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GASTRO-INTESTINAL STUDIES. I. GASTRIC JUICE INPERNICIOUS ANEMIA

By 0. M. HELMER, PAUL J. FOUTS, AND L. G. ZERFAS(From the Lilly Laboratory for Clinical Research, Indianapolis City Hospital and the

Department of Medicine, Indiana University School of Medicine, Indianapolis)

(Received for publication June 20, 1932)

GASTRIC JUICE IN PERNICIOUS ANEMIA

Several authors have indicated that the determination of the hydro-chloric acid, in itself, is not an adequate measure of the impairment of thesecretory activity of the gastric mucosa. Michaelis (1) demonstratedthis fact by the simultaneous determination of acid and enzymes ingastric juice from cases of low and high acidity and achylia. Pollandand Bloomfield (2) stated that a low pepsin output is a more delicateindex of gastric function than low acid values. Davies (3) similarlyreported low pepsin values in the achylia of pernicious anemia.

Since the determination of the enzyme content of the gastric juicemay be a valuable aid in the differential diagnosis of certain types ofanemia, we are presenting the data that we have obtained in a series ofpernicious anemia cases and normal controls. In a later paper data willbe presented on secondary anemia and some obscure types of anemia.

In this series of experiments we have determined the gastric enzymes,pepsin and rennin, the free and total acid, the hydrogen ion concentrationand the nitrogen, chloride, and phosphorus content, in order to determineas fully as possible the extent of the dysfunction of the gastric glands inthe achylia gastrica occuring in patients with pernicious anemia.

MATERIAL AND METHODS

Ten young, healthy adults, who worked in the hospital and who had noevidence of disease, were used as controls in these experiments. All of theforty-seven patients with pernicious anemia were proven cases, and a moredetailed description of them will be found later in the paper.

No food or drink was given to the subjects between the evening meal andthe morning of the test. Early in the morning, the fasting contents wereremoved by means of a Rehfuss tube, after which 0.5 mgm. of histamine hydro-chloride was injected. All patients developed a generalized erythema followingthe injection. Subsequent samples of the gastric juice were collected attwenty minute intervals over a period of eighty minutes, great care beingtaken to remove as completely as possible all of the gastric contents at eachwithdrawal. The patients were advised to take particular care not to swallowsaliva.

To fourteen of the forty-seven patients with pernicious anemia, intravenousliver extract was given at the end of the sixty minute period, and two more

1129


samples of gastric juice were collected. Each specimen of gastric juice wasmeasured in a graduated cylinder and was filtered through paper before usingit for analysis. The presence or absence of bile, blood, and mucus was noted.

The pH was determined by means of the colorimetric method of Brown (4)using one drop of gastric juice, the free acid by Sahli's method using 1 cc. ofjuice, and the total acid on 1 cc. of juice, titrating with N/IOO sodium hydroxideusing phenolphthalein as an indicator. Chlorides were measured by Wilsonand Ball's modification (5) of the Van Slyke method, and nitrogen was deter-mined, using 0.5 cc. of gastric juice, by the gasometric method of Van Slyke (6).

Phosphorus was determined by Fiske and Subbarow's method (7). One-half cc. of the samples of gastric juice obtained from patients with perniciousanemia were digested in pyrex test tubes graduated to a volume of 10 cc.Larger samples, depending upon the acidity, were used when the subjects werenormals. When normal juice was precipitated with trichloracetic acid, thefiltrate contained practically the same amount of phosphorus as the wholejuice, whereas, with samples from patients with pernicious anemia, we wereunable to get clear filtrates after trichloracetic acid precipitation, and, therefore,the total phosphorus was determined on the whole juice.

Pepsin was determined by the method of Koch and McMeekin (8)-asimple, rapid, and accurate method, for which only one cc. of gastric juice wasrequired. The substrate employed in this method was coagulated egg white,which is particularly useful in the determination of pepsin in gastric juice,since trypsin is not able to act upon it to an appreciable extent.

The coagulated egg white was prepared as follows: a 20 per cent solutionof Merck's powdered egg white (or albumin) was prepared and coagulated withheat by placing it in a boiling water bath for fifteen minutes, stirring vigorouslythroughout the coagulation period. The coagulated egg white was filteredthrough cheese cloth, and was broken up into small pieces by passing it througha fine meat chopper. It was then shaken with an excess of 0.3 per cent hydro-chloric acid for two hours, at 400 C., to remove inorganic salts, colored ex-tractives, and other substances which are soluble in 0.3 per cent hydrochloricacid, thereby reducing the soluble nitrogen blank of the egg white. Thecoagulated egg white was filtered through gauze with the aid of suction and wasthoroughly washed with distilled water. It was then spread out on trays anddried in a current of warm air. After drying, it was ground so as to pass throughan eighty mesh sieve.

