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Effect of Time, Temperature, and Peptic Activity on Intrinsic Factor Content of Acid Gastric Juice
Vol. 61. No.6
GASTROENTEROLOGY
Printed in U.S.A.
Copyright ©1971 by The Williams & Wilkins Co.
EFFECT OF TIME, TE-1\IPERATURE, AND PEPTIC ACTIVITY ON INTRINSIC FACTOR CONTENT OF ACID GASTRIC JUICE A. DoscHERHOLMEN, M.D., PH .D.,
AND
S. E. SILvis, M.D.
Sections of Hematology and Gastroenterology, Department of Medicine, Veterans Administration Hospital, and University of Minnesota, Minneapolis, Minnesota
The effect of time and temperature on the intrinsic factor (IF) activity of acid in native human gastric juice was investigated using a standardized radioimmunoassay. The following observations were made on the loss of IF activity: at body temperature, the loss was very rapid over a period of 4 hr; at room temperature, the loss occurred at a much slower rate over a 7-day period; at + 1 C the loss was almost imperceptible over a week's time; at - 20 C the deterioration was relatively rapid and led to an almost total loss in only 7 days; and at -70 C, after an initial fall, no further deterioration occurred for the next 6 days. The initial loss at -70 C could be minimized by rapid freezing and thawing of the gastric juice. When acid gastric juice was depepsinized, reacidified to its original pH value, and kept at body temperature, practically no loss of IF activity occurred over a 4-hr period. Thus the loss of IF at body temperature would seem to be caused by the peptic activity. Depepsinized, reacidified gastric juice stored at - 20 C showed only a minor loss of IF activity over several days. Therefore, the relatively rapid loss of IF activity when native acid gastric juice is stored at -20 C would seem to be due mainly to the peptic activity. Gastric intrinsic factor (IF) can be readily quantitated by radioimmunoassay. 2 • 3 The IF has been found relatively resistant to storage at neutral pH. 4 However, using the radioimmunoassay conflicting results have been reported regarding the stability of IF activity of gastric juice stored at an acid pH. Thus, lrvine 3 reported no loss of IF activity whereas Ashworth and associates5 found that it deteriorated rapidly. This unexplained discrepancy has been of concern to investigators. 5 Since the test conditions of these studies differed, the discrepancy in their results may have been caused by factors other than the pH. Received April 13, 1971. Accepted June 28, 1971. A preliminary report of this work was presented at the 13th Congress of the International Society of Hematology held in Munich, Germany, August 2 to 8, 1970, and an abstract has been published. 1 Address requests for reprints to: Dr. A. Doscherholmen, Veterans Administration Hospital, Minneapolis, Minnesota 55417.
Using a standardized radioimmunoassay for quantitation, we studied the IF activity in acid gastric juice taken from human subjects and stored at different temperatures for varying lengths of time. The findings show great variation in IF activity, depending on the storage conditions of gastric juice, and appear to explain the discrepancy in results reported by previous investigators.
Methods Twenty patients with peptic ulcer were selected for study. After the patients received betazole hydrochloride stimulation, samples of gastric juice were collected and placed in an ice-water bath in a cold storage room until used. Temperature measurements of the samples of gastric juice, ranging in pH from 1.05 to 1.5 did not exceed + 1C. The time sequence studies were started immediately after the gastric juices were collected in four experiments, after 1 to 7 days in 20, between 7 and 14 days in four, and after 24 days in one. Daily measurements started 844
December 1971
845
INTRINSIC FACTOR IN ACID GASTRIC JUICE
from the time of collection showed that gastric juice, stored in this manner, loses its IF activity very slowly and imperceptibly, and it may take 5 to 7 days before a real loss becomes evident. The contents of IF in the gastric juices ranged from 22 to 111 U per ml, 1 U being that amount of IF capable of binding 1 ng of Co 57-labeled vitamin B 12 . Experiment 1. Two portions of gastric juice were labeled A and B, respectively, and placed in a water bath at +37 C. Portion A was incubated in its native state, and portion B was incubated after depepsinization and reacidification to the original pH. Depepsinization was carried out at pH 11.0 for 20 min • before the readjustment of the pH. When the temperature of the two portions of gastric juice had equilibrated with the temperature of the water bath, 5-ml aliquots were removed at stated intervals and immediately prepared for IF assay. Experiment 2. Nondepepsinized acid gastric juice was kept at a room temperature of +22 C for various lengths of time. At stated intervals, 5-ml samples were removed and immediately prepared for IF analysis. Experiment 3. Nondepepsinized acid gastric juice was stored at + 1 C, - 20 C, and -70 C. When stored at + 1 C, the gastric juice was kept in an Erlenmeyer flask; when stored at colder temperatures, the gastric juice was divided into 5-ml samples and frozen separately. At time zero and at stated intervals thereafter, 5-ml portions of gastric juice were removed from storage and prepared for IF analysis. The 5-ml samples frozen at -20 C were placed in the freezer in liquid form and then were thawed in the air at room temperature, usually in 45 min. Some samples stored at -70 C were handled in a similar manner (frozen and thawed slowly); others were frozen in an acetone- C02 bath for 3 min before being placed in the freezer and then were thawed in a water bath at room temperature (frozen and thawed quickly), which took about 3 min. Experiment 4. Acid gastric juice was divided into two portions, A and B, respectively. Samples containing 5 ml each of portion A in its natural state were placed in a freezer at -20 C. Portion B was depepsinized as in experiment 1, and after the pH had been readjusted to its original value, individual 5-ml aliquots were removed and frozen at - 20 C. At time zero and at stated intervals, samples were removed, thawed in the air at room temperature, and prepared for IF assay. In all the samples prepared for IF assay, the pH was readjusted to 7.0 and the analysis took place at room temperature. The charcoal
method 7 as adapted for quantitative IF assay 2 was used, with the following minor modifications: 2 ml of normal saline of pH 7.0 served as a vehicle for the gastric juice and serum and the supernatant rather than the charcoal was counted. Comparison with buffered normal saline with a pH of 7.0 as vehicle gives identical results. In no instance did the total B 12 binding capacity of the gastric juice exceed 65 % of the Co 57 vitamin B 12 that had been added.' The IF assays were performed within 1 to 2 weeks after the samples were prepared and stored at -20 C. Experiments have shown that there is no significant deterioration of the IF content of neutralized gastric juice by such storage for many months. The IF content of the gastric juices was always determined at the beginning of each time sequence study (100% activity at time zero). All subsequent measurements were expressed in percentage of this original value. By having a zero-hour control value for each sequence study for comparison, the small dilutional changes brought about by the depepsinization and reacidification procedure (performed with 10% NaOH and 10% HCl solutions) were obviated. Peptic activity, measured according to the method of Klotz, 8 was completely destroyed after depepsinization at pH 11.0 for 20 min. Co 57 was counted by scintillation spectrometry in a well-type Nuclear-Chicago Model 4233 automatic counter. The counting error was less than 4% with a 95 % confidence limit.
