Ghrelin in diseases of the gastric mucosa associated with "Helicobacter pylori" infection

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Ghrelin in diseases of the gastric mucosa associated with Helicobacter pylori infection

Anna Zub-Pokrowiecka

¹

BCDEF, Kazimierz Rembiasz

¹

ABCDG,

Stanislaw J. Konturek

²

ADE, Andrzej Budzyński

¹

E, Peter C. Konturek

³

BCD, Piotr Budzyński

¹

E

1

2

^nd

Department of General Surgery of Jagiellonian University Medical College, Cracow, Poland

2

Department of Physiology of Jagiellonian University Medical College, Cracow, Poland

3

Department of Medicine, Thuringia Clinic Georgius Agricola Saalfeld Teaching Hospital University Jena, Saalfeld, Germany

Source of support: This work was supported by The Ministry of Science and Higher Education in Poland Grant K/PBP/000012 for 2006–2008 and by Jagiellonian University Medical College Research Project for 2006–2009

Summary

Backround:

Ghrelin is a hormone produced by neuroendocrine cells of gastric mucosa. Its concentration chang- es in cases of Helicobacter pylori (H. pylori) infection of stomach, but the relation between the ex- pression of plasma and mucosal ghrelin content and H. pylori infection is not completely defined.

This study was designed to determine the changes in plasma and gastric mucosa ghrelin concen- trations in diseases of gastric mucosa depending upon the H. pylori infection.

Material/Methods:

The following groups were included; Group 1. patients with gastric cancer and concomitant H.

pylori infection (N=25); Group 2. patients with antral gastritis with H. pylori infection and concom- itant duodenal peptic ulcer (N=18); Group 3. patients with atrophic gastritis of both the fundus and the body of the stomach without H. pylori infection (N=10); Group 4. control group consisting of patients without morphological and histological lesions of gastric mucosa and without H. pylori infection (n=25). Endoscopic biopsies of gastric mucosa of fundus, body and pyloric region were obtained in all tested groups. In patients suffering from gastric cancer biopsies of tumor were also taken. The ghrelin concentrations were measured by specific RIA. Biopsy specimens were examined to assess ghrelin mRNA expression in intact gastric mucosa and gastric cancer.

Results:

The study showed significant influence of H. pylori infection on ghrelin plasma concentrations. The highest ghrelin concentrations were found in patients of the group 2 (average 503 pg/mL (95%

CI: 285–886). Ghrelin concentrations were found to fall to the lowest values in the group 3 (average 144 pg/mL (95% CI: 93–222). In the group 1, the ghrelin concentration averaged 203 pg/mL (95% CI: 161–257), while in the group 4 (control group) – 255 pg/mL; 95% (CI: 160–406). The study proved that gastric cancer does not exert any ghrelin-production activity, as confirmed by RT-PCR examination of biopsy specimens of the cancer.

Conclusions:

This study shows that the presence of H. pylori in the stomach with peptic ulcer increases plasma ghrelin levels, whereas in gastric cancer and atrophic gastritis it is accompanied by a marked decrease in plasma and cancer tissue levels of ghrelin.

key words: ghrelin • Helicobacter pylori • gastric cancer • peptic ulcer, gastritis Full-text PDF: https://www.medscimonit.com/abstract/index/idArt/881200

Word count: 4706 Tables: 1 Figures: 4 References: 35

Author’s address: Anna Zub-Pokrowiecka, 2

^nd

Department of General Surgery of Jagiellonian University Medical College, Kopernika 21 Str., 31-501 Cracow, Poland, e-mail: zuban@poczta.onet.pl

Authors’ Contribution:

A Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection Received: 2010.04.03 Accepted: 2010.04.07 Published: 2010.10.01

Clinical Research

PMID: 20885354

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B

ackground

Ghrelin, a natural ligand for growth hormone secretagogue receptor (GHR-S), was identified in rat gastric mucosa by Kojima and coworkers in 1999 [1,2] and found to represent an independent gastric regulator of growth hormone release. Immunohistochemical studies revealed that ghrelin-producing cells (Gr cells) are compatible with the X/A cells of the mucosa of gastric fundus, but their hor- monal and physiological function has not been clarified.

Location, population, and ultrastructural characteristics of Gr cells suggest that they are identical to the X/A cells.

