Pepsinogens, Pepsins, and Pepsin Inhibitors

Vol. 60, No. 4 Printed in U.S. A .

G ASTROENTEROLOGY

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1971 by The Williams & Wilkins Co.

PROGRESS IN GASTROENTEROLOGY PEPSINOGENS, PEPSINS, AND PEPSIN INHmITORS 1.

MICHAEL SAMLOFF, M.D.

Division of Gastroenterology, Departments of Medicine , Harbor General Hospital, Torrance, California , and University of California School of Medicine, Los Angeles, California

The subject of gastric proteases and protease inhibitors was last reviewed here by Turner, Miller, and SegaP in 1967. Two additional reviews, one in the French literature by Cheret and Bonfils 2 and one by Knowles 3 on the mechanism of pepsin action, appeared in 1970. This review emphasizes the literature which has appeared since mid-1967 and attempts to cover the heterogeneity, distribution, and nomenclature of the pepsinogens and pepsins, the measurement of pepsin, the secretion of pepsin, and the pepsin inhibitors. Reference is also made to some earlier works in an attempt to relate recent developments to past observations. The zymogens or precursors of the pepsins are found in both the oxyntic gland and the pyloric gland areas of the stomach, in the proximal duodenum, in seminal fluid, where they presumably originate from the seminal vesicles, in amniotic fluid where they are probably of fetal origin, and in blood and urine. The gastric peptic cells are the major source of the pepsinogens in blood and urine, but it is uncertain whether they enter the circulation through endocrine secretion or through release from degenerating cells. During the conversion process to pepsin, which begins below pH 5, several basic peptides are Received September 24, 1970. Address requests for reprints to: Dr. 1. Michael Samloff, Harbor General Hospital, 1000 West Carson Street, Torrance, California 90509. This study was supported by United States Public Health Service Grant AM 13233 from the National Institutes of Health.

cleaved from the zymogen molecules. The pH optima of the pepsins vary from about 1.8 to 3.5 depending upon the species of pepsin, the ionic strength of the incubation mixture, and the type and concentration of substrate used. 4 , 5 The generally accepted criteria for classifying a protease as a pepsin are that it exists as an inactive alkali-stable zymogen from which it is formed in the presence of acid, that it clots milk, that it is active at acid pH, and that it is inactivated at neutral or slightly alkaline pH. Human Pepsinogens Heterogeneity, distribution, and nomenclature. During the past 8 years, several groups of investigators have demonstrated from two to eight proteolytic fractions in extracts of human gastric mucosa. Each group has used its own nomenclature and some have presented evidence or implied that certain of their fractions are homogeneous. However, in light of recent studies, it seems fair to state that none of the pepsinogen or pepsin fractions obtained thus far is homogeneous. Since studies of the proteolytic and molecular characteristics of these preparations have assumed a homogeneous population, the conclusions drawn from these investigations may need revision in the future. In 1963, Tang and Tang 6 isolated two zymogen fractions, designated IA and lB, from extracts of gastric mucosa by column chromatography on diethylaminoethyl cellulose. Upon activation with acid, each zymogen gave rise to two enzymes6 , 7 586

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which corresponded in chromatographic behavior to the "pepsin" and "gastricsin" previously isolated from gastric juice. s Although zymogen IA was found to be homogeneous by several criteria, recent studies indicate that it is separable into at least six electrophoretically distinct fractions. 9 Also in 1963, Seijffers et at. 10 reported the isolation of three pepsinogen fractions by diethylaminoethyl cellulose chromatography. These were designated pepsinogens I, II, and III in order of their elution from the column. Pepsinogens II and III were found only in fundic mucosa, but pepsinogen I was found in fundic, antral, and duodenal mucosa. A fourth minor fraction, which eluted ahead of pepsinogen I, was not studied. On activation with acid, pepsinogens I and III gave rise to chromatographically distinct enzymes, pepsins I and Ill, but pepsinogen II produced two pepsins, suggesting that the latter fraction was heterogeneous. 11 It is now known that pepsinogens I, n, and III are heterogeneous. 9 In 1964 Kushner et al. 12 identified four proteolytic fractions in human gastric mucosa by agar gel electrophoresis. These were designated P I, P n, P Ill, and P IV. The fraction with the slowest electrophoretic mobility, P IV, unlike the other fractions, was not inactivated by sequential acidification and neutralization. This failure to satisfy one of the criteria for pepsinogen, together with the recent demonstration that a protease having similar electrophoretic mobility and resistance to inactivation as P IV is also present in a jejunal mucosa,13 suggests that this fraction is not a pepsinogen. On acid activation, P III appeared to give rise to two pepsins. Although immunological studies suggested that both pepsins were derived from a single precursor, recent studies indicate that both P II and P III are heterogeneous. 13 Fundic mucosa contained all four fractions, but the pyloric gland area contained only P III and P IV. This was confirmed by Hirsch-Marie,14 who also found P III and P IV in extracts of proximal duodenal mucosa. Cheret and Bonfils 15 found five proteolytic fractions in human gastric mucosa by

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agar gel electrophoresis. Demonstrating considerable restraint, these investigators did not name these fractions, but noted that the two with the slowest electrophoretic mobility were also found in antral mucosa. Hanley and colleagues 16 identified three proteolytic fractions by starch gel electrophoresis which they termed PG I, PG II, and PG III. Preliminary data suggested that antral mucosa contained only PG II. In 1969 Samloff1 3 reported the resolution of eight proteolytic fractions in human gastric mucosa by agar gel electrophoresis. The seven fastest migrating fractions were inactivated by sequential acidification and neutralization and were termed Pg 1 through Pg 7 in order of decreasing electrophoretic mobility. The eighth fraction was not inactivated by this procedure and was also found in jejunal mucosa. Since this fraction may not be a pepsinogen, it was given the descriptive and noncommittal designation of slow moving protease (SMP). Fundic mucosa contained all eight fractions, while the distal antrum and proximal duodenum contained only Pg 6, Pg 7, and SMP. Although there is uncertainty about the number of human pepsinogens, all investigators agree that some fractions are limited to fundic mucosa while others are found in fundic, antral, and proximal duodenal mucosa. In addition, all have found that the former fractions behave chromatographically and electrophoretically as more negatively charged molecules than the latter. Based upon this concordance and upon further similarities which are presented below, it seems reasonable to separate the human pepsinogens into two groups and to propose that they are related as shown in table 1. It has been suggested that those fractions which are limited to fundic mucosa be designated the group I pepsinogens and that the fractions which are also present in the antral and proximal duodenal mucosa be termed the group II pepsinogens.17 This nomenclature is used hereafter. At this writing, then, seven human pepsinogen fractions have been identified, five of which are group I pepsinogens (Pg 1

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1. Heterogeneity, nomenclature, and proposed relationships of the human pepsinogens

Investigator

and year

Tang and Tang,· 1963 Seijffers et al.,'· 1963

Kushner et al.,12 1964 Hanley et al.,'· 1966 Samloff,13 1969

Method

No.'

