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Intrinsic Factor, Nonintrinsic Factor Vitamin B12 Binder, and Pepsinogen Secretion in Normal Subjects
Vol. 57, No.5 Printed in U.S .A .
GASTRO ENTEROLOGY
Copyright© 1969 by The Williams & Wilkins Co.
INTRINSIC FACTOR, NONINTRINSIC FACTOR VITAMIN B12 BINDER, AND PEPSINOGEN SECRETION IN NORMAL SUBJECTS Quantification using intragastric neutralization and inorganic phosphate as a marker R. G. STRICKLAND, M .D ., L. A. E. AsHWORTH, PH.D., N. C. Koo, AND K. B. TAYLOR, D .M . Division of Gastroenterology, Stanford University School of Medicine, Stanford, California
A technique of quantifying gastric secretion in normal subjects while maintaining the gastric contents at neutral pH is described. Phosphate buffer was used to achieve continuous intragastric neutralization during stimulated gastric secretion using maximal doses of histalog. Inorganic phosphate in the buffer served as a gastric marker, and the evidence to support its use is reported in detail. The method was applied to the quantification of intrinsic factor, nonspecific B12 binder, and pepsinogen secretion in 6 normal subjects. Differing patterns of secretion of these three constituents of gastric juice were observed. The output of intrinsic factor was not sustained and appeared to represent a true washout phenomenon. The secretion of nonspecific B12 binder was not influenced by histalog stimulation. The pattern of pepsinogen secretion clearly differed from that of intrinsic factor and appeared to be one of true stimulation. It is suggested that this technique may have a wider application, particularly in the quantification of other gastric juice macromolecules which are susceptible to intragastric proteolysis and/or acid degradation. Several studies of intrinsic factor secretion in normal subjects in response to a variety of gastric stimulants have been reported.1-9 Earlier studies of the response of pepsin secretion to histamine stimulation in normal subjects are cited by Piper. 10 More recent observations are those of Hunt, 11 Hirschowitz, 12 and Makhlouf et al. 13' 14 None of these measurements
has taken into consideration the volume of secretion lost through the pylorus, the possibility of incomplete recovery of gastric contents by current techniques of collection, or the significant quantitative reduction in intrinsic factor activity produced by a short period of acid-peptic action which we have recently demonstrated. 15 The utilization of continuous intragastric neutralization in the quantification of these macromolecular components of gastric secretion requires a suitable marker which will accurately reflect gastric juice recovery and transpyloric loss. We describe here the observations which led us to the use of inorganic phosphate, both as a buffer and as a gastric marker, and the application of this marker to the quantifi-
Received May 2, 1969. Accepted June 24, 1969. Address requests for reprints to: Dr. K. B. Taylor, Division of Gastroenterology, Stanford University Medical Center, Stanford, California 94305. This investigation was supported by Grant AM08282-05 of the National Institutes of Health and Training Grant AM-05418 from the United States Public Health Service. The authors gratefully acknowledge the technical assistance of Miss Mozetta Taylor. 511
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cation of gastric secretion of the vitam B,2binding substances and pepsinogen in normal subjects. Materials and Methods The subjects studied were healthy medical students or members of the hospital staff who showed no clinical evidence of gastrointestinal disease. Comparative studies of inorganic phosphate and polyethylene glycol. Polyethylene glycol (PEG, Carbowax 4000) was used in preliminary studies. The resting gastric secretions of 4 fasting normal subjects were aspirated after the passage of a no. 16 FR Levin type tube . Thirtymilliliter volumes of 0.07 M phosphate buffer (pH 7.8), with and without 1% PEG, were introduced separately into the stomach, mixed rapidly, and aspirated. PEG in this and subsequent experiments was estimated by the method of Hyden. 16 The mean (±so) PEG recovery in these four studies was 95 ± 5%. Nearly complete mixing of the two solutions in the stomach occurred as evidenced by the immediate dilution of PEG to half its original concentration (48% ± 5.6% so). However, PEG over a wide concentration range interfered with the immunoassay of the B, z-binding substances, thus precluding its use in their quantification. The use of inorganic phosphate, present in the buffer, as a marker was then investigated by comparing its recovery with that of PEG in the following manner. A no. 16 FR Levin type tube was introduced into the stomach in 4 fasting normal subjects. Mter the gastric residue was emptied, betazole hydrochloride (histalog, Eli Lilly and Company), 1. 7 mg per kg of body weight, was given intramuscularly and gastric juice collected over 90 min in 15-min periods using continuous intragastric neutralization with 0.07 M phosphate buffer (pH 7.8) to which 1% PEG had been added. PEG concentration in the recovered whole gastric juice was estimated, and inorganic phosphate was measured by the method of Fiske and SubbaRow 17 in the supernatant following trichloroacetic acid precipitation of protein and PEG. The concentrations of both phosphate and PEG in recovered gastric juice were compared by expressing them as percentages of the concentration of each in the original buffer instilled. Quantification of secretory constituents. Six normal subjects were studied following an overnight fast. A no. 16 FR Levin type nasogastric tube was passed and the subject positioned
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semirecumbent on the left side. Resting gastric juice was aspirated and its pH was checked by Hydrion pH paper. Saliva was expectorated throughout the test period. Mter the stomach was emptied, 0.07 M phosphate buffer (pH 7.8) was instilled in 20to 30-ml volumes, mixed twice by hand suction, and aspirated, and its pH was tested by Hydrion pH paper. This procedure was repeated continuously throughout the entire test period in order to maintain the gastric contents close to pH 7.0. Following an initial 15-min period of gastric washout with buffer, basal secretion was collected for 15 min, and then histalog was administered intramuscularly in a dosage of 1. 7 mg per kg of body weight. Gastric juice was then collected for 90 min in six 15-min periods. At the end of each collection period the stomach was emptied as completely as possible before proceeding further. Two technicians were utilized for each study, one to carry out the intragastric neutralization and collection and the other to record the volumes of buffer introduced and the final volume recovered from each collection period. The average volume of buffer used for each study was 1440 ml and the range in the 6 individuals was 1180 to 1604 ml. Continuous intragastric neutralization could be achieved using this technique, provided that increased volumes of buffer were used during the period of maximal flow and acid secretion after stimulation. An aliquot of gastric juice from each collection period was saved for phosphate estimation, and the remainder was filtered through heayy duty paper tissues (Kimwipes, Kimberley-Clark Corporation) concentrated at 4 C by ultrafiltration using 1/4-inch diameter dialysis tubing to a final volume of 10 ml and stored at -20 C until ready for analysis. The pH of each concentrated collection was measured electrometrically before storage. All juices were in the pH range 6.8 to 7.2. Inorganic phosphate was determined as before, both in the buffer used and in a sample of whole gastric juice from each collection period. Intrinsic factor (IF) and nonintrinsic factor (NIF) B, z binding was determined in each concentrated collection by immunoassay 18 using 25 .ul of the gastric juice, 200 ng of Co 60 B,z and 100 .uliter of pernicious anemia serum containing intrinsic factor antibody in high titer. Total B, z-binding capacity of the gastric juice never exceeded 65% of the 200 ng of Co60 B, z using this system. Content of IF and NIF B, z binder
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INTRINSIC FACTOR, NONINTRINSIC FACTOR
in each concentrated gastric juice collection was expressed in units, 1 unit being equal to that amount which binds 1 ng of Co 60 B 12. Pepsinogen content of each concentrated gastric juice collection was measured by a modification of the method of Die and Spray, 19 omitting the use of charcoal, using 25 ~tl of gastric juice and a 1-hr incubation with hemoglobin substrate (Sigma Chemical Company) at pH 2.0. A pepsinogen standard (Sigma Chemical Company) and substrate control were run with each sample. The total amount of tyrosine released in micrograms per milliliter per hour at pH 2.0 for each collection period was estimated and peptic units were calculated from the pepsinogen standard (1 peptic unit produced an increase in OD2so of 0.060 per hr at pH 2.0 and 37 C).
Results Comparison of PEG and inorganic phosphate as gastric markers. The concentrations of PEG and phosphate in recovered gastric juice in each of six 15-min stimulated collection periods in 4 subjects were compared. The results revealed that PEG and phosphate concentrations are in close agreement. The coefficients of variation between them in individual collection periods were consistently below 5%, as were the mean coefficients of variation for each of the four studies undertaken. In addition, the small differences found did not show any consistent trend. Recent studies of the secretion of inorganic phosphate by the human stomach20' 21 indicate a low basal output of 3 to 6 mg per hr and no enhancement of output following maximal stimulation. The phosphate buffer used in the present study contains approximately 2 g of inorganic phosphate per liter, and, since more than 1 liter has been necessary in order to achieve continuous intragastric neutralization in normal subjects, the contribution of endogenous phosphate secretion during maximal stimulation was less than 1%. This fact, together with the close agreement between phosphate recovery and that of PEG, a substance neither absorbed nor secreted by the gastric mucosa, provides indirect evidence for there being little significant net transfer of phosphate in these studies.
The quantities of vitamin B12 binders and pepsinogen secreted have thus been estimated from the recovery of inorganic phosphate in each 15-min collection period and are based on the two following assumptions. (a) Mixing of the buffer and secreted gastric juice is complete throughout the test period and that, therefore, the content of phosphate in gastric juice leaving the stomach via the pylorus will be the same as that recovered and measured in the gastric aspirate. Our preliminary observations using PEG indicated that mixing of two volumes in the stomach occurred rapidly and was virtually complete. (b) The stomach is emptied at the end of each collection period. In these studies a residual volume in excess of 10%, indicating incomplete collection, occurred in 1 subject in the last three collection periods. Isolated instances of a significant residual volume were observed in 2 other individuals, again in the latter stages of each study. The following formulas were applied to e.:;timate secretory volume and the secretion of the gastric constituents studied: (1)
Vs = VF- VI
where Vs is volume secreted per 15 min; VI is volume of buffer introduced per 15 min; and V F is the final volume after mixing, made up of that recovered and that lost through the pylorus. Since no significant net transfer of phosphate occurs, the amount of phosphate in the final volume must equal the amount of phosphate introduced, or V F [PF] = VI [PI]
(2)
where [P F] is concentration of phosphate in the final volume and [PI] is concentration of phosphate in the buffer instilled and V [PrJ 1 [PF] Substituting this for VF in equation (1), V s = Vr
[~:]]
- Vr
(3)
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30000
and (4)
where Cs is the total output of C per 15 min when C is any measurable gastric secretory component, and CF is the concentration of C in the final volume. Secretion of IF B12 binder, NIF B12 binder, and pepsinogen. There was a mean increase in secreted volume from basal to stimulated periods, but the variation in basal secretion was such that these figures did not reach statistical significance in the small group of subjects studied. However, it is of note that the standard errors in secreted volume were much smaller during the period of maximal stimulation. The mean (±SE) secretion of IF B12 binder basally and following maximal histalog stimulation is shown in figure 1. The secretion of NIF B1 2 binder was not significantly altered from basal levels following histalog, whereas IF secretion was significantly increased above basal levels in the first (P < 0.01) , second (P < 0.001), 8000
t
HISTALOG
MINUTES
FIG. 1. Mean ( ± s E) secretion of intrinsic factor (0) and nonspecific B12-binder ~) basally and following intramuscular histalog, 1. 7 mg per kg body weight, in 6 healthy human subjects.
.~ c:
" .'E<>. "'a. f-
::>
a. f-
::> 0
10000
0 ~~15~~30~~4~5--~6~0--7~5--~9~0--~100
t HISTALOG
MINUTES
FIG. 2. Mean (±SE) secretion of pepsinogen basaily and following intramuscular histalog, · 1.7 mg per kg body weight, in the same 6 subjects shown in figure 1.
third (P < 0.001), and fourth (P < 0.01) periods after stimulation. The ratio of IF to NIF B12-binder in basal secretions was 1: 1.5 and following stimulation increased to 6 : 1 and fell progressively thereafter to 1.5: 1, 90 min after histalog. Peak output of IF occurred in the first 15 min following stimulation when the mean secretion reached 5,500 units. The mean hourly IF output after stimulation was 13,700 units with a range of 8,500 to 22,900 units. The corresponding pepsinogen content in the same basal and posthistalog collections is shown in figure 2. Pepsinogen secretion in the first (P < 0.05), second (P < 0.001), third (P < 0.001), and fourth (P < 0.02) periods following stimulation was significantly increased over basal levels. The mean maximal hourly output of pepsinogen after stimulation was 98,000 peptic units with a range of 83,000 to 114,000. The pattern of pepsinogen secretion was different from that of intrinsic factor. Peak output of pepsinogen following histalog occurred in the 30- to 45-min collection period, whereas the peak output of IF occurred in the first 15 min after stimulation. In addition, there was no correlation between the maximal output of pepsinogen
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INTRINSIC FACTOR, NONINTRINSIC FACTOR
and that of IF, nor were their secretions significantly correlated in any of the individual 15-min collections following histalog. Discussion In the present investigation, an attempt
has been made to quantify IF secretion in normal human subjects during maximal histalog stimulation, utilizing continuous in vivo neutralization to overcome the partial intragastric degradation of this molecule which results from a short period of acid-peptic action. 15 This effect has not always been considered in previous studies; the in vitro neutralization of the aspirated gastric juice frequently is delayed for a variable time period. Further, the use of inorganic phosphate present in the buffer as a gastric marker has provided a more valid assessment of the actual secretion of gastric juice constituents by correcting for volume loss through the pylorus. Polyethylene glycol introduced intragastrically at low infusion rates has been validated as a marker of gastric juice recovery during basal and maximally stimulated secretion in man. 22 Our preliminary observations using larger volumes of buffer and PEG confirmed the excellent recovery of the latter. However, PEG could not be used in the present studies because of its interference with the immunoassay of the B1 2 binders. Falsely elevated counts in the supernatant fluid were observed with PEG in the system, suggesting that PEG in the gastric juice adsorbs to the albumin-coated charcoal and impedes the uptake of free Co60 B1 2 . The close agreement between the recoveries of PEG and phosphate observed in a comparative study of four separate time courses in normal subjects demonstrated the feasibility of utilizing phosphate as a marker. The possibility that endogenous secretion of phosphate might interfere is ruled out by the results of studies in man which show such secretion to be low, constant, and unaffected by gastric stimulants.20 , 2 1 The present study describes for the first time the secretion of NIF B1 2 binder in response to gastric stimulation and compares
515
the secretion of the B1 2 binders of gastric juice with that of pepsinogen in the same subjects. We have found that the technique of continuous intragastric neutralization used in this study will maintain the pH of aspirated gastric juice between 6.8 and 7.2 in most normal subjects, even during periods of maximal secretory activity following stimulation. The immunoassay of B1 2 binders is not affected in this pH range, and spontaneous conversion of pepsinogen to pepsin should not occur. Restoration of the pH to 2.0, however, will restore maximal peptic activity. 23 The secretion of IF in normal subjects using conventional collection techniques and in vitro neutralization of gastric juice has been studied using a variety of gastric secretagogues both in single dose form 1 - 7 and as constant infusions. 8 ' 9 In only two studies2 ' 3 has histalog been used, and in only one of these 3 was the time course of IF secretion studied. The average hourly output of IF following stimulation in normal subjects has varied widely in previous studies. Although this may partly reflect variation in volume collection from study to study, 5 lack of uniform handling of collected gastric juice, particularly with respect to the time when neutralization is carried out, may also account for some of this variation. Our results in terms of mean IF output after stimulation agree closely with similar studies in which immediate neutralization of aspi4 7 rated gastric juice was carried out. ' In other studies where neutralization appears to have been delayed, the mean hourly IF output after stimulation was significantly lower than in the present study. 1 • 8 Despite the variation in total IF output in response to maximal stimulation, there is general agreement with respect to the pattern of its secretion. Peak output occurs in the first 30 min following stimulation, returning within 40 to 60 min to a low constant level of secretion. This pattern is regarded as being due to an initial washout of stored IF from the parietal cells, which is followed by a much lower secretion rate close to, but significantly different from, basal levels. 9
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The time course of IF secretion observed in our 6 normal subjects did not differ from that seen in previous studies. However, it is interesting that no enhancement in the secretion of NIF B12 binding occurred following stimulation, an observation which has not been previously reported but which supports a nonparietal origin of this gastric juice constituent. The response of pepsinogen secretion to histalog in normal subjects has not been investigated previously. Makhlouf et al. 13 ' 14 have demonstrated an earlier peak in pepsin output than that seen in the present study in response to single large doses of histamine and gastrin. Maximal histalog stimulation has been shown to induce a later peak in acid output than that obtained using the augmented histamine test in the same subjects. 24 The later peak of pepsinogen output observed in the present study in response to maximal doses of histolog may represent a similar phenomenon, but direct comparison with histamine in the same subjects is needed in order to confirm this suggestion. The pattern of pepsinogen secretion in these 6 subjects differed from that of IF measured in the same gastric juice collections. Pepsinogen secretion was more sustained, with the peak response occurring 30 to 45 min after stimulation; no significant correlation emerged between IF and pepsinogen outputs in any individual collection period. Bitsch et al. 25 studied pepsin and IF secretion in patients with duodenal ulcer and observed that their pattern of secretion was similar in resP-onse to histamine and insulin. They concluded that gastric stimulation leads to a "washout" of stored pepsinogen similar to that which appears to occur in the case of IF. This mechanism of pepsinogen response to gastric stimulation had been proposed in earlier studies. 26 - 28 However, comparative studies of pepsin and acid secretion in man 10· 12 · 14 · 29 - 31 indicate that the pattern of pepsin secretion in response to a variety of secretagogues is one of true stimulation. Our finding of a clear difference between the patterns of IF and pepsinogen secretion
supports the current belief that an initial flushing out of preformed IF occurs in response to stimulation, whereas the same stimuli of gastric secretion result in the elaboration of pepsinogen. REFERENCES 1. Ardeman, S., I. Chanarin, and J. C. Doyle. 1964. Studies on secretion of gastric intrinsic factor in man. Brit. Med. J. 2: 600-603. 2. Ardeman, S., and I. Chanarin. 1965. Stimulation of gastric intrinsic factor secretion. Brit. Med . J . I : 1417-1418. 3. Jeffries, G. H., and M. H. Sleisenger. 1965. The pharmacology of intrinsic factor secretion in man. Gastroenterology 48: 444-448. 4. Wangel, A. G., m.d S. T . Callender. 1965. Effect of gastrin I and II on the secretion of intrinsic factor. Brit. Med. J. 1: 1409-1411. 5. R.fdbro, P ., P. M. Christiansen, and M. Schwartz. 1965. Intrinsic factor secretion in stomach diseases. Lancet 2: 1200-1203. 6. Rfldbro, P., and P. M. Christiansen. 1966. Quantitative estimation of parietal cell secretory function in man. Scand. J. Gastroent. I: 292-298. 7. R;dbro, P., and P . M. Christiansen. 1967. Quantitative determination of gastric intrinsic factor after large histamine doses in healthy persons. Scand. J. Clin. Lab. Invest. 19: 186-189. 8. Lawrie, J. H., and N. M. Anderson. 1967. Secretion of gastric intrinsic factor. Lancet 1: 68-71. 9. Weir, D. G., I. J. Temperley, and D. Collery. 1967. Intrinsic factor secretion in -response to continuous histamine infusion. Gastroenterology 52: 23-28. 10. Piper, D. W. 1960. The effect of histamine on pepsin secretion. Amer. J. Dig. Dis. 5: 880-888. 11. Hunt, J. N. 1950. An interpretation of the histamine test of gastric secretion. Gastroenterology 16: 231-240. 12. Hirschowitz, B. I. 1961. Electrolytes in human gastric secretion. Observations and a theory. Amer. J. Dig. Dis. 6: 199-228. 13. Makhlouf, G. M., J . P. A. McManus, and W. I. Card. 1966. Action of the pentapeptide (ICI 50,123) on gastric secretion in man. Gastroenterology 51: 455-465. 14. Makhlouf, G. M., J . P. A. McManus, and W. I. Card. 1967. Comparative effects of gastrin II and histamine on pepsin secretion in man. Gastroenterology 52: 787-791. 15. Ashworth, L.A. E. , R. G. Strickland, N . C. Koo, and K. B. Taylor. 1969. Effect of pH on intrinsic factor and nonintrinsic factor vitamin Bw
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16.
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19. 20.
21.
22.
