1 GASTROENTEROLOGY Cpyright 1970 by The Williams & Wilkins C. Vl. 58, N.3 Printed in U.S.A. UNDISSOCIATED ACIDITY OF HUMAN GASTRIC JUICE Measurelllent and relatinship t prtein buffers GABRIEL M. MAKHLOUF, M.D., PH.D., ANDRE L. BLUM, M.D., AND EDWARD W. MOORE, M.D. Metablism and Gastrenterlgy, Lemuel Shattuck Hspital and Tufts University Medical Schl, Bstn, Massachusetts, and Divisin f Gastrenterlgy, University f Alabama Medical Center, Birmingham, Alabama The undissciated acidity f gastric juice represents the difference between titratable acidity and H+ in cncentratin as measured by the glass electrde. An estimate f titratable acidity was btained by extending the titratin t that end pint at which the psitive charges cntributed by Na+, K+, H+ ins and buffered prtns matched the negative charges cntributed by Cl-. In additin, equatins were derived based n the significant crrelatin between H+ in cncentratin and titratable acidity which permit direct estimatin f undissciated acidity frm either value. The psitive relatin nted between undissciated acidity and the cncentratin f nnpepsin prtein suggests that the latter is the main buffer in gastric juice. The hydrgen secreted by the parietal cells is present in gastric juice in bth dissciated (hydrgen in) and undissciated (buffered) frms. Mre and Scarlata1 have shwn clearly that, in gastric juice, H + in cncentratin can be determined with accuracy nly by direct measurement f hydrgen in activity with the glass electrde. H + in cncentratin is btained as the rati f H + in activity and the mean activity cefficient. The latter is a functin f the inic strength f the slutin; its values fr slutins f different inic strength have been established and are available fr general use.2 The cncentratin f undissciated acid Received Nvember 25, Accepted Octber 8,1969. Address requests fr reprints t: Dr. Gabriel M. Makhluf, Divisin f Gastrenterlgy, University f Alabama Medical Center, Birmingham, Alabama This wrk was supprted in part by Research Grants AM-07417, AM-10377, and AM frm the Natinal Institute f Arthritis and Metablic Diseases, United States Public Health Service. 345 represents the difference between titratable acidity and H + in cncentratin. Its value s far has defied measurement because the end pint f titratable acidity remains unknwn. The difficulty f lcating the end pint is cmpunded by the absence f infrmatin n the nature f the prtein r prtein fragments that culd act as buffers in gastric juice. This study is a preliminary attempt t reslve this prblem. Methds and Materials The data were btained as part f a study" n the pepsin respnse in man t cntinuus intravenus infusin f varying dses f gastrin II r histamine acid phsphate. The study was cnducted in 1 male subject, weighing 66 kg, n whm a large bdy f gastric secretry data is available. Details f intubatin and cllectn f secretin have been described in a previus cmmunicatin.' Salivary cntaminatin was prevented by the insertin and frequent changing f dental cttn pledgets in the sulci f the cheek and under the tngue. Basal secretin was cllected fr 30 min prir t each f eight separate tests. Five-minute cllectins were
2 346 MAKHLOUF ET AL. Vl. 58, N.2 btained during the curse f all infusins and were pled int samples crrespnding t I5-min intervals. A number f estimatins were made n each sample, including thse f Na+, K+, Ct, ttal and inized calcium, the details f which were reprted earlier:' 5 In all f the 70 samples btained, titratable acidity was measured by electrmetric titratin using a Crning mdel I2-B ph meter. A full titratin curve was develped fr each sample by the additin f discrete amunts f 0.1 N NaOH t 2 ml f sample. At least 30 readings were btained fr each sample which allwed an accurate plt f the titratin curve. Phenlphthalein was placed in each sample and the ph at which the first develpment f pink clr ccurred was nted. In 41 f these samples, H+ in cncentratin was determined with the glass electrde by the methd f Mre and Scarlata' using an Orin Research, Inc. digital ph meter, mdel 801. Ttal prtein cncentratin was measured by a mdificatin f the biuret methd." Pepsin was measured by the methd f Hunt 