Diethylaminoethyl cellulose chromatography Diethylaminoethyl cellulose chromatography

2

Zymogen lAd Zymogen IE

4

Agar gel electrophoresis Starch gel electrophoresis Agar gel electrophoresis

4

Prepepsinogen I Pepsinogen I Pepsinogen II Pepsinogen III P I, P II, P III, PIV PG I, PG II, PG III Pg 1, Pg 2, Pg 3, Pg 4, Pg 5, Pg 6, Pg 7, SMP

3

8

Nomenclature

Group 1"" pepsinogens

Group ITe pepsinogens

N onpepsino-

gen protease

Pepsinogen II Pepsinogen III

Pepsinogen I Prepepsinogen I

PI, P II

PIlI

PIV

PG I

PG lIe

PG III

Pg 1, Pg 2, Pg 3, Pg 4, Pg 5

Pg 6, Pg 7

SMP

Number of fractions isolated or identified. Limited to fundic mucosa. e Present in fundic, antral, and proximal duodenal mucosa. d Contains both group I and group II pepsinogens. e Not demonstrated in duodenal mucosa.

a b

through Pg 5) and two of which are group II pepsinogens (Pg 6 and Pg 7). No claim has been made for the homogeneity of any of these fractions. Indeed, gel filtration and electrophoretic studies have shown that Pg 1, although distinguishable from the other pepsinogen fractions, is itself heterogeneous. 13 In addition, there is an acidactive protease in human gastric mucosa which differs from the pepsinogens in its resistance to sequential acidification and neutralization and in its presence in jejunal mucosa. This protease, which recent studies in this laboratory suggest consists of at least two fractions, has been given the descriptive designation of slow moving protease or SMP. It may represent a tissue cathepsiI}. While modifications of existing methods and -the development of new techniques will undoubtedly demonstrate further heterogeneity of the human pepsinogens, considerable additional information is available to support the concept that they are separable into two groups. Serum and urinary pepsinogens. All investigators agree that the group I pepsinogens are invariably present in urine and that the group II pepsinogens are rarely found in normal urine. Seijffers et al. 18

were unable to detect the group II pepsinogens in the urine of any of 12 subjects. In other studies the group II pepsinogens were found in the urine of approximately 2% of normal individuals 17 . 19 and in the urine of approximately 87% of subjects with proteinuria. 17 An explanation for the usual absence of the group II pepsinogens in normal urine is not available. Since both groups are present in serum 17 and an inhibitor of the group II pepsinogens has not been found in urine,17, 18 possible mechanisms include a failure of glomerular filtration through polymerization or protein binding in the serum or tubular reabsorption in the kidney. The usual absence of the group II pepsinogens in urine indicates that uropepsin activity cannot be used as an indicator of either the endocrine or exocrine component of total gastric pepsinogen secretion. A relationship may exist between uropepsin activity and the secretion of the group I pepsinogens, but this remains to be shown. Serum pepsinogen activity, however, measures both the group I and group II pepsinogens, but the relative amounts of each group in serum and gastric juice are unknown. A low level of serum pepsin-

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ogen appears to be of some use in the differentiation of megaloblastic anemias and in the detection of atrophic gastritis,20 but, in the individual patient, an elevated level is of little diagnostic value. The separate determination of the group I and group II pepsinogens in serum may prove to have clinical value. One might anticipate finding an increased level of the group II pepsinogens in inflammatory conditions of the antrum and duodenal bulb. Seminal pepsinogens. The presence of pepsinogen in seminal fluid was reported by Lundquist and Seedorff21 in 1952. Subsequent studies have shown that all of the proteolytic activity in seminal fluid at low pH is attributable to the group II pepsinogens. 22 . 23 Seijffers and associates' data suggested that seminal pepsinogen activity may be diminished in sterile males with normal sperm counts 24 ; Hirsch-Marie and Conte 23 reported approximately identical activities in normal, oligospermic, and azoospermic fluids. It is unknown whether seminal pepsinogen enters the circulation. Do the group II pepsinogens have a role in fertility? Esophageal pepsinogens. Hirsch-Marie 14 reported finding the group II pepsinogens in esophageal mucosa. Amniotic fluid pepsinogens. Wagner 25 found traces of milk-clotting activity in amniotic fluid beginning at 28-week gestation. This activity, which was attributed to pepsinogen originating from the fetus, increased rapidly during the last month of pregnancy. In this laboratory (unpublished observations), only the group I pepsinogens have been detected in each of five specimens of amniotic fluid obtained between 15- and 20-week gestation, but in none of the specimens obtained before 15 weeks. The group II pepsinogens have been detected in amniotic fluid for the first time at 33-week gestation, but fluid has not been available between 21 and 32 weeks. The time of appearance of the group I and group II pepsinogens in fetal gastric mucosa has not been determined. Is it possible that the level of pepsinogen in amniotic fluid might serve as an index of fetal maturity?

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Immunochemical relationships. The results of immunoelectrophoretic studies indicate that the group I and group II pepsinogens are immunologically unrelated: antiserum against both groups of pepsinogens produces precipitin arcs of nonidentity against a mixture of the group I and group II pepsinogens 12. 14, 19; rabbit antibodies to the group I pepsinogens do not form a precipitin arc against the group II pepsinogens.26 A recent study has shown that the five group I pepsinogens, although electrophoretically heterogeneous, are immunochemically similar. 26 The nonpepsinogen protease in gastric mucosa is immunochemically distinct from the group I and group II pepsinogens. 12, 14, 19 Cellular origins. The limitation of the group I pepsinogens to fundic mucosa has been taken as evidence that these zymogens are synthesized by the chief cells. Recent immunofluorescent studies in this laboratory (unpublished obseruations) , using rabbit antiserum specific for the group I pepsinogens, have localized this group to both the chief and mucous neck cells in fundic mucosa. These findings are similar to those reported by Yasuda et al. 27 for the cellular localization of hog pepsinogen and support the conclusion of a recent study that the mucous neck cell is the precursor of the chief cell. 28 In keeping with the anatomic distribution of the group I pepsinogens, fluorescent cells were not seen in antral or duodenal mucosa. Inasmuch as the antiserum was known to react against each of the five group I pepsinogens, it is not possible to conclude that every fraction is present in each cell or in each cell type. There are several recent studies of the fine structural characteristics of the .mucous neck cell and the chief cell in man and other species. 2~31 The cellular origins of group II pepsinogens have not been established. Genetic polymorphism. Samloff and Townes 17 reported that Pg 5, one of the group I pepsinogens, is absent from both the gastric mucosa and urine of some individuals. By contrast, individuals with Pg 5 in their mucosa always had Pg 5 in their urine. Population and family studies have shown that approximately 14% of the

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white non-ulcer bearing population is deficient in Pg 5 and that this deficiency is inherited as a simple autosomal recessive trait. 32 Individuals with Pg 5 have been designated phenotype A and those without Pg 5 have been designated phenotype B. It will be of interest to learn whether an association exists between pepsinogen phenotype, blood group, secretor status, and peptic ulcer. The finding that Pg 5 is immunochemically related to the other members of its group suggests that all of the group I pepsinogens are determined at the same chromosomal locus but that the polymorphism is reflected only in Pg 5. This proposal is based on the observations that isozymes controlled by allelic genes are usually immunochemically indistinguishable from each other. 33 By contrast, multi genic nonallelic isozymes are generally immunochemically distinguishable. Accordingly, if the group I and group II pepsinogens can be considered as isozyme sets, it would seem that they are controlled by genes at different loci.