To determine the amount of peptic activity, 4 grams of egg white weresuspended in 99 cc. of 0.3 per cent hydrochloric acid in a 125 cc. Erlenmeyerflask, at room temperature. Then 1 cc. of gastric juice was added. Thesolution was shaken and placed in an incubator at 400 C. for two hours whereit was rotated vertically during the entire period of incubation.' After thetwo hour incubation period, the solution was filtered by decantation through18 cm. quantitative filter paper. The refractive index was determined at250 C. on the filtrate by means of an emersion refractometer, and the readingswere converted into "milligrams of pepsin" (1 to 4,000) by means of a tableprepared by Koch and McMeekin (8). Since the concentration of pepsin is solow in gastric juice obtained from patients with pernicious anemia, only twograms of egg white were used as a substrate, and the total volume of acid wasmade up to 50 cc. instead of 100 cc. Otherwise, the method was identical withthat used with normal juice.

1 The water bath and shaker, designed by Doctor Koch, was obtained fromthe Arthur H. Thomas Company, Philadelphia.

1130

0. M. HELMER, PAUL J. FOUTS AND L. G. ZERFAS

Rennin was determined by a method similar to the one described byMichaelis (1). Forty-five cc. of fresh, raw, cows' milk and 5 cc. of a 10 per centsolution of crystalline calcium chloride were mixed. Test tubes containing2.5 cc. of this reagent were placed in the water bath at 400 C., and, when theyhad attained the temperature of the bath, gastric juice equivalent to 1/100 cc.to 1 cc., depending upon the rennit activity of the juice, was added to the tubesand the clotting time noted. The rennit activity was converted into "milli-grams of rennin" by referring to a table made by using the above techniqueon known concentrations of dried commercial rennin. The clotting time withthe known concentrations of rennin are shown in Table 1.

TABLE 1

The clotting time required for known amounts of dry commercial renninRennin Average clotting time Average clotting timemgm. seconds minutes1.5 91.0 10.50.5 210.3 310.2 440.1 730.075 980.050 145 1.630.030 222 3.700.020 334 5.900.010 650 10.83

RESULTS IN NORMALSUBJECTS

In Table 2 are tabulated the results of the analyses of the gastricjuice obtained from ten normal subjects.

In Chart I, the values obtained in an average normal subject areplotted. With the exception of one case, the fasting samples containedonly a trace of mucus. After histamine stimulation, the juice becamemore watery, filtering with the greatest rapidity at the height of the acidsecretion. The greatest volumes of juice were obtained at the twentyminute period and ranged from 35 to 80 cc., with an average of 56 cc.

In the fasting samples the pH varied from 1.6 to 7.2. The peak ofthe acid secretion was in the forty minute period in the majority of cases,with pH values of 1.1 or less. The free acid at its peak ranged from 72 to140 cc. N/10 per 100 cc., and the total acid varied from 77 to 149.

The pepsin and rennin were secreted at the same rate, with the peakat twenty minutes. At the height of secretion the pepsin varied from1.4 to 7.1 mgm. per cc., with an average of 4.6 mgm., and the renninvaried from 15 to 70 mgm. The concentration of enzymes in the gastricjuice did not follow the curve of acid secretion.

The nitrogen concentration fell after stimulation, which is in accordwith the data in the literature. The lowest values for nitrogen were foundat the peak of the acid secretion. The curve of the nitrogen concentrationdid not follow that of the enzymes. This is contrary to the findings ofPolland and Bloomfield (9), who stated that in normal persons the con-

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VALUES FROM GASTRIC JUICE OF NORMALSUBJECT NUMBER4 (A TYPICALNORMAL) FOLLOWINGTHE SUBCUTANEOUSINJECTION OF 0.5 MGM. OF

HISTAMINE HYDROCHLORIDE

secretion of enzymes apparently influenced the nitrogen concentration inCase 2, Case 5, and Case 8; after stimulation, there was the usual increasein acid, with a simultaneous increase in pepsin and rennin of greaterproportions than usual, but with a comparatively smaller decrease innitrogen concentration.

1134


The total chloride concentration, in general, followed the acid con-centration; however, the increase in chlorides was not in proportion tothe change in acidity, and could not, in itself, account for the increasedacidity. Gamble and McIver (10) and Austin and Gammon (11),using dogs with gastric pouches, stated that the chloride concentrationremained almost constant during their experiments.

The total phosphorus concentration decreased as the acidity increased,reaching its low point at the height of the acid secretion. As the aciddecreased, the phosphorus values returned to values approaching thoseof the fasting samples. Bulger, Stroud, and Heideman (12) point outthat, if much acid is produced, the phosphates decrease, and they attributethe fluctuations to relative amounts of secretion from the mucous glands.Wehave also found that if stimulation did not produce a secretion of freehydrochloric acid, the concentration of phosphate often increased.Hoesch (13), too, showed that the phosphorus content was low when theacidity was high, and, in cases of anacidity or achylia, the phosphoruscontent of the gastric juice was high. Hoesch believes that the antago-nism between chloride and phosphate may be an important factor in thesecretory mechanism that produces free hydrochloric acid.

Bolton and Goodhart (14) believe that mucus is an important factorin controlling the acidity of the stomach, especially when there is only asmall flow of gastric juice. Since the nitrogen and phosphorus of thegastric juice are probably secreted mainly by the mucous glands, thefall in both nitrogen and phosphorus with active acid secretion combinedwith their subsequent rise as the acid secretion diminishes, might furtherpoint to an important rOle of gastric mucus in the control of the acidityof the stomach.

RESULTS IN PERNICIOUS ANEMIA

Table 3 records the results of the forty-seven individual analyses,while Chart II shows the average of the results obtained in the forty-seven cases of pernicious anemia. The gastric juice obtained in patientswith pernicious anemia was usually small in volume and contained muchmucus, but the samples with the greatest volumes usually containedthe least mucus. Vineburg and Babkin (15), and Gilman and Cowgill(16) stated that histamine did not stimulate the gastric enzymes but thatpilocarpine did. We did separate, complete gastric analyses on onepatient, after 0.5 mgm. histamine given subcutaneously, after 3.2 mgm.pilocarpine subcutaneously, and after 20 cc. of liver extract intravenously.There was practically no difference in the three analyses, so we con-sidered histamine an adequate stimulus. Two patients also had gastricanalyses after histamine stimulation, first while in relapse and again soonafter remission had been induced by liver extract, and there were nodifferences in the gastric findings.

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Volume. Histamine stimulation caused an increase in volume overthe fasting sample in twenty-five of the forty-seven cases examined.At the height of secretion, the volume varied from 4 to 83 cc., with anaverage of 22 cc. The total volume secreted in sixty minutes varied

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AVERAGEVALUES FROM GASTRIC JUICE OF FORTY-SEVEN PATIENTS WITHPERNICIOUS ANEMIA, FOLLOWINGTHE SUBCUTANEOUSINJECTION OF 0.5

MGM. OF HISTAMINE HYDROCHLORIDE

from 9 to 193 cc., with an average of 41 cc. Following the injectionof liver extract there was an increase in volume in all but one of fourteencases.

The patients who had lower red blood cell counts tended to havesmaller volumes than those having higher counts. Seven of eight patients

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1147

A


with red blood cell counts below three million had sixty minute volumesof less than 25 cc. Patients with early or no central nervous systeminvolvement more often had a greater volume of gastric juice than thosewith moderate or advanced central nervous system involvement. Itwould seem that the patients with larger volumes more frequently hadpH values of the gastric juice that decreased after histamine stimulation,although the volume had no relation to the exact pH value.

The percentage of pepsin in the individual samples bore little or norelationship to the volume; however, the patients with larger total volumestended to have the higher total pepsin values, although a few of thepatients with the highest volumes had the smallest amounts of pepsin.The amounts of rennin present seemed to have no relation to the volumeof the sample. Contrary to expectations, the percentage of phosphoruswas higher in the samples with the greater volumes. Likewise, in thecases with greater volumes, there was a higher percentage of cases inwhich there was an increase in phosphorus values over the fasting.The samples having larger volumes usually had a higher percentage ofchlorides. The amount of nitrogen in a sample bore no relationship tothe volume of the sample.

