Accelerated peptic activity in ice

Volume

Accelerated

Peptic

A. DOSCHERHOLMEN

Department of

Medicine,

AND

This communication, showing that the IF of native acid gastric juice is destroyedat an acceler-

ated rate in ice, besidesthe theoretical considerations, hasalsopractical implication. METHODS Gastric juice was obtained from patients with peptic ulcer diseasein a basal state or after betazole hydrochloride stimulation. The gastric juice was aspiratedby continuoussuction into a submerged

in ice water

where

it re-

mained until the time-sequencestudies were started, usually within a week after the time of collection. Under these circumstances,the measured temperature of the juices did not exceed +l” C. Studies have shownthat the IF content of gastric

juice under

these conditions

deterio-

6, Lkxmber, Nil I’riulrd jr& U.S. A.

in Ice1

S. E. SILVIS

Veterans Adminibation Hospifal. Minneapolis, Minnesota 5641;

It is known that freezing may accelerateenzyme react,ions(7) aswell as catalyzed and noncatalyzed simple chemicalreactions (6, 8). Several explanationsfor acceleratedchemical reactions in ice have been considered,but no single theory ran satisfactorily explain all the observed phenomena(2,3,8, 11). During in vitro studiesof the gastric intrinsic factor (IF) and the non-IF (NIF) vitamin B, bindersunder various conditions, an accelerated lossof activity was observed in ice in the presenceof peptic activity. To our knowledgethis is the first report of acceleratedpeptic activity in ice. The radioimmunoassayapplied in these studies,being a most reliable in vitro assayfor quantitative determination of the IF (9), has gained wide clinical acceptancein recent years.

container

Activity

8, No.

ruid

University

of Miwnesoftr,

fined as the vitamin B, binder in gast,ric juice that can be blocked by an anti-IF antibody (12). The NIF vitamin B, binder, on t,he other hand, cannot be blocked by this antibody and representsthe rest of the B,, binding capacity of the gastric juice (12). Three types of experiment’swere carried out: (A) Native gastric juices were kept in Erlenmeyer flasks in the ice-water bath in a cold storage room for up to a week from the beginning of each time-sequencestudy. Each day a 5 ml sample was removed and prepared for IF assay. (B) Individual 5 ml portions of native acid gastric juices were placed in a freezer at -20” C. Each day for a week an aliquot, was removed, thawed at air temperature (22” C) and immediately prepared for IF assay. (C) Naiivc acid gastric juices were depepsinizedfor 20 min at pH 11.0 by adding 10% NaOH, whereupon t’he pH was readjustedto it,s original raluc with 10% HCl. Individual 5 ml portions were then stored in the freezer at -20” C. A samplewas removed daily for a week, thawed at 22” C :md immediately prepared for IF assay. No peptic act’ivity could be detected in the gastric juices after depepsinization.The zero-hour samplesin experimentsB and C were not frozen, but, prepared for IF assay immediately before the rest of the correspondingsampleswere plared in the freezer. The pH of all samplesprepared for IF assay wasadjusted to 7.0 and the sampleswere stored at -20” C. The IF assaywasperformed at ronm temperature within 1-2 weeksafter this preparation. The IF content of neutralized gastric juice st’oredat -20” C remainsst#ablefor man)

rates very slowly and imperceptibly, requiring about 5-7 days from the time of collection before a real lossbecomesevident. Depepsinization months. The charcoal method (5) as adupt,ed for (16) and pept,ic activity determinations (10) quant,itative IF assay (12) was used, with 111r were carried out according to methods pre- following minor modifications: 2 ml of normal viously described. saline with a pH of i.0 served as a \-chicle for For the purpose of this study, the IF is de- the gastric juice and the serum. and the supernate rather than the charcoal was count& by Received July 21, 1971. ’ A brief abstract of this work has appeared Clin. Res. 19, 390 (1971).

scintillation spectrometry in a well-tyl)c, Xu(~l~x Chicago Model 4233 automatic countcsr. (‘om-

in 577

575

DOSCHERHOLMEN

Dnys FIG. 1. Percentage Ioss of intrinsic factor vitamin B,, binding capacities of native acid human gastric juices stored at $1” C (A) and -20” C (B) and dcpepsinized, reacidified gastric juices stored at -20” C (C). Values indicate means (2 S.E.) of 7 (A). 15 (B) and 8 (C) gastric juices obtained after betazole hydrochloride stimulation. parison with the use of buffered saline with a pH of 7.0 as a vehicle gave identical results. The “‘Co-B12 used had a specific activity of about 1 &i/~g vitamin B,,. The reproducibility of the B,? binding capacities with this method is very good and generally within the range of 22 ng units for binding capacities below 50 units/ml of gastric juice, and *3 unit’s for those over 50 units/ml, which is in agreement with the experience of Irvine (9). In all time-sequence studies, the loss of the IF and NIF B,, binders was expressed as a perccnta.ge of the activity found at the beginning of each experiment. By having a zero-hour control value for each sequence st,udy for comparison, no adjustment for dilutional changes due to depepsinization and reacidification was therefore necessary. In no instance did the total vitamin B,, binding capacity of the gastric juice exceed 65% of the added “CO--B~~