7 and the results were expressed in grams f pepsin prtein using a 3 X crystalline pepsin standard (Nutritinal Bichemicals Crpratin). The biuret values f pepsin were similar t thse f the prtein (albumin) used as standard. N npepsin prtein was calculated as the difference between ttal and pepsin prtein J... i 120 E r::: :a 100 > W...J ID 40 «20,,,,/ /:1 SID". / ',-,- ",,,, TITRATABLE ACIDITY (ph 8.3) meq/l FIG. 1. Crrelatin between the titratable acidity t ph 7 and the titratable acidity t ph 8.3 (psthistamine values, 0; pstgastrin and basal values,.). Interrupted line represents the 1: 1 slpe. Results The relatinship between hydrgen in cncentratin and titratable acidity. There was a highly significant crrelatin between H + in cncentratin as measured by the glass electrde and titratable acidity t any given ph. The fllwing representative equatins give the numerical estimate f the relatinship fr titratable acidities at ph 7 and ph 8.3, where y represents H + in cncentratin and x represents titratable acidity t a given ph: at ph 7, y = 1.1Ox 14.0 and r 0.99 (1) and at ph 8.3, y = 1.12x and r = 0.99 (2) As these equatins shw, there was a discrepancy between titratable acidity and H + in cncentratin which increased as the cncentratin f acid decreased. At the limit, H + in cncentratin culd be nil (y = 0) with titratable acidities f 12.7 meq per liter at ph 7 and 17.1 meq per liter at ph 8.3. In nearly pure parietal secretin, hwever, H+ in cncentratin (150 meq per liter) was similar t titratable acidity: meq per liter at ph 7 and meq per liter at ph 8.3. Relatinship between titratable acidities t different ph(s). As expected frm the significant crrelatin between H + in cncentratin and titratable acidities t varius ph levels, there existed a significant crrelatin between titratable acidities t any tw given ph values. This is prtrayed in figure 1 fr titratin t ph 7 and ph 8.3. The crrelatin hlds at all cncentratin levels and fr basal, histamine, and gastrin data. The values reach near equality arund 150 meq per liter, but diverge t a maximum f 5.5 meq per liter represented by the intercept n the hrizntal axis. The latter value als culd be predicted by cmbining equatins 1 and 2 fr that fractin f the results fr which H+ in cncentratin data were available. The end pint f titratable acidity. In
3 March 1970 ACIDITY OF GASTRIC JUICE 347 rder t satisfy cnditins f electrical neutrality in the sample f gastric juice, it may be argued that the best estimate f t i t r a a ble acidity is btained at that ph WhICh gives equality t catins (N a +, K +, and H+ bth dissciated and undissciated) and' anins (Cl-). This f curse neglects the small effects prduced by Ca + + and Mg++ r S04= and H 2P0 4 - which were assumed t cancel ut. In all but three f the 41 samples, the value fr the catins calculated frm the sum f N a +, K +, and H + ins as measured by the glass electrde was less than the crrespnding Cl- cncentratin, the discrepancy widening with decreasing acid cncentratin. This strngly suggested the presence f psitive charge in the undissciated acid fractin presumably dnated by buffer prtns. The sum f Na+, K+, and titratable acidity t ph 7 was als lwer than the C cncentratin in 80% f the samples, while the sum f Na+, K+, and titratable acidity t ph 9.4 was higher than the Cl- cncentratin in 90% f the samples. The best tally at all levels and fr the average f the cmbined data was btained fr titratable acidity t ph 8.3. Table 1 prvides a summary f the average values btained. It is f interest t nte that the first appearance f a faint pink clr with phenlphthalein ccurred in the range f ph 8.1 t 8.7, with a median value f ph h e cincidence between the bserved end pmt fr phenlphthalein and the ph at which titratable acidity gave the best tally f catins and anins is purely frtuitus. Indeed, it is nt uncmmn practice fr titratin using phenlphthalein t prceed t a higher ph beynd the first transient develpment f clr. Undissciated acidity. If, as nted abve, ph 8.3 is adpted as an average end pint, then undissciated acidity wuld represent in each sample the difference between titratable acidity t ph 8.3 and H + in cncentratin as