Relationships Among Serum Pepsinogen I, Serum Pepsinogen II, and Gastric Mucosal Histology

GASTROENTEROLOGY 1982;83:204-9 Relationships Among Serum Pepsinogen I, Serum Pepsinogen II, and Gastric Mucosal Histology A Study in Relatives of Patients with Pernicious Anemia I. MICHAEL SAMLOFF, KALLE VARIS, TIMO IHAMAKI, MAX SIURALA, and JEROME I. ROTTER Research and Medical Services, Veterans Administration Medical Center, Sepulveda, California; Second Department of Medicine, University of Helsinki, Helsinki, Finland; and Departments of Medicine, Harbor-UCLA Medical Center. Torrance, and UCLA School of Medicine, Los Angeles. California To examine the possible effect of gastritis on the endocrine component of pepsinogen secretion, we determined relationships among gastric mucosal histologic characteristics and serum levels of pepsinogen I and pepsinogen II in 170 first-degree relatives of patients with pernicious anemia. Sixtyfour had normal fundic gland mucosa, 66 had superficial gastritis, 17 had mild or moderate atrophic gastritis, and 23 had severe atrophic gastritis. In superficial gastritis, serum pepsinogen I and pepsinogen II were both significantly higher than normal, but the percentage rise in pepsinogen II was significantly greater than the rise in pepsinogen I. In mild/ moderate atrophic gastritis, pepsinogen I did not differ from normal, but pepsinogen II was significantly ele vated, while in severe atrophic gastritis, pepsinogen II did not differ from normal, but pepsinogen I was significantly decreased. The nonparallel changes in serum pepsinogen I and pepsinogen II levels resulted in a decrease in the pepsinogen I to pepsinogen II ratio from 6.2 ± 0.2 in subjects with normal fundic gland mucosa to 4.3 ± 0.2,2.9 ± 0.4, and 0.7 ± 0.2, respectively, in those with superficial gastritis, mild to moderate atrophic gastritis, and Received October 9, 1981. Accepted February 22, 1982. Address requests for reprints to: 1. Michael Samloff, M.D., Research Service (151), Veterans Administration Medical Center, 16111 Plummer Street, Sepulveda. California 91343. This work was supported by Research Grant AM 13233. Cent er Grant AM 17328. and Clinical Investigator Award AM 00523 from the National Institutes of Health, Bethesda. Maryland and by grants from the Yrjo [ahnsson Foundation and Dumex Ltd. 1982 by the American Gastroenterological Association 0016-5085/8 2/07204-06$02.50 severe atrophic gastritis. The pepsinogen I to pepsinogen II ratio in combination with the absolute level of serum pepsinogen I correctly predicted the histologic status of the gastric mucosa in 119 of the 170 subjects, 70.0%. The results indicate that (a) increasing severity of gastritis is associated with nonparallel alterations in serum levels of pepsinogen I and pepsinogen II, (bj that these changes lead to a progressive decrease in the pepsinogen I to pepsinogen II ratio, and (c) that the pepsinogen I to pepsinogen II ratio, in combination with pepsinogen I, is predictive of the histologic status of the gastric mucosa. We have recently described the development of a radioimmunoassay for pepsinogen II (PG II) and an improved radioimmunoassay for pepsinogen I (PG I), and have reported on the relationship between serum levels of PG I and PG II in healthy control subjects and in patients with pernicious anemia (1). Of particular interest was the finding that pernicious anemia is associated with a significant decrease in serum PG I and a normal level of serum PG II. It is known from results of previous studies that PG II is derived from the pyloric glands in the gastric antrum and from the chief and mucus neck cell components of the fundic glands in the gastric body (2), while PG I is derived only from the fundic glands (3). It is also known that the loss offundic glands in pernicious anemia is associated with preservation of the pyloric glands in the gastric antrum (4,5) and with pyloric gland metaplasia of the proximal stomach (4-6), and it has been reported that metaplastic

July 1982 SERUM PEPSINOGENS I AND II IN GASTRITIS 205 pyloric glands contain PG II (7). Thus, we attributed the marked decrease in serum PG I in pernicious anemia to the loss of fundic glands and the normal level of serum PG II to an increase in the mass of pyloric glands (1). If this proposal were correct, one would anticipate that less severe forms of chronic gastritis, which are also associated with a loss of fundic glands, and expansion of the pyloric gland area (8-10), might also produce nonparallel changes in serum levels of PG I and PG II. Specifically, our hypothesis was that increasing severity of chronic gastritis of the fundic gland mucosa would result in a progressive decrease in the ratio of PG