Some pathogenetic relationships in gastroesophageal reflux disease in children

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Introduction

The reflux of gastric contents into the esophagus is the main pathogenetic link in the development of gastroesophageal reflux disease (GERD), leading to the formation of chronic inflammation, which is a key factor in the progression of the disease [1; 2]. The mucous and submucous layers, which are enriched with connective tissue, one of the main components of which is collagen, are subject to a chronic inflammatory process [3–6]. However, the metabolism of collagen and the mechanisms of its damage in GERD remain unexplored [7–10].

The aim of the study was to evaluate the relationship between the acidity of the upper gastrointestinal tract and collagen metabolites, as well as proteoglycans, in children with GERD.

Materials and methods

Children with gastroesophageal reflux disease, who were included in the study, were treated in the pediatric gastroenterology department of the State Budgetary Healthcare Institution of the Ministry of Health of the Udmurt Republic “Republican Children's Clinical Hospital” in Izhevsk, Ministry of Health of the Udmurt Republic.

A stratified randomization method was used. Patients were divided into two groups. The main group included 62 children with gastroesophageal reflux disease aged 9 to 17 years, of which 27 (44 %) were boys and 35 (56 %) were girls; 24 (39 %) children were aged 9–12 years, and 38 (61 %) children were aged 13–17 years.

The control group included 32 people – 11 (34 %) boys and 21 (66 %) girls (p = 0.390). In the age groups of 9-12 and 13-17 years, there were 13 (41 %) and 19 (59 %) children (p = 0.862), respectively. The diagnosis of GERD in the control group was excluded by clinical and endoscopic signs, the main diagnoses were chronic gastritis or chronic gastroduodenitis (in the period of incomplete clinical remission of the disease).

To verify the diagnosis, clinical data and the results of esophagogastroduodenoscopy (with simultaneous determination of the acidity of the environment in the esophagus and stomach) were used. The endoscopic classification of gastroesophageal reflux disease by G. Tytgat (1996) as modified by V.F. Privorotsky et al. (2007) was used.

Inclusion criteria for the study: patients aged 9 to 17 years, clinical signs of GERD, presence of esophagitis. Exclusion criteria: age under 9 years, absence of esophagitis, patients suffering from food allergy and atopic dermatitis. Exclusion criteria: suspected acute surgical pathology, suspected acute intestinal infection.

Parietal endoscopic pH-metry was performed using the AGM-03 device (ZAO Scientific and Production Enterprise Istok-Sistema, c. Fryazino, Russian Federation) to study the acidity of the environment of the esophagus and stomach (in the first 2 days from the moment the child was admitted for examination). The method is based on determining the pH of the internal environment using a pH probe, which is inserted into the gastrointestinal tract through the biopsy channel of the endoscope. The pH data were used at the following points: in the esophagus, the "lake" of the stomach, the fundus of the stomach, the body of the stomach, the antral section of the stomach, and in the bulb of the duodenum.

The activity of acid formation was assessed in the stomach based on pH data: pH > 5.0 was considered an anacid state; рН 5.0–2.1 – hypoacid state; рН 1.2–2.0 – normacid state; рН < 1.2 – hyperacid state. The acidity level of the environment in the esophagus is taken as normal at a pH value of > 4.

The state of collagen and proteoglycans of the mucous membrane and submucosa was studied by determining the concentration of glycosaminoglycans (GAG), peptidyl-linked hydroxyproline, free hydroxyproline, and α-mannosidase activity in gastric acid. The study was conducted at the Department of Biochemistry and Laboratory Diagnostics of the Federal State Budgetary Educational Institution of Higher Education “IGMA”. Gastric acid was collected (the “fasted” portion was collected using a disposable 10 ml Apexmed probe in test tubes) in the first three days after the children were admitted to the gastroenterology department.

Hydroxyproline fractions were studied using the method of P.N. Sharaev (1981, 2009). The following biochemical analyzers were used in the study: FP-901 Labsystems and humalyser 2000 Huma. The activity of α-mannosidase (EC 3.2.1.24) was determined by the amount of free 4-nitrophenol released during the enzymatic cleavage of the substrate p-nitrophenyl-α-D-mannose. The Anson method was used as modified by N.A. Yusipova, P.N. Sharaev (2009).

Statistical processing of the study results was performed using the Statistica 7.0 software package (StatSoftInc, USA). Nonparametric methods were used in the analysis. The frequency of qualitative features in the groups was compared using the c² criterion. Differences were considered statistically significant at p < 0.05. Spearman correlation analysis was used to determine the presence of a relationship between the features. Binary logistic regression was used. ROC analysis was used to assess the quality of the logistic regression model by constructing ROC curves (Receiver Operator Characteristic). Sensitivity and Specificity were plotted on the axes. The AUC (Area Under Curve) indicator was used for interpretation. The quality of the model was determined by the AUC indicator: 0.9–1.0 – excellent; 0.8–0.9 – very good; 0.7–0.8 – good; 0.6–0.7 – average; 0.5–0.6 – unsatisfactory.

Results and discussion

Typical complaints of epigastric burning were presented by 27 (71 %) children aged 13–17, which bothered the majority (20 children) 2–3 times a week, epigastric burning was noted daily by 7 patients. 15 (63 %) children aged 9–12 years noted unpleasant sensations in the throat or behind the breastbone, periodically complained of belching, nausea, which intensified after eating. Many patients aged 9–12 years did not understand the term "epigastric burning", as a result of which they could not describe their complaints.

Various degrees of damage to the esophageal mucosa were diagnosed in all children with gastroesophageal reflux disease. At the age of 9–12 years, grade I esophagitis was detected in 20 children (32 %), grade II – in 4 (6.5 %). In patients aged 13–17 years, grade I esophagitis was detected in 16 children (26 %), grade II – in 20 (32 %), and grade III – in 2 (3.2 %).

All patients underwent esophagogastroduodenoscopy with parietal endoscopic pH-metry.

The indices of various sections of the upper gastrointestinal tract of patients are presented in Table 1.

 

Table 1

PH Values ​​in different parts of the upper gastrointestinal

Part	Group		р
	Base Group, n = 83	Control Group, n = 77
Distal segment of esophagus	2.04 ± 0.1	3.015 ± 0.15	0.0022
“Lake” of the stomach	1.48 ± 0.056	1.82 ± 0.12	0.0052
Gastric fundus	1.4 1 ± 0.062	1.72 ± 0.12	0.032
Body of the stomach, posterior wall	1.2 3 ± 0.05	1.56 ± 0.12	0.038
Body of the stomach, anterior wall	1.12 ± 0.062	1.4 2 ± 0.11	0.038
Antrum	2.8 ± 0.094	3.07 ± 0.12	0.062
Bulb of dodecadactylon	5.75 ± 0.077	5.85 ± 0.085	0.067

Note: n is the number of children, p is the reliability of differences in relation to the indicators of patients in the control group.

 

The results of the study revealed the presence of a hyperacid state in the esophagus, body of the stomach, and "lake" of the stomach. A highly acidic pH level was determined in 33 (39.8 %) people, in the esophagus (pH < 2); in 40 (48.2 %) people, the pH level corresponded to the acid value – from 2.1 to 2.9; in 10 (12 %) people, the acidity in the esophagus had faintly acid values ​​– from 3.0 to 6.0. It was found that in the control group, a faintly acid environment was detected significantly more often – in 42 (54.5 %) people (c² = 10.3; p = 0.001), in 22 (28.6 %) (c² = 10.3; p = 0.001) – an acid environment, in 13 (16.8 %) (c² = 12.3; p = 0.0003) children – a highly acid environment.

The results of the correlation analysis of the acidity indices of the upper digestive tract are presented in Table 2. Analysis of the obtained data showed that a direct positive dependence of the acidity indices in all studied sections of the stomach was determined, and there was also a correlation between the acidity indices in different sections of the stomach itself (“lake”, fundus, posterior wall of the body of the stomach).

 

Table 2

Spearman correlation analysis of the acidity indices of the upper gastrointestinal of children in the index group

Part	Esophagus	“Lake” Of the Gaster	Gastric fundus	Body of the Gaster, Posterior Wall	Body of the Stomach, Anterior Wall	Antrum	Bulb of Dodecadactylon
Esophagus	1.0000	0.554324	0.363590	0.478665	0.394826	0.248037	0.399207
“Lake” of the gaster	0.554324	1.0000	0.608983	0.748905	0.652745	0.381603	0.16858
Gastric fundus	0.363590	0.608983	1.0000	0.624118	0.526753	0.288324	0.234391
Body of the gaster, posterior wall	0.478665	0.748905	0.624118	1.0000	0.815286	0.360185	0.183940
Body of the stomach, anterior wall	0.394826	0.652745	0.526753	0.815286	1.0000	0.192250	0.093868
Antrum	0.248037	0.381603	0.288324	0.360185	0.192250	1.0000	0.425542
Bulb	0.399207	0.16858	0.234391	0.183940	0.093868	0.425542	1.0000

Note: Correlations significant at p < 0.05000 are marked in red.

 

Based on the obtained data, it can be concluded that in childhood GERD, the breakdown of collagen in the mucous membrane of the gastroesophageal zone is activated, in connection with which high levels of GAG, free hydroxyproline, peptidyl-linked hydroxyproline α-mannosidase are detected in the gastric acid (Table 3).

 

Table 3

Indicators of collagen and proteoglycan metabolites in gastric acid of children with gastroesophageal reflux disease

Indicator	Index Group, n = 62	Control Group, n = 32	p
Free hydroxyproline, umol/l	29.6 ± 3.2	20.2 ± 2.4	0.032
Peptidyl-linked hydroxyproline, umol/l	25.5 ± 2.1	16.3 ± 1.8	0.0042
GAG, umol/l	73.8 ± 4.5	31.9 ± 2.8	0.0016
α-mannosidase, umol/l	82.6 ± 5.2	38.1 ± 3.4	0.0014

Note: n is the number of children, p is the reliability of changes in relation to the control group.

 

Thus, in children with GERD, the structural integrity of the connective tissue of the gaster esophagus is impaired. High concentrations of the enzyme α-mannosidase in the gastric acid, which promote the breakdown of GAG, indicate the presence of pronounced destruction of connective tissue (Spearman correlation index 0.82 at p < 0.05).

The structure of the mucous membrane of the submucosa of the esophagus and stomach is affected by the acidity of the environment. The ROC analysis method showed the relationship between the level of acidity of the esophagus and the level of GAG and free hydroxyproline in the gastric acid (Fig. 1).

 

Fig. 1. ROC curve relationships between GAG concentrations in gastric acid and esophageal pH in children with GERD

 

The area under the ROC curve was 0.86, which determines the quality of the pattern as very good.

ROC analysis also showed a sufficient relationship between the level of esophageal acidity and the level of free hydroxyproline in gastric acid in children with GERD (Fig. 2).

 

Fig. 2. ROC curve relationships between free hydroxyproline concentrations in gastric acid and рН esophagus in children with GERD

 

The area under the ROC curve was 0.72, which determines the quality of the pattern as good.

The ROC curve of the relationship between the levels of peptidyl-linked hydroxyproline, α-mannosidase in gastric acid and esophageal pH determined the quality of the model as average.

Conclusions

Degradation of collagen of the mucous membrane of the submucosa of the gaster esophagus occurs as a result of a violation of the dynamic balance of aggressive-protective factors. It has been shown that in children with GERD, glycosaminoglycans, peptide-linked hydroxyproline, and free hydroxyproline, which are components of connective tissue, accumulate in the gastric acid. The damaging effect that promotes the breakdown of connective tissue is exerted by the high level of acidity in the gaster and esophagus. An increased concentration of the enzyme α-mannosidase in gastric acid can be considered as a pathogenetic link in the development of GERD, which indicates the depth of damage to the mucous membrane and submucosa.

About the authors

N. V. Butorina

Izhevsk State Medical Academy

Author for correspondence.
Email: naili5@yahoo.com

PhD (Medicine), Associate Professor of the Department of Childhood Diseases and Neonatology of the Pediatric Faculty

Russian Federation, Izhevsk

M. N. Repetskaya

E.A. Vagner Perm State Medical University

Email: naili5@yahoo.com

DSc (Medicine), Professor, Head of the Department of Childhood Diseases of the Medical Faculty

Russian Federation, Perm

Ya. M. Vakhrushev

Izhevsk State Medical Academy

Email: naili5@yahoo.com

DSc (Medicine), Professor, Head of the Department of Propaedeutics of Internal Diseases of the Medical Faculty

Russian Federation, Izhevsk

References

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