Correlation between a proteolytic method and a radioimmunoassay for porcine serum pepsinogen concentrations

Research in Veterinary Science 80 (2006) 260–266 www.elsevier.com/locate/rvsc

Correlation between a proteolytic method and a radioimmunoassay for porcine serum pepsinogen concentrations D.I. Sidikou a, H. Banga-Mboko a, H.H. Tamboura b, J.L. Hornick c, B. Remy a, J.F. Beckers a,* a

b

Department of Animal Physiology of Reproduction, Faculty of Veterinary Medicine, University of Lie`ge, Bd de Colonster n
20 B41, 4000 Sart Tilman, Belgium Department of Research on Animal Production, Institute of Environment and Agricultural Research, P.B. 8645, Ouagadougou, Burkina Faso c Department of Nutrition, Faculty of Veterinary Medicine, University of Lie`ge, Bd de Colonster B43, 4000 Sart Tilman, Belgium Accepted 10 June 2005

Abstract The measurement of serum pepsinogen concentrations by enzymatic method and immunoassay provides diagnostic values and should be helpful in the detection of gastric diseases related to a rise of blood pepsinogen. In the present study, the correlation between a conventional enzymatic method and a recently developed radioimmunoassay (RIA) for serum pepsinogen A was investigated. A total of 123 sera samples of porcine foetuses (n = 28), adult healthy pigs (n = 56), pigs with parakeratosis (n = 25) and pigs with ulceration of the pars oesophagea (n = 14) were tested. Overall, there was a slight correlation between the two methods (r = 0.60). In relation to individual animal groups, the correlations (r) were 0.39 (P > 0.05), 0.74 (P < 0.001), 0.19 (P > 0.05) and 0.34 (P > 0.05) in foetuses, healthy pigs, pigs with parakeratosis and pigs with ulcers, respectively. In both methods, pepsinogen concentrations (means ± SE) were significantly higher (P < 0.05) in pigs with parakeratosis (1778 ± 86.00 mUTyr/L; 690 ± 53.00 ng/mL) and in pigs with ulcers (2026 ± 153.00 mUTyr/L; 1747 ± 94.00 ng/mL) when compared to healthy pigs (935 ± 58.00 mUTyr/L; 275 ± 35.00 ng/mL). The proteolytic method gave a significant increased activity (P < 0.05) in foetuses (1150 ± 82.00 mUTyr/L) vs. (935 ± 58.00 mUTyr/L) in healthy adult pigs, indicating an additional proteolytic activity in the sera of foetuses or neonates.  2005 Elsevier Ltd. All rights reserved. Keywords: Porcine pepsinogen; Radioimmunoassay; Proteolytic activity; Correlation

1. Introduction The gastric juice of all vertebrates contains various proteases such as pepsinogens involved in the digestion of dietary proteins. Pepsinogens as well as pregnancy associated glycoproteins (PAG) belong to the aspartic protease family (Szecsi, 1992), but it is known that pepsinogens have proteolytic activity while PAGs isolated from the placenta of various ruminant species, as far as has been reported, are enzymatically inactive (Beckers *

Corresponding author: Tel.: +3241564161; fax: +3241564165. E-mail address: [email protected] (J.F. Beckers).

0034-5288/$ - see front matter  2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2005.06.004

et al., 1999). As regards to pepsinogen secretion, Gritti et al. (2000) reported that, in the resting state, pepsinogens are stored in granules of the chief cells, which inhibit further synthesis. After appropriate physiological or external chemical stimuli, pepsinogens are secreted in the stomach lumen where hydrochloric acid, secreted by parietal cells, converts them into corresponding active pepsins, via an autocatalytic process that requires proteolytic removal of the prosegment (Richter et al., 1998). Different zymogens with proteolytic activity have been identified and are classified into the following groups: pepsinogen A (or I from human) (EC 3.4.23.1)