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binding in human gastric juice neutralized in vivo. Gastroenterology 57: 506-510. Hyden, S. 1955. A turbidimetric method for the determination of higher polyethylene glycols in biologic materials. Ann. Roy. Agr. Call. Sweden22: 139-143. Fiske, C. H., and Y. SubbaRow. 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66: 375-400. Gottlieb, C., K. Lau, L. R. Wasserman, and V. Herbert. 1965. Rapid charcoal assay for intrinsic factor (IF), gastric juice-unsaturated B, 2 binding capacity, antibody to IF, and serum-saturated B, 2 binding capacity. Blood 25: 875-883. Die, V., and G. H. Spray. 1966. Estimation of serum pepsinogen. Gut 7: 415-419. Burhol, P. G., J. Myren, and 0. P. Foss. 1966. Electrolytes in gastric juice of man. I. A statistical analysis of calcium and inorganic phosphorus before and after subcutaneous injection of large doses of histamine. Scand. J. Clin. Lab. Invest. 18: 325-330. Semb, L. S., J . Myren, and 0. P . Foss. 1966. Magnesium, calcium, and inorganic phosphorus in gastrin-stimulated gastric secretion of man. Scand . J. Gastroent. 1: 33-39 Gundry, R. K. , R. M. Donaldson, Jr., C. A. Pinderhughes, and E. Barrabee. 1967. P!!tterns of gastric acid secretion in patients with duodenal ulcer: correlations with clinical and
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personality features. Gastroenterology 52: 176---184. Piper, D. W., and B. H. Fenton. 1965. pH stability and activity curves of pepsin with special reference to their clinical importance. Gut 6: 506-508. Ward, S., I. E. Gillespie, E. P. Passaro, and M. I. Grossman. 1963. Comparison of histalog and histamine as stimulants for maximal gastric secretion in human subjects and in dogs. Gastroenterology 44: 620-626. Bitsch, V. , P. M . Christiansen, V. Faber, and P. Rodbro. 1966. Gastric secretory patterns before and after vagotomy. Lancet 1: 1288-1291. Babkin, B. P . 1930. The value of histamine as a test of gastric secretion from a physiological point of view. Canad. Med. Assn. J . 23: 268272. lhre, B. J . E. 1938. Human gastric secretion. Acta Med. Scand., suppl. 95, 1. Toby, C. G. 1939. Does histamine stimulate the secretory activity of the peptic cells in man? Amer. J . Dig. Dis. Nutr. 3: 902-903. Ashford, C. A., H. Heller, and G. A. Smart. 1949. The action of histamine on hydrochloric acid and pepsin secretions in man. Brit. J . Pharmacal. 4: 1032-1036. Wyllie, J. E., and G. Smith. 1965. Histamineinfusion test. Lancet 2: 823-824. Gillespie, I. E ., and D. J . Bowen. 1962. The gastric secretion of pepsin in man. Gut 3: 255-259.
GASTRO ENTEROLOGY
Copyright© 1969 by The Williams & Wilkins Co.
INTRINSIC FACTOR, NONINTRINSIC FACTOR VITAMIN B12 BINDER, AND PEPSINOGEN SECRETION IN NORMAL SUBJECTS Quantification using intragastric neutralization and inorganic phosphate as a marker R. G. STRICKLAND, M .D ., L. A. E. AsHWORTH, PH.D., N. C. Koo, AND K. B. TAYLOR, D .M . Division of Gastroenterology, Stanford University School of Medicine, Stanford, California
A technique of quantifying gastric secretion in normal subjects while maintaining the gastric contents at neutral pH is described. Phosphate buffer was used to achieve continuous intragastric neutralization during stimulated gastric secretion using maximal doses of histalog. Inorganic phosphate in the buffer served as a gastric marker, and the evidence to support its use is reported in detail. The method was applied to the quantification of intrinsic factor, nonspecific B12 binder, and pepsinogen secretion in 6 normal subjects. Differing patterns of secretion of these three constituents of gastric juice were observed. The output of intrinsic factor was not sustained and appeared to represent a true washout phenomenon. The secretion of nonspecific B12 binder was not influenced by histalog stimulation. The pattern of pepsinogen secretion clearly differed from that of intrinsic factor and appeared to be one of true stimulation. It is suggested that this technique may have a wider application, particularly in the quantification of other gastric juice macromolecules which are susceptible to intragastric proteolysis and/or acid degradation. Several studies of intrinsic factor secretion in normal subjects in response to a variety of gastric stimulants have been reported.1-9 Earlier studies of the response of pepsin secretion to histamine stimulation in normal subjects are cited by Piper. 10 More recent observations are those of Hunt, 11 Hirschowitz, 12 and Makhlouf et al. 13' 14 None of these measurements
has taken into consideration the volume of secretion lost through the pylorus, the possibility of incomplete recovery of gastric contents by current techniques of collection, or the significant quantitative reduction in intrinsic factor activity produced by a short period of acid-peptic action which we have recently demonstrated. 15 The utilization of continuous intragastric neutralization in the quantification of these macromolecular components of gastric secretion requires a suitable marker which will accurately reflect gastric juice recovery and transpyloric loss. We describe here the observations which led us to the use of inorganic phosphate, both as a buffer and as a gastric marker, and the application of this marker to the quantifi-
Received May 2, 1969. Accepted June 24, 1969. Address requests for reprints to: Dr. K. B. Taylor, Division of Gastroenterology, Stanford University Medical Center, Stanford, California 94305. This investigation was supported by Grant AM08282-05 of the National Institutes of Health and Training Grant AM-05418 from the United States Public Health Service. The authors gratefully acknowledge the technical assistance of Miss Mozetta Taylor. 511
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STRICKLAND ET AL .
