W. Bravton Batson and Patty H. Laswicki Clarton State College Clarion. PA 16214
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Pepsin and Antacid Therapy: A Dilemma
Analysis of commercial antacid products has been the suhject of experiments appearing in a number of sources including laboratory manuals (1,2), Chemistry ( 3 , 4 )and this Journal (6.6.7). However, recent puhlished research on the mechanism of digestion implicitly suggests skepticism and renders the conclnsions from most of these student experiments incomplete or misleading. This paper will illustrate some areas of uncertainty in the current literature and will present a modified experiment for first year chemistry students or nonscience majors which is more consistent with current hiocbemical research. It has been stated in this Journal (7) and the pharmaceutical and medicalliterature that a goal of an ideal antacid is to raise the stomach contents to a pH a t which pepsin, now believed to be a contributing factor in peptic ulcer formation, would he rendered proteolytically inactive. Perhaps the question of desirable antacid characteristics should he reexamined for healthy persons not suffering from ulcers who casually buy antacids to relieve the medically undefined conditions of "heartburn" or "acid indigestion" and "sour stomach."These conditions may not always he caused or accompanied hy excess acid. Even when they are, however, the proper degree of treatment is probably less than the average person suspects, for reasons we shall clarify. T o discuss the best conditions for antacid functioning in a healthy individual suffering temporary hyperchlorhydria, a key question should he the importance, if any, of pepsin to the body. Research indicates that "pepsin" actually consists of a t least two enzymes in the stomach of man, pepsin A-the principal enzymatic component-and pepsin C. These are produced from slightly different structures of an inactive precursor pepsinogen. Both become active under acidic conditions in the stomach and hoth contain carhoxyl groups in their active sites; hoth also cleave bonds adjacent to aromatic amino acids in protein substrates. Most articles do not take the minor component pepsin C into consideration hut merely refer to "pepsin," which implies pepsin A. Its structure was elucidated by Andreeva et al. in 197fi using single crystal X-ray diffraction Concerning the role of pepsin, Buchs has suggested that its purpose is to split off certain amino acids from proteins in the stomach. These amino acids stimulate a further secretion of pepsin and also that of pancreozymin after transfer to the intestine (9). Since pancreozymin causes the secretion of enzymes from the pancreas, the main role of pepsin, Buchs indicates, consists of triggering the digestion of fats and carhohydrates in the gastrointestinal tract. Wade has suggested recently a somewhat similar theory (10). He notes that pepsin is most active in hydrolyzing honds which yield peptones containing N-terminal aromatic amino acids (phenylalanine, tryptophan, tyrosine) but that it also acts a t other points, including honds involving leucine and methionine. These five amino acids have a lipophilic side-chain in common. Wade cites evidence that pepsin "unmasks" the stimulant amino acids present in dietary protein. "As the chyme enters the duodenum," he states, "these amino acids, whether still bound to peptones or liberated from them by mucosal N-terminal end-peptidases, initiate the release of amounts of pancreatic enzyme appropriate to the protein content of the meal (lo).'' He concludes that the function of pepsin is to participate in the regulation of pancreatic enzyme secretion, and not simply to play a minor "and apparently unnecessary" part in the 484 1 Journal of Chemical Education
hvdrolvtic " . direstion of oroteins in readiness for ahsorntion. The natureof the prokin substrate itself plays an important part in determining the "ideal" pH a t which pepsin operates. A 1969 study of acidic and neutral peptide suhstrates indicated that the rate of ~ e ~ scatalvzed in hvdrolvsis is controlled by the ionization of t&ogroups; neutraland acidic substrates are bound to the enzyme most strongly a t differing pH's (11). Similarly, a 196fi study by other workers, who synthesized several new peptide substrates for swine pepsin, revealed different pH optima in peptide bond cleavage by pepsin (12, 13). These differences in optima were accounted for by the hvoothesis that an alnha-carhoxvl to the . . . . eroun . adiacent . wnsitive p q ~ t i d rIrmd is inhibitory to pepsir action. This liypothrsi> was nmfirmrd furthrr nnd additional kinrrir 'tudies reporred in a 1YW article hy i o t h u rr.e;lrchers ( 1 11. Kinef~cr ~ r n s t a ~of ~ t two s series o i peptide suosrrates were ~ ~lrtrrminvdat 1111 2.0 and -I>; in one series t h suhstmte rontainvd an :~lpha-carta,xvlgroup. Ctunlnrlinns of the kinetic constants and of thr activuv tm,file< indicated that little conformational change occurred in the binding region in the active center of oensin . . when the DH was raised from 2.0-4.5: y t suhstr:#tt:swith an alpha-cnrlr,uyl grwlp were Iunlnd much less s t r ~ ~ wat l vthe hieher DH hecuuse sirnlficnnt ~onizatim ~ of this group occurredat p ' 4.5. Thus there is no "ideal" DH for oeosin activitv; it reallv depends on the nature of the meal ingested. In light of t h k knowledge, a significant number of statements in print have been miileading or contradictory. For example; a recent textbook of biochemistry states that pepsin A is most active in a o H ranee of 1.3-3 and neosin C in a ranee of 3-3.5 (16). A textbook of medicinal chemistry claims t h z the activity df pepsin is greatest a t pH 1.5 (16).In this Journal, a 1975 article on the physicochemical properties of antacids states that " n. e.