Human Pepsins Chromatographic and electrophoretic studies have shown that, upon acidification, the group I and group II pepsinogens give rise to chromatographically and electrophoretically distinct pepsins. 11 , 12 This indicates that the heterogeneity of the pepsinogens is due to differences in the pepsins. It is unknown whether differences also exist in the basic peptides which are cleaved from the zymogens during the activation process. The finding that acidification of an apparently homogeneous pepsinogen yielded two pepsins has been interpreted as indicating that a single precursor yields two pepsins s, 12 or that the precursor was probably heterogeneous. 11 The recent demonstration of considerable heterogeneity of the pepsinogens suggests that the latter interpretation is correct. The demonstrations that the chromatographic and electrophoretic characteristics of the pepsins derived from acidified mucosal extracts and from gastric juice are similars , 15, 34 indicate that all the pepsinogens which have been found in gas-

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tric mucosa are secreted into the gastric lumen. We have in a sense come almost full circle to the earlier concept of a "pyloric" pepsin and a "fundic" pepsin,35 with the realization that each is heterogeneous and that the pyloric pepsin is also present in fundic and proximal duodenal mucosa. Etherington and Taylor 3S , 37 found eight proteolytic zones in human gastric juice by agar gel electrophoresis, seven of which were found in acidified extracts of gastric mucosa. 3S Recent studies in this laboratory (unpublished observations) suggest that, upon acidification, each of the seven electrophoretically distinct pepsinogen fractions gives rise to an electrophoretically distinct pepsin fraction with greater anodal mobility than its precursor. It is of interest that acidification also increases the electrophoretic mobility of the SMP. However, the number of proteolytic fractions observed after acidification is dependent upon the conditions of activation; as many as 11 have been found. This may be due to autodigestion of the pepsins, with the formation of several proteolytically active products, to the formation of pepsin-pepsin inhibitor complexes, or to further heterogeneity of the pepsinogens. The gastricsin of Tang and co-workers S-S and the pepsin I of Seijffers et al. 11 have been stated to have similar characteristics,39, 40 and it is probable that they are the same. Since pepsin I is derived from pepsinogen I,ll a zymogen having the characteristics of a group II pepsinogen (table 1), it follows that gastricsin and pepsin I may be classified as group II pepsins. Similarly, the "pepsin" of Tang: and associates S-s is probably a mixture of pepsins IIA, lIB, and III of Seijffers et al. 11, 34 These pepsins may logically be designated group I pepsins since they arise from zymogens having the characteristics of group I pepsinogens. The two groups of pepsins differ in their abilities to hydrolyze certain peptide bonds. 7, 41, 42 In addition, the group II pepsins are less resistant to heat,7, 39 are more resistant to alkali,39, 40, 43 and have a higher pH optimum 7, s, 35 than the group I pepsins, but differences may exist among members of a group. 39

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fractions in monkey gastric mucosa by agar gel electrophoresis; these were designated P I, P II, P III, P IVa, and P IVb. Immunoelectrophoretic studies using an antiserum against monkey gastric mucosa revealed that P I and P II were immunochemically indistinguishable from each other and immunologically unrelated to P III and P IV. Chiang et aI. 53 isolated porcine gastricsin and demonstrated that it, like human gastricsin (group II pepsin), did not hydrolyze the synthetic substrate, N-acetyl-Lphenylalanyl-L-diiodotyrosine (APDT). By Nonhuman Pepsinogens and Pepsins contrast, both porcine and human pepMultiple forms of pepsinogen have been sins hydrolyzed this substrate. Similarly, found in the pig, 15, 16 rat,15 rabbit,15, 16, 45 Ryle and Hamilton 54 have shown that pepguinea pig,15 monkey, 46 chicken, 47 dog- sin C, the enzyme obtained by acidificafish,48 and, probably, the COW.49 As with tion of pepsinogen C which is the predomthe human pepsinogens, a uniform nomen- inant zymogen in swine pyloric mucosa, clature has not been adopted. Some au- has little activity against APDT. By conthors 47 , 48 have used the system adopted trast, porcine pepsins A and D hydrolyze by Ryle 50 for the porcine pepsinogens as this substrate. 55 Furthermore, on agar gel recommended by the Commission on En- electrophoresis, porcine pepsinogen A has zymes of the International Union of Bio- greater electrophoretic mobility than porchemistry. 51 The order of elution of the cine pepsinogen C and immunochemical zymogens from diethylaminoethyl cellu- studies suggest that the former zymogen lose is pepsinogen B, D, A, and C. shares antigenic determinants with the huThe results of investigations of the het- man group I pepsinogens but not with the erogeneity, anatomic distribution, sub- group II pepsinogens. 12 strate specificities, and immunochemical These data, when taken together, sugrelationships of the pepsinogen fractions gest that homologies exist among certain in several species suggest that they are, pepsinogens in man and other species. Obas in man, separable into two main groups. viously, systematic studies will be needed Agar gel electrophoresis of extracts of fun- to establish the relationships, but the dic and pyloric gland mucosa of the rat and available information allows the speculadog has revealed that some pepsinogen tion that porcine, chicken, and dogfish fractions are limited to fundic mucosa and pepsinogens A and D are the counterparts that others are found both in fundic and of the human group I pepsinogens and that antral mucosa (unpublished observations). the pepsinogen C of these species is the Similar findings have been reported by counterpart of the human group II pepsinCheret and Bonfils. 15 ogens. Donata and Van Vunakis 47 , 52 have found Agar gel electrophoresis of a preparation that chicken pepsinogens A and Dare im- of hog pepsinogen, reputedly homogeneous munochemically identical and immunolog- by chromatography, has revealed the exically unrelated to chicken pepsinogen C. istence of four proteolytic fractions. 13 Since These investigators suggested that two agar gel electrophoresis appears to be the main types of pepsinogen exist in the most discriminating of the several methods chicken. The immunochemical relation· used to separate the human pepsinogens ships of dogfish pepsinogens A, D, and C and pepsins,9, 13, 36, 37 it seems reasonable to each other are identical to that found to suggest that the homogeneity of pepsinfor the respective chicken pepsinogens. 48 ogen preparations obtained from other speSaint Martin 46 identified five proteolytic cies should be ascertained by this method.

The physiological significance of having two groups of pepsin precursors in fundic mucosa and one group in antral and proximal duodenal mucosa is not clear. There is no evidence at present for the existence of any abnormal proteases in patients with peptic ulcer. Taylor 44 has reported that the frequency of occurrence of pepsin zone 1 (a group I pepsin) in histamine-stimulated gastric juice is higher in patients with gastric or duodenal ulcer than in normal subjects.