Results Figure 1 shows the results of experiment 1, which was performed with gastric juices obtained from 3 patients and kept at body temperature. The IF activity in nondepep-
.,
- - - Non- d•p•psi ni zed
co"'
--------- Depepsiniz•d
I
"' cO. ~
~
+10 0
~
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----------~
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-40
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.
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/pHt.s
5 L-7.~7--~==~~~~~==~~==~ ""-100 )! 3 4 ~ Hours
FIG. 1. Intrinsic factor activity of nondepepsinized and depepsinized acid gastric juice taken from 3 human subjects and stored at +37 C.
DOSCHERHOLMEN AND SILVIS
846
sinized acid gastric juice decreased rapidly: the average loss was 46% after 1 hr and 92% after 4 hr. When the same gastric juices were depepsinized and reacidified to the original pH and then were simultaneously incubated, the loss in IF activity averaged 2 and 5% after 1 and 4 hr, respectively. As seen in the figure, the rate of loss of IF in native gastric juices varied slightly and was greatest in the most acid and smallest in the least acid juice. Figure 2 shows the results of experiment 2, which was performed on nondepepsinized acid gastric juice taken from 3 patients and kept at room temperature. The IF activity deteriorated quite slowly over a 7-day period and the average loss in IF activity was 7 and 39% after 4 and 24 hr, respectively, in three samples of gastric juice; 73% after 72 hr in two samples; and 93 % after 168 hr in one sample. The figure shows that the rate of loss of IF was greatest in the most acid and smallest in the least acid gastric juice. Figure 3 shows the results of experiment 3, in which samples of native acid gastric juice were stored at various cold t~m~e~a tures. When the samples of gastnc JUICe from 3 patients were stored at +1 C, the IF activity deteriorated very slowly. After 1, 3, and 7 days, the average IF loss was 5, 7 and 15%, respectively. When the samples of native gastric juices from 9 patients were stored at - 20 C, the IF activity showed relatively rapid deterioration, the loss averaging 48% after 1 day, 83% after 3 days, and 99% after 7 days.
Hours
FIG. 2. Intrinsic factor activity of nondepepsinized acid gastric juice taken from 3 human subjects and stored at room temperature ( +22 C).
+ZO
+l"c
IZ345&7
Days
Vol. 61 , No . 6
-zo'c
IZ345o7
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FIG. 3. Intrinsic factor activity of nondepepsinized acid gastric juices taken from human subjects and stored at various cold temperatures ranging from +1 C to - 70 C.
When the samples of native gastric juice from 2 patients were frozen slowly at - 70 C and thawed slowly at room temperature, the average initial loss of IF activity was 67.5% after 1 day, but no further deterioration occurred during the next 6 days of storage. When the samples of native gastric juice from 2 other patients were frozen quickly, stored at -70 C, and thawed rapidly, the IF activity showed an initial but minor loss averaging 10 ~;, after 1 day, with no further deterioration during the next 6 days. Figure 4 shows the results of experiment 4 in which samples of gastric juice from 4 ~tients were used. In the depepsinized samples, the loss of IF activity was minor, ranging from 5 to 36 ~(, after 5 or 6 days of storage, whereas in the nondepepsinized samples, the loss of activity ranged from 64 to lOOS;, over the same period. To learn the effect of acid alone on IF activity, a sample of depepsinized gastric juice containing 49 U of IF activity per ml was reacidified to a pH range of 0.5 to 7.0. Individual portions of this sample and a sample of nondepepsinized gastric juice were then stored at - 20 C. Individual samples were removed daily and assayed for IF activity. The results are shown in figure 5. The nondepepsinized control samples, as was expected, revealed a rapid loss of IF activity. The depepsinized samples showed little, if any, actual loss of IF
December 1971
0"1
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...... _____ .......... . .. _ _ _ _ _ _ _ _ 4
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1
847
INTRINSIC FACTOR IN ACID GASTRIC JUICE
2
345o
Days
1234So
Days
FIG. 4. Intrinsic factor activity of nondepepsinized
and depepsinized acid gastric juice taken from 4 human subjects and stored at - 20 C.
activity until the pH fell below 3.0. As the pH values dropped, the loss of IF activity increased until a pH value of 1.5 was reached. Below this point, the actual loss of IF activity was, if anything, a little less pronounced. The influence that the storage age of gastric juice at the time of freezing might have on IF activity was investigated. This was done by slowly freezing samples of a native gastric juice containing 31 and 14 U of IF activity per ml after storage for 4 and 24 days respectively, in an ice-water bath. Samples were removed daily from the freezer, thawed slowly, and analyzed for IF activity. Table 1 shows that the loss of IF activity was more rapid in samples of gastric juice that had been stored in an ice-water bath for 4 days than those that had been stored for 24 days.
phasized the excessive production of IF in normal man. Only 1% or less of the daily production of IF is needed for the absorption of the daily minimum requirement of vitamin B, 2 • 12 The rapid digestion of IF by normal gastric juice at body temperature may explain the need for the apparent oversupply of this vital factor. The small loss of the IF activity in depepsinized, reacidified gastric juice kept at body temperature may indicate that the acid alone damages the IF. This is consistent with previous findings. s, 13 It is understandable that lrvine 3 did not report a slow loss rate in IF activity of nondepepsinized acid gastric juice kept at room temperature, since he did no!; carry out his investigation beyond 2 hr of storage. Our studies showed an average loss of only 1% of the IF activity after 2 hr of storage, which is within the error of the assay system. At room temperature the destruction of IF activity was much slower than at body temperature. The relatively rapid deterioration of IF activity in samples of gastric juice stored at - 20 C was in striking contrast to the relative stability of IF when the acid gastric juice was kept for a prolonged time in an ice-water bath. Apparently the peptic activity is most responsible for the accelerated loss of IF activity in nondepepsinized acid gastric juice storage at - 20 C. To our knowledge, this observation has not been made before, but it is consistent with the
w... "' u
Discussion These studies reveal a profound effect of time and temperature on the stability of IF activity of nondepepsinized acid gastric juice from human subjects. The rapid destruction of the IF activity in native gastric juice at +37 C must be due to the peptic activity since practically no IF loss occurred in the corresponding depepsinized juices (fig. 1). This is in agreement with the findings of Ashworth and associates. 5 Undoubtedly this type of autodigestion of IF also occurs in vivo. 9 • 10 ROdbro 11 has em-
- - Non-depepsini ze d
'-
.eu · ;;:;
c: .E .,."" c:
~ c: c: I<>
"' c: ..c: u
'if~~~~~";-;-;~:~--~;; jj
·20 -40
"""'!:!
-&o
-so
"'
·------• D~pepsinind
+10 0
··-pH 1.5
-100 0
3
4
"
7
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FIG. 5. Intrinsic factor activity of nondepepsinized acid gastric juice taken from a human subject versus that of the same gastric juice depepsinized and reacidified to pH values ranging from 0.5 to 7.0.
848
DOSCHERHOLMEN AND SILVIS TABLE
Vol . 61, No. 6
1. Comparison of intrinsic factor loss in a gastric juice k ept in an ice-water bath for 4 days and 24 days before freezing at -20 C Percentage of intrinsic factor loss after freezing
Gastric juice kept in ice·water bath
Day 0
Day I
Day 2
Day 3
Day 4
0 0
26 17
33 30
53 27
50 32
Day 5
Day 6
Day 7
Day 8
63
66 36
77 47
Day 9
days
4 24
concept of accelerated enzyme activity in ice, which is a newly described phenomenon.14 Apparently hydrochloric acid alone also increases the rate of destruction of the IF activity of gastric juices stored at -20 C. This observation is in accord with the concept that simple (:hemical reactions are accelerated in ice, a phenomenon also previously described. 15 There may be an optimum pH for the damaging effect of acid on the IF activity. In figure 5, the results of the experiment show an optimal pH of 1.5, because at pH values of 1.0 and 0.5 no further increase in the rate of loss of IF occurred. Another possible cause is that the gastric juice may have contained an enzyme with optimal activity at pH 1.5 and with the ability to withstand alkalinization. Such a gastric protease has recently been described. 16 Whether it can withstand a pH of 11.0 for 20 min and still be damaging to the IF remains to be seen. Several possible causes for individual variations in loss rate of IF when nondepepsinized acid gastric juices were stored at - 20 C (fig. 3) are: differences in pH values, content of pepsin, and qualitative variations in pepsin. At least eight different proteolytic fractions in human gastric mucosa exist. 16 It is not unlikely that they have individual functions and that one or more of them are more active than others in the degradation of the IF. Finally, the storage age of the gastric juice at the time of freezing also may have influenced the rate of IF loss (table 1). The slower rate of loss of IF after prolonged storage at + 1 C may possibly be due to alterations in the IF or to quantitative or qualitative changes in the peptic activity of the gastric juice. At - 70 C the initial loss of IF activity may have been due to the damaging effect
46
of the periods of moderately low temperature as the gastric juice was frozen and thawed. This concept is supported by the finding that this loss could be minimized by freezing and thawing quickly. The acceleration of chemical reactions in ice is not clearly understood. 17 Increased concentrations of the reactants in highly structured frozen water at moderately low temperatures, 18 catalytic action by ice structure itself, 19 i1,;,;reased proton mobility in ice, 20 and favorable substrate-catalyst positional constraint, 20 have all been considered causative factors. At moderately low temperatures, water in the frozen state is highly structured with ice crystals and tiny pockets of water containing the reactants. 17 Under these circumstances, chemical reactions may accelerate. In biological systems, water is solidly frozen at - 65 C to - 70 C, 21 which may explain the stability of IF activity when gastric juice is stored at - 70 C. Ardeman et al. 22 reported a mean value of 8900 U for IF activity in the 1st hr after histamine stimulation in normal subjects, whereas ROdbro and Christiansen 2 reported a mean value of 16,000. Although Chanarin 23 ascribed this difference largely to the increased volume of gastric juice that ROdbro and Christiansen obtained by continuous suctioning, the latter investigators also found a 10 ~;, higher mean concentration of IF activity. This difference in mean concentration may have been due to the fact that Ardeman and Chanarin at times used intermittent suctioning, which could have permitted in vivo autodigestion of IF. These observations on the collecting and storing of gastric juice to minimize the loss of IF activity have practical use in the quantitative determination of this factor. As soon as the gastric juice is secreted, it
December 1971
INTRINSIC FACTOR IN ACID GASTRIC JUICE
should be removed from the stomach and immediately lowered to + 1 C. This can be done by continuous suctioning of the gastric juice into a container submerged in ice water, where it should remain until it is depepsinized. Acid gastric juice should never be frozen before it is depepsinized and prepared for routine quantitative IF assay. REFERENCES
11. 12.
13.
14.
1. Doscherholmen A: Effect of pH, time and tem-
2.
3.
4.
5.
6.
7.
8.
9. 10.
perature on the intrinsic factor activity of human gastric juice (abstr) . J Lab Clin Med 76:882, 1970 Rodbro P, Christiansen PM: Quantitative determination of gastric intrinsic factor after large histamine doses in healthy persons. Scand J Clin Lab Invest 19:186-189, 1967 Irvine WJ : hnmunoassay of gastric intrinsic factor and the titration of antibody to intrinsic factor. Clin Exp hnmun 1:99-118, 1966 Glass GBJ: Gastric intrinsic factor and its function in the metabolism of vitamin Bu . Physiol Rev 43:529-849, 1963 Ashworth LAE, Strickland RG, Koo NC, et al: Effect of pH on intrinsic factor and nonintrinsic factor vitamin Bu binding in human gastric juice neutralized in vivo. Gastroenterology 57:506-510, 1969 Sullivan LW, Herbert V, Castle WB: In vitro assay for human intrinsic factor. J Clin Invest 42:14431458, 1963 Gottlieb C, Lau K, Wasserman LR, et al : Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated Bu binding capacity, antibody to IF, and serum unsaturated B u binding capacity. Blood 25:875-883, 1965 Klotz AP: The laboratory determination of gastric proteases. Ann New York Acad Sci 140:697-708, 1967 Abels J, Schilling RF: Protection of intrinsic factor by vitamin Bu . J Lab Clin Med 64:375-384, 1964 Grasbeck R. Simons K, Sinkkonen 1: Isolation of
15.
16.
17.
18.
19.
20.
21.
22.
23.
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intrinsic factor and its probable degradation product, as their vitamin B, 2 complexes, from human gastric juice. Biochim Biophys Acta 127:47-58, 1966 Rodbro P: Human gastric intrinsic factor secretion. Scand J Gastroent 4:473-475, 1969 Ardeman S, Chanarin 1: Assay of gastric intrinsic factor in the diagnosis of Addisonian pernicious anemia. Brit J Haemat 11 :305-314, 1965. Castro-Cure) Z: Effects of peptides and proteolysis on intrinsic factor (IF) activity. Clin Res 12:205, 1964 Grant NH, Album HE: Acceleration of enzyme reactions in ice. Nature (London) 212:194, 1966 Grant NH, Album HE: Fast reactions of ascorbic acid and hydrogen peroxide in ice, a presumptive early environment. Science 150:1589-1590, 1965 Samloff IM : Slow moving protease and the seven pepsinogens. Electrophoretic demonstration of the existence of eight proteolytic fractions in human gastric mucosa. Gastroenterology 57:659-669, 1969 Larsen SS : Kemiske reaksjoner i frosne oplosninger. En oversigt. Dansk T Farm 41 :177- 190, 1967 Butler AR. Bruice TC: Catalysis in water and ice. A comparison of the kinetics of hydrolysis of acetic anhydride, tl-propiolactone, and p-nitrophenyl acetate and the dehydration of 5-hydro-6-hydroxydeoxyuridine in water and in ice. J Amer Chern Soc 86:313-319, 1964 Bruice TC, Butler AR: Catalysis in water and ice. II. The reaction of thiolactones with morpholine in frozen systems. J Amer Chern Soc 86:4104-4108, 1964 Grant NH, Clark DE, Album HE: hnidazole- and base-catalyzed hydrolysis of penicillin in frozen systems. J Amer Chern Soc 83:4476-4477, 1961 Sussman MV, Chin L: Liquid water in frozen tissue: Study by nuclear magnetic resonance. Science 151:324-325, 1966 Ardeman S, Chanarin I, Doyle JC: Studies on secretion of gastric intrinsic factor in man. Brit Med J 2:600-603, 1964 Chanarin I: Intrinsic factor, Clin Chim Acta 22: 85-90, 1968
GASTROENTEROLOGY
Printed in U.S.A.
Copyright ©1971 by The Williams & Wilkins Co.