~ s i nis larrelv .. . inactivated above D H 3 (7)." Yet a clinical rhrmi.;rry trxtl~nnkstares rhnt "the optimum pH ior pepsin t pII 4.5 is 2.0. hut 70 11ercent of th1.nctivity is still p r r s ~ n at r I;).''A 197:j honk ofdrug evaluation by the American \ledirnl Association savs that the goal of imtii~idtherapy IS to ralst. t hr IIH uf the. nastric contents 10 5. hecause ar this lewl "the proteolytic activity of pepsin is almost completely abolished (la)." A hook on nonnrescriotion drugs hv the - ouhlished . American Pharmaceutical Association states that "the ideal pH to which the gastric contents are buffered by the antacid is between pH 4 and 5. This optimum range is sufficiently high to inhibit the ~roductionof ~ e o s i n(19)." The above discussion impiiei to us that the role of pepsin is i m ~ o r t a nhut t that more research in this com~lexarea needs to bidone and the inconsistencies resolved. By assimilating the hits and pieces gleaned from the literature, we conclude that the consumption of an antacid product by any person not suffering from ulcers is undesirable if it causes significant
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Presented at the annual meeting of the Pennsylvania Association of College Chemistry Teachers, The University of Pittsburgh at Johnstown, April 29,1978. Author to whom correspondence should be addressed. The pepsin molecule is approximately ellipsoidal, with molecular axes of 55.35, and 40 A; there is an extended depression along part of its surface as well as a large internal cavity which divides the protein into two unequal portions. The catalytically active aspartate residues are located in the surface depression (8).
.. maximum." W e feel t h a t if these two criteria-quantity of acid neutralized a n d final pH-are n o t met, a healthy individual suffering temporary stomach distress might h a r m himself. If t o o m u c h acid is "consumed" and/or t h e p H rises too much, pepsin activity will cease a n d t h e entire digestive process of f a t s a n d carbohydrates, a s well as proteins, m a y be hindered. T h e individual may t h e n t a k e one tablet after another in hope of feeline hetter. h u t each time feeline somewhat worse. T h i s
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nach-I lrallon of mreacted nCI with sfandard.red 0 1 MNaOd, aflcr in.1 al rcac on A lh var 08,s orands of antacid tan em Fth) m of 0.244 MdC 7 10 0 rn H 2 0 used for all samples but Maalox (used 60.0 rnl HCI)and Alka-Seltzer Gold (used 110 ml HCI). Band T represent endpoints of the respective indicators. lnoredients listed for various brands: Alka41CaCOd Alka-Seltzer Goldlbi-
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If pepsin does play a key role in digestibn, a n antacid product, presumably t a k e n after a meal, m u s t allow p e p s i n to r e m a i n proteolytically active. Illustrative Experiment a n d Procedures
I n u i t m student experiments far the direct titration of antacid tablets with HCI x h t i o n have been published. Some are simple ( 4 , 6) whereasanother involves elaborate temperature control and constant additions of knnwn increments of acid (7). We find that simple direct titration to a particular endpoint works well for several brands but is not feasible for some products (such as crushed tablets of Maalox or Gelusil) because of slow kinetics. In our laboratory these latter products have consistently yielded falsely low titers when titrated directly to a methyl orange endpoint. Back-titration procedures in student experiments have also been described; they involve reaction a t ambient temperature with the antacid followed by titration of excess HCI with standardized sodium hydroxide t o a brombphenol hlue endpoint (1,2,3). As can he seen from the illustration, bmmophenol
:'A recent textbook of medicine states that stomach acidity in the fasting state is normally around a pH of 0.9 t o 1.5 (20); a book of clinical chemistry notes that the pH of the gastric contents after a meal may rise to 4 (21). It is recommended, generally in the literature that antacids be taken within an hour after a meal to increase retention time in the stomach from 10-20 min to greater than 1 hr (22,23). Wne reference states that after gastric stimulation the normal stomach should secrete a maximum of 130 mmoles HCIII. Since the typical gastric volume is less than 50 ml, less than 13 mmoles HC1 should he present after a meal (24).Other references note that HCl produced by gastric stimulation in test patients varies typically from 1-20 mmoleslhr (21); maximum stimulation produces slightly less than 23 mmoleslhr (20). Though these figures vary widely from individual