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Measurement of Pepsin

The estimation of pepsinogen and pepsin in biological fluids has suffered from several deficiencies including the presence of other proteolytic enzymes active at acid pH, the absence of an acceptable and available reference standard, and the use of protein substrates of varying composition. Recently reported methods for the estimation of proteolytic activity at acid pH include a nephelometric assay using bovine albumin as substrate,56 a colorimetric method using an insoluble substrate covalently labeled with Remazolbrilliant Blue,57 a method using autologous serum as substrate,58 a radial diffusion assay in which the hemoglobin substrate is incorporated into agar gel, 59 a microassay based on the detection of amino groups liberated from N, N -dimethylhemoglobin, 60 a titrimetric assay based on the consumption of hydrogen ions during the hydrolysis of peptide bonds,61 and a method using human hemoglobin as substrate. 62 A semiautomated procedure for serum pepsinogen has also been reported. 63 Recent studies indicate that pepsin hydrolyzes ester as well as peptide bonds. Pepsin has been shown to hydrolyze selected esters of j3-phenyl-L-Iactic acid 64-66 and certain sulfite esters. 67 ' 69 Robinson and White 69 have reported that p-nitrophenyl sulfite is a suitable substrate for the spectrophotometric assay of peptic activity in canine gastric juice. The relative specificities of the human group I and group II pepsins for these substrates are not known. Considerable information has continued to accumulate on the hydrolysis of various synthetic peptides by hog pepsin. 70-73 These studies have been concerned mainly with delineating the mechanism of pepsin action and determining the active site of the enzyme. The synthetic substrates are not in general use. Three approaches have been taken to develop an assay for the separate determination of the group I and group II pepsinogens and pepsins. Chiang et al. 41 have reported that the group I pepsins, but not the group II pepsins (gastricsin), hydrolyze APDT. The

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activity of the group II pepsins in gastric juice was determined by subtracting the activity obtained against APDT from the activity obtained against hemoglobin. Huang and Tang 42 have recently discovered a series of synthetic substrates apparently specific for the group II pepsins. The relative insolubility of these substrates and the prolonged incubation time required may limit their usefulness. A second approach is based on the differential heat lability of the two groups of pepsins. At pH 7.25 and 25 C for 2 min,40 or, at pH 7.1 and 20 C for 90 min!3 the group II pepsins are stable but the group I pepsins are inactivated. A third potentially useful approach is based on the findings that the group I and group II pepsinogens are immunochemically distinguishable. This has not been definitely shown for the respective pepsins. Hirsch-Marie 74 has reported preliminary results with an electrophoretic immunoprecipitation technique using antiserum apparently specific for the group II pepsinogens and pepsins. Considerably more work must be done in this area since proteolytically inactive hydrolysis products of pepsin may retain immunological reactivity. Both histamine and histalog have been reported to increase the output of the group I and the group II pepsins, 1, 41 but no systematic studies of the effects of different stimuli are available. Pepsin Secretion The effect of different stimulants on pepsin secretion is subject to many variables. These include differences in purity, dose, timing, and route of administration of the agent or agents under study, species differences, variations in the type of experimental preparation used within a species, and differences in duration and method of collection of the gastric secretion. The subject of pepsin secretion was reviewed by Hirschowitz 75 in 1967. In this section, an attempt is made to summarize some recent studies on the effects of different stimulants, neural, hormonal, and chemical, on gastric pepsin secretion. To

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allow comparison among studies, all doses of histamine have been converted to histamine base. The expression WJ per kg-hr implies that a drug was given intravenously by continuous infusion. Other routes of administration are specified as required. The expression maximal pepsin output indicates the maximal response observed to a given stimulant under the conditions of a particular experiment and implies that a larger dose of the stimulant did not or would not increase the response. Fundic pouches of the Heidenhain and Pavlov types are referred to as vagally denervated and innervated pouches, respectively. Makhlouf and co-workers76 . 77 have proposed two models for pepsin secretion in man. One is identical with the model proposed earlier for acid secretion 78 ,79 and predicts that the maximal secretory capacity of pepsin is determined by the peptic cell mass and us independent of the stimulant used to induce secretion. An alternate model, which also fits the data available for histamine and gastrin stimulation,76, 77 correlates pepsin output with parietal volume flow. This latter construct is clearly not applicable to pepsin secretion stimulated by secretin, which is accompanied by diminished parietal secretion. 80 Little information is available on pepsin secretion in infants and children. R0dbro and associates81 determined basal pepsin output in 18 children, 9 to 30 months of age, and found significant correlations with age and body surface area but not with body weight. Mean pepsin output was not significantly increased by histamine, 14.5 J.l,g per kg subcutaneously. In a study of infants 12 hr to 3 months of age by Agunod et a1.,82 the pepsin output in response to histalog, 1 mg per kg subcutaneously or intramuscularly, was very low on the 1st day of life but gradually increased into the 2nd and 3rd months with an unexplained decline in the 3rd and 4th weeks. When corrected for body weight, mean pepsin output at 2 to 3 months of age was calculated to be less than half that in adults. Malpress83 found pepsin in the gastric juice of human infants from birth to 6 weeks of

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age, but activity comparable with calf rennin was not found. Basal secretion. Long84 determined basal pepsin secretion in the chicken and calculated that, on the basis of body weight, basal pepsin output was highest in the chicken and progressively less in rat, man, monkey, and dog. It was suggested that this variation is related to the feeding pattern behavior of the species, the chicken characterized as a "continuous" feeder and the dog as a "once a day" feeder . In a further study of the relationship of food intake to gastric secretion, Long85 observed that the increased food intake which occurs in lactating rats is accompanied by a significant increase in basal pepsin output which could be inhibited by atropine or vagotomy. A parallel observation is the finding of a significant increase in the peptic cell population of rats during pregnancy and lactation. 86 Cholinergic effects. Cholinergic mechanisms play a central role in pepsin secretion, producing the strongest stimulation of pepsin output in most species and increasing the responsiveness of the peptic cells to other stimulants. Cooke 87 compared the effects of endogenous cholinergic stimulation, induced by insulin or 2-deoxy Dglucose, and exogenous cholinergic stimulation with bethanecol on pepsin secretion in gastric fistula dogs. The highest pepsin output in response to bethanecol was only half that achieved for insulin, but side effects prevented the administration of larger doses of the former. Peak pepsin outputs in response to insulin and 2-deoxyD-glucose were not significantly different, but were significantly greater than the response to pentagastrin. Isenberg et a1. 88 have shown that pepsin output in man increases with graded doses of insulin; a maximal response was not observed at a dose of 0.4 U per kg, twice that required for maximal acid output. In the chicken, insulin, 4 or 8 U per kg, inhibited acid output but did not affect pepsin output. 84 Caridis and Smith89 have reported that, in man, inhibition of histamine-stimulated pepsin output by hexamethonium does

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not occur when the hypotension induced by the latter is prevented by the infusion of dextran. The administration of sustained release tablets of i-hyoscyamine to patients with chronic duodenal ulcer has been reported to decrease 90 and to have no effect91 on histamine-stimulated pepsin output. The addition of glycopyrrolate to the drinking water of rats for 5 months produced a significant decrease in both spontaneous and histamine-stimulated pepsin secretion. 92 The effect of vagotomy on stimulated pepsin secretion varies with the species and with the dose and type of stimulant. In the dog, vagotomy abolishes the pepsin response to vagal stimulation induced by insulin, but increases the sensitivity of the peptic cells to Urecholine (shift of doseresponse curve to the left) without any change in maximal pepsin output. 93 Vagotomy reduces the responsiveness of the peptic cells to gastrin (shift of dose-response curve to right) without any reduction in the maximal response. 94 The doseresponse curve to histamine is also shifted to the right by vagotomy, but the maximal pepsin response obtainable is slightly less than that observed before vagotomy. 94 In addition, doses of histamine which were supramaximal and which inhibited pepsin output before vagotomy stimulated pepsin output after vagotomy. Thus vagotomy reduced the observed pepsin response to a small dose of histamine but increased the response to a large dose of histamine. In the cat, vagotomy virtually abolished the responsiveness of the peptic cells to both histamine and gastrin. 95 Mter vagotomy, pepsin output in response to graded doses of histamine and gastrin did not exceed basal output before vagotomy. In gastric fistula dogs receiving background stimulation with histamine in a dose supramaximal for pepsin output, truncal vagotomy was found to decrease the peak pepsin response to a large intravenous dose of gastrin. 96 This finding is at variance with an earlier study, also in dogs, which concluded that vagal denervation was attended by an increased pepsin response to a large dose of gastrin. 97 How-

