA(1 oi'v,r 5() vears oldl Smtoke ± drinik n 13 11ii19 1i 10 ni 13 (11-Plt(mig/100 ml) 317 ± 14 328 ±_ 19 322 ± 27 317 ± 23 (L, Mt(mig/1I00 ml) 194 + 13 229 ±_ 1 200 + S225 ± 17 TIC§(Y inhib lOp][) 39.2 + 2.1 37.6 + 2. 40.7 ± 3.2 34.9 ± 3.6 ELCII(% inhib/20p.i) 73.1 ±2.9 76.1 + 3.3 80. + 3.0 81.9 + 2 *Dait;tcxprcssedissmeain ±SE; 0 -Protcasc inhibitor; ct-,-MacroglobuIin;§Trypsin-inhibity cpity; IlElastase inhibitory capacity. Gut: first published as 10.1136/gut.28.3.330 on 1 March 1987. Downloaded from Circulating enzymes in smokers 333 with respect to assessment of elastase-inhibitory Although the smokers used in the present study capacity of a,-PI (Table 1). In most cases, however, were apparently healthy, their basal circulating con- trypsin inhibitory capacity of a,-PI was slightly centrations of amylase and immunoreactive elastase higher in the non-smokers, though the differences 2 were found to be significantly higher than in the were not statistically significant. In addition none non-smoking controls. Furthermore, a single of these parameters were significantly affected by injection of secretin produced a profound rise secretin in any group. Thus, these data translate into in serum amylase, immunoreactive cationic a two to three fold increase in the protease-to- trypsin(ogen) and elastase 2. This effect was not seen protease inhibitor ratio in serum from heavy smokers in the non-smokers. Balidin et al` and Andriulli et al' after secretin administration (Figure, Table 1). have also observed a rise in serum immunoreactive Furthermore, no significant differences were noted trypsin concentrations in smokers after secretin in circulating concentrations of lysosomal hydrolases injection. Whether these results are because of between the smokers and non-smokers during the increased synthesis and/or enhanced secretion of course of the study (Table 2). Acid phosphatase and enzymes, is at present unknown. We have recently 3-glucuronidase activity was within normal limits in observed, however, that in rats, administration of all but two smokers. Again these two individuals acetaldehyde, the primary metabolite of ethanol and were heavy drinkers as well. y-glutamyl transpepti- a constituent of tobacco smoke,'9 significantly dase activity was, however, higher in all groups of reduced the amylase and trypsinogen content of the smokers than in controls (Table 2). Raised y- pancreas and increased serum amylase concentra- glutamyl transpeptidase activity was particularly pro- tions."3 Further, it was observed that in dispersed nounced in individuals who were also heavy drinkers. pancreatic acini from acetaldehyde treated rats, Smokers also had lower concentra- basal secretion of preformed exportable hydrolases, tions compared with non-smokers (Table 2), and no amylase, trypsinogen and chymotrypsinogen was methemalbumin was detected in serum from smokers increased 40-50% over control levels and nicotine- either before or after secretin administration. induced enzyme release was enhanced." These data suggest that acetaldehyde treatment enhances pan- Discussion creatic enzyme secretion. Since similar results have been observed after nicotine infusion in rats The exocrine pancreas is one of the most metabolic- (Majumdar, Dubick, 'esenka and Geokas- unpub- http://gut.bmj.com/ ally active organs in the body, as evidenced by its high lished observations), it appears that heavy cigarette rate of protein synthesis. Its major function is to smoking may result in an accelerated secretion of secrete into the gut, on demand, large quantities of pancreatic enzymes. bicarbonate, water and digestive enzymes." Under Because pancreatic secretion into the duodenum normal conditions, pancreatic enzymes enter the was, however, not measured in this study, the precise duodenum via the pancreatic duct, with a small mechanism(s) for the increased serum enzyme con- fraction being directly secreted into the blood.2" In centrations observed remain unknown. Previous serum from normal healthy adults, fasting levels of studies have shown that nicotine infusion'~'4 or on October 1, 2021 by guest. Protected copyright. immunoreactive trypsinogen and elastase 2 averaged cigarette smoking3' alter interdigestive pancreatic 26 ng/ml3"' and 71 ng/ml,"3 respectively, with no exocrine secretion into the duodenum. As bile secre- significant variation between adults of various tion into the duodenum is also affected by cigarette ages.213"'' 3' This observation is consistent with data smoking," it is possible that smoking may induce an obtained from the non-smokers in this study. In 'exit block' at the level of the sphincter of Oddi. At contrast, it is known that pancreatic inflammation present we are unaware of any studies on the direct results in a profound rise in circulating concentra- effects of nicotine or cigarette smoking on sphincter tions of pancreatic hydrolases.'2 of Oddi motility.

Table 2 Serum albumin lysosomalhydrolases and GGTP* acdivityk .Smokers Notn-s4nokers A 11 Smtokerx over 50 vears old Smoke + (lrinik n =13 11=9 11=10 n1=13 Albumin (g/dl) 4.5 + 0(1 3.7 + -2 39 + (2 3 ± (1.2`: Acid phosphatase (pmole p-nitrophcnol/h/ml) 0(27 ± 0(02 0(34 ± 0(03 0(37 + 0-05 0.37 ± ()(14 IiGlucuronidasc(pgphenophthlcin/h/ml) 215 + 28 25 1 + 3.3 19.2 + 22 21 ±6 28 GGTP(units/ml) 14.1 + 18 329 + 7-8- 42.2 +± 125 4(10) ±+ 10.5--- 'G.Immna glutmanl transpcptidasc; tData cxprcsscd a.s mcan [t SE; 'p<0-05. Gut: first published as 10.1136/gut.28.3.330 on 1 March 1987. Downloaded from 334 Dubick, Conteas, Billy, Majunldar, and Geokas