cation of gastric secretion of the vitam B,2binding substances and pepsinogen in normal subjects. Materials and Methods The subjects studied were healthy medical students or members of the hospital staff who showed no clinical evidence of gastrointestinal disease. Comparative studies of inorganic phosphate and polyethylene glycol. Polyethylene glycol (PEG, Carbowax 4000) was used in preliminary studies. The resting gastric secretions of 4 fasting normal subjects were aspirated after the passage of a no. 16 FR Levin type tube . Thirtymilliliter volumes of 0.07 M phosphate buffer (pH 7.8), with and without 1% PEG, were introduced separately into the stomach, mixed rapidly, and aspirated. PEG in this and subsequent experiments was estimated by the method of Hyden. 16 The mean (±so) PEG recovery in these four studies was 95 ± 5%. Nearly complete mixing of the two solutions in the stomach occurred as evidenced by the immediate dilution of PEG to half its original concentration (48% ± 5.6% so). However, PEG over a wide concentration range interfered with the immunoassay of the B, z-binding substances, thus precluding its use in their quantification. The use of inorganic phosphate, present in the buffer, as a marker was then investigated by comparing its recovery with that of PEG in the following manner. A no. 16 FR Levin type tube was introduced into the stomach in 4 fasting normal subjects. Mter the gastric residue was emptied, betazole hydrochloride (histalog, Eli Lilly and Company), 1. 7 mg per kg of body weight, was given intramuscularly and gastric juice collected over 90 min in 15-min periods using continuous intragastric neutralization with 0.07 M phosphate buffer (pH 7.8) to which 1% PEG had been added. PEG concentration in the recovered whole gastric juice was estimated, and inorganic phosphate was measured by the method of Fiske and SubbaRow 17 in the supernatant following trichloroacetic acid precipitation of protein and PEG. The concentrations of both phosphate and PEG in recovered gastric juice were compared by expressing them as percentages of the concentration of each in the original buffer instilled. Quantification of secretory constituents. Six normal subjects were studied following an overnight fast. A no. 16 FR Levin type nasogastric tube was passed and the subject positioned
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semirecumbent on the left side. Resting gastric juice was aspirated and its pH was checked by Hydrion pH paper. Saliva was expectorated throughout the test period. Mter the stomach was emptied, 0.07 M phosphate buffer (pH 7.8) was instilled in 20to 30-ml volumes, mixed twice by hand suction, and aspirated, and its pH was tested by Hydrion pH paper. This procedure was repeated continuously throughout the entire test period in order to maintain the gastric contents close to pH 7.0. Following an initial 15-min period of gastric washout with buffer, basal secretion was collected for 15 min, and then histalog was administered intramuscularly in a dosage of 1. 7 mg per kg of body weight. Gastric juice was then collected for 90 min in six 15-min periods. At the end of each collection period the stomach was emptied as completely as possible before proceeding further. Two technicians were utilized for each study, one to carry out the intragastric neutralization and collection and the other to record the volumes of buffer introduced and the final volume recovered from each collection period. The average volume of buffer used for each study was 1440 ml and the range in the 6 individuals was 1180 to 1604 ml. Continuous intragastric neutralization could be achieved using this technique, provided that increased volumes of buffer were used during the period of maximal flow and acid secretion after stimulation. An aliquot of gastric juice from each collection period was saved for phosphate estimation, and the remainder was filtered through heayy duty paper tissues (Kimwipes, Kimberley-Clark Corporation) concentrated at 4 C by ultrafiltration using 1/4-inch diameter dialysis tubing to a final volume of 10 ml and stored at -20 C until ready for analysis. The pH of each concentrated collection was measured electrometrically before storage. All juices were in the pH range 6.8 to 7.2. Inorganic phosphate was determined as before, both in the buffer used and in a sample of whole gastric juice from each collection period. Intrinsic factor (IF) and nonintrinsic factor (NIF) B, z binding was determined in each concentrated collection by immunoassay 18 using 25 .ul of the gastric juice, 200 ng of Co 60 B,z and 100 .uliter of pernicious anemia serum containing intrinsic factor antibody in high titer. Total B, z-binding capacity of the gastric juice never exceeded 65% of the 200 ng of Co60 B, z using this system. Content of IF and NIF B, z binder
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in each concentrated gastric juice collection was expressed in units, 1 unit being equal to that amount which binds 1 ng of Co 60 B 12. Pepsinogen content of each concentrated gastric juice collection was measured by a modification of the method of Die and Spray, 19 omitting the use of charcoal, using 25 ~tl of gastric juice and a 1-hr incubation with hemoglobin substrate (Sigma Chemical Company) at pH 2.0. A pepsinogen standard (Sigma Chemical Company) and substrate control were run with each sample. The total amount of tyrosine released in micrograms per milliliter per hour at pH 2.0 for each collection period was estimated and peptic units were calculated from the pepsinogen standard (1 peptic unit produced an increase in OD2so of 0.060 per hr at pH 2.0 and 37 C).
Results Comparison of PEG and inorganic phosphate as gastric markers. The concentrations of PEG and phosphate in recovered gastric juice in each of six 15-min stimulated collection periods in 4 subjects were compared. The results revealed that PEG and phosphate concentrations are in close agreement. The coefficients of variation between them in individual collection periods were consistently below 5%, as were the mean coefficients of variation for each of the four studies undertaken. In addition, the small differences found did not show any consistent trend. Recent studies of the secretion of inorganic phosphate by the human stomach20' 21 indicate a low basal output of 3 to 6 mg per hr and no enhancement of output following maximal stimulation. The phosphate buffer used in the present study contains approximately 2 g of inorganic phosphate per liter, and, since more than 1 liter has been necessary in order to achieve continuous intragastric neutralization in normal subjects, the contribution of endogenous phosphate secretion during maximal stimulation was less than 1%. This fact, together with the close agreement between phosphate recovery and that of PEG, a substance neither absorbed nor secreted by the gastric mucosa, provides indirect evidence for there being little significant net transfer of phosphate in these studies.
The quantities of vitamin B12 binders and pepsinogen secreted have thus been estimated from the recovery of inorganic phosphate in each 15-min collection period and are based on the two following assumptions. (a) Mixing of the buffer and secreted gastric juice is complete throughout the test period and that, therefore, the content of phosphate in gastric juice leaving the stomach via the pylorus will be the same as that recovered and measured in the gastric aspirate. Our preliminary observations using PEG indicated that mixing of two volumes in the stomach occurred rapidly and was virtually complete. (b) The stomach is emptied at the end of each collection period. In these studies a residual volume in excess of 10%, indicating incomplete collection, occurred in 1 subject in the last three collection periods. Isolated instances of a significant residual volume were observed in 2 other individuals, again in the latter stages of each study. The following formulas were applied to e.:;timate secretory volume and the secretion of the gastric constituents studied: (1)
Vs = VF- VI
where Vs is volume secreted per 15 min; VI is volume of buffer introduced per 15 min; and V F is the final volume after mixing, made up of that recovered and that lost through the pylorus. Since no significant net transfer of phosphate occurs, the amount of phosphate in the final volume must equal the amount of phosphate introduced, or V F [PF] = VI [PI]
(2)
where [P F] is concentration of phosphate in the final volume and [PI] is concentration of phosphate in the buffer instilled and V [PrJ 1 [PF] Substituting this for VF in equation (1), V s = Vr
[~:]]
- Vr
(3)
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30000
and (4)
where Cs is the total output of C per 15 min when C is any measurable gastric secretory component, and CF is the concentration of C in the final volume. Secretion of IF B12 binder, NIF B12 binder, and pepsinogen. There was a mean increase in secreted volume from basal to stimulated periods, but the variation in basal secretion was such that these figures did not reach statistical significance in the small group of subjects studied. However, it is of note that the standard errors in secreted volume were much smaller during the period of maximal stimulation. The mean (±SE) secretion of IF B12 binder basally and following maximal histalog stimulation is shown in figure 1. The secretion of NIF B1 2 binder was not significantly altered from basal levels following histalog, whereas IF secretion was significantly increased above basal levels in the first (P < 0.01) , second (P < 0.001), 8000
t
HISTALOG
MINUTES
FIG. 1. Mean ( ± s E) secretion of intrinsic factor (0) and nonspecific B12-binder ~) basally and following intramuscular histalog, 1. 7 mg per kg body weight, in 6 healthy human subjects.
.~ c:
" .'E<>. "'a. f-
::>
a. f-
::> 0
10000
0 ~~15~~30~~4~5--~6~0--7~5--~9~0--~100
t HISTALOG
MINUTES
FIG. 2. Mean (±SE) secretion of pepsinogen basaily and following intramuscular histalog, · 1.7 mg per kg body weight, in the same 6 subjects shown in figure 1.
third (P < 0.001), and fourth (P < 0.01) periods after stimulation. The ratio of IF to NIF B12-binder in basal secretions was 1: 1.5 and following stimulation increased to 6 : 1 and fell progressively thereafter to 1.5: 1, 90 min after histalog. Peak output of IF occurred in the first 15 min following stimulation when the mean secretion reached 5,500 units. The mean hourly IF output after stimulation was 13,700 units with a range of 8,500 to 22,900 units. The corresponding pepsinogen content in the same basal and posthistalog collections is shown in figure 2. Pepsinogen secretion in the first (P < 0.05), second (P < 0.001), third (P < 0.001), and fourth (P < 0.02) periods following stimulation was significantly increased over basal levels. The mean maximal hourly output of pepsinogen after stimulation was 98,000 peptic units with a range of 83,000 to 114,000. The pattern of pepsinogen secretion was different from that of intrinsic factor. Peak output of pepsinogen following histalog occurred in the 30- to 45-min collection period, whereas the peak output of IF occurred in the first 15 min after stimulation. In addition, there was no correlation between the maximal output of pepsinogen
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INTRINSIC FACTOR, NONINTRINSIC FACTOR
and that of IF, nor were their secretions significantly correlated in any of the individual 15-min collections following histalog. Discussion In the present investigation, an attempt
has been made to quantify IF secretion in normal human subjects during maximal histalog stimulation, utilizing continuous in vivo neutralization to overcome the partial intragastric degradation of this molecule which results from a short period of acid-peptic action. 15 This effect has not always been considered in previous studies; the in vitro neutralization of the aspirated gastric juice frequently is delayed for a variable time period. Further, the use of inorganic phosphate present in the buffer as a gastric marker has provided a more valid assessment of the actual secretion of gastric juice constituents by correcting for volume loss through the pylorus. Polyethylene glycol introduced intragastrically at low infusion rates has been validated as a marker of gastric juice recovery during basal and maximally stimulated secretion in man. 22 Our preliminary observations using larger volumes of buffer and PEG confirmed the excellent recovery of the latter. However, PEG could not be used in the present studies because of its interference with the immunoassay of the B1 2 binders. Falsely elevated counts in the supernatant fluid were observed with PEG in the system, suggesting that PEG in the gastric juice adsorbs to the albumin-coated charcoal and impedes the uptake of free Co60 B1 2 . The close agreement between the recoveries of PEG and phosphate observed in a comparative study of four separate time courses in normal subjects demonstrated the feasibility of utilizing phosphate as a marker. The possibility that endogenous secretion of phosphate might interfere is ruled out by the results of studies in man which show such secretion to be low, constant, and unaffected by gastric stimulants.20 , 2 1 The present study describes for the first time the secretion of NIF B1 2 binder in response to gastric stimulation and compares
515