EFFECT OF TIME, TE-1\IPERATURE, AND PEPTIC ACTIVITY ON INTRINSIC FACTOR CONTENT OF ACID GASTRIC JUICE A. DoscHERHOLMEN, M.D., PH .D.,
AND
S. E. SILvis, M.D.
Sections of Hematology and Gastroenterology, Department of Medicine, Veterans Administration Hospital, and University of Minnesota, Minneapolis, Minnesota
The effect of time and temperature on the intrinsic factor (IF) activity of acid in native human gastric juice was investigated using a standardized radioimmunoassay. The following observations were made on the loss of IF activity: at body temperature, the loss was very rapid over a period of 4 hr; at room temperature, the loss occurred at a much slower rate over a 7-day period; at + 1 C the loss was almost imperceptible over a week's time; at - 20 C the deterioration was relatively rapid and led to an almost total loss in only 7 days; and at -70 C, after an initial fall, no further deterioration occurred for the next 6 days. The initial loss at -70 C could be minimized by rapid freezing and thawing of the gastric juice. When acid gastric juice was depepsinized, reacidified to its original pH value, and kept at body temperature, practically no loss of IF activity occurred over a 4-hr period. Thus the loss of IF at body temperature would seem to be caused by the peptic activity. Depepsinized, reacidified gastric juice stored at - 20 C showed only a minor loss of IF activity over several days. Therefore, the relatively rapid loss of IF activity when native acid gastric juice is stored at -20 C would seem to be due mainly to the peptic activity. Gastric intrinsic factor (IF) can be readily quantitated by radioimmunoassay. 2 • 3 The IF has been found relatively resistant to storage at neutral pH. 4 However, using the radioimmunoassay conflicting results have been reported regarding the stability of IF activity of gastric juice stored at an acid pH. Thus, lrvine 3 reported no loss of IF activity whereas Ashworth and associates5 found that it deteriorated rapidly. This unexplained discrepancy has been of concern to investigators. 5 Since the test conditions of these studies differed, the discrepancy in their results may have been caused by factors other than the pH. Received April 13, 1971. Accepted June 28, 1971. A preliminary report of this work was presented at the 13th Congress of the International Society of Hematology held in Munich, Germany, August 2 to 8, 1970, and an abstract has been published. 1 Address requests for reprints to: Dr. A. Doscherholmen, Veterans Administration Hospital, Minneapolis, Minnesota 55417.
Using a standardized radioimmunoassay for quantitation, we studied the IF activity in acid gastric juice taken from human subjects and stored at different temperatures for varying lengths of time. The findings show great variation in IF activity, depending on the storage conditions of gastric juice, and appear to explain the discrepancy in results reported by previous investigators.
Methods Twenty patients with peptic ulcer were selected for study. After the patients received betazole hydrochloride stimulation, samples of gastric juice were collected and placed in an ice-water bath in a cold storage room until used. Temperature measurements of the samples of gastric juice, ranging in pH from 1.05 to 1.5 did not exceed + 1C. The time sequence studies were started immediately after the gastric juices were collected in four experiments, after 1 to 7 days in 20, between 7 and 14 days in four, and after 24 days in one. Daily measurements started 844
December 1971
845
INTRINSIC FACTOR IN ACID GASTRIC JUICE
from the time of collection showed that gastric juice, stored in this manner, loses its IF activity very slowly and imperceptibly, and it may take 5 to 7 days before a real loss becomes evident. The contents of IF in the gastric juices ranged from 22 to 111 U per ml, 1 U being that amount of IF capable of binding 1 ng of Co 57-labeled vitamin B 12 . Experiment 1. Two portions of gastric juice were labeled A and B, respectively, and placed in a water bath at +37 C. Portion A was incubated in its native state, and portion B was incubated after depepsinization and reacidification to the original pH. Depepsinization was carried out at pH 11.0 for 20 min • before the readjustment of the pH. When the temperature of the two portions of gastric juice had equilibrated with the temperature of the water bath, 5-ml aliquots were removed at stated intervals and immediately prepared for IF assay. Experiment 2. Nondepepsinized acid gastric juice was kept at a room temperature of +22 C for various lengths of time. At stated intervals, 5-ml samples were removed and immediately prepared for IF analysis. Experiment 3. Nondepepsinized acid gastric juice was stored at + 1 C, - 20 C, and -70 C. When stored at + 1 C, the gastric juice was kept in an Erlenmeyer flask; when stored at colder temperatures, the gastric juice was divided into 5-ml samples and frozen separately. At time zero and at stated intervals thereafter, 5-ml portions of gastric juice were removed from storage and prepared for IF analysis. The 5-ml samples frozen at -20 C were placed in the freezer in liquid form and then were thawed in the air at room temperature, usually in 45 min. Some samples stored at -70 C were handled in a similar manner (frozen and thawed slowly); others were frozen in an acetone- C02 bath for 3 min before being placed in the freezer and then were thawed in a water bath at room temperature (frozen and thawed quickly), which took about 3 min. Experiment 4. Acid gastric juice was divided into two portions, A and B, respectively. Samples containing 5 ml each of portion A in its natural state were placed in a freezer at -20 C. Portion B was depepsinized as in experiment 1, and after the pH had been readjusted to its original value, individual 5-ml aliquots were removed and frozen at - 20 C. At time zero and at stated intervals, samples were removed, thawed in the air at room temperature, and prepared for IF assay. In all the samples prepared for IF assay, the pH was readjusted to 7.0 and the analysis took place at room temperature. The charcoal
method 7 as adapted for quantitative IF assay 2 was used, with the following minor modifications: 2 ml of normal saline of pH 7.0 served as a vehicle for the gastric juice and serum and the supernatant rather than the charcoal was counted. Comparison with buffered normal saline with a pH of 7.0 as vehicle gives identical results. In no instance did the total B 12 binding capacity of the gastric juice exceed 65 % of the Co 57 vitamin B 12 that had been added.' The IF assays were performed within 1 to 2 weeks after the samples were prepared and stored at -20 C. Experiments have shown that there is no significant deterioration of the IF content of neutralized gastric juice by such storage for many months. The IF content of the gastric juices was always determined at the beginning of each time sequence study (100% activity at time zero). All subsequent measurements were expressed in percentage of this original value. By having a zero-hour control value for each sequence study for comparison, the small dilutional changes brought about by the depepsinization and reacidification procedure (performed with 10% NaOH and 10% HCl solutions) were obviated. Peptic activity, measured according to the method of Klotz, 8 was completely destroyed after depepsinization at pH 11.0 for 20 min. Co 57 was counted by scintillation spectrometry in a well-type Nuclear-Chicago Model 4233 automatic counter. The counting error was less than 4% with a 95 % confidence limit.