I to PG II in serum. The results of this study support this hypothesis and indicate that the PG IIPG II ratio in combination with the absolute level of PG I is predictive of the histologic status of the gastric mucosa. Materials and Methods Subjects The original subject population consisted of 183 first-degree relatives of 68 probands with pernicious anemia who had participated in an endoscopic biopsy study of the prevalence of chronic gastritis in family members of patients with pernicious anemia. Data on gastric morphologic characteristics and gastric acid output in this population have been reported previously (4). Serum was available for the determination of PG I and PG II in 173 of the 175 relatives who had biopsies of fundic and pyloric gland mucosa and from 62 probands. Of the 173 relatives, three were excluded from analysis because an elevated serum PG I level was associated with a high serum urea nitrogen level (11). The 170 subjects who form the basis of this report consisted of 72 men and 98 women ranging in age from 15 to 87 yr old. None had clinical symptoms suggestive of peptic ulcer disease and none were found to have an ulcer at endoscopy. Gastric peak acid output had been determined in response to pentagastrin, 6 JLg/kg body wt, in 169 subjects. Gastric morphologic characteristics had been ascertained from endoscopic biopsies of the midbody and antrum. The median number of biopsy specimens obtained from each site was 6 (range 2-12) and 3 (range 1-7), respectively. The histologic status of each biopsy specimen had been classified without knowledge of the secretory or serologic data. Subjects with nonconcordant biopsy specimens were assigned to a histologic category based on the average of all specimens or if two categories were represented equally to the more severe. Sixty-four subjects had normal fundic gland mucosa and 55 of these also had normal pyloric gland mucosa. There were 66 subjects with superficial gastritis of the body mucosa, only three of whom had normal pyloric gland mucosa. The pyloric gland mucosa was normal in five of 17 subjects with mild or moderate atrophic gastritis and in 17 of 23 subjects with severe atrophic gastritis of the fundic gland mucosa. Serum Pepsinogens I and II Serum PG I and PG II levels were determined by specific radioimmunoassays using reference standards that had been prepared from gastric mucosa (1). Statistical Tests The significance of differences in mean values between histologic groups was determined by Student's t test for unpaired samples and the equality of proportions was tested by z-approximation (12). In all analyses, mild and moderate atrophic gastritis were combined because the latter category included only four subjects. Discriminant Function Analysis To determine the ability of each test to predict the histologic status of the gastric mucosa, discriminant values were generated by computer using established statistical programs (13,14). For this purpose, serum PG I, serum PG II, and their derived values were normalized by square root transformation. The discriminant value of each test was used to calculate its sensitivity (percentage of subjects with the specified trait who have a positive test), specificity (percentage of subjects without the trait who have a negative test), positive predictive value (percentage of subjects with a positive test who have the trait), negative predictive value (percentage of subjects with a negative test who do not have the trait), and efficiency (percentage of subjects correctly ascertained by positive and negative test results). For the simultaneous assignment of all subjects to each histologic category, only sensitivity and positive predictive value could be calculated. Results Pepsinogen I, Pepsinogen II, and Gastric Mucosal Histology Mean (±SE) levels of serum PG I and PG II in the subjects with normal fundic gland mucosa were 66.3 ± 3.2 and 11.1 ± 0.5 ng/ml, respectively. Total immunoreactive serum pepsinogen (TISP) was 77.4 ± 3.6 ng/ml and the PG IIPG II ratio was 6.2 ± 0.2. The distribution of values for the PG IIPG II ratio is shown in Figure 1. Mean TISP in the subjects with superficial gastritis was significantly higher than in those with normal fundic gland mucosa due to significant increases in serum PG I and PG II (Table 1). The rise in PG II, as a percentage of normal, was 91%,while the rise in PG I was only 30%. This resulted in a significant fall in the PG IIPG II ratio to 4.3 ± 0.1. Within the group of subjects with superficial gastritis, the PG IIPGII ratio was 4.5 ± 0.2 in those with mild involvement and 3.5 ± 0.5 in those with severe involvement (Figure 1). Both values differed significantly from normal, but not from each other.

206 SAMLOFF ET AL. GASTROENTEROLOGY Vol. 83. No.1. Part 2 Figure 1. Distribution of values for the PG IIPG II ratio according to the hi stologic status of the fundic gland mu cosa. The symbols indicate mean ± SE. 14 12 10 o ~ 8 4 2 o I - I t".., If ~f - t I.. NORMAL N'28 -N-'2"'-j- -N-'"-- --N-.1-7-* ~ N'62 MILD MODERATE SEVERE MILD MOO. SEVERE PERNICIOUS SUPERFICIAL GASTRITIS ATROPHIC GASTRITIS ANEMIA........ I I CASE o.10 CASES Serum PG I was lower than normal in the subjects with mild to moderate atrophic gastritis, but the difference was not statistically significant. Serum PG II, however, was significantly higher than normal (Table 1). The PG I1PG II ratio was 2.9 ± 0.4, significantly less than in normal subjects and in subjects with superficial gastritis. Serum PG I was significantly lower than normal in the subjects with severe atrophic gastritis, but serum PG II did not differ from normal. This led to a further decrease in the PG I/PG II ratio to 0.7 ± 0.2 (Table 1). The values for serum PG I, serum PG II, TISP, and the PG I/PG II ratio in the patients with pernicious anemia were all lower than in the subjects with severe atrophic gastritis, but none of the differences were statistically significant (Table 1). With the exception of serum PG II, all values were significantly lower than normal. Age and Sex Mean levels of serum PG I and PG II did not differ significantly in men and women matched for the same histologic status of the gastric body mucosa. The PG I1PG II ratio was 5.2 ± 0.2 in subjects who were 50 yr of age or less and 3.0 ± 0.2 in those over age 50 yr (p < 10-5 ). This was attributable to an increase in the prevalence of atrophic gastritis from 13.8% to 37.7% (p < 10-3 ) in the respective age groups. Gastric Acid Output The mean level of gastric peak acid output in each histologic category paralleled the change in the PG I/PG II ratio (Table 1). Table 1. Mean (±SE) Levels of Serum Pepsinogen I (PG I), Serum Pepsinogen II (PG II), Total Immunoreactive Serum Pep sinogen (TISP), PG IIPG II Ratio, and Peak A cid Output Peak Histology Serum Serum acid of fundic PG I PG II TISP PG IIPG II output gland mucosa (ng/ml) (ng/rnl] [ng/ml] ratio (rnmol/h] Normal mu cosa 66.3 ± 3.2 11.1 ± 0.5 77.4 ± 3.6 6.2 ± 0.2 30.0 ± 1.6 Superficial gastritis 86.5 ± 4.6 b 21.2 ± 1.2 c 107.7 ± 5.6 c 4.3 ± 0.2 c 23.3 ± 1.6 U Mild to mo derate atr ophic gastr itis 55.5 ± 8.6 x 18.2 ± 1.6 c 73.7 ± 9.6 x 2.9 ± 0.4 c x 6.1 ± 1.6 d z Severe atr oph ic gastritis 9.6 ± 2.2 d,z 12.5 ± 1.1 x 22.1 ± 2.9 d,z 0.7 ± 0,2 d,z 0.4 ± o.a-> Pernicious ane mia 4.8 ± 0.6 d 10.2 ± 0.6 15.0 ± 1.0 d 0.5 ± 0.1< O.Od Signi fican ce of difference from normal: up < 10-2 bp < 10-3 "p < 10-4 d p < 10-5, Signi fican ce of difference from preceding value: xp < 10-2, Yp < 10-3, zp < 10-4

July 1982 SERUM PEPSINOGENS I AND II IN GASTRITIS 207 Table 2. Classification Matrix of Gastric Mucosal Histology by Pepsinogen I/Pepsinogen II Ratio in Combination with Pepsinogen I Classification by PG IIPG II ratio and PG I Mild to moderate Severe Classification Normal Superficial atrophic atrophic by biopsy No. mucosa gastritis gastritis gastritis Normal mucosa 64 51 7 6 0 Superficial gastritis 66 10 39 16 1 Mild to moderate atrophic gastritis 17 1 6 8 2 Severe atrophic gastritis 23 0 0 2 21 Discriminant Function Analysis Discriminant function analysis revealed that the PG I/PG II ratio in combination with the absolute level of PG I had a higher sensitivity and positive predictive value for the morphologic status of the fundic gland mucosa than the ratio alone. This combination correctly assigned 119 (70.0%) of the 170 subjects (Table 2). When classification was made by gastric peak acid output, 86 (50.9%) of 169 subjects were assigned to the correct histologic category (Table 3). A comparison of Table 2 and Table 3 reveals that the combined determination of PG I and PG II was better able to predict normal mucosa and superficial gastritis than peak acid output, but that they did not differ in their ability to predict atrophic gastritis. The operational characteristics of the PG I/PG II ratio in combination with the level of PG I were also better than those for the ratio alone in distinguishing between subjects with and without normal fundic mucosa. In this instance, the sensitivity for normal mucosa was 87.5% and the positive predictive value was 77.8% (Table 4). The respective values for peak acid output were 67.2% and 57.3%. Discussion This study has shown that in subjects with normal fundic gland mucosa the concentration of PG I in serum is about six times higher than that of PG II. This finding implies that the rate of endocrine release of PG I from the chief cells is greater than the rate of release of PG II from both the chief cells and the pyloric glands or that PG I has a longer metabolic clearance rate than PG II. There is no information about the clearance rates of PG I and PG II, but it has been reported that the ratio of the concentrations of PG I and PG II in fundic gland mucosa and PG II in pyloric gland mucosa is in the order of 9:3:1 (15). Thus, there is reason to suppose that the higher concentration of PG I in serum is due, at least in part, to a higher rate of synthesis and release of this zymogen into the circulation. Since the fundic gland area of the stomach is normally larger than the pyloric gland area and contains a higher concentration of PG II (15), there is also reason to suppose that the chief cells are normally the major source of circulating PG II. A diagrammatic representation of the relationships found among nsp, PG I, and PG II in the 64 subjects with normal fundic gland mucosa Table 3. Classification Matrix of Gastric Mucosal Histology by Peak Acid Output Classification by peak acid output Mild to moderate Severe Classification Normal Superficial atrophic atrophic by biopsy No. mucosa gastritis gastritis gastritis Normal mucosa 64 38 19 6 1 Superficial gastritis 66 27 19 19 1 Mild to moderate atrophic gastritis 17 0 2 8 7 Severe atrophic gastritis 22 0 0 1 21

208 SAMLOFF ET AL. GASTROENTEROLOGYVol. 83, No.1, Part 2 Table 4. Discriminant Values of Tests and Their Operational Characteristics for Normal Gastric Mucosa Discriminant Sensitivity Specificity PVpos" PVNeg b Efficiency Test value (%) (%) (%) (%) (%) TISpc <74.4 ng/ml 43.8 44.3 32.2 56.6 44.1 Serum PG I d >62.0 ng/ml 59.4 43.4 38.8 63.9 49.4 PAO e >22.8 mmol/h 67.2 69.5 57.3 77.6 68.6 Serum PG lit <14.0 ng/ml 81.3 67.0 59.8 85.5 72.4 PG IIPG II ratio >4.3 84.4 69.8 62.8 88.1 75.3 PG IIPG II & PG Ig 87.5 84.9 77.8 91.8 85.9 a Positive predictive value. b Negative predictive value. C Total immunoreactive serum pepsinogen. d Pepsinogen I. e Peak acid output. t Pepsinogen II. g Discriminant value for the combination: 5.49YPG IIPG II - 0.76YPG I >5.60. is shown in Figure 2A. The values shown for the fundic and pyloric gland components of serum PG II were calculated on the assumption that 80% of total serum PG II is normally derived from the fundic glands and 20% is derived from the pyloric glands. These are hypothetical values for the purpose of discussion. Superficial gastritis of the fundic gland mucosa was associated with significant increases in serum PG I and PG II, but the rise in serum PG II, as a percentage of normal, was about threefold greater than that of PG I. These changes could be due to a greater increase in the rate of release of PG II than PG I from the chief cells or to an increased rate of PG II release from the pyloric glands and an increased but parallel rate of PG I and PG II release from the chief cells. The latter hypothesis is consistent with the observation that all but three subjects with superfi- Figure 2. Diagrammatic representation of relationships among total immunoreactive serum pepsinogen (TISP), serum pepsinogen I (PG I), serum pepsinogen II (PG II), and gastric mucosal histologic characteristics. The area of each circle is proportional to the level of total immunoreactive serum pepsinogen. The mean levels of serum PG I and PG II found in each histologic category are shown within their respective segments. Each PG II segment is divided by a dashed line into fundic and pyloric gland components. The numbers adjacent to the fundic and pyloric gland components were derived as follows: A. The values were calculated on the premise that the fundic gland contribution to serum PG II is normally four times greater than that of the pyloric glands; B, C and D. The values for the fundic gland component of serum PG II were calculated on the premise that a change in serum PG I, as a percentage of normal, is associated with a parallel percentage change in the fundic gland contribution to serum PG II. The values for the pyloric gland component of serum PG II were calculated by subtracting the fundic gland component from total serum PG II. TISP (no/mil PGIIPGII RATIO NORMAL 77.4 6.2 141LD/MODERATE ATROPHIC GASTRITIS TlSP (no/ml) PGIIPGII RATIO 73.7 2.9 SUPERFICIAL GASTRITIS D. 22.1 0.7 9.6 107.7 4.3 SEVERE ATROPHIC GASTRITIS o PGI [i) Fundic PGll rn Pyloric PGlI

July 1982 SERUM PEPSINOGENS I AND II IN GASTRITIS 209 cial gastritis of the gastric body mucosa also had antral gastritis. Since the percentage rise in serum PG I above normal was 30%, the model for this hypothesis (Figure 2B) attributes 30% of the observed increase in serum PG II to the chief cells and 70% to the pyloric glands. The small decrease in serum PG I and the persistent and significant increase in serum PG II in mild to moderate atrophic gastritis can be explained by extending the general concepts developed for superficial gastritis as follows: (a) The chief cells, although reduced in number, continue to release PG I into the circulation at an increased rate due to the surrounding inflammatory changes. The net result is a modest reduction in serum PG I. (b) The inflammatory changes in the antral mucosa, which were found in most of these subjects, together with an increase in the number of pyloric glands due to pyloric gland metaplasia results in an elevation in serum PG II. The model for this proposal (Figure 2C), in concert with the one proposed for superficial gastritis (Figure 2B), assumes a parallel rate of PG I and PG II release from the chief cells. Thus, the calculated value for the fundic gland contribution to circulating PG II is less than normal. The marked fall in serum PG I and the return of serum PG II to normal in severe atrophic gastritis can be explained by the marked loss of fundic glands and a further increase in the number of pyloric glands. The calculated values for the fundic and pyloric gland contributions to total serum PG II are shown in Figure 2D. The results of previous endoscopic biopsy studies suggest that the topology of gastritis in relatives of patients with pernicious anemia is similar to that found in other populations (16,17). For example, in a study of 50 healthy volunteers, Kreuning et al. (17) found only two with gastritis limited to one mucosal area; both gland areas were abnormal in 16 and both were normal in 32 subjects. Since a determinant of the PG I/PG II ratio appears to be the topology of gastritis, there is reason to suppose that the results of this investigation