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ever, different doses of histamine, different preparations of gastrin, and different types of experimental preparations were used in the two studies. Tovey et al. 98 have reported that, in man, vagotomy diminished pepsin output to histamine, 14.5 f.lg per kg subcutaneously, but not to antral irrigation with 10% peptone broth. It is clear from these studies that, depending upon the particular species and the type and dose of stimulant, vagotomy may increase, decrease, abolish, or have no effect on pepsin output. Histamine. The relatively small increase of pepsin output in dogs to histamine has been attributed to the washout of preformed enzyme rather than to the stimulation of pepsin secretion. While this mechanism may be operative, the demonstration that the prolonged infusion of histamine produces a sustained increase in pepsin output indicates that histamine is a definite but relatively weak stimulant of pepsin secretion in the dog. 99-101 Histalog has also been reported to stimulate pepsin output in the dog. 102 The dose of histamine which produces maximal pepsin output in the dog is submaximal for acid output and has varied from 6 to 30 f.lg per kg-hr. 94, 103, 104 Maximal pepsin output to histamine in the cat,103 miniature pig,105 the rat, 106 and man 107, 108 is reported to occur at approximately 6, 60, 250, and 14.5 f.lg per kg-hr, respectively. In the latter three species, the dose of histamine which produces maximal pepsin output is close to that for maximal acid output. Gastrin. Gastrin stimulates pepsin output in man, 109, 110, dog,94, 104, 111 cat,95, 98 rat,112-114 rabbit,115 and frog. 116 Cooke 104 compared the relative potencies of several preparations of gastrin and related peptides in gastric fistula dogs and observed that the maximal pepsin outputs obtained with natural hog gastrin II and synthetic human gastrin I were the same, but were approximately 40% less than that obtained with crude hog gastrin. It is possible that a pepsigogue other than gastrin may be present in crude extracts of hog antrum, but

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none has been isolated to date. The dose of gastrin required for maximal pepsin response in dogs is close to that for maximal acid response. 103 , 104 In conjunction with vagal stimulation, exogenous gastrin inhibits pepsin secretion in dogs. Olbe et al. 117 reported that the infusion of gastrin extract inhibited pepsin output to sham feeding in antrectomized dogs with vagally innervated fundic pouches. The degree of inhibition was directly related to the dose of gastrin. By contrast, acid secretion was increased. Similarly, Vagne and Grossman 118 found that the pepsin response to gastric distension in gastric fistula dogs was inhibited by exogenous gastrin. Pentagastrin and tetragastrin. Cooke 104 has reported that, in gastric fistula dogs, pentagastrin and tetra gastrin produced maximal pepsin output at doses of 1 ~g per kg-hr, but that the pepsin response to tetragastrin was almost twice that for pentagastrin, almost 4 times that for natural hog gastrin II and synthetic human gastrin I, and approximately 12-fold that for histamine. The pepsigogic effect of pentagastrin in dogs has also been reported by other investigators, 99, 119-121 but it has not been found by Nakajima and Magee.122-124 The different results appear to arise from methodological differences in collecting the gastric secretion. In man, maximal pepsin output to pentagastrin was obtained at a dose of 0.5 ~g per kg-hr, half that required for maximal acid output. 107 In two studies, the peak pepsin output at 15 min to subcutaneously administered pentagastrin, 6 J-Lg per kg, or tetragastrin, 20 J-Lg per kg, was greater than that obtained with histamine, 14.5 ~g per kg,125, 126 but there did not appear to be any significant difference in the 1-hr pepsin outputs. In another study in man, pentagastrin, 6 J-Lg per kg; tetragastrin, 10 or 20 J-Lg per kg; gastrin I, 2 or 4 J-Lg per kg; and histamine, 14.5 J-Lg per kg intramuscularly, produced equivalent hourly pepsin outputs. 109 In gastric fistula rats, pentagastrin in doses ranging from approximately 42 to 1315 J-Lg per kg increased acid but not pepsin output. 127 These rather large doses may

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have inhibited the peptic cells. A possible parallel observation is that of Crean et al. 128 who found a significant increase of the gastric parietal cell population but not of the chief cell population in rats given pentagastrin, 2 mg, twice daily (approximately 480 J-Lg per kg per hr assuming a body weight of 350 g) in a depot preparation for 21 days. By contrast, duodenal obstruction produced a significant increase in both the total parietal cell and chief cell populations,129 whereas vagotomy produced a significant decrease in only the peptic cell population. 130 Sewing131 has commented that tetragastrin in doses ranging from 0.1 to 3.2 J-Lg per min did not stimulate pepsin output in anesthetized cats with gastric fistula. Caerulein. Caerulein is a decapeptide with the same five carboxy-terminal amino acids as gastrin and cholecystokinin (CCK) . On both a molar and weight basis, caerulein is more active than human gastrins I and II in stimulating pepsin secretion in the dog. 132, 133 Unlike gastrin, which has identical potencies in its sulfated and desulfated forms, desulfated caerulein has only one-fifth the potency on pepsin secretion as natural caerulein. 134 Secretin. Secretin has been known to inhibit gastric acid secretion for many years, but its pepsigogic activity has only recently been documented. The strong stimulating effect of secretin on pepsin secretion has been shown in the dog,124, 135, 136 cat,136 and man. 80 , 137-140 Brooks et al. 80 observed that the peak 30-min pepsin output to secretin in man was only slightly less than that found after insulin 0.4 U per kg intravenously. 88 Glucagon. Glucagon resembles secretin in structure. In a dose of 25 ~g per kg intravenously, it had no effect on pepsin output in man.80 Earlier studies had shown inhibition of pepsin secretion by glucagon (see reference 80). Cholecystokinin. Variable results have been reported for the effect of CCK on gastric pepsin secretion. In two studies, 120, 141 preparations of CCK were found to increase pepsin output in dogs, but one preparation contained a small amount of secretin and the purity of the other was in doubt. Naka-

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jima and Magee 142. 143 have reported 'that, in dogs with vagally denervated fundic pouches, separated duodenal pouches, and a gastrojejunostomy, CCK in doses of approximately 2 to 8 U per kg-hr inhibited both unstimulated pepsin output and pepsin output in response to secretin, and, in doses of approximately 3 to 4 U per kg-hr, it inhibited pepsin output to Mecholyl. By contrast, Stening and Grossman 133 have reported that CCK in graded doses from 2 to 8 U per kg-hr stimulated pepsin secretion in gastric fistula dogs. The pepsin output to CCK, 8 U per kg-hr, was similar to that produced by gastrin, 1 Ilg per kg-hr. In 13 of 15 human subjects studied by WormsleY,139 CCK, 16 U per kg-hr, stimulated pepsin output. Brooks and Grossman 137 found that in man the pepsin response to pentagastrin, 4 Ilg per kg-hr, was not altered by CCK, 4 U per kg-hr. It is clear that no general conclusions are possible at present regarding the effect of CCK on pepsin secretion. Duodenal perfusion. Variable results also have been obtained for the pepsin response to duodenal acidification. Nakajima and Magee 143 found that acidification of duodenal pouches to pH 3 in dogs with denervated fundic pouches increased pepsin output in response to pentagastrin, histamine, or methacholine. During acidification to pH 1, pepsin output was the same or less than control levels. These authors suggested that acidification to pH 3 released secretin which stimulated pepsin output, while acidification to pH 1 also released CCK which inhibited pepsin output. In other studies, the pH has not been controlled. Duodenal acidification in cats with denervated fundic pouches has been reported to increase pepsin output. 136 Wormsley144 studied the pepsin response to duodenal acidification in normal subjects and in patients with duodenal ulcer. Duodenal perfusion fbr 10 min with 100 ml of 100 mM HCI or for 30 min with 220 ml of 50 mM HCI produced no significant change in mean pepsin output. Brooks et al. 80 perfused the duodenum of normal subjects for 90 min with 600 ml of 100 mM HCl. In this study, the gastric aspirate was obtained

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during continuous perfusion of the stomach with 100 mM HCl. Under these conditions, there was over a 2-fold increase in pepsin output. Ward et al,l45 have reported that perfusion of the duodenum with either hypotonic or hypertonic saline or with hypertonic glucose inhibits pentagastrin-stimulated pepsin output in both duodenal ulcer patients and control subjects. The insertion of long or medium chain triglycerides into the duodenum of pylorusligated rats inhibited pepsin output. 146 In chickens, the infusion of fat into the duodenum did not affect pepsin output. 84 Prostaglandins. The prostaglandins are a group of long chain, unsaturated, oxygenated fatty acids which have been isolated from a variety of tissues including the gastrointestinal tract. 147 Robert and associates 148 have reported that, in dogs, PGE l and PGE 2 inhibited pepsin output to histamine and eating. PGA l and PGF 2<> did not affect histamine-induced pepsin secretion, but PGA l did inhibit the pepsin response to eating. PGE l has also been found to inhibit unstimulated pepsin output in pylorus-ligated rats and to reduce the incidence and severity of ulcers induced either by pylorus ligation and restraint or by prednisolone. 149 The mechanism of inhibition of gastric secretion by PGE l does not appear to be secondary to alterations in mucosal blood flow. 150. 151 Calcium and magnesium. Calcium, administered by intravenous infusion or by single intravenous injection, increases pepsin output in man. 152. 153 Barreras and Donaldson 153 observed that the mean pepsin output in response to calcium was 43% of the peak histalog response, but that hypercalcemia did not alter the peak response to histalog. Atropine, 152. 153 vagotomy,152 and pentolinium 153 abolished the hypercalcemic effect on pepsin secretion. Induced hypermagnesemia lowered both unstimulated pepsin output and pepsin output in response to calcium. 153 In gastric fistula rats, hypercalemia had no effect on pepsin output, decreased acid output, lowered gastric tissue pepsinogen, and increased blood pepsinogen. 154

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Adrenal hormones. The influence of adrenocortical steroids on gastric secretion was reviewed here by Cooke l55 in 1967. In the dog, bilateral adrenalectomy did not decrease pepsin output to histamine and gastrin, 156, 157 but the administration of hydrocortisone to adrenalectomized dogs or to dogs with intact adrenals produced an increase in the maximal pepsin output to histamine or gastrin. 156, 158 Neither the chronic administration of corticotropin gel to dogs l59 nor the administration of cortisone acetate, 100 j.tg per kg for 10 days, to guinea pigsl60 altered pepsin secretion. In the gastric fistula monkey, norepinephrine, 1 to 3 j.tg per kg-min, inhibited both basal pepsin output and pepsin output in response to histamine, 0.55 j.tg per kg-hr. 161 However, in the gastric fistula dog receiving histamine, 36 j.tg per kg-hr, pepsin secretion was stimulated by norepinephrine and epinephrine in doses of approximately 0.15 j.tg per kg-min. 162 Aldosterone, administered by acute intravenous infusion or by chronic daily injection to dogs with denervated fundic pouches, had no effect on pepsin output.163 Gonadal hormones. The administration of large doses of estrogen 164, 165 or progesterone 165 to man 164, 165 and monkey 164 did not alter basal or histamine-stimulated pepsin output. Oophrectomy or estrogen administration in female rats and castration or testosterone administration in male rats had no effect on pepsin concentration. 166 Identical results were found in dogs, with the single exception that estrogen decreased histamine-stimulated pepsin concentration. A significant increase in basal pepsin output was found in rats during lactation. 85 Thyroid. Two studies on the relationship of thyroid hormone to gastric secretion in rats have appeared. Unstimulated pepsin output and pepsin output to hio;tamine, 250 j.tg per kg-hr, in gastric fistula rats treated with thiouracil were found to be significantly less than in control rats. 167 Increased pepsin output was found to histamine, 18.2 j.tg per kg subcutaneously, in rats made hyperthyroid with i-thyroxine, but pepsin output in rats made hypothy-

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roid with carbimazole did not appear to be altered. 16B The histology of the gastric muc:osa was normal in both groups.

Pepsin Inhibitors Recent studies of pepsin inhibitors have been concerned with their mechanism of action, their ability to prevent the development of ulcers in the experimental animal, and their ability to promote the healing and decrease the recurrence rate of peptic ulcer in man. In addition, these substances have been found to have the potentially deleterious effect of accelerating the conversion of pepsinogen to pepsin. Inhibition of peptic hydrolysis by macroan ions has been found to result from the interaction of the negatively charged inhibitor with the positively charged protein substrate. 6l , 169-171 The protein-inhibitor complex, which has been demonstrated by electrophoresis and by gel diffusion , 172, 173 is not hydrolyzed by pepsin. When substrate-inhibitor interaction does not occur, as when the synthetic dipeptide APDT is used as the substrate, inhibition is not observed. 170 Under certain conditions, the high molecular weight sulfated polysaccharide, undegraded A-carrageenan, inhibits peptic activity by physical encapsulation of the enzyme. l74 The effect of pH, order of addition of the reactants, ratio, and nature of the interacting species of different macroanions and substrates on the inhibition of peptic activity have been extensively studied by Anderson and associates.169-171, 174 Barnes et al. 175 studied the effects of chondroitin sulfate, heparin, carragheen, and dextran sulfate on peptic activity in vitro and on the mortality and incidence of ulcers in pylorus-ligated rats. Equivalent amounts of heparin, carragheen, and dextran sulfate produced equivalent inhibition of peptic activity in vitro, but dextran sulfate was more effective both in decreasing mortality and the incidence of ulcers. Dextran had no beneficial effect. When administered intravenously, however, dextran has been found to reduce both pepsin output and the ulcer index in pylorus-ligated rats l76 and to reduce pepsin output in gastric fistula rats. 177