Results Figure 1 shows the results of experiment 1, which was performed with gastric juices obtained from 3 patients and kept at body temperature. The IF activity in nondepep-
.,
- - - Non- d•p•psi ni zed
co"'
--------- Depepsiniz•d
I
"' cO. ~
~
+10 0
~
'~~;.,.....i~;;.;.~==::::::.-:._.~·......:!1-·----
..., -20
----------~
·;;;
E."
-40
" ~
-(,0
.
:?'-so
/pHt.s
5 L-7.~7--~==~~~~~==~~==~ ""-100 )! 3 4 ~ Hours
FIG. 1. Intrinsic factor activity of nondepepsinized and depepsinized acid gastric juice taken from 3 human subjects and stored at +37 C.
DOSCHERHOLMEN AND SILVIS
846
sinized acid gastric juice decreased rapidly: the average loss was 46% after 1 hr and 92% after 4 hr. When the same gastric juices were depepsinized and reacidified to the original pH and then were simultaneously incubated, the loss in IF activity averaged 2 and 5% after 1 and 4 hr, respectively. As seen in the figure, the rate of loss of IF in native gastric juices varied slightly and was greatest in the most acid and smallest in the least acid juice. Figure 2 shows the results of experiment 2, which was performed on nondepepsinized acid gastric juice taken from 3 patients and kept at room temperature. The IF activity deteriorated quite slowly over a 7-day period and the average loss in IF activity was 7 and 39% after 4 and 24 hr, respectively, in three samples of gastric juice; 73% after 72 hr in two samples; and 93 % after 168 hr in one sample. The figure shows that the rate of loss of IF was greatest in the most acid and smallest in the least acid gastric juice. Figure 3 shows the results of experiment 3, in which samples of native acid gastric juice were stored at various cold t~m~e~a tures. When the samples of gastnc JUICe from 3 patients were stored at +1 C, the IF activity deteriorated very slowly. After 1, 3, and 7 days, the average IF loss was 5, 7 and 15%, respectively. When the samples of native gastric juices from 9 patients were stored at - 20 C, the IF activity showed relatively rapid deterioration, the loss averaging 48% after 1 day, 83% after 3 days, and 99% after 7 days.
Hours
FIG. 2. Intrinsic factor activity of nondepepsinized acid gastric juice taken from 3 human subjects and stored at room temperature ( +22 C).
+ZO
+l"c
IZ345&7
Days
Vol. 61 , No . 6
-zo'c
IZ345o7
Days
FIG. 3. Intrinsic factor activity of nondepepsinized acid gastric juices taken from human subjects and stored at various cold temperatures ranging from +1 C to - 70 C.
When the samples of native gastric juice from 2 patients were frozen slowly at - 70 C and thawed slowly at room temperature, the average initial loss of IF activity was 67.5% after 1 day, but no further deterioration occurred during the next 6 days of storage. When the samples of native gastric juice from 2 other patients were frozen quickly, stored at -70 C, and thawed rapidly, the IF activity showed an initial but minor loss averaging 10 ~;, after 1 day, with no further deterioration during the next 6 days. Figure 4 shows the results of experiment 4 in which samples of gastric juice from 4 ~tients were used. In the depepsinized samples, the loss of IF activity was minor, ranging from 5 to 36 ~(, after 5 or 6 days of storage, whereas in the nondepepsinized samples, the loss of activity ranged from 64 to lOOS;, over the same period. To learn the effect of acid alone on IF activity, a sample of depepsinized gastric juice containing 49 U of IF activity per ml was reacidified to a pH range of 0.5 to 7.0. Individual portions of this sample and a sample of nondepepsinized gastric juice were then stored at - 20 C. Individual samples were removed daily and assayed for IF activity. The results are shown in figure 5. The nondepepsinized control samples, as was expected, revealed a rapid loss of IF activity. The depepsinized samples showed little, if any, actual loss of IF
December 1971
0"1
·=
-Non-depepsinized o .... -----Dtpepsiniud
~
:::
rii
·oO
-.. . . .