the secretion of the B1 2 binders of gastric juice with that of pepsinogen in the same subjects. We have found that the technique of continuous intragastric neutralization used in this study will maintain the pH of aspirated gastric juice between 6.8 and 7.2 in most normal subjects, even during periods of maximal secretory activity following stimulation. The immunoassay of B1 2 binders is not affected in this pH range, and spontaneous conversion of pepsinogen to pepsin should not occur. Restoration of the pH to 2.0, however, will restore maximal peptic activity. 23 The secretion of IF in normal subjects using conventional collection techniques and in vitro neutralization of gastric juice has been studied using a variety of gastric secretagogues both in single dose form 1 - 7 and as constant infusions. 8 ' 9 In only two studies2 ' 3 has histalog been used, and in only one of these 3 was the time course of IF secretion studied. The average hourly output of IF following stimulation in normal subjects has varied widely in previous studies. Although this may partly reflect variation in volume collection from study to study, 5 lack of uniform handling of collected gastric juice, particularly with respect to the time when neutralization is carried out, may also account for some of this variation. Our results in terms of mean IF output after stimulation agree closely with similar studies in which immediate neutralization of aspi4 7 rated gastric juice was carried out. ' In other studies where neutralization appears to have been delayed, the mean hourly IF output after stimulation was significantly lower than in the present study. 1 • 8 Despite the variation in total IF output in response to maximal stimulation, there is general agreement with respect to the pattern of its secretion. Peak output occurs in the first 30 min following stimulation, returning within 40 to 60 min to a low constant level of secretion. This pattern is regarded as being due to an initial washout of stored IF from the parietal cells, which is followed by a much lower secretion rate close to, but significantly different from, basal levels. 9
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The time course of IF secretion observed in our 6 normal subjects did not differ from that seen in previous studies. However, it is interesting that no enhancement in the secretion of NIF B12 binding occurred following stimulation, an observation which has not been previously reported but which supports a nonparietal origin of this gastric juice constituent. The response of pepsinogen secretion to histalog in normal subjects has not been investigated previously. Makhlouf et al. 13 ' 14 have demonstrated an earlier peak in pepsin output than that seen in the present study in response to single large doses of histamine and gastrin. Maximal histalog stimulation has been shown to induce a later peak in acid output than that obtained using the augmented histamine test in the same subjects. 24 The later peak of pepsinogen output observed in the present study in response to maximal doses of histolog may represent a similar phenomenon, but direct comparison with histamine in the same subjects is needed in order to confirm this suggestion. The pattern of pepsinogen secretion in these 6 subjects differed from that of IF measured in the same gastric juice collections. Pepsinogen secretion was more sustained, with the peak response occurring 30 to 45 min after stimulation; no significant correlation emerged between IF and pepsinogen outputs in any individual collection period. Bitsch et al. 25 studied pepsin and IF secretion in patients with duodenal ulcer and observed that their pattern of secretion was similar in resP-onse to histamine and insulin. They concluded that gastric stimulation leads to a "washout" of stored pepsinogen similar to that which appears to occur in the case of IF. This mechanism of pepsinogen response to gastric stimulation had been proposed in earlier studies. 26 - 28 However, comparative studies of pepsin and acid secretion in man 10· 12 · 14 · 29 - 31 indicate that the pattern of pepsin secretion in response to a variety of secretagogues is one of true stimulation. Our finding of a clear difference between the patterns of IF and pepsinogen secretion
supports the current belief that an initial flushing out of preformed IF occurs in response to stimulation, whereas the same stimuli of gastric secretion result in the elaboration of pepsinogen. REFERENCES 1. Ardeman, S., I. Chanarin, and J. C. Doyle. 1964. Studies on secretion of gastric intrinsic factor in man. Brit. Med. J. 2: 600-603. 2. Ardeman, S., and I. Chanarin. 1965. Stimulation of gastric intrinsic factor secretion. Brit. Med . J . I : 1417-1418. 3. Jeffries, G. H., and M. H. Sleisenger. 1965. The pharmacology of intrinsic factor secretion in man. Gastroenterology 48: 444-448. 4. Wangel, A. G., m.d S. T . Callender. 1965. Effect of gastrin I and II on the secretion of intrinsic factor. Brit. Med. J. 1: 1409-1411. 5. R.fdbro, P ., P. M. Christiansen, and M. Schwartz. 1965. Intrinsic factor secretion in stomach diseases. Lancet 2: 1200-1203. 6. Rfldbro, P., and P. M. Christiansen. 1966. Quantitative estimation of parietal cell secretory function in man. Scand. J. Gastroent. I: 292-298. 7. R;dbro, P., and P . M. Christiansen. 1967. Quantitative determination of gastric intrinsic factor after large histamine doses in healthy persons. Scand. J. Clin. Lab. Invest. 19: 186-189. 8. Lawrie, J. H., and N. M. Anderson. 1967. Secretion of gastric intrinsic factor. Lancet 1: 68-71. 9. Weir, D. G., I. J. Temperley, and D. Collery. 1967. Intrinsic factor secretion in -response to continuous histamine infusion. Gastroenterology 52: 23-28. 10. Piper, D. W. 1960. The effect of histamine on pepsin secretion. Amer. J. Dig. Dis. 5: 880-888. 11. Hunt, J. N. 1950. An interpretation of the histamine test of gastric secretion. Gastroenterology 16: 231-240. 12. Hirschowitz, B. I. 1961. Electrolytes in human gastric secretion. Observations and a theory. Amer. J. Dig. Dis. 6: 199-228. 13. Makhlouf, G. M., J . P. A. McManus, and W. I. Card. 1966. Action of the pentapeptide (ICI 50,123) on gastric secretion in man. Gastroenterology 51: 455-465. 14. Makhlouf, G. M., J . P. A. McManus, and W. I. Card. 1967. Comparative effects of gastrin II and histamine on pepsin secretion in man. Gastroenterology 52: 787-791. 15. Ashworth, L.A. E. , R. G. Strickland, N . C. Koo, and K. B. Taylor. 1969. Effect of pH on intrinsic factor and nonintrinsic factor vitamin Bw
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