Acidity. In all samples there was an absence of free hydrochloricacid and the pH varied from 6.9 to 8.6. Only five of the forty-sevenpatients had not received liver extract medication previous to the gastricanalysis. The length of time the others had been on liver extract medi-cation varied from a few months to five years. This continued absenceof free acid in the gastric juice of patients with pernicious anemia, afteroral liver therapy, is very well known, and it is interesting to note thatthe seventeen patients who have received liver extract by injection forfrom one to eight months still have an achylia gastrica. The amount oftotal acid excreted in sixty minutes varied between 0 and 30 cc. N/100.In only fourteen cases was there a measurable drop in pH value afterhistamine stimulation. After liver extract injection there was no con-stant change in pH value. The presence of bile in the samples caused anincrease in the amount of total acid present and a lowering of the pH.There was no relation between the amount of total acid present or theheight of the pH and the clinical condition of the patient, except thatonly one of eight patients with red blood cell counts below 3.0 millionhad a pH value below 7.5 in any sample. However, in the fourteeninstances in which the pH of the gastric juice decreased after histamine,nine of the patients had only early or no central nervous system involve-ment. The height of the pH bore no relation to the percentage of pepsin,rennin, phosphorus, chlorides, or nitrogen in the samples; but, in thecases with a decrease in pH value after histamine stimulation, there was agreater percentage with an increase in phosphorus, chlorides, and nitrogen.

Pepsin. The pepsin in the gastric juice was either totally absent or

148


present in essentially negligible amounts, varying between 0.0 and 0.20mgm. per cc., with an average of 0.04 mgm. The total amount of pepsinsecreted in sixty minutes varied between 0.0 and 7.4 mgm., with anaverage of 1.8. There was a slight increase in the amount of pepsinafter histamine stimulation in thirty-three of the forty-seven casesexamined. The injection of liver extract caused an increase in pepsinin seven of fourteen cases. The clinical condition of the patient did notseem to show any relationship to the percentage of pepsin present in thesamples. Likewise, the amount of pepsin showed no relation to theheight of pH, amount of phosphorus, chlorides, or nitrogen, althoughthirteen of seventeen cases with an increase in nitrogen after histaminealso had an increase in pepsin. A trace of bile in the specimen occasion-ally caused a slight increase in peptic activity, but even then the amountof pepsin found was not over 0.17 mgm. per cc. Occasionally, when bilewas present, there was a total absence of peptic activity.

Rennin. The amount of rennin present varied from 0.0 to 0.19 mgm.per cc., with an average of 0.013. The total amount of rennin secreted insixty minutes varied from 0 to 4.0 mgm. (average 0.42 mgm.). Becauseof the very small amounts of rennin present-the maximum in the patientswith pernicious anemia being only 1/350th of the maximum in the normals-it was decided to express the rennin in Chart II as either being presentor absent. The presence of bile in the sample usually caused an increasein amount of rennin, but again there occasionally was no rennin insamples containing bile. There seemed to be no relation between theamount of rennin in the gastric sample and the clinical condition of thepatient or the pH value, volume, amount of phosphorus, nitrogen, orchlorides. The determination of rennin was a good check on the pepsindetermination, as there was always a proportionately greater decrease inthe amount of rennin when there was a decrease in pepsin.

Nitrogen. The amount of nitrogen present was definitely high in allsamples. The presence of bile in the samples caused a great increase inthe amount of nitrogen, and, therefore, the nitrogen determinations wereof no value in samples containing bile. The amount of nitrogen presentin the samples of gastric juice had no relation to the clinical condition ofthe patient. An increase in the concentration of nitrogen was noted insix of eight patients who received liver extract intravenously. Theamount of nitrogen present showed no relationship to pH values or tothe amounts of phosphorus, chlorides, pepsin, or rennin.

Chloride. The chloride values were definitely low, but they did notseem to be as low as would be expected with a total absence of free hydro-chloric acid-a few high values, obtained in patients with achylia, weregreater than some of the lower values obtained in normal persons.There was an increase in chlorides after histamine stimulation in onlynine of twenty-five cases. As has been shown above, there was a tendency

1 149


for specimens with larger volumes to have higher percentages of chlorides.There was no correlation between the amounts of chlorides and the pH,amounts of nitrogen, pepsin, rennin, and phosphorus in the samples.