----.•...,
...... _____ .......... . .. _ _ _ _ _ _ _ _ 4
___
·~---·
-&0 · 100
~'------1="""'1---...L_~~~~~.w
1
847
INTRINSIC FACTOR IN ACID GASTRIC JUICE
2
345o
Days
1234So
Days
FIG. 4. Intrinsic factor activity of nondepepsinized
and depepsinized acid gastric juice taken from 4 human subjects and stored at - 20 C.
activity until the pH fell below 3.0. As the pH values dropped, the loss of IF activity increased until a pH value of 1.5 was reached. Below this point, the actual loss of IF activity was, if anything, a little less pronounced. The influence that the storage age of gastric juice at the time of freezing might have on IF activity was investigated. This was done by slowly freezing samples of a native gastric juice containing 31 and 14 U of IF activity per ml after storage for 4 and 24 days respectively, in an ice-water bath. Samples were removed daily from the freezer, thawed slowly, and analyzed for IF activity. Table 1 shows that the loss of IF activity was more rapid in samples of gastric juice that had been stored in an ice-water bath for 4 days than those that had been stored for 24 days.
phasized the excessive production of IF in normal man. Only 1% or less of the daily production of IF is needed for the absorption of the daily minimum requirement of vitamin B, 2 • 12 The rapid digestion of IF by normal gastric juice at body temperature may explain the need for the apparent oversupply of this vital factor. The small loss of the IF activity in depepsinized, reacidified gastric juice kept at body temperature may indicate that the acid alone damages the IF. This is consistent with previous findings. s, 13 It is understandable that lrvine 3 did not report a slow loss rate in IF activity of nondepepsinized acid gastric juice kept at room temperature, since he did no!; carry out his investigation beyond 2 hr of storage. Our studies showed an average loss of only 1% of the IF activity after 2 hr of storage, which is within the error of the assay system. At room temperature the destruction of IF activity was much slower than at body temperature. The relatively rapid deterioration of IF activity in samples of gastric juice stored at - 20 C was in striking contrast to the relative stability of IF when the acid gastric juice was kept for a prolonged time in an ice-water bath. Apparently the peptic activity is most responsible for the accelerated loss of IF activity in nondepepsinized acid gastric juice storage at - 20 C. To our knowledge, this observation has not been made before, but it is consistent with the
w... "' u
Discussion These studies reveal a profound effect of time and temperature on the stability of IF activity of nondepepsinized acid gastric juice from human subjects. The rapid destruction of the IF activity in native gastric juice at +37 C must be due to the peptic activity since practically no IF loss occurred in the corresponding depepsinized juices (fig. 1). This is in agreement with the findings of Ashworth and associates. 5 Undoubtedly this type of autodigestion of IF also occurs in vivo. 9 • 10 ROdbro 11 has em-
- - Non-depepsini ze d
'-
.eu · ;;:;
c: .E .,."" c:
~ c: c: I<>
"' c: ..c: u
'if~~~~~";-;-;~:~--~;; jj
·20 -40
"""'!:!
-&o
-so
"'
·------• D~pepsinind
+10 0
··-pH 1.5
-100 0
3
4
"
7
Days
FIG. 5. Intrinsic factor activity of nondepepsinized acid gastric juice taken from a human subject versus that of the same gastric juice depepsinized and reacidified to pH values ranging from 0.5 to 7.0.
848
DOSCHERHOLMEN AND SILVIS TABLE
Vol . 61, No. 6
1. Comparison of intrinsic factor loss in a gastric juice k ept in an ice-water bath for 4 days and 24 days before freezing at -20 C Percentage of intrinsic factor loss after freezing
Gastric juice kept in ice·water bath
Day 0
Day I
Day 2
Day 3
Day 4
0 0
26 17
33 30
53 27
50 32
Day 5
Day 6
Day 7
Day 8
63
66 36
77 47
Day 9
days
4 24
concept of accelerated enzyme activity in ice, which is a newly described phenomenon.14 Apparently hydrochloric acid alone also increases the rate of destruction of the IF activity of gastric juices stored at -20 C. This observation is in accord with the concept that simple (:hemical reactions are accelerated in ice, a phenomenon also previously described. 15 There may be an optimum pH for the damaging effect of acid on the IF activity. In figure 5, the results of the experiment show an optimal pH of 1.5, because at pH values of 1.0 and 0.5 no further increase in the rate of loss of IF occurred. Another possible cause is that the gastric juice may have contained an enzyme with optimal activity at pH 1.5 and with the ability to withstand alkalinization. Such a gastric protease has recently been described. 16 Whether it can withstand a pH of 11.0 for 20 min and still be damaging to the IF remains to be seen. Several possible causes for individual variations in loss rate of IF when nondepepsinized acid gastric juices were stored at - 20 C (fig. 3) are: differences in pH values, content of pepsin, and qualitative variations in pepsin. At least eight different proteolytic fractions in human gastric mucosa exist. 16 It is not unlikely that they have individual functions and that one or more of them are more active than others in the degradation of the IF. Finally, the storage age of the gastric juice at the time of freezing also may have influenced the rate of IF loss (table 1). The slower rate of loss of IF after prolonged storage at + 1 C may possibly be due to alterations in the IF or to quantitative or qualitative changes in the peptic activity of the gastric juice. At - 70 C the initial loss of IF activity may have been due to the damaging effect
46
of the periods of moderately low temperature as the gastric juice was frozen and thawed. This concept is supported by the finding that this loss could be minimized by freezing and thawing quickly. The acceleration of chemical reactions in ice is not clearly understood. 17 Increased concentrations of the reactants in highly structured frozen water at moderately low temperatures, 18 catalytic action by ice structure itself, 19 i1,;,;reased proton mobility in ice, 20 and favorable substrate-catalyst positional constraint, 20 have all been considered causative factors. At moderately low temperatures, water in the frozen state is highly structured with ice crystals and tiny pockets of water containing the reactants. 17 Under these circumstances, chemical reactions may accelerate. In biological systems, water is solidly frozen at - 65 C to - 70 C, 21 which may explain the stability of IF activity when gastric juice is stored at - 70 C. Ardeman et al. 22 reported a mean value of 8900 U for IF activity in the 1st hr after histamine stimulation in normal subjects, whereas ROdbro and Christiansen 2 reported a mean value of 16,000. Although Chanarin 23 ascribed this difference largely to the increased volume of gastric juice that ROdbro and Christiansen obtained by continuous suctioning, the latter investigators also found a 10 ~;, higher mean concentration of IF activity. This difference in mean concentration may have been due to the fact that Ardeman and Chanarin at times used intermittent suctioning, which could have permitted in vivo autodigestion of IF. These observations on the collecting and storing of gastric juice to minimize the loss of IF activity have practical use in the quantitative determination of this factor. As soon as the gastric juice is secreted, it
December 1971
INTRINSIC FACTOR IN ACID GASTRIC JUICE
should be removed from the stomach and immediately lowered to + 1 C. This can be done by continuous suctioning of the gastric juice into a container submerged in ice water, where it should remain until it is depepsinized. Acid gastric juice should never be frozen before it is depepsinized and prepared for routine quantitative IF assay. REFERENCES
11. 12.