to individual, the average figure for "excess"HCI production is mughly 10 mmoles greater than that of normal HCI production. Therefore one dose of an antacid product should react with no more than about 10 mmoles of HClIhr. For many people this upper limit should be much less. V h o u g h not discussed as one of the main points of this paper, we emphasize that some gastric distress may not involve the production of excessive hydrochloric acid; therefore, in these situations antacids should be avoided. For instance, gastric HCI output appears to be less than normal in diseases such as superficial chronic gastritis (25,26! and carcinoma of the stomach. The common condition "heartburn" is thought to be caused by regurgitation of the gastric contents into the esophagus, and "acid indigestion" may be merely due to overeating or to an irritating food (27). This concentration of HCI was chosen rather than the 0.1 M HCI described in several articles in order to keep the total volume under 100 ml. The normal volume of gastric juice is reported to be between 50 and 100 ml, usually under 50 ml(20,28). We ran titration curves of earbonate-containing products either hoiled or heated for 2CL50min at 40-50°C and found that boiling did not alter the shape of the curves. Since boiling is easier and gives more consistency in sample handling, we recommend boiling for a short hut unifnrm time. This elevated temperature takes care of the slow kinetics of some products and does not necessitate controlling, say, a 37°C constant temperature bath for 1hr.
blue changes color s t a pH of 3.5-4.1, whieh for some products is on the "knee-bend of the buffer region; hence, endpoint determinations are somewhat indefinite and product comparisons difficult. One of the direct titration procedures ( 6 )utilizes bramocresol green as indicator, which changes color a t an even higher pH (4.0-5.fi) than bromophenol blue and, for some products, should cause a poorer endpoint or none a t all. Worse, bromocresol green and perhaps also hnmophenol blue yield endpoints above the pH limit for favorable pepsin activity. In light of our concerns about the importance of pepsin and noting the experimental problems mentioned above, we have designed a simple two-part experiment involving back-titration of an antacid1 HCI solution but utilizing thymol hlue (which undergoes a color change around pH 3) as indicator. Directions are as follows: 1) Crush a weighed antacid tablet between glass plates. Transfer quantitatively to a beaker usingafew milliliters ofwater toaid in the transfer. Add 50.0 ml (sorhe products may require more) of approximately 0.24 M HCI and stir for several minute^.^ 2) Cover with a watch glass and boil gently for 5-10 m h S Allow to cool. red to yellow. 4) Calculate the millimoles of HCI added initially, the millimoles of NaOH needed, the millimoles of HCI which reacted with the antacid, the millimoles and milligrams of "active ingredient" per tablet, and the weight percent of inert hinder. Finally, note which products react with too many moles of hydrochloric acid-more than approximately 10 mmole HCl/tablet.5 A variation of this experiment involves performing steps (1) and (2) as written above. The directions are then-Calibrate a pH meter and perform a back-titration of the unreacted HCI using small increments of standardized 0.1 M NaOH. For each antacid, make a curve of pH versus ml of NaOH (or millimoles of neutralized HCI). Discussion Both portions of the experiment are illustrated in the figure.Four popular brands clearly can produce tob high a pH per tablet; one product seems too potent, reacting with far too much HCI a t too high a pH. Noting t h e recommended doses listed on t h e bottle labels of thevarious brands, i t becomes clear t h a t these doses a r e illadvised. For instance, statements such as these are found: "chew 2 or 4 tablets. . ."(Phillips); "chew 2 tablets. . ." (DiGel); "2 t o 4 tablets chewed or swallowed.. ." (Maalox). Other factors which might be noted are that two products in the figure contain significant quantities of sodium ion, a systemic hazard for some individuals; several brands contain carbonate, which may cause momentarv relief bvaidine in the exoulsion of t r a ~ o e d air hut renal problems; aluminum ion can produce nausea in some individuals (29). Furthermore, the adsorptive properties of antacids containing AI(OHh should be considered. Preparations containing this eompound have been shown to adsorb pepsin significantly (30.31)-as well as aspirin and other salicylates (32). Preparations containing Volume 56, Number 7, July 1979 1 485
Al(II1) also interfere with the absorption of barbiturates (33) and tetracycline (34);antacids containing Ca(l1) or Mg(l1) are believed also to interfere with the effectiveness of tettacvcline hecause of the chelating action of the antibiotic (27). In addition to the students becoming thoughtful consumers from this experiment, we hope that they gain same important chemical principles.For example, given pH curves for the titrations, they might wonder why for some compounds the vertical portion near the equivalence point is longer and steeper than for others; another question is haw Al(II1) functions as a RMnsted acid in a buffer.