measured by the glass electrde. Equatin 2 culd be transfrmed t give an estimate f undissciated acidity in terms f y, the H+ in cncentratin, r f x, the titratable acidity t ph 8.3: Undissciated acidity titratable acidity x - H + in cncentratin Y y (3) = x (4) Relatinship between prtein buffers and undissciated acidity. Since the H+ in cncentratin and titratable acidity t any ph Were linearly related, it was elected t e s ure the difference between titratable acidity t ph 8.3 and ph 7 fr which results were available ver the whle range (fig. 1) and fr all 70 samples. The difference b e t w e n the tw titratable acidities shuld prvide a direct index althugh nt an actual measure f the true undissciated acidity and shuld be sufficiently accurate fr cmparisn with prtein cnstituents f.the juice: The relatinship between the difference m titratable acidity t ph 8.3 and ph 7 and the crrespnding cncentratin. f t.tal prtein is shwn in figure. 2. The m c r a I. n difference between the titratable acidities with increasing ttal prtein cncentratin was related t the nnpepsin prtein fractin (fig. 3), since the difference in titratin was unrelated t the pepsin cncentratin (fig. 4). In table 2, a summary f all f the results, the samples were gruped fr each TABLE 1. Cmparisn between Cl- cncentratin and the sum f the catins (N a+ + K+ + titratable acidity t ph 7, ph 8.3, r ph 9.4) Sum f catins Acidity N. f Clrange samples ph 7 I ph 8.3 I ph meg/liter meg/liter meg/liter < > a Classificatin f the ranges accrdlllg t titratable acidity at ph 7.
4 348 MAKHLOUF ET AL. Vl. 58, N I... 4 Z 3 -I c H (ph 8.3-pH7) meq IL FIG. 2. Relatinship between ttal prtein cncentratin in grams per liter and the difference between titratable acidities t ph 8.3 and ph 7 (psthistamine values, 0; pstgastrin and basal values,.). Slpe fr mean values as shwn in figure 5. 5.J ';;; 4 E (!) z 3 jjj b If 2 U i= fbi 0- I Z z t.ch ('lh 8.3-pH 7) meq/l FIG. 3. Relatinship between the nnpepsin prtein cncentratin in grams per liter and the difference between titratable acidities t ph 8.3 and ph 7 (psthistamine values, 0; pstgastrin and basal values,.). Slpe fr mean values as shwn in figure 5. milliequivalent f difference between the titratable acidities and the average value f the difference cmpared with the crrespnding average values fr ttal prtein, pepsin, and nnpepsin prtein cncentratin. These results are given in figure 5. Discussin Undissciated acidity and prtein buffers. Of the tw cmpnents f gastric acidity (hydrgen in (H +) and undissciated r buffered hydrgen), nly H+ ins can be measured directly. This requires measurement f H + activity with the glass electrde as described by Mre and 8carlata. 1 Undissciated hydrgen cannt be measured directly but can be estimated nly as the difference between titratable acidity and H + in cncentratin. T measure titratable acidity, an end pint fr the titratin must be identified. This in turn requires the fllwing assumptins: (1) gastric juice is electrically neutral at all times and (2) electric charge carried by unmeasured catins (Ca + +, Mg+ +, etc.) cancels ut the charge carried by unmeasured anins (804=, H 2P04-, etc.). Alteratins in cmpsitin by smtic r electrchemical frces acrss the mucsa are nt relevant immediately t measurements made n the juice fllwing its withdrawal frm the stmach. The first assumptin cannt be challenged seriusly fr all real slutins appear t bey the law f electrical neutrality. Similarly, the cncentratins f unmeasured ins are t lw with the catins clsely matching anins5, 8, 9 t have large effects n electrical neutrality cnsideratins. It was fund that the sum f N a +, K +, and H + ins (determined by the glass electrde) was always less than that f CI-, the difference increasing with decreasing acidity. This difference clsely crrelated with the cncentratin f nnpepsin prtein. The latter is believed t represent in part -l... III E (!) z If) 0- IL! Q e... eo I 0 0,..,.....,1> e t.c H (ph 8.3- ph7) meq/l FIG. 4. Absence f crrelatin between pepsin cncentratin in grams per liter and the difference between titratable acidities t ph 8.3 and ph 7 (psthistamine values, 0; pstgastrin and basal values,.).