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Vocac and Alphin 17S . 179 have studied the pepsin inhibitor and antiulcerogenic properties of a number of lignosulfonates. These compounds, when administered orally to rats following pylorus ligation, inhibited peptic activity in the gastric juice but did not significantly change volume or acid output. However, the degree of peptic inhibition in vitro did not parallel the degree of protection against ulcer. illhibition of peptic activity by carbenoxolone has also been reported. ISO Ellis et al. lSI have studied the effect of several pepsin inhibitors on peptic ulcers produced in dogs by placing the gastric antrum on the colon as an antiperistalic diverticulum. SN-263, carrageenan, and powdered charcoal significantly reduced the incidence of ulceration and also decreased the incidence of perforation, mean ulcer size, and mean number of ulcers per dog. An antacid consisting of aluminum hydroxide and magnesium trisilicate had no protective effect. Goldberg et al. 182 have reported that the pepsin inhibitor SN-263 prevented the development of experimental esophagitis in cats when added to an acid-pepsin solution known to cause esophagitis. Heparin, ribonucleic acid, chondroitin sulfate, and a sulfated glycoprotein fraction from human gastric juice have been shown to promote the conversion of pepsinogen to pepsin. 1s3 • lS4 This action occurs at concentrations of the inhibitors less than those required for peptic inhibition, does not occur at pH 5 or above, is maximal at about pH 4, and has been attributed to dissociation of the pepsin-pepsin inhibitor complex by the polyanion. It has been suggested that, in conditions associated with increased permeability of the gastric mucosa to H + and decreased concentration of the naturally occurring sulfated polyanions in gastric mucosa, intracellular pepsinogen might be activated to pepsin and further damage the gastric mucosa. 183. 184 This is a provocative concept, particularly in regard to the pathogenesis of diffuse hemorrhagic gastritis. ill double blind studies, the sulfated polysaccharide SN-263 has been shown to accelerate the healing of gastric ulcers 185

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and to decrease the recurrence of duodenal ulcers. 186 Zimmon et al. 185 found a mean decrease in ulcer size of 9.14 sq mm per day in 10 patients given SN-263 and a mean decrease of 2.21 sq mm per day in 9 patients given placebo. Antacids and anticholinergics were not prescribed, and dietary restrictions were not instituted. ill 2 patients studied sequentially, SN-263 appeared to have an advantage over antacid therapy. Sun and Ryan IS6 studied the effect of SN-263, an anticholinergic, and combined therapy on the recurrence rate of duodenal ulcer. Seventy patients were followed for 1 year or until they developed a recurrent ulcer. These authors found a recurrence of 75% in the placebo group, 39% in the anticholinergic group, 16% in the SN263 group, and 12% in the combined SN263 and anticholinergic group. Although SN-263 appears to be more effective than placebo in treating gastric ulcer, there are no well controlled double blind studies which compare this substance with other modalities of treatment. Furthermore, the advantage of SN-263 over the anticholinergic in preventing the recurrence of duodenal ulcer could have been due to the use of the latter in standard rather than optimally effective doses. Additional clinical studies are needed to delineate the efficacy of the pepsin inhibitors in the treatment of peptic ulcer. REFERENCES 1. Turner MD, Miller LL, Segal HL: Gastric proteases and protease inhibitors. Gastroenterology 53:967-983, 1967 2. Cheret AM, Bonfils S : Pepsine et pepsinogene. Origine, proprietes, preparation. Path Bioi (Paris) 18:317-342, 1970 3. Knowles JR: On the mechanism of action of pepsin. Phil Trans Roy Soc London [Bioi] 257: 135-146, 1970 4. Taylor WH: Biochemistry of pepsins, chap 120, Handbook of Physiology, sect 5: Alimentary Canal, vol 5. Edited by CF Code. Washington, American Physiological Society, 1968, p 25672587 5. Seijffers MJ, Miller LL, Segal HL: Effect of concentration of substrate and of chloride on the optimum pH for the proteolysis of bovine serum albumin by human pepsin m. Biochemistry

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(Wash) 3:5-9, 1964 6. Tang J, Tang KI: Purification and properties of a zymogen from human gastric mucosa. J Bioi Chern 238:606-612, 1963 7. Tang J, Mills J, Chiang L, et al: Comparative studies on the structure and specificity of human gastricsin, pepsin and zymogen. Ann NY Acad Sci 140:688-696, 1967 8. Richmond V, Tang J, Wolf S, et al: Chromatographic isolation of gastricsin, the proteolytic enzyme from gastric juice with pH optimum 3.2. Biochim Biophys Acta 29:453-454, 1958 9. Turner MD, Mangla JC, Samloff 1M, et al: Studies on the heterogeneity of human gastric zymogens. Biochem J 116:397-404, 1970 10. Seijffers MJ, Segal HL, Miller LL: Separation of pepsinogen I, pepsinogen II, and pepsinogen III from human gastric mucosa. Amer J Physiol 205:1099-1105, 1963 11. Seijffers MJ, Segal HL, Miller LL: Separation of pepsin I, pepsin IIA, pepsin lIB, and pepsin III from human gastric mucosa. Amer J Physiol 205:1106-1112, 1963 12. Kushner I, Rapp W, Burtin P: Electrophoretic and immunochemical demonstration of the existence of four human pepsinogens. J Clin Invest 43:1983-1993, 1964 13. Samloff 1M: 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 14. Hirsch-Marie MH: Mise en evidence et separation de pepsinogenes et hydrolyses acides extragastriquel!. Bioi Gastro-Ent 2:109-122, 1968 15. Cheret AM, Bonflls S: Etude electrophoretique des proteases du suc et de la muqueuse gastique de differentes especes animales. Path BioI (Paris) 16:1061-1070, 1968 16. Hanley WB, Boyer SH, Naughton MA: Electrophoretic and functional heterogeneity of pepsinogen in several species. Nature (London) 209: 996-1002, 1966 17. Samloff 1M, Townes PL: Electrophoretic heterogeneity and relationships of pepsinogens in human urine, serum, and gastric mucosa . Gastroenterology 58:462-469, 1970 18. Seijffers MJ, Segal HL, Miller LL: Separation of pepsinogen II and pepsinogen ill from human urine. Arner J Physiol 206:1106-1110, 1964 19. Hirsch-Marie H, Conte M : Etude du pepsinogene urinaire et de ses correlations avec la secretion proteolytique gastrique. Rev Franc Etud Clin Bioi 14:977-983, 1969 20. Singh AK, Shinton NK: Serum pepsinogen in the differentiation of megaloblastic anemia. J Clin Path 18:349-352, 1965 21. Lundquist F, Seedorff HH : Pepsinogen in hu-

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man seminal fluid. Nature (London) 170:11151116, 1952 22. Seijffers MJ, Miller LL, Segal HL: Pepsinogen I in semen. Chromatographic separation of pepsinogen I from human semen. Proc Soc Exp Bioi Med 118:405-409, 1965 23. Hirsch-Marie H, Conte M: Etude de la protease acide du liquide seminal humain. Bull Soc Chim Bioi (Paris)49:147-155, 1967 24. Seijffers M.J, Segal HL, Miller LL: Preliminary appraisal of the role of seminal pepsinogen I in human sterility. Fertil Steril 16:202-207, 1965 25. Wagner H : The development to full functional maturity of the gastric mucosa and the kidneys in fetus and newborn. Bioi Neonat 3:257-274, 1961 26. Samloff 1M: Immunological studies of human group I pepsinogens. J Immun (in press) 27. Yasuda K, Suzuki T, Takano K: Localization of pepsin in the stomach, revealed by fluorescent antibody technique. Okajima Folia Anat Jap 42:355-367, 1966 28. Matsuyama M, Suzuki H: Differentiation of immature mucous cells into parietal, argyrophil, and chief cells in stomach grafts. Science 169:385- 387, 1970 29. Rubin W, Ross LL, Sieisenger MH : The normal human gastric epithelia. A fine structural study. Lab Invest 19:598-626, 1968 30. Helander HF: Ultrastructure and function of gastric mucoid and zymogen cells in the rat during development. Gastroenterology 56:5370, 1969 31. Stephens RJ, Pfeiffer CJ: Ultrastructure of the gastric mucosa of normal laboratory ferrets. J Ultrastruct Res 22:45-62, 1968 32. Samloff IM, Townes PL: Pepsinogens: Genetic polymorphism in ,man. Science 168:144-145, 1970 33. Ogita Z: Genetic control of isozymes. Ann NY Acad Sci 151 :243-262,1968 34. Seijffers MJ, Segal HL, Miller LL: Chromatographic separation of pepsins from human gastric juice. Arner J Physiol 207:8-12, 1964 35. Taylor WH: Studies on gastric proteolysis. 3. The secretion of different pepsins by fundic and pyloric glands of the stomach. Biochem J 71:384-388, 1959 36. Etherington DJ, Taylor WH: Nomenclature of the pepsins. Nature (London) 216:279-280, 1967 37. Etherington DJ, Taylor WH: The pepsins of normal human gastric juice. Biochem J 113: 663-668, 1969 38. Etherington DJ, Taylor WH: The pepsins from human gastric mucosal extracts. Biochem J 118:587-594, 1970 39. Seijffers MJ, Miller LL, Segal HL: Partial

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characterization of human pepsin I, pepsin ITA, pepsin lIB, and pepsin III. Biochemistry (Wash) 3:1203-1209, 1964 40. Turner MD, Tuxill JL, Miller LL, et al: Measurement of pepsin I (gastricsin) in human gastric juice. Gastroenterology 53:905-911, 1967 41. Chiang L, Sanchez-Chiang L, Wolf S, et al: The separate determination of human pepsin and gastricsin. Proc Soc Exp BioI Med 122:700704, 1966 42. Huang WY, Tang J: On the specificity of human gastricsin and pepsin. J BioI Chern 244: 1085-1091, 1969 43. Seijffers MJ, Tkatch R: Assay of two pepsin fractions in human gastric juice by alkali inactivation. Gastroenterology 59:528-533,1970 44. Taylor WH: Pepsins of patients with peptic ulcer. Nature (London) 227:76-77, 1970 45. Samloff 1M, Barnett EV, Turner MD: Immunization of rabbits with hog pepsinogen. I. Autoantibodies to pepsinogen and elevated serum pepsinogen levels. Gastroenterology 52:165175, 1967 46. Saint Martin Jde: Etude immunologique des pepsinogenes de la muqueuse gastrique du singe Papio papio. Ann Inst Pasteur (Paris) 117: 828-838, 1969 47. Donata ST, Van Vunakis H: Chicken pepsinogens and pepsins. Their isolation and properties. Biochemistry (Wash) 9:2791-2797, 1970 48. Merrett TG, Bar-Eli E, Van Vunakis H: Pepsinogens A, C, and D from the smooth dogfish. Biochemistry (Wash) 8:3696-3702, 1969 49. Chow RE, Kassell B: Bovine pepsinogen and pepsin. I. Isolation, purification, and some properties of the pepsinogen. J BioI Chern 243:17181724, 1968 50. Ryle AP: Pepsinogen B: The zymogen of pepsin B. Biochem J 96:6-16, 1965 51. Report of the Commission on Enzymes of the International Union of Biochemistry. London, Pergamon Press, Ltd, 1961 52. Donata ST, Van Vunakis H: Immunochemical relationships of chicken pepsinogens and pepsins. Biochemistry (Wash) 9:2798-2802, 1970 53. Chiang L, Sanchez-Chiang L., Mills IN, et al: Purification and properties of porcine gastricsin. J BioI Chern 242:3098-3102, 1967 54. Ryle AP, Hamilton MP: Pepsinogen C and pepsin C. Further purification and amino acid composition. Biochem J 101:176-183, 1966 55. Lee D, Ryle AP: Pepsinogen D. A fourth proteolytic zymogen from pig gastric mucosa. Biochern J 104:735-741, 1967 56. Gerring EL, Allen EA: A nephelometric pepsin method. Clin Chim Acta 24:437-443, 1969 57. Rinderknecht H, Geokas MC, Silverman P, et al: A new ultrasensitive method for the determination of proteolytic activity. Clin Chim Acta

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21:197-203, 1968 58. Uete T, Wasa M, Shimogami A: A simplified method for the determination of pepsinogen in blood and urine. Clin Chern 15:42-55, 1969 59. Samloff 1M, Kleinman MS: A radial diffusion assay for pepsinogen and pepsin. Gastroenterology 56:30-34, 1969 60. Lin Y, Means GE, Feeney RE: The action of proteolytic enzymes on N,N-dimethyl proteins. Basis for a microassay for proteolytic enzymes. J BioI Chern 244:789-793, 1969 61. Martin F, Vuez JL, Berard A, et al: A study of the mechanisms of inhibition of peptic proteolysis by a sulphated polysaccharide. Digestion 1:165-174, 1968 62. Berstad A: A modified hemoglobin substrate method for the estimation of pepsin in gastric juice. Scand J Gastroent 5:343-348, 1970 63. Wenger J, Munro M: Semiautomated method for serum pepsinogen determination. Clin Chern 16:207-211, 1970 64. Lokshina LA, Orekhovich VN, Sklyankina VA: Esterase activity of pepsin. Nature (London) 204:580, 1964 65. Inouye K, Fruton JS: Pepsin as an esterase. J Amer Chern Soc 89:187-188, 1967 66. Inouye K, Fruton JS: Studies on the specificity of pepsin. Biochemistry (Wash) 6:1765-1777, 1967 67. Reid TW, Fahrney D: The pepsin-catalyzed hydrolysis of sulfite esters. J Amer Chern Soc 89:3941-3943, 1967 68. Reid TW, Stein TP, Fahrney D: The pepsincatalyzed hydrolysis of sulfite esters. II. Resolution of alkyl phenyl sulfites. J Amer Chern Soc 89:7125-7126, 1967 69. Robinson LA, White TT: A spectrophotometric method for the analysis of pepsin. Gastroenterology 58:798-800, 1970 70. Hollands TR, Fruton JS: Kinetics of the hydrolysis of synthetic substrates by pepsin and by acetyl-pepsin. Biochemistry (Wash) 7:20452053, 1968 71. Clement GE, Snyder SL, Price H, et al: The pH dependence of the pepsin-catalyzed hydrolysis of neutral dipeptides. J Amer Chern Soc 90:5603-5610, 1968 72. Jackson WT, Schlamowitz M, Shaw A, et al: The effect of pH on the kinetic constants of peptic substrates and inhibitors. Arch Biochem 131:374-385, 1969 73. Hollands TR, Voynick 1M, Fruton JS: Action of pepsin on cationic synthetic substrates. Biochemistry (Wash) 8:575-585, 1969 74. Hirsch-Marie H: Dosage immunochimique du pepsinogene et de la pepsine gastrique et urinaire par la methode de Laurell. Clin Chern Acta 24:411-416, 1969 75. Hirschowitz B1: Secretion of pepsinogens, Hand-

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