Acknowledgment The authorsare grateful to Mr. Bruce Stauffer of the Clarion State College Department of Geography & Earth Science for making the illustration.
(121 Innuye, K.,Voyniek. 1. M..Delpierro.G.R.,and Fruton.J. S.,Riorhrmirtry, 5.2478 llqefil ,..... ,. 11x1 Frulim,.J.S., i n "Adv F-nl.Relrt Amas M d . Rid," (Editor: Mcistpr.A.1 John Wiloy and Snnr. New Vork. 1976,Vnl. 44, p. 3. 1141 .laeknm W.T..Schlamowitz.M..Shaw.A..andT~ujillo. R..Arch. Riochm? Riophyn., 131,874 (19691. 1151 Karbon. P.,"Modern Biwhemiatry,"4th Ed.. Aesdemie Preu. New York, 1975. pp. 165-7. R.. "lntmductinn of Medicinal Ch~mistry," (181 Macl.~an. D.. Evans. D.. and .Jones. .I. Harper snd Row, Inc. Now York. 1976.p.209. I171 Tietr, N. W. IIldiinrI, "Fundamentsls of Clinical Chemistry." W. R. Saunders Co.. Philadelphia, 1970.p.796. I l R l "AMA Drua Evaluations: 2nd Ed., premred h y American Medical Asrocistion, Puhlirhing Sciencea Group. Inc., Aelion. Mass.. 1973. p. 786. 1191 Penns, R. P.. in "Handbcnk of Non~PrcrcripLinnD~ugs,"1Edcfor: Griffenhagen. G. %and Hawkina.1.. L.l,American Pharmaceutical Asroeiation, Wsrhinqtnn.lX.. ,a,, " 9 ".-. I201 Reeson. P. R.. and McDermntt, W. IEdilors1,"Terthnnk of Medicine," 14th Ed.. W. R. Ssunderr Co.. Philadelphia. 1975, p 1889. I211 R d l i p ) . P.794. 1221 Ref. 1201. 1207. I291 Rdlmn..J.S;and Cnllyna. J. A. H . N u E n d J. Med. 274.921 119661. 1241 Ref. 117). o. 800. (251 Ref. 1171; 804. 1261 K ~ t e r G.. , Tnrreinn. G.. Riel, F.. and Pachaly,L.. A m r r J. D M Direorer. 12.607 118671. I271 "AMA Drug Eunluationr," 8rd Ed..prepared by American Medical Asmiatinn. Puhlishing Sriencor Gmup, Inc.. I.iflleton. Mars.. 1977, p. 1026. 1281 Re( 117),p.799. 0 9 1 Ref. ilCI. o. 788. 12. m o l ner. ITPI, (:,I1 Andarscm. W.,and Hanhill..J. E . J . Pharm. Pharmoroi.. 24. ISuppl.), 166P (19721. Daabis. N. A..Phormaiie. 31.461 119761. i i 2 l Naerar.V. F.. Khalil.. S. A..and . ~ ~ l ' i i n )p. . 787. 1341 "Selecting YourOwn Medicines-Nonpwseription AltsUd Pmduds," U S . Dept. af Health. F.amtion, and Welfare. Public Health Service. Fond and Drue Administration, Reekuill~.Maryland, H E W Puhlicatian No. (FDA1 77~3025.
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