13.
14.
1. Doscherholmen A: Effect of pH, time and tem-
2.
3.
4.
5.
6.
7.
8.
9. 10.
perature on the intrinsic factor activity of human gastric juice (abstr) . J Lab Clin Med 76:882, 1970 Rodbro P, Christiansen PM: Quantitative determination of gastric intrinsic factor after large histamine doses in healthy persons. Scand J Clin Lab Invest 19:186-189, 1967 Irvine WJ : hnmunoassay of gastric intrinsic factor and the titration of antibody to intrinsic factor. Clin Exp hnmun 1:99-118, 1966 Glass GBJ: Gastric intrinsic factor and its function in the metabolism of vitamin Bu . Physiol Rev 43:529-849, 1963 Ashworth LAE, Strickland RG, Koo NC, et al: Effect of pH on intrinsic factor and nonintrinsic factor vitamin Bu binding in human gastric juice neutralized in vivo. Gastroenterology 57:506-510, 1969 Sullivan LW, Herbert V, Castle WB: In vitro assay for human intrinsic factor. J Clin Invest 42:14431458, 1963 Gottlieb C, Lau K, Wasserman LR, et al : Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated Bu binding capacity, antibody to IF, and serum unsaturated B u binding capacity. Blood 25:875-883, 1965 Klotz AP: The laboratory determination of gastric proteases. Ann New York Acad Sci 140:697-708, 1967 Abels J, Schilling RF: Protection of intrinsic factor by vitamin Bu . J Lab Clin Med 64:375-384, 1964 Grasbeck R. Simons K, Sinkkonen 1: Isolation of
15.
16.
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18.
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21.
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23.
849
intrinsic factor and its probable degradation product, as their vitamin B, 2 complexes, from human gastric juice. Biochim Biophys Acta 127:47-58, 1966 Rodbro P: Human gastric intrinsic factor secretion. Scand J Gastroent 4:473-475, 1969 Ardeman S, Chanarin 1: Assay of gastric intrinsic factor in the diagnosis of Addisonian pernicious anemia. Brit J Haemat 11 :305-314, 1965. Castro-Cure) Z: Effects of peptides and proteolysis on intrinsic factor (IF) activity. Clin Res 12:205, 1964 Grant NH, Album HE: Acceleration of enzyme reactions in ice. Nature (London) 212:194, 1966 Grant NH, Album HE: Fast reactions of ascorbic acid and hydrogen peroxide in ice, a presumptive early environment. Science 150:1589-1590, 1965 Samloff IM : Slow moving protease and the seven pepsinogens. Electrophoretic demonstration of the existence of eight proteolytic fractions in human gastric mucosa. Gastroenterology 57:659-669, 1969 Larsen SS : Kemiske reaksjoner i frosne oplosninger. En oversigt. Dansk T Farm 41 :177- 190, 1967 Butler AR. Bruice TC: Catalysis in water and ice. A comparison of the kinetics of hydrolysis of acetic anhydride, tl-propiolactone, and p-nitrophenyl acetate and the dehydration of 5-hydro-6-hydroxydeoxyuridine in water and in ice. J Amer Chern Soc 86:313-319, 1964 Bruice TC, Butler AR: Catalysis in water and ice. II. The reaction of thiolactones with morpholine in frozen systems. J Amer Chern Soc 86:4104-4108, 1964 Grant NH, Clark DE, Album HE: hnidazole- and base-catalyzed hydrolysis of penicillin in frozen systems. J Amer Chern Soc 83:4476-4477, 1961 Sussman MV, Chin L: Liquid water in frozen tissue: Study by nuclear magnetic resonance. Science 151:324-325, 1966 Ardeman S, Chanarin I, Doyle JC: Studies on secretion of gastric intrinsic factor in man. Brit Med J 2:600-603, 1964 Chanarin I: Intrinsic factor, Clin Chim Acta 22: 85-90, 1968