D.I. Sidikou et al. / Research in Veterinary Science 80 (2006) 260–266

(Ichihara et al., 1985; Eckersall et al., 1987; Foltmann et al., 1992; Mostofa et al., 1990); pepsinogen B (EC 3.4.23.2) (Nielsen and Foltmann, 1995; Suchodolski et al., 2002); pepsinogen C or progastricsin (or II from human) (EC 3.4.23.3) (Bobe and Goetz, 2001; Richter et al., 1998); pepsinogen D (no available EC) (Lee and Ryle, 1967); pepsinogen F (no available EC) (Chen et al., 2001; Kageyama, 2002) and prochymosin (EC 3.4.23.4) (Foltmann et al., 1977; Baudys et al., 1988; Houen et al., 1996). Previous investigations about the ontogeny of aspartic proteases showed that pepsin A and pepsin C are found in the gastric juice of adult vertebrates, while chymosin and pepsin F are found in large amount in foetuses/neonates (Antonini and Ribadeau-Dumas, 1971; Foltmann et al., 1998; Kageyama, 2002). The presence of pepsinogen F in visceral yolk sac and in gastric chief cells of preweaned neonates, suggests that this form of pepsinogen is specialized in digestive functions (Kageyama, 2002). Pepsinogens are also found in blood circulation in measurable quantities in healthy subjects. Although the mechanism whereby a small amount of pepsinogen enters the blood circulation is not well understood, there is a consensus that blood pepsinogen reflects the morphological and functional status of the gastric mucosa (Titchener et al., 1974), and, more specifically, the mass of chief cells, which are the major source of pepsinogens. Serum pepsinogens have clinical importance in the study of gastric and duodenal disorders. In humans, a high ratio of pepsinogen A/C (more than 4) is associated with duodenal ulcer with Helicobacter pylori infection and low A/C ratio (less than 1.8) is a predictor of gastric cancer, atrophic gastritis or gastric neoplasia (Biemond et al., 1993; Miki et al., 2003). In farm animals, parasites such as Ostertagia in ruminants (Berghen et al., 1993) and Hyostrongylus rubidus in pigs (Enight et al., 1972), during their histotrophic phase may damage the abomasum or the fundic area, especially the parietal cells resulting in a decrease of acid production and a subsequent reduction of the pepsinogen transformation into pepsin. Consequently, the accumulated pepsinogen may escape into the broken cell junctional complexes and reach the blood stream (Murray, 1969). The estimation of serum pepsinogen concentration by means of proteolytic assays (Dorny and Vercruysse, 1998) and ELISA (Turner and Shanks, 1982) has been proposed as an aid to the detection of clinical or sub clinical infestations with abomasal nematodes. Beside the proteolytic methods, radioimmunoassays (RIA) using purified commercial pig pepsinogen A and polyclonal antisera anti-pig pepsinogen A were developed. Data obtained by both RIA indicated that serum pepsinogen was significantly higher in pigs with ulcers compared to healthy subjects (Nappert et al., 1990;

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Banga-Mboko et al., 2003c). Despite the coexistence of the two methods for measuring serum pepsinogen in studies related to gastric diseases in pigs, the two methods have never been compared. Such investigation may improve our knowledge on the usefulness of each of them in studies related to chronic gastric diseases in pigs in intensive breeding (outbreak of ulcers) and also in developing countries where parasitism is more frequent, and could help to validate easy methods useful in small laboratories. The present study was designed to establish a correlation between the proteolytic and immunoassay methods in serum samples collected in foetuses, healthy pigs and pigs with lesions of the pars oesophagea.

2. Materials and methods 2.1. Animals and blood collection 2.1.1. Foetal sera Four gravid uteri from sows were collected at the slaughterhouse and immediately exposed through a surgical incision of the uterine horns. A total of 28 viable foetuses, aged between 5 and 15 days prepartum, were selected. Blood samples were collected from the heart or the umbilical cord and stored overnight at room temperature. Thereafter, sera were separated by centrifugation and frozen at 20
C until pepsinogen assays. 2.1.2. Adult sera Adult pigs of both sex, 10 ± 2 month old (range 8–16 months) and weighing approximately 60 kg were used. Pigs originated from the traditional breeding system in Burkina Faso. The farming system was characterized by pigs scavenging during the day, followed with overnight housing in rudimentary boxes built with local material. The sampling took place in the municipal slaughterhouse of Ouagadougou. Blood samples were taken from the jugular vein and were processed in the same way as the foetal samples. 2.2. Post-mortem examination As previously reported by Nappert et al. (1990), the stomachs were collected in the slaughterhouse and examined within 2 h. They were opened along the greater curvature, from the cardia to the pylorus. The contents were removed and the gastric mucosa was lightly rinsed with tap water. Representative tissue sections were taken from the pars oesophagea and fixed in 10% buffered formalin. The tissues were then embedded in paraffin, sectioned and stained with hematoxylin, phloxin and safran (HPS). The classification of pig gastric lesions observed was based on gross and histological findings as previously