These cells, representing about 20% of the population of endocrine cells of gastric mucosa, are located in the lower parts of the oxyntic glands of the fundus and body of the stomach, which is also the only source of gastric acid production. Surrounded by a network of blood vessels these cells have no contact with the lumen of the stomach and are ac- tivated by paracrine, endocrine and possibly autocrine stim- uli. Ghrelin is secreted into the circulation but not into the lumen of the gastrointestinal (GI) tract in the absence of food in the stomach. Ghrelin plays a physiological role in initiation of food intake in humans. Plasma ghrelin levels was found to increase nearly twofold before each meal, but falls to low levels within about 1 h after eating. Serum levels of ghrelin is typically about 160±10 pg/ml [3–5]. Ghrelin is probably most important anabolic hormone. High expression of this hormone in the stomach indicates a presence of the gastro-hypothalamic-pituitary axis, which affects the secretion of GH, the growth processes of the body and the stimulation of appetite [6]. Ghrelin has been also reported to exert a protective influence on gastric mucosa. It shows some anti-inflammatory effect mediated via prostaglandin which was presented in experimental model of the expo- sure of gastric mucosa to ethanol. Gastroprotective action of ghrelin is accompanied by significant increase in mucosal blood flow which is well-known defense mechanism of the gastric mucosa. Ghrelin-induced gastric mucosal hyper- emia is probably related to the direct vasodilatory action of the peptide [7]. In the case of impaired production of ghrelin, the signaling pathway to the brain may be disturbed, resulting in alterations of food intake and energy homeo- stasis of the organism [8]. It may be, thus, very important to disclose which diseases of gastric mucosa that affect the expression of ghrelin and which mechanisms are involved in the release of ghrelin. H. pylori infection is involved in the pathogenesis of atrophic gastritis, gastric or duodenal peptic ulcer, gastric cancer and MALT lymphoma [9–11].

The impact of H. pylori infection on gastric ghrelin has not been so far fully elucidated. There are diverse opinions of the researchers on the influence of H. pylori infection on ghrelin secretion. In the Turkish study, Gokcel and colleagues [12]

did not find any significant differences in plasma and gastric mucosa contents of ghrelin between subjects with and with- out H. pylori infection. In contrast, British study of Nwokolo and coworkers [13], demonstrated the increase in the con- centration of ghrelin in plasma after H. pylori eradication.

Further studies are required to fully elucidate this problem.

The aim of this study was the determination of changes in ghrelin plasma levels and gastric mucosa concentrations of this peptide in cases of diseases of gastric mucosa related to H. pylori infection.

M

aterialand

M

ethods

The study group consisted of 78 adults. The subjects were re- cruited from the group of 3000 patients undergoing gastroscopy from January 2006 to May 2008 in the 2^nd Department of Surgery, Jagiellonian University, Medical College because of clinical suspicion of upper GI tract pathology. Exclusion criteria were; age below 18 years, history of diabetes mel- litus, diseases of the thyroid, neuroendocrine tumors, administration of glucocorticoids, progesterone or testoster- one; kidney and/or liver failure, pervious chemotherapy;

drug and/or alcohol addiction; pretreatment within past 4 weeks with proton pump inhibitors (PPI), histamine H₂-blockers, antacids or antibiotics, body mass index (BMI) over 30 kg/m² and pregnancy.

Patients fulfilling recruiting criteria and included into the study program were assigned to one of four study groups according the initial diagnosis (Table 1). In all 78 subjects tested, the factors such as age, sex, BMI, presence of H. pylori infection in gastric mucosa, ghrelin and gastrin levels in peripheral blood plasma were analyzed.

All subjects underwent gastroscopy. The study was performed on an empty stomach, that is after at least 12 hrs of fasting.

Topical lidocain (Lignocainum aerosol 10%, Glaxo-Wellcome) was used for local throat anesthesia if there was no history of allergy to it. All gastroscopies were performed by the same endoscopist. During each endoscopy visual assessment of the mucosa of esophagus, cardia, body and antral part of the stomach, pylorus, first portion of the duodenum and fundus of the stomach in inverted gastroscopy was performed.

In all patients rapid urease test was performed. The biopsy samples from antral mucosa (or in cases of involvement of the distal stomach by cancer from any unchanged mucosa above) were placed on the yellow colored gel containing urea. The change in the color of indicator contained in the gel from yellow to pink documented the presence of urease in the specimen. In accordance with the attached instructions (Urease test “GUTplus”, Lencomm Trade International), the results were read half an hour and three hours after biopsy sample collection.

During endoscopy in control subjects, the samples from the gastric mucosa of the fundus, body and antrum were obtained for histopathological examination and to determine the mucosal expression of mRNA for ghrelin and gastrin.

In patients with gastric cancer, biopsy specimens were taken from the tumor for histopathological examination and also for mucosal mRNA for ghrelin and gastrin. In patients with gastric cancer, the lesion was described according to the Bormann classification system. In patients with atrophic gastritis and in the group with antral gastritis and duodenal ulcer, the biopsy specimens for histopathological examination were collected from fundus, body and antrum of the stomach.

Biopsy samples for evaluation of mucosal ghrelin were immediately immersed in a solution stabilizing RNA (“RNAlater”, Ambion). Later those were stored until final analysis at -80°C at the conditions providing highest possible quality in terms of RT-PCR testing. Venous blood was collected in all patients from an empty stomach (i.e. at least after night 10–12 hrs without eating solid food or liquid), at 8 am, and

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posprandially at 60 and 90 min after a meal (at 10 and 10.30 am, respectively).

Venous blood was collected into tubes containing 10% aque- ous solution of disodium ethylene diamine-tetra-acetate (EDTA) to prevent clotting (0.05 ml 10% EDTA in 5 ml of blood). The centrifugation was performed within 10 min after collection of blood. The blood was centrifuged for 10 min at 3000 rpm (centrifuge MPW-340, Precision Mechanics, Warsaw, Poland). Plasma obtained after the separation was divided into 3 portions and frozen (–80°C) until quantifi- cation analysis of hormones such as ghrelin and gastrin by specific radioimmunoassays.

Laboratory tests were performed in the Laboratory of Isotopic Diagnostics of the Department of Physiology, Jagiellonian University Medical College. The concentration of ghrelin in the test plasma samples was determined by RIA, using kits (S – 2227; RIKU4864 purchased from Peninsula Laboratories, Inc., Division Bachem, CA, USA).

All measurements were performed in duplicate. Standard curve was prepared by appropriate dilution of 12.8 µL lyophilized peptide. Initial incubation of 100 µL plasma samples and subsequent dilutions of ghrelin standards (1, 2, 4, 8, 16, 32, 64, to 128 pg) was carried out for 24 hours at 4°C after addition of 100 µL highly specific antibodies for

the human ghrelin (Rabbit Anti-Ghrelin, Human, Serum).

Latter 100 µL of “tracer” (¹²⁵I-Ghrelin, 10000–15000 cpm) was added to each and incubation was continued for anoth- er 24 hrs at 4°C. The immunoprecipitation by addition of second antibody (Goat anti-rabbit IgG serum) and centrifugation after 90 mins incubation at 25°C was performed for final separation of the free and bound fraction. The concentrations of ghrelin in plasma samples were calculated based on the calibration curve that was obtained by „Spline” method based on measurements of radioactivity (gamma counter as above) for consecutive concentrations of the standards.

Essay sensitivity was 3.0 pg/ml, and the specificity of the antibodies for the labeled human ghrelin was 100%. In accordance with the attached attestation (Peninsula Laboratories, USA) antibodies did not (0%) in cross-reacted with moti- lin, GHRF, orexine, secretin, VIP and galanin.

Assessments of gastrin levels were performed using a com- mercial kit GAS-PR RIA (CIS Bio – International, France) following the manufacturer’s recommendations. The plasma samples (100 µL) were incubated in duplicate at 25°C for 3 hrs with 100 µL of “tracer”, ie (¹²⁵I-gastrin) and 300 µL of anti G-17 Antibody. Antibody equally recognizes and had affinity to the gastrin 17 and gastrin 34. Points of the standard curve at concentrations of 11.2, 28.4, 68.4, 255.8, 651.2

Grup no Histology

H. pylori infection N Gender and age BMI (body mass index)

(kg/m²)

Male Female Mean age (years)

1 Gastric cancer,

H. pylori positive

(100%) 25 64.6

(min. 43; max. 81) 23.1

(min. 18.7; max. 29.7)

(56%) 14 67.5

(min. 56; max. 81) 23.05

(min. 18.7; max. 28.6)

(44%) 11 6.5

(min. 43; max. 78) 23.2

(min. 20.2; max. 29.7)

2 Superficial gastritis, duodenal ulcer,

H. pylori positive

(100%) 18 55.7

(min. 23; max. 77) 22.9

(min. 20.9; max. 24.2)

(56%) 10 50.5

(min. 23; max. 63) 23.1

(min. 22.2; max. 24.2)

(44%) 8 62.6

(min. 50; max. 77) 22.7

(min. 20.9; max. 23.8)

3 Chronic atrophic gastritis of the fundus and body,

H. pylori negative

(100%) 10 63.8

(min. 43; max. 79) 22

(min. 19.5; max. 25)

(50%) 5 71

(min. 68; max. 75) 21

(min. 19.9; max. 21.6)

(50%) 5 59.6

(min. 43; max. 79) 22.5

(min19.5; max. 25)

4 Health gastric mucosa,

H. pylori negative

(controls)

(100%) 25 51

(min. 22; max. 67) 23.4

(min19.5; max. 30)

(48%) 12 50.4

(min. 22; max. 65) 23.4

(min. 21.5; max. 24.8)

(52%) 13 51.6

(min. 35; max. 67) 23.4

(min. 19.5; max. 30)

Table 1. Characteristics of the study groups.

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pmol/L were prepared from lyophilized synthetic gastrin G – 17 and incubated as above. Separation of the free from bound fraction was obtained by immunoprecipitation. Final radioactivity in the samples was assayed, and standard curve points were measured in gamma counter (1574 Clinigamma, Wallac-LKB, Sweden), using the computer program the

“Spline” in order to calculate the concentration of gastrin.

Extraction of total RNA from mucosal biopsies was performed by single-stage method as described by Chomczyński and Sacchi using guanidine/phenol thiocyanate and chloroform according to the manufacturer’s instructions (Stratagene, Heidelberg, Germany). After precipitation, RNA was immersed in RNase-free TE buffer and its concentration was measured by absorption at the wavelength 260 nm. Then, the quality of each RNA sample was checked during separation in agarose denaturing gel. RNA material was stored at –80°C until analysis. Single-strand cDNA was prepared from 5 µg total cellular RNA using reverse transcriptase Moloney leu- kemia virus (MMLV-RT) (Stratagene, Heidelberg, Germany) and oligo-(dT)-primers (Stratagene, Heidelberg, Germany).

5 µg of total RNA denatured by heat (65°C for 5 min) and then reverse transcription reaction to complementary DNA (cDNA) was performed at 50 µL volume of reaction mixture containing 50 U MMLV-RT, 0.3 mg oligo-(dT)-prim- er, 1 µl RNase Block Ribonuclease Inhibitor (40U/µL), 2 µL 100 mM/L mixture of dATP, dTTP, dGTP and DTP, 10 mM/L Tris-HCl (pH=8.3), 50 mM KCl, 5 mM MgCl₂. Obtained cDNA (2 µL) was subjected to amplification in a reaction volume of 50 µL containing 2U Taq polymerase, dNTPs (200uM each) (Pharmacia, Germany), 1.5 mM MgCl₂, 5 µL 10× buffer of polimerase chain reaction (50mm KCl, 10mM Tris-HCl, pH=8) and specific primers to a final concentration of 1 mM (all reagents from Takara, Shiga, Japan).

The solution was covered by 25 µL of mineral oil to prevent evaporation. Amplification conditions were as follows: de- naturation at 94°C for 1 min, aniling at 60°C for 45 s and elongation at 72°C for 2 min. PCR reaction products were determined by electrophoresis in 1.5% agarose gel containing ethylene bromide. Optical density of the reaction products was determined using image analysis system by the Kodak Digital Science. The intensity of the signal originat- ing from the products of genes was standardized according to the intensity measured for GAPDH in each sample and expressed as the ratio between the two.

Histopathological examinations were performed at the Department of Pathomorphology, Jagiellonian University Medical College. Specimens were fixed in 10% buffered for- malin and used for paraffin preparations stained with hema- toxylin and eosin, alcian blue and p.a. S at pH 2.5 and the Giemza method in the standard way. Fixed specimen was immersed in a series of alcohols and xylenes with increasing concentrations, and then sunk in the paraffin cube. The paraffin blocks were sliced to the thickness of 3 microns, depa- raffined in a series of xylenes and alcohols with decreasing concentration, and stained by pathological methods. The Sydney classification system was used for the assessment of inflammation and atrophy and Lauren classification system for the evaluation of the type of gastric cancer.

Comparison of average concentrations of hormones in the groups of measurements was performed using the so-called

general linear model (GLM), and specifically the analysis of covariance (ANCOVA). In this study if a factor was statistically significant (at alpha=0.05) the post-hoc Tukey test (according to unequinoumerous groups variant) was used to compare all possible pairs of mean resulting from cate- gorization according to factor.

r

esults

Analyzis of the plasma levels of ghrelin in the peripheral blood of patients in each group in the subsequent collec- tions, i.e. under fasting conditions and at 60 and 90 min after a meal in all study groups, showed that the highest hormone concentrations were observed under fasting conditions (Figure 1).

The highest concentrations of fasting ghrelin was observed in the group 2 (patients with antral gastritis, active H. pylori infection and duodenal ulcer) reaching 585 pg/ml, (95% CI:

320–1070). Ghrelin concentration in group 4 (controls) averaged 293 pg/ml (95% CI: 179–480), in group 1 (patients with gastric cancer and H. pylori infection) prior to surgery it averaged 218 pg/ml (95% CI: 170–280), while in group 3 (patients with atrophic gastritis in the fundus and body of the stomach without concomitant H. pylori infection) was 148 pg/ml, 95% (CI: 93–235) (Figure 1).

Postprandially the ghrelin concentration measured one hour after meal was reduced compared to the baseline in all groups. The strongest decline occurred in groups 2 and 4. Those decreases were, however, not significant when compared to baseline. The highest concentrations of ghrelin 60 min after meal were observed in group 2, reaching about 469 pg/ml, 95% (CI: 269–818), and they were higher than those recorded in group 4–234 pg/ml, (95% CI: 148–368), in group 1 (before surgery) – 198 pg/

Figure 1. Plasma ghrelin concentration v. H. pylori infection in

consecutive groups of patients. (group 1 before operation) regarding the times of blood collection. p H. pylori infection and fasting plasma ghrelin =0.00008; p H. pylori infection and postprandial plasma ghrelin at 60 min and 90 min after meal =0.3572; * p for differences in fasting concentrations between group 2 and 3 =0.0211;

^#

p for differences in fasting concentrations between group 1 and 4 =0.0578;

##

p for differences in fasting concentrations between group

3 and 4 =0.0648.

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ml (95% CI: 157–250) and in group 3–144 pg/ml (95%

CI: 94–220) (Figure 1).

In the last measurement at 90 min after the meal, the average concentration of plasma ghrelin slightly increased in group 4 (controls) to 243 pg/ml (95% CI: 151–392). Concentration in group 2 was 463 pg/ml (95% CI: 258–828). In the re- maining groups ghrelin concentrations decreased; in group 1 (before surgery) ghrelin concentrations 90 min after the meal were – 195 pg/ml, 95% (CI: 153–248), while in group 3 they averaged 139 pg/ml (95% CI: 89.2–218) (Figure 1).

The plasma gastrin levels were determined in all studied groups. Statistically significant correlation between the H.

pylori infection and gastrin levels has been demonstrated in the treatment groups in the primary (fasting) measurement (H. pylori infection/fasting gastrin plasma levels, p=0.0457).

The highest concentrations of fasting plasma gastrin were observed in group 3 and group 2, followed by group 1 before surgery. The lowest level of fasting gastrin was noted in control group 4 (Figure 2).

Analyzing simultaneous effects of time and H. pylori infection on gastrin levels in the various intervals after it was found that both of these two factors significantly affected the lev- els of plasma gastrin in the treatment groups (H. pylori in- fection and subsequent times intervals v. plasma gastrin p=0.0128). Gastrin levels were as follows: in group 3 fasting plasma levels were 56.4 pM/L, 95% CI: 27.4–115.2, 60 min after a meal they increased to 62 pM/L, 95% CI: 32.1–121 and 90 min after a meal to 53 pM/L, 95% CI: 28–101. In group 2, the average fasting gastrin level was 56.1 pM/L, 95%

CI: 22–144, 60 min after a meal – 54 pM/L, 95% CI: 23–128 and 90 min after a meal – 61 pM/L, 95% CI: 26.2–140.1.

In group 1: fasting – 29.1 pM/L, 95% CI: 20–43, 60 min after a meal – 38 pM/l, 24.5–54.2 and 90 min after a meal – 37 pM/L, 95% CI: 26.4–53. In group 4 (controls): fasting

concentrations were 18.3 pM/L, 95% CI: 8.4–39.5, 60 min after a meal in 39 pM/L, 95% CI: 19.1–79 and 90 min after a meal 40 pM/L, 95% CI: 20–79 (Figure 2).

The expression of mRNA for ghrelin and gastrin was measured by RT-PCR. The highest mucosal gene expression of ghrelin mRNA among controls was observed in the body and fundus of the stomach, the lowest in the antrum (Figure 3).

The highest mucosal expression of mRNA for gastrin was found in antrum, while markedly lower in the body and fundus of the stomach (Figure 4). The specimens obtained from gastric cancer did not show any expression of mRNA for ghrelin, while concomitantly very strong expression of mRNA for gastrin was noticed (Figures 3, 4).

d

iscussion

Ghrelin is a peptide hormone released into the circulation mainly by the endocrine cells X/A located in the gastric mucosa [4]. The fact that ghrelin is produced mainly in the stomach is supported by our as yet unpublished studies [5,8].

It is well established that alterations in the production of ghrelin, influence the information reaching the brain, resulting in the changes in food uptake and body energy ho- meostasis [8]. The concentration of ghrelin in plasma can be modulated by other endogenous hormones [14,15].

Therefore, we excluded from our study patients with a history of endocrine disorders. In patients enrolled into the study, plasma gastrin levels were analyzed concomitantly with ghrelin in order to document their mutual influences.

The opinions about relationship between Hp infection and gastric ghrelin are conflicting. As mentioned in the intro- duction, according to the Turkish study of Gokcel et al. [12], the presence of H. pylori infection has no effect on ghrelin concentrations when measured in either in the plasma or in gastric mucosa. It should be, however, noted that the authors did not evaluate the histological conditions of gastric mucosa and the degree of mucosal atrophy in the study group.

In the Turkish population, CagA positive H. pylori strains re- sponsible for atrophy are relatively rare, so it is possible that geographical variations such as difference in H. pylori strains affect the concentration of ghrelin after H. pylori eradication [16]. Also in the study by Cindoruk et al. [16] it has been proposed that H. pylori infection has no influence on plas- ma ghrelin concentrations in the Turkish population. They enrolled patients without atrophic gastritis and with normal gastric acidity and they did not observe any significant dif- ference in plasma ghrelin levels between the antrum dominant and corpus dominant gastritis [16]. In contrast, a study which aimed to determine plasma ghrelin level variations according to topographical distribution of gastritis, showed that in the antrum predominant gastritis, ghrelin levels were lower than that on the corpus predominant gastritis [17].

In the British study, Nwokolo et al. [13] demonstrated that the concentration of ghrelin actually increases after H. pylori eradication. Similar results were presented by scientists from Korea; Jang et al. [18], who showed elevated levels of ghrelin in plasma as well as increasing expression of ghre- lin in gastric mucosa after eradication H. pylori in patients with peptic ulcer disease but also in patients with superficial gastritis. The group of Lee et al. [19] showed a significant Figure 2. Plasma gastrin concentration v. H. pylori infection in

consecutive groups of patients. (group 1 before operation) regarding the times of blood collection. p H. pylori infection and fasting plasma gastrin =0.0457; p H. pylori infection and consecutive postprandial plasma ghrelin =0.0128;

* p for differences in fasting and postprandial concentration at 60 min in group 4 =0.0027; ** p for differences in fasting and postprandial concentration at 90 min in group 4 =0.0036.

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increase in the expression of ghrelin in the mucosa of the gastric fundus without any significant influence of H. pylori eradication on the concentration of this hormone in plasma, independently of gastric inflammatory changes.

The concentration of ghrelin in plasma may, however, not accurately reflect the concentration of ghrelin in gastric mucosa. In experimental studies on gerbils significantly lower levels of ghrelin were found in gastric mucosa infected with H. pylori infected as compared to those without H. pylori in- fection. In contrast, plasma gastrin concentrations in the same group was significantly increased [20].

In our study for the analysis of the impact of H. pylori infec- tion on ghrelin levels in plasma, two groups of patients with H. pylori infection at study entry were selected. These were patients with superficial antral gastritis coexisting with duodenal ulcers and patients with stomach cancer. Interestingly, in our study ghrelin concentrations in plasma were found to reach the highest levels in patients with H. pylori infec- tion and superficial antral gastritis with coexisting duodenal ulcer. This can be explained by the fact that in these patients mucosa of the gastric body remained unchanged, in contrast to antral mucosa, which is supported by the find- ing of high gastric acid secretion.

In the analysis of the impact of H. pylori infection on plas- ma concentrations of ghrelin in the consecutive samples (on an empty stomach, at 60 min and 90 min after a meal), we demonstrated its significant influence only on the basal

levels measured under fasting conditions. Here again the highest concentrations was found in the group with H.

pylori infection and duodenal ulcer and to a lesser degree in the control group.

Data supporting our results are scarce. Fukuhara et al. [21]

showed markedly elevated plasma ghrelin levels in mice who received cysteamine, compared with the controls regardless of the presence or absence of duodenal ulcers. Thus, it is possible that the increase in plasma levels of ghrelin pre- cedes the development of duodenal ulcers resulting from administration of cysteamine. Cysteamine is a potent somatostatin inhibitor. It could be assumed, that cysteamine-induced peptic ulcers are associated with the loss of gastric somatostatin release, resulting in the observed rise in plas- ma gastrin level. It could be that in the course of H. pylori infection, an increase in plasma ghrelin concentrations could be due to the fall in of somatostatin release by this infection with subsequent increase in ghrelin secretion. PPI, such as lansoprazole lead to healing of duodenal ulcer induced by cysteamine in mice, but does not affect plasma ghrelin plasma. This suggests that the increase in the plasma ghrelin level is not caused by cysteamine-induced duodenal ulcers [21].

Suzuki and coworkers [22] showed a significantly higher basal plasma ghrelin levels in patients with duodenal or gastric ulcers compared to the patients with chronic gastritis but no ulcers. Interestingly, the concentration of ghrelin was not reduced significantly after recovery from ulcer.

Figure 3. Expression of mRNA for ghrelin in relation to GAPDH in

the intact gastric mucosa of the antrum, body, fundus and cancer cells.

Figure 4. Expression of mRNA for gastrin in relation to GAPDH in

the intact gastric mucosa of the antrum, body, fundus and

cancer cells.

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According to Suzuki et al. these results may indicate an as- sociation between elevated levels of ghrelin in plasma and mucosal susceptibility to damage, which makes ghrelin a valuable non-invasive serological prognostic risk factor for the development of peptic ulcer [22].

In the study of Stec-Michalska et al [23] patients with H.

pylori infection and early gastritis also showed higher ghre- lin levels than patients without H. pylori infection. Supposed explanation of this observation may be the activation of defense processes in the mucosa in response to the early stage of inflammation. Commenting discrepancies with the afore- mentioned studies Stec-Michalska et al. [23] argue that in fact these studies examined the levels of ghrelin in patients with more advanced histopathological changes in the gastric mucosa (ie, chronic atrophic gastritis, peptic ulcers).

The lack of separate analysis for gender could influence the results of analysis as well. According to Stec-Michalska female gender predisposes to higher levels of ghrelin [23].

In our study influence of gender was not taken into ac- count. It, however, supports the observation that histopath- ologically more advanced lesions i.e. atrophic gastritis and gastric cancer correlated with low levels of ghrelin. In patients with gastric cancer its basal concentration was significantly lower compared to the control, but the lowest levels were observed in patients with atrophic gastritis in the body of the stomach.

Also, in the studies of Salles et al [24] ghrelin levels in the gastric mucosa of patients with H. pylori infection accompa- nied by chronic gastritis were reduced proportionally to the severity of inflammation and degree of atrophy. The level of plasma ghrelin in these patients with H. pylori infection was also significantly reduced [24].

The studies on reduced levels of ghrelin concentrations both in plasma and gastric mucosa in patients with atrophic gastritis were performed by Osawa [25], Tatsuguchi [26] and Isomoto [27]. In their observations the concentration of plasma ghrelin, expression of mRNA for ghrelin and the number ghrelin-positive cells in the gastric mucosa were significantly lower in patients with atrophic gastri- tis related to H. pylori infection. As ghrelin was reported to attenuate the inflammation through the activation of NO pathway [28,29], the reduction in ghrelin release could ex- plain the development of atrophic gastritis in ghrelin-de- ficient subjects.

In our study we evaluated the expression of ghrelin mRNA by RT-PCR in the gastric cancer and in the healthy mucosa from the fundus, body and antrum of the stomach in the control group, irrespectively from the plasma ghrelin. It was shown that cancer cells have virtually no ghrelin activity.

Among healthy subjects from the control group the RT-PCR analysis showed the highest expression of ghrelin mRNA in mucosa of the body, lesser in the fundus and the lowest in the antrum of the stomach.

For patients with gastric cancer the levels of ghrelin both in plasma as well as in gastric mucosa were found to be significantly lower than in controls, probably due to the ex- tent of infiltration of cancer, whose cells did not show any

ghrelin production activity and presumably because of concomitant atrophy of gastric mucosa.

Like in our study, Mottershead et al [30], in their RT-PCR study, also showed the lack of expression of mRNA for ghrelin in gastric cancer cells. In contrast the healthy non-neoplastic gastric mucosa was proved to be rich in ghrelin-positive cells and able to express gene mRNA for ghrelin. The highest expression was noted in non-neoplastic mucosa of the gastric body, significantly higher than in the cardia and in the antrum, which is confirmed by our study [30].

Gastrin was the next hormone analyzed in our study. The overall impact of H. pylori infection on the concentration of gastrin plasma among our patients was quite pronounced in the group with H. pylori infection, antral gastritis and du- odenal ulcers, who showed the highest levels of this hormone. Only slightly lower concentrations were found in patients without active H. pylori infection at the time of the study (after an earlier eradication), but with mucosal atrophy in the fundus and body of the stomach. Significant el- evation of plasma gastrin was documented in patients with duodenal ulcer and in patients with atrophic gastritis as in the studies [31]. Hypergastrinaemia is directly related to the increasing mucosal atrophy in the body and fundus of the stomach, parallelly to the decrease in production of HCl, increase in gastric pH with subsequent stimulation of G cells.

Gastrin levels are higher in patients with H. pylori infection, particularly involving the body and fundus of the stomach.

Also, patients with gastric cancer tend to have higher levels of gastrin, which can be both a result of the H. pylori infec- tion and accompanying atrophy of the mucous membrane, and increased production of the hormone possibly by the tumor cells themselves [11,32,33].

Elevated basal plasma gastrin levels as compared to the control group were also found in our study in patients with gas- tric cancer and coexisting H. pylori infection.

The expression of mRNA for gastrin in our RT-PCR analysis was very high in gastric cancer tissue, even in quanti- ties higher than in the healthy antral mucosa of the controls. High expression of gastrin in gastric cancer cells have been demonstrated also previously [34,35]. The lowest basal concentration of plasma gastrin was observed in the control group. This was the only group with statistically significant postprandial increase in this hormone levels found in plasma samples collected at 60 min and 90 min after meal.

In healthy gastric mucosa RT-PCR revealed the highest expression of mRNA for gastrin in gastric antrum, lower in the body and the lowest in the fundus of the stomach.

c

onclusions

In our observation of highest concentrations of plasma ghre- lin was found in patients with H. pylori infection, superficial gastritis and concomitant duodenal ulcer disease and this could result from the activity of ghrelin as a gastroprotective factor. Lower ghrelin levels in patients with gastric cancer and H. pylori infection than in the control group may result from the negligible expression of ghrelin in cancer cells and associated mucosal atrophy. Higher plasma gastrin levels, compared to the control group, observed in patients with H. pylori infection are associated with an increase in gastrin

CR

(8)

secretion caused by these bacteria. In this study expression of mRNA for ghrelin evaluated in cancer cells by RT-PCR was virtually undetectable with concomittant strong expression of mRNA for gastrin. Results of RT-PCR for expression of mRNA for ghrelin in healthy gastric mucosa suggests that the main site of its production is the body and fundus of the stomach. By contrast, the highest expression of mRNA for gastrin was detected in antrum, lower in the body and the lowest in the fundus of the stomach.

r

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