may be applicable to other populations. Preliminary results in a control population support this proposal (18). Discriminant function analysis revealed that the expression of serum PG I and PG II that best correlated with the histologic status of the fundic gland mucosa was the PG I/PG II ratio in combination with the absolute level of serum PG I. For the simultaneous assignment of all subjects, the overall sensitivity and positive predictive value of this combination was 70%, i.e., 119 of the 170 subjects were classified correctly. This was significantly higher (p < 0.005) than the value for peak acid output, which was only 51%. For the dichotomous assignment, normal vs. any degree of gastritis, the combined determination of serum PG I and PG II had a sensitivity of 87.5% and a positive predictive value of 77.8%. The corresponding values for peak acid output were again about 20% lower. These observations suggest that the combined determination of serum PG I and PG II provide a "serologic biopsy" of the fundic gland mucosa. As such, they may be useful for studies of the genetic, immunologic, epidemiologic, and clinical correlates of chronic gastritis. References 1. Samloff 1M. Pepsinogens I and II. Purification from gastric mucosa and radioimmunoassay in serum. Gastroenterology 1981;82:26-33. 2. Samloff 1M,Liebman WM. Cellular localization of the group II pepsinogens in human stomach and duodenum by immunofluorescence. Gastroenterology 1973;65:36-42. 3. Samloff 1M. Cellular localization of group I pepsinogens in human gastric mucosa by immunofluorescence. Gastroenterology 1971;61:185-8. 4. Varis K, Ihamaki T, Harkonen M, et al. Gastric morphology, function, and immunology in first-degree relatives of probands with pernicious anemia and controls. Scand J Gastroenterol 1979;14:129-9. 5. Siurala M, Eramaa E, Nyberg W. Pernicious anemia and atrophic gastritis. Acta Med Scand 1960;166:213-3. 6. Lewin KJ, Sawling F, Wright JR, et al. Gastric morphology and serum gastrin levels in pernicious anemia. Gut 1976;17:551 60. 7. Lechago J, Black C, Samloff 1M. Immunofluorescence studies of gastric heterotopia of the small intestine in Crohn's disease. Gastroenterology 1976;70:429-32. 8. Kimura K. Chronological transition of the fundic-pyloric border determined by stepwise biopsy of the lesser and greater curvatures of the stomach. Gastroenterology 1972; 63:584-92. 9. Whitehead R. Mucosal biopsy of the gastrointestinal tract. Philadelphia: W. B. Saunders, 1979. 10. Siurala M, Isokoski M, Varis K, et al. Prevalence of gastritis in a rural population. Bioptic study of subjects selected at random. Scand J GastroenteroI1968;3:211-23. 11. Samloff 1M, Liebman WM, Panitch NM. Serum group I pepsinogens by radioimmunoassay in control subjects and patients with peptic ulcer. Gastroenterology 1975;69:83...,90. 12. Dixon WI. Massey SJ, Jr. Introduction to statistical analysis. 3rd ed. New York: McGraw Hill, 1969. 13. Afifi AA, Azen SP. Statistical analysis. A computer oriented approach. New York: Academic Press, 1972. 14. Dixon WI. Brown MB. BMDP-77, Biomedical computer programs P-series. Berkeley: University of California Press, 1977. 15. Libman LJ, Samloff 1M. Quantitative distribution of pepsinogens I and II in gastric mucosa (abstr). Gastroenterology 1978;74:1132. 16. Ihamaki T, Kekki M, Varis K, et al. Family patterns of antrofundal gastritis. Acta Hepatogastroenterol 1976;23:345-50. 17. Kreuning I. Bosman FT, Kuiper G, et al. Gastric and duodenal mucosa in "healthy" individuals. An endoscopic study of 50 volunteers. J Clin Pathol1978;31:69-77. 18. Samloff 1M,Varis K, Ihamaki T, et al. Determination of gastric mucosal histology by serological and secretory tests (abstr). Gastroenterology 1981;80:1269.