Phosphorus. The amount of phosphorus present was greatly in-creased in nearly all specimens. In eighteen of twenty-five cases thephosphorus value increased after histamine stimulation. As has beenshown, the specimens with larger volumes more often had higher phos-phorus values, and a higher percentage of the specimens with largervolumes had an increase in phosphorus values after histamine stimulation.There was no correlation between the phosphorus values and the pH,amounts of nitrogen, chlorides, pepsin, or rennin.

It is evident from these findings that, while in normal persons thereis a direct relationship between the free acid, total acid, chlorides, phos-phorus, and nitrogen in the gastric juice after histamine stimulation,there is absolutely no relationship between these substances in the gastricjuice of patients with pernicious anemia.

Gastric juice findings in relation to maintenance dosage of liver extractAll of the forty-seven patients in this group were clinically and

hematologically typical of pernicious anemia, and either had had orsubsequently have had good responses to liver extract administered bymouth or by injection. The length of time that these patients havebeen on liver extract has varied from 0 to 5 years. Twenty-one of thepatients have been followed at monthly intervals by this department forfrom over one to five years, and the remaining patients for only a fewmonths. Seventeen were in relapse (either mild or severe) at the timethe analyses were made. The red blood cell counts varied from 0.5million to 6.0 million and the hemoglobin percentages from 13 to 115per cent. All degrees of central nervous system involvement wererepresented-from the very slightest to the most advanced stage wherethe patient was completely bedridden. In this group of patients, somewere able to maintain their blood at normal levels on a very small dailyamount of potent liver extract by mouth, while others were not able todo this on large amounts and required liver extract by injection.

An attempt was made to determine whether the quantity or qualityof the gastric juice bore any relation to the amount of liver extractrequired to maintain the blood at normal levels. There were thirty-onepatients in the group who gave a history of having taken liver extractfor a sufficient period of time to be considered. Six of the thirty-onewere in relapse at the time the gastric analyses were taken. Since it hasbeen shown that patients with a low red blood cell count have smalleramounts of gastric juice than those with a high red blood cell count, it isdifficult to correlate the gastric juice findings of the patients in relapseand the maintenance dosage; therefore these patients were excluded.

1150


It would seem that patients with larger volumes of gastric juice, afterhistamine stimulation, more often were able to maintain the blood atnormal levels on smaller amounts of liver extract. However, all but twoof the sixteen patients requiring more than the amount of liver extractderived from 300 grams of whole liver daily had one or more complications,such as: moderate to advanced central nervous system involvement,generalized arteriosclerosis, infections, non-infectious complications(other than arteriosclerosis), or chronic diarrhea. Such complications,in themselves, have been shown by Beebe and Lewis (17) and by Foutsand Zerfas (18) to be present in a high percentage of patients requiringlarge daily amounts of potent liver extract. These facts make it difficultto correlate the gastric juice findings with the maintenance dosage ofliver extract; however, it is interesting to note that, of nine patients withno complications, six had large total volumes of gastric juice and onlyone required more than 300 grams of liver daily, while one of the threepatients having small total volumes required more than that amount ofliver.

The amount of pepsin per cc. of gastric juice had no bearing on theamount of liver extract required by a patient. Since the total amount ofpepsin secreted in sixty minutes varied approximately as did the volume,the total amount of pepsin bore the same relationship to maintenancedosage of liver extract as did the volume. The amount of rennin, like-wise, had no relation to the maintenance dose of liver extract. The pHvalue, amount of total acid, amount of phosphorus, chlorides, andnitrogen had no correlation with the amount of liver extract required tomaintain the blood at normal levels.

From these facts it is evident that the quantity and quality (as tested)of the gastric juice have little or no relationship to the maintenance dosageof liver extract. The only positive finding was that larger volumes andlarger total amounts of pepsin have a questionable correlation with themaintenance dosage. Davies (3) found that two of his patients, whowere able to maintain their blood at normal levels with very smallamounts of liver extract, had large amounts of pepsin. Wewere notable to find appreciable amounts of pepsin or rennin in any of the forty-seven cases of pernicious anemia examined. Also, we could not find anycorrelation between the small variations in the amount of pepsin and themaintenance dosage of liver extract. Wewere unable to test the gastricjuice for the presence of, or for the quantitative changes in, the amountsof intrinsic factor of Castle. If there are no qualitative or quantitativedifferences in the proteolytic enzymes of the small intestines of patientswith pernicious anemia or in the amounts of the intrinsic factor, it isevident that the ability of such patients to absorb the active principlecontained in liver extract largely governs the amount of liver extractrequired to maintain the blood at normal levels, when the extract is

1151


administered orally. Castle (19) has suggested that there is a defectiveabsorption of liver extract in some patients having pernicious anemia andfurther suggests that the inability to absorb the active principle from foodis of etiological importance in the disease.

CONCLUSIONS

1. The gastric juice of forty-seven patients with pernicious anemiaand of ten normal persons, after histamine stimulation, has been studied.

2. The complete dysfunction of the gastric glands in patients withpernicious anemia was demonstrated by the following facts: (a) Smallvolumes of juice containing large amounts of mucus; (b) Absence of freehydrochloric acid, with an actual alkaline reaction; (c) Practical absenceof the gastric enzymes, pepsin and rennin; (d) Low chloride concentration;(e) High nitrogen and phosphorus values; (f) No relation between totalacid, pH, chlorides, nitrogen, and phosphorus as found in normal controls.

3. The quality and quantity of the gastric juice of patients withpernicious anemia has little or no relationship to the maintenance dosageof liver extract.

BIBLIOGRAPHY

1. Michaelis, L., Deutsche med. Wchnschr., 1918, xliv, 685. Die Bestimmungund Bedeutung der Fermente im Magensaft.

2. Polland, W. Scott, and Bloomfield, A. L., J. Clin. Invest., 1931, ix, 107.The Diagnostic Value of Determinations of Pepsin in Gastric Juice.

3. Davies, Daniel T., Quart. J. Med., 1931, xxiv, 447. Studies on Achlor-hydria and Anemia.

4. Brown, J. H., J. Lab. and Clin. Med., 1924, ix, 239. The ColorimetricDetermination of the Hydrogen Ion Concentration of Small Amounts ofFluid.

5. Wilson, D. W., and Ball, E. G., J. Biol. Chem., 1928, lxxix, 221. A Studyof the Estimation of Chloride in Blood and Serum.

6. Van Slyke, Donald D., J. Biol. Chem., 1927, lxxi, 235. GasometricMicro-Kjeldahl Determination of Nitrogen.

7. Fiske, C. H., and Subbarow, R., J. Biol. Chem., 1925, lxvi, 375. TheColorimetric Determination of Phosphorus.

8. Koch, F. C., and McMeekin, T. L., Personal Communication.9. Polland, W. Scott, and Bloomfield, A. L., J. Clin. Invest., 1929, vii, 57.

Quantitative Measurements of Pepsin in Gastric Juice Before and AfterHistamine Stimulation.

10. Gamble, James L., and McIver, M. A., Proc. Soc. Exper. Biol. and Med.,1926, xxiii, 439. Fixed Base in Gastric Juice.

11. Austin, J. H., and Gammon, G. D., J. Clin. Invest., 1931, x, 287. GastricSecretion after Histamine: Sodium and Potassium Content and PepsinEstimation.

12. Bulger, H. A., Stroud, C. M., and Heideman, M. L., J. Clin. Invest., 1928,v, 547. Studies of the Chemical Mechanism of Hydrochloric AcidSecretion. I. Electrolyte Variations in Human Gastric Juice.

13. Hoesch, K., Deutsches Arch. f. klin. Med., 1929, clxv, 201. Beitrage zurMangenfunktion. I. tYber die Phosphate im reinen Magensaft.

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0. M. HELMER, PAUL J. FOUTS AND L. G. ZERFAS 1153

14. Bolton, Charles; and Goodhart, Gordon W., J. Physiol., 1931, lxxiii, 115.The Variations in the Acidity of the Gastric Juice During Secretion.

15. Vineburg, Arthur M., and Babkin, B. P., Am. J. Physiol., 1931, xcvii, 69.Histamine and Pilocarpin in Relation to the Gastric Secretion.

16. Gilman, Alfred, and Cowgill, George R., Am. J. Physiol., 1931, xcvii, 124.Effect of Histamine upon the Secretion of Gastric Pepsin.

17. Beebe, Richard T., and Lewis, E. G. Eric, Am. J. Med. Sci., 1931, clxxxi,796. The Maintenance Dose of Potent Material in Pernicious Anemia.

18. Fouts, Paul J., and Zerfas, L. G., Ann. Int. Med., In Press. MaintenanceDosage of Liver Extract in the Treatment of Pernicious Anemia.

19. Castle, William B., and Taylor, F. H. Laskey, J. Am. Med. Assoc., 1931,xcvi, 1198. Intravenous Use of Extract of Liver.

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