It also appears that the effect of secretin on and elastase 2 in human abdominal aortic pancreatic enzyme secretion is multifactorial. After aneurysms,"' these studies suggest that circulating secretin infusion in six patients, Gullo, et alP con- pancreatic proteolytic enzymes could be operative, at cluded that secretin induced pancreatic enzyme leatst in pairt, in the initiation of pulmonary secretion through a direct action on the acinar cells, emphysema and aortic aneurysms, diseases often while Carr-Locke et al observed that secretin infu- associated with heavy cigarette smoking. Work sion selectively relaxed the pancreatic duct sphincter is now in progress in order to investigate this without any effect on the common bile duct or bile hypothesis. duct sphincters. DiMagno et afl also observed that secretin dilated the pancreatic duct and decreased the H T Billy is the recipient of a student fellowship flow of pancreatic juice into the duodenum. A from the American Heart Association, California differential effect of secretin on duct and sphincter Affiliate. pressures could also result in an 'exit block' and may explain, in part, the observed effects of secretin on This work was supported by the Medical Research serum amylase and lipase in patients with biliary and Service of the Veterans' Administration and by a pancreatic disease. " Further studies in this area are Grant-in-Aid from the American Heart Association, clearly indicated. California Affiliate and with funds contributed by the In addition, the present study showed that the American Heart Association, Santa Clara County secretin stimulated serum levels of pancreatic hydro- Chapter. lases tended to be only slightly higher in the subjects References who smoked and imbibed alcohol regularly, as compared with the cigarette smokers group as a I Carp H, Janoff A. Possible mechanisms of emphysema whole. Although numerous studies have shown that in smokers. In vitro suppression of serum elastase- ethanol affects exocrine pancreatic function both in inhibitory capacity by fresh cigarette smoke and its vitro and in vivo,4" the present data do not support prevention by antioxidants. Am Rev Respir Dis 1978; an additive effect of cigarette smoking and ethanol 118: 617-21. consumption on exocrine 2 Hoidel JR, Niewoehrer DE. Cigarette smoke inhalation pancreatic secretion. potentilates elastase-induced emphysema in hamsters. Our study also shows that trypsin and elastase Amn Rev Respir Dis 1983; 127: 478-8 1. inhibitory capacity and circulating levels of ca,-PI and 3 Janoff A. Carp H. Laurent Raju L. The P, role of http://gut.bmj.com/ ut,-M were not significantly different between oxidative processes in emphysema. Am Rev Respir Dis smokers and non-smokers. Carp and Janoff' found 1983; 127: S31-S38. that fresh cigarette smoke could suppress elastase- 4 Kaufman DW, Helmrich SP, Rosenberg L, Miettinen inhibitory capacity in vitro and Beatty et a43 reported OS. Shapiro S. Nicotine and carbon monoxide content the presence of oxidised ct-PI in serum from of cigarette smoke aind the risk of myocardial infiarction cigarette smokers. Other investigators,"" however, in young men. N EniglJ Med 1983; 308: 409-13. did not observe decreased protease 5 Auerbach 0, Garfinkel L. Atherosclerosis and inhibitory aneurysm of aorta in relation to smoking habits and age. capacity in cigarette smokers. Although protease Chest 1980; 78: 805-9. on October 1, 2021 by guest. Protected copyright. inhibitory capacity was not significantly compro- 6 Hill P, Haley NJ, Wynder EL. Cigarette smoking: mised in the smokers of this study, the data show an , plasma nicotine, cotine and increase in the protease to inhibitor ratio in serum thiocyanatc vs self-reported smoking data and cardio- from cigarette smokers. This condition has been vascular disease. J Clhronic Dis 1983; 36: 439-49. hypothesised as a major factor in the pathogenesis of 7 Hartz AJ. Anderson AJ, Brooks HL, Manley JC, pulmonary emphysema and aortic aneu'rysms.- II l' Parent GT. Barboria JJ. The association of smoking Thus, the results of the present study as well as with cardiomyopathy. N EnglJ Med 1984; 311: 1201-6. those of others"'' indicate that secretin 8 Yen S, Hsieh C-C, MacMahon B. Consumption of induces alcohol and tobacco and other risk factors for pancre- increatsed circulating levels of pancreatic proteases in atitis. Am J Epidemniol 1982; 116: 407-14. heavy cigarette smokers, without affecting enzyme 9 Schmidt W, Popham RE. The role of drinking and levels in non-smokers. Therefore, it is tempting to smoking in mortality from cancer and other causes in speculate that an enhanced responsiveness of the male alcoholics. Cancer 198 1; 47: 1031-41. pancreas to physiological stimuli - for example, in 10 Kuhn C, Senior RM. The role of elastases in the response to food intake resulting in hormonal secre- development of emphysema. Lunzg 1978; 155: 185-97. tion, may result in episodic increases in circulating 11 Cannon DJ, Reaid RC. Blood elastolytic activity in protease levels in chronic cigarette smokers. With the paticnts with aortic aneurysm. Ann Thorac Slurg 1982; increased ratio 34: 10-15. protease/inhibitor observed in serum 12 Busuttil RW, Rinderbriccht H, Flesher A, Carmack C. in our present study, and our recent detection of Elastase activity: The role of elastase in aortic aneurysm immunoreactive pancreatic cationic trypsin(ogen) formation. J Slurg Res 1982; 32: 214-7. Gut: first published as 10.1136/gut.28.3.330 on 1 March 1987. Downloaded from Circulating ('e t7zv1CS in smokers 335

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