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described by Zamora et al. (1975), (Banga-Mboko et al., 2003c). The pathological changes observed in the collected stomachs showed that 56 pars oesophagea were noted to be normal, 25 with parakeratosis and 14 with ulcers.

3. Assays 3.1. Proteolytic method (Dorny and Vercruysse, 1998) 3.1.1. Reagent and equipment Glycine, sodium chloride, chloridric acid, sodium hydroxide, Folin–Ciocalteus Phenol reagent and trichloro acetic acid (Merck kGaA, Darmstadt, Germany); L-tyrosin (Sigma–Aldrich, Germany); bovine serum albumin (Fraction V) (INC Biochemicals, OH, USA); polypropylen conical tubes (Greiner bio-one, Germany) and a spectrophotometer GENESYS 5 (Spectronics, Rochester, New York). 3.1.2. Buffers Stock A = glycine (0.1 M) in NaCl (0.1 M); Stock B = HCl (0.27 N); Tyrosin stock solution = L-tyrosin (10 mM) in HCl (0.1 N); Stock C = stock A + stock B (2/1:v/v); Albumin substrate solution (1 h before use) = stock C + bSA (2%). 3.1.3. Procedure Tyrosine standard was constituted by a set of three points 0.1–0.2 and 0.3 mmol/L. Samples contained 100 lL of sera (or H2O for blanks) and 500 lL substrate bSA. All tubes were closed, vortexed and incubated for 24 h at 37
C. After incubation, 1 mL of TCA (4%) was added to the substrate blank and sample tubes were vortexed and then settled for 10 min. After centrifugation at 3000g for 20 min, a volume of 200 lL of the supernatants and standards was taken and added to 2 ml of NaOH (0.25 M) and 300 lL of Folin reagent (in H2O) (1/3:v/v). Tubes were vortexed and incubated for 30 min at room temperature. The optical density (OD) was measured at 680 nm and the following formula was used to calculate the pepsinogen concentrations: UTyr = (ODsample ODbSA substrate blank) · F · 11.11 (UTyr: units of tyrosine/L); F (calculation factor) = (0.1/a + 0.2/b + 0.3/c)/3 where a = OD0.1 mmol/L; b = OD0.2 mmol/L; c = OD0.3 mmol/L; 11.11 = conversion factor. The intra- and inter-assay coefficients of variation ranged between 2.93–15.8% and 6.28–15.7%, respectively. 3.1.4. Radioimmunoassay (RIA) Pepsinogen A concentrations were measured according to the radioimmunoassay procedure as recently described by Banga-Mboko et al. (2003a). The detection

limit was The intra 3.9% and 8.8% and

0.2 ng/mL. The recovery was close to 95%. assay coefficient of variation ranged between 7.5%, whereas the interassay ranged between 12%.

3.1.5. Statistical analyses The pepsinogen concentrations were expressed as means ± standard error. The data obtained were subjected to one-way analysis of the variance by the GLM procedure of SAS. For each method, the means were compared by using StudentÕs t-test. The correlation between the two methods was evaluated by the SpearmanÕs rank correlation coefficients.

4. Results 4.1. Correlation The correlations between the two methods are presented in Fig. 1. Overall, there was a slight correlation (r = 0.60) between the two methods (Fig. 1(e)). The correlation increased if the results from foetal samples were not considered (data not shown). In healthy pigs a significant correlation (r = 0.74; P < 0.001) was observed in the range of 300–2420 mUTyr/L corresponding to 112–499.8 ng/mL in RIA (Fig. 1(b)). The regression between proteolytic method measurements (mUTyr/L) and immunoassay (ng/mL) can be expressed: Y(mUTyr/L) = 2.77X(ng/mL) + 169.91. However, no significant correlation was observed between the two methods for foetuses (r = 0.39) (Fig. 1(a)), pigs with parakeratosis (r = 0.19) (Fig. 1(c)) and pigs with ulcers (r = 0.34) (Fig. 1(d)). 4.1.1. Pepsinogen concentrations The results are presented in Table 1. In the proteolytic method, pepsinogen concentration was significantly higher (P < 0.0001) in pigs with ulcers (2026.25 ± 153.00 mUTyr/L) or parakeratosis (1778 ± 87.00 mUTyr/L) when compared to adult healthy pigs (935.71 ± 58.00 mUTyr/L). There was no difference (P > 0.05) in pepsinogen concentration between pigs with ulcers and those with parakeratosis (1150–2840 vs. 1170–2730 mUTyr/ L). By contrast, pepsinogen concentrations were significantly higher in foetuses (P < 0.05) than in adult healthy pigs (1150.71 ± 82.00 vs. 935 ± 58.00 mUTyr/L). As regards the RIA, pepsinogen concentration in sera were significantly (P < 0.05) lower (2.55 ± 5.00 ng/mL) in foetuses than in other groups. Healthy pigs had intermediate concentrations (275.68 ± 35.37 ng/mL) between those observed in foetuses and pigs with parakeratosis (689.70 ± 53.00 ng/mL) or ulcers. The highest values were observed in pigs with ulcers (1747.83 ± 93.59 ng/mL).

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Y = 2.77x + 169.91 n = 56

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Fig. 1. Correlation between proteolytic measurement and Radioimmunoassay for porcine pepsinogen. Y: trend line equation; n: number of animals: (a) foetuses; (b) healthy adult pigs; (c) adult pigs with parakeratosis; (d) adult pigs with ulcer; (e) total population. Table 1 Pepsinogen concentrations measured by the proteolytic method and the RIA in pig sera samples Sera samples

n

Proteolytic method (mUTyr/L)

Radioimmunoassay (ng/mL)

Means ± SE

Range

Means ± SE

Range

Fetuses Healthy pigs Parakeratosis Ulcers

28 56 25 14

1150a ± 82 935b ± 58 1778c ± 86 2026c ± 153

14–1890 300–2420 1150–2840 1170–2730

2.5a ± 50.2 275.7b ± 35.4 689.7c ± 53 1747.8d ± 93.6

0.2–21.12 112–499.8 530.4–954.5 1058–3809

In each column two values that are not followed by the same superscript letter are different (P < 0.05).

5. Discussion The main objective of this work was to study the correlation between an indirect method (proteolytic method) and RIA for porcine serum pepsinogen A. The correlation observed in adult healthy pigs may suggest that the antiserum in RIA and pepsin activity in the proteolytic method probably target the same pepsinogen

molecule, or that pepsinogen A is the most abundant zymogen found in this group. In foetuses, the high concentrations observed by the proteolytic method point out the presence of high levels of chymosin and probably the other isoforms of pepsinogen. The proteolytic method showed a significant difference in pepsinogen concentrations between the healthy pigs (935 ± 58 mUTyr/L) and pigs with ulcers

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(2026 ± 153 mUTyr/L) in contrast with previous reports (Zamora et al., 1975; Bunn et al., 1981). A similar finding was also observed in cows infected by parasite nematodes (Chiejina, 1977; Kerboeuf et al., 1982). The lack of significant differences between pigs with parakeratosis and pig with ulcers is in agreement with Nappert et al. (1990) who found that serum pepsinogen probably depends more on the depth of the damage to the gastric mucosa than on the extent of gastric mucosa lesions. Surprisingly, the enzymatic method indicated that pepsinogen concentration was higher in foetuses than in healthy adults. This paradox may be explained by the presence in the foetal sera of several gastric protease precursors such as pepsinogen F (Kageyama, 2002), prochymosin (Foltmann et al., 1998; Houen et al., 1996) that are believed to be more expressed in foetuses or in neonatal subjects, and other forms of pepsinogen (Banga-Mboko et al., 2002). Preliminary observations in our laboratory showed that calf chymosin B has a proteolytic activity of 0.19 UTyr/mg and the commercial pure preparation of porcine pepsinogen A gave a proteolytic activity of 4.22 ± 0.37 UTyr/mg (unpublished data). It is known that the main principle of the proteolytic method consists to acidifying sera samples in order to convert zymogens into active enzymes. Gastric proteases show a similarity in their activation to active enzymes when the optimum pH is lower than 4 (range 2 and 3.5) (Foltmann et al., 1995). As the protease activity depends essentially on pH and on the nature of the substrate used, the enzymatic method is thus not specific for a determined gastric protease but measures the cumulative concentration of different proteases, while radioimmunoassay is able to quantify a specific molecule. A similar discrepancy was also observed in several studies, where the level of serum proteolytic activity in patients with total gastrectomy has been in the range of one-fourth to over one-third of the mean activity found in control subjects. This high background activity has usually been attributed to acid proteases other than pepsinogen I (Samloff et al., 1975). Human pepsinogen A (PgA) is secreted by fundic mucosa, while pepsinogen C (PgC) is secreted by fundic, pyloric and proximal mucosa (Foltmann, 1981), and the ratio between the two forms (A/C) is around five in normal subjects (Biemond et al., 1989). Both serum pepsinogen A and C increase in duodenal ulcer disease, but the ratio (A/C) decreases (ratio: 2.1). The eradication of Helicobacter pylori provokes changes in serum pepsinogen values: it reduces both pepsinogens A and C, and elevates the PgA to PgC ratio (Wagner et al., 1994; Bermejo et al., 2001). To our knowledge only pepsinogen A has been investigated by radioimmunoassay in pigs showing gastric diseases (Nappert et al., 1990). Pig pepsinogen B and pepsinogen C have been partially sequenced (Nielsen and Foltmann, 1995), the release of pepsinogen A is significantly higher (Heim et al., 1997)

and pepsinogen B and C did not cross react with pepsinogen A by immunochemical determination (Foltmann et al., 1995; Nielsen and Foltmann, 1995). Consequently, the RIA used in this study was probably specific to pepsinogen A. There were large differences within the present data obtained with RIA (healthy pigs: 275 ± 35.4 ng/mL; pigs with ulcers: 1747.8 ± 93.6 ng/mL) than those reported by Nappert et al., 1990) (healthy pigs: 1.5 ng/ mL; pigs with ulcers: 5.15 ng/mL), these differences are maybe due to methodological parameters as shown in the very high inter-assay coefficient of variation (15.7 to 35.7%) in their system. The RIA developed in our laboratory was improved by the use of a specific polyclonal antiserum and the incorporation of pepstatin and tween in the buffer (Banga-Mboko et al., 2003b). Pepstatin is a protease inhibitor that helps to stabilise protease (Umezawa et al., 1979). In addition, pepsinogen values observed in healthy pigs agreed with the suggestion put forward by Knight et al. (1995) that there is a variability in the concentration of serum pepsinogen in asymptomatic populations. Further studies dealing with more samples and taking into account other parameters such as sex, age and body weight are necessary to confirm these first observations.

6. Conclusion In the present study, it was found that the proteolytic method and the RIA for determination of serum concentration of pepsinogen A showed a good correlation in healthy pig populations. Furthermore, each method was able to distinguish healthy pigs and diseased animals. This work validates the two methods as efficient tools for clinical investigations. Although the proteolytic method diverged with the RIA in foetal samples, the two methods are useful in clinical investigations, while they should be associated in laboratory research on gastric aspartic proteases, in order to facilitate the interpretation of the results.

Acknowledgements The authors thank Dr. I. Youssao for the statistical analysis, Mrs. R. Noucairi-Fares for secretary assistance. The Belgian Ministry of Agriculture and the FNRS granted this study.

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