(12) RESULTS The IF activity of seven different gastric juices, obtained after betazole stimulation, and stored in the ice-water bath, is shown in Fig. 1A. Under these circumst,ances the IF act,ivity determined at daily intervals was quite stable with an average loss of 8.6% (SE 3.8) at the end of a week. The IF activity of individual samples of 15

AND SILVIS different gastric juices that were stored at 20” C for a week is also shown in Fig. 1B. Under this condition a relatively rapid loss of IF activity occurred. Thus, after a day the IF loss averaged 56.3% (SE 5.8), and after a week 98.2% (SE 0.7) . In eight instances the above gastric juices were depepsinized followed by reacidification to the original pH values before individual samples were frozen at -20” C. The IF content of the samples treated in this manner is shown in Fig. IC. The IF loss of these aliquots was considerably smaller than that of native gastric juices stored under similar circumstances, but slightly Iarger than that of gastric juices stored in the ice-water bath. Thus, the average IF loss amounted to 8.0% (SE 2.9), and 46.4% (SE 9.8) after 1 and 7 days, respectively, in these 8 depepsinized, reacidified juices. Gastric juice obtained after stimulation contains very little NIF B,, binder. In contrast, basal gastric juice has a relatively high concentration of this type of B,, binder (15). Consequently, similar studies were carried out on 3 gastric juices obtained in the fasting state. The results of the IF studies in fasting gastric juices were entirely similar t,o those obt,ained in juices after betazole hydrochloride stimulation (Fig. 2, upper-half). The behavior of the NIF B, binder was similar to that of the IF B,, binder when the juices were stored at +l” C (Fig. 2, lowerhalf A). At -20” C the behavior of these two vit,amin B,, binders, however, differed. Thus, at this temperature the NIF B, binder of native gast’ric juices deteriorated at a slightly slower rate than did the IF B,, binder (Fig. 2, lowerhalf B) . The average loss for the NIF B, binder after a week amounted to 68.0% (SE 13.1) compared with 96.1% (SE 1.9) for the IF B,, binder. In the depepsinized, reacidified gastric juices stored at -20” C, the NIF B, binder was stable (Fig. 2, lower-half C) Thus, after a week an average activity of 105.1% (SE 5.2) was found compared with an average IF loss of 51.1% (SE 10.8) during the same period of time. DISCUSSION These st,udies show an accelerated loss of IF and NIF B,, binders of gastric juice stored in its nat,ive acid state at -20” C as compared with storage at +I” C. Intrinsic factor from fasting

ACCELERATED

PEPTIC ACTIVITY

gastric juice behaved in a manner similar to that obtained after betazole hydrochloride stimulation. The observation of a more rapid destruction of IF and NIF B, binders in native gastric juices stored at -20” C as compared with depepsinized, reacidified gastric juices suggests an acceleration of peptic activity in ice. The reason for some loss of IF activity in the depepsinized, reacidified juices is not entirely clear. It is possible that IF, being somewhat unstable in an acid environment (4, 16), was more rapidly destroyed at -20” C due to the known acceleration of simple chemical reactions in ice. Another possibility is that some enzymatic, although not peptic, activity might have remained after depepsinization. A gastric protease with maximal pH activity at 1.8 and with the ability to resist depepsinization at pH 8.3, in contrast to the pepsins, has recently been described (13). It remains to be shown, however, whether this enzyme can degrade the IF or even withstand depepsinization at pH 11.0 for 20 min as used in our studies. The greater stability of the NIF B, binder in native acid gastric juices is of considerable interest. At differing temperatures the NIF B,, binder has also been found to be more resistant than the IF to peptic digestion (1, 14). The difference in the behavior of the two B, binders after freezing of native acid and depepsinized, reacidified gastric juices probably reflects basic st*ructural differences between them. The reason for acceleraGon of chemical reactions in ice is incomplet,ely understood (11). At moderately low temperatures, water in the frozen state is highly structured with ice crystals and tiny pockets of water containing the reactants in a highly concentrated form (3, 11). Under these circumstances, chemical reactions may accelerate. Increased concentration of the reactants (3), catalytic action by ice structure itself (a), increased proton mobility in ice (8), and favorable substrate-catalyst positional constraint (8) have all been considered causative fart)ors behind this phenomenon. The present study with its findings of accelerated pept’ic artivity in ire has practical clinical implication for workers using t,his radioimmunoassay insofar that native acid gastric juice should never be frozen before it, is depepsinized and prepared for routine quantitative IF assay.

+I0

>

+100

.t

579

IN ICE Intrinsic Factor

Non-Intrinsic

Factor C

A

H

-2O-

2 ;

-4O-GO5 -BO-

-loo3 Days Percentage loss of intrinsic factor and non-intrinsic factor vitamin Bu binding capacities of acid human gastric juice obtained from 3 patients without any stimulation. Values represent means (* SE) of non-depepsinized samples kept at +l” C (A) and -20” C (B) and depepsinized, rcacidified samples stored at -20” C (C). FIG.

2.

SUMMARY When native acid human gastric juice was kept at +l” C for a week, the intrinsic factor (IF) and t’he non-IF (NIF) B, binding capacities as determined by a standardized radioimmunoassay were quite stable. In contrast, a relatively rapid deterioration of the IF B,, binder and slightly less rapid decrease in the NIF B,? binder occurred when native acid gastric juice was kept at -20” C for a simila,r period of time. In depepsinized gastric juices, reacidified to their original pH values and kept at -20” C, the NIF B,, binder was stable, whereas the IF B,, binding capacity showed some loss, but always less than that seen in the corresponding nondepepsinized gastric juices. It was therefore concluded that, peptic activity was accelerated in ice. The practical clinical implication of t,his observation for the immunoassay of TF is stressed.

DOSCHERHOLMEN

580 REFERENCES

1. Ashworth, L. A. E., Strickland, R. G., Koo, N. C., and Taylor, K. B. Effect of pH on intrinsic factor and non-intrinsic factor vitamin B1e binding in human gastric juice neutralized in viva Gastroenferofog’$J 57, 506510 (1969). 2. Bruce, T. C., and Butler, A. R. Catalysis in water and ice. II. The reaction of thiolactones with morpholine in frozen systems. J. Amer. Chem. sot. 86,4104-4108 (1964). 3. Butler, A. R., and Bruce, T. C. Catalysis in water and ice. A comparison of the kinetics of hydrolysis of acetic anhydride, /3-propiolactone, and p-nitrophenyl acetate and the dehydration of 5-hydro-6-hydroxy-deoxuridine in water and in ice. J. Amer. Chem. sot. 86,313319 (1964). 4. Castro-Curel, Z. Effects of peptides and proteolysis on intrinsic factor (IF) activity. CZin.Res.

12,205 (1964).

5. Gottlieb, C., Lau, K. S., Wasserman, L. R., and Herbert, V. Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated B,z binding capacity, antibody to IF, and serum unsaturated BU binding capacity. Blood 25,875-883 ( 1965). 6. Grant, N. H., and Alburn, H. E. Fast reactions of ascorbic acid and hydrogen peroxide early environment. in ice, a presumptive Science 150,15891590(1965). 7. Grant. N. H., and Alburn, H. E. Acceleration of enzyme reactions in ice. Nature London 212, 194 (1966).

AND SILVIS 8. Grant, N. H., Clark, D. E., and Album, H. E. Imidazole- and base-catalyzed hydrolysis of penicillin in frozen systems. J. Amer. Chem. Sot. 83,4476-4477 (1961). 9. Irvine, W. J. Immunoassay of gastric intrinsic factor and the titration of antibody to intrinsic factor. Clin. Exp. Immunol. 1, QQ118 (1966). 10. Klotz, A. P. The laboratory determination of gastric proteases. Ann. N. Y. Acad. Sci. 140, 697-708 (1967). 11. Larsen, S. S. Kemiske reaksjoner i frosne opIosninger. En oversigt. Dan. Tidsskr. Farm. 41, 177-190 (1967). 12. RGdbro, P., and Christiansen, P. M. Quantitative determination of gastric intrinsic factor after large histamine doses in healthy persons. Stand. J. Clin. Lab. Invest. 19, 186189 (1967). 13. Samloff, I. M. Slow moving protease and the seven pepsinogens. Electrophoretic demonstration of the existence of eight proteolytic fractions in human gastric mucosa. Gastroen terologw 57,659-669 ( 1969). 14. Simons, K. Vitamin B= binders in human body fluids and blood cells. Sot. Sci. Fenn., Commentat. Biol. 27,5 (1964). 15. Strickland, R. C., Ashworth, L. A. E., Koo, N. C., and Taylor, K. B. Intrinsic factor, non-int,rinsic factor vitamin B, binder, and pepsinogen secretion in normal subjects. Gastroenterology 57,511-517 (1969). 16. Sullivan, L. W., Herbert, V., and Castle, W. B. In vitro assay for human intrinsic factor. J. Clin. Invest. 42, 1443-1458 (1963).