5 March 1970 ACIDITY OF GASTRIC JUICE 349 TABLE 2. Range and average difference between titratable acidities t ph 8.3 and ph 7 and the crrespnding titratable acidities and cncentratins f ttal prtein, pepsin, and nnpepsin prtein ATitratable acidity (ph 8.3 t ph 7) Titratable acidities N. f samples Range Mean ph 7 ph 8.3 I Ttal prtein Pepsin Nnpepsin prtein meg/liter g/liter prtein f interstitial fluid rigin and its degradatin prducts.3 In rder t satisfy electrical neutrality cnditins, the substantial unmeasured psitive charge in the juice has t be accunted fr. We pstulate that this charge is accunted fr by undissciated acidity and represents prtns taken up by the nnpepsin prteins and their degradatin prducts. These prtns were titrated ff the prteins by alkalinizatin with NaOH. Since electrical neutrality cnditins usually were satisfied by titratin t ph 8.3, this was taken as the average end pint fr titratable acidity. Of curse, nt all buffered prtns are in the prtein fractin, since ther buffer anins, rganic and inrganic, exist in small quantities in gastric juice10 and electrical neutrality cnsideratins must apply t these als. Fr example, H + and buffer anin A - may react t frm an electrically neutral mlecule: H+ + A- HA The end pint fr titratable acidity based n electrical cnsideratins thus wuld include all bund prtns, but present evidence indicates that mst bund prtns are attached t prtein and fragments f prtein. The view expressed in this paper n the nature f undissciated acidity therefre may be summarized as fllws: it is assumed in agreement with Michaelis ll that the smth titratin curves bserved in gastric juice between ph 4 and ph 8 t 9 represent the fusin f many discrete pk's re- 6 5 II> 4 VI Z i;j 3 a.. 2 TOTAL PROTEIN 6 PEPSIN NON-PEPTIC PROTEIN i!.ch (ph8.3-ph7) meq/l FIG. 5. Relatinship between the average cncentratins f ttal prtein (.), pepsin (ll.), and nnpepsin prtein (.) and the average difference between titratable acidities t ph 8.3 and ph 7. suiting principally frm fragmentatin f prteins in the juice. It is well knwn that the titratin curve f a prtein is characterized by a striking flatness when cmpared with the titratin curve f a mixture f its cnstituent amin acids. This effect is caused by electrstatic interactins between the ingenic grups within the prtein mlecule.12 Fllwing enzymatic fragmentatin which alters the prtein, these ingenic (negatively charged) grups are freed t react with H + ins in slutin. These additinal negatively charged grups by binding additinal H + ins wuld require that titratin with NaOH be carried t an end pint beynd the riginal ph at which the unaltered prtein was secreted. These pep-
6 350 MAKHLOUF ET AL. Vl. 68, N.2 tide fragments presumably have variable pk's which accunt fr the smth titratin curves bserved in gastric juice. It is, f curse, unlikely that a unique ph wuld represent the end pint f titratable acidity fr each and every sample because f variatin f prtein cntent and acidity. It is clear frm t b1, l hwever, e that the adptin f a ph between 8 and 8.5 as an average end pint fr titratin will nt result in significant errrs prvided that the riginal assumptins are crrect. Estimates f undissciated acidity. In practice, a direct estimate f undissciated acidity culd be btained n the basis f equatin 3 frm the value f H + in cncentratin r f equatin 4 frm the value f titratable acidity t ph 8.3. Althugh the predictive eql.latins were develped n the basis f data btained frm a single individual, indirect evidence may be advanced which suggests that they may have wider applicability. Fr example, equatin 2 relating H + in cncentratin and titratable acidity was similar t that develped by Mre and Scarlata l fr psthistalg samples btained frm 3 subjects. The average difference between H+ in cncentratin and titratable acidity t phenlphthalein reprted by McEwan-Alvarad et alp in 45 subjects was 5.8 meq per liter and crrespnds clsely t the difference calculated frm equatin 2 when titratin was extended t ph 8.3. Finally, as shwn in an earlier study,s the cncentratins f nnpepsin prtein presumed respnsible fr the levels f undissciated acidity were similar in the subj ect f this study and a grup f 6 ther subj ects. l4 Practical aspects f acidity measurements. Mre and Scarlata l have nted previusly that "the frmatin f undissciated hydrgen must be cnsidered in evaluating parietal cell functin and in defining 'achlrhydria.' Any hydrgen secreted by the parietal cells remains in a dissciated r inized state (althugh nt all such ins are 'active') nly after all prtn-binding sites have becme saturated." The measurement f parietal cell activity shuld take int accunt all f the hydrgen including that riginally secreted and subsequently buffered. Althugh undissciated acidity largely represents buffered hydrgen it may t sme extent, depending n the accuracy with which the end pint has been defined, i.e., by electrneutrality, represent cnstituent hydrgen frm the peptide fragments. At the usual cncentratins encuntered in secretry studies, the difference between H + in cncentratin and titratable acidity is t small (rarely exceeding 2 t 3%) t be f significance. If parietal cell activity is lw and the cncentratin f buffer is high, as in sme pathlgical cnditins, enugh secreted hydrgen may be remved frm slutin t reduce substantially the H + in cncentratin, i.e., increase sample ph. In these instances, a high ph in the gastric aspirate cannt be equated with the absence f parietal cell activity and the measurement f H+ in cncentratin, titratable acidity, and prtein cncentratin shuld prvide a better indicatin f the secretry status f the subject. REFERENCES 1. Mre, E. W., and R. W. Scarlata The determinatin f gllbtric acidity by the glllbs electrde. Gastrenterlgy 49: Mre, E. W Determinatin f ph by the glass electrde: ph meter calibratin fr gastric analysis. Gastrenterlgy 54: Makhluf, G. M., E. W. Mre, and A. L. Blum Mdels fr the secretin f pepsin and ther prteins by the human stmach. Gastrenterlgy 55: Makhluf, G. M., J. P. A. McManus, and W. I. Card A quantitative statement f the tw-cmpnent hypthesis f gastric secretin. Gastrenterlgy 51: Mre, E. W., and G. M. Makhluff Calcium in nrmal human gllbtric juice: a 4-cmpnent mdel with speculatin n the relatin f calcium t pepsin secretin. Gastrenterlgy 55: Grnall, A. C., C. J. Bardawill, and M. M. David Determinatin f serum prteins by means f biuret reactin. J. Bil. Chem. 177: Hunt, J. N A methd fr estimating peptic activity in gastric cntents. Bichem. J. 42:
7 March 1970 ACIDITY OF GASTRIC JUICE Vagne, M Les electrlytes du suc gastrique humain. Dctral thesis, Faculty f Medicine, Paris. 9. Demand, H. A., H. V. Grss, and G. Berg Effects f cntinuus insulin infusins n unstimulated human gastric secretin. II. Quantitative changes f the gastric juice pattern. Gastrenterlgy 54: Piper, D. W., B. H. Fentn, and L. R. Gdman Lactic, pyruvic, citric and uric acid and urea cntent f the human gastric juice. Gastrenterlgy 53: Michaelis, L Sme prblems cncerning the gastric juice. Harvey Lectures 22: Schultze, H. E., and J. F. Heremans The mlecular bilgy f human prteins, Vl. 1, p. 94. American Elsevier Publishing Cmpany, New Yrk. 13. McEwan-Alvarad, G., J. Lasater, and N. C. Hightwer Cmparisn f values f gastric acidity determined by the glass electrde t values btained by titratin and indicatr techniques. Gastrenterlgy 52: Makhluf, G. M., J. P. A. McManus, and W. 1. Card Cmparative effects f gastrin II and histamine n pepsin secretin in man. Gastrenterlgy 52: