General Biochemical Methods to be Used in Gastrointestinal Mucosa in Animal Experiments and in Human Observations Done on Gastrointestinal Resecates (after Surgical Interventions)

General aims of these observations were to biochemically examine all the gastrointestinal tissues in both animals and humans. The outstanding point was that from 1965 to date, we have been unable to know the exact details of different regulatory mechanisms (by neural, hormonal, pharmacological, immunological and nutritional pathways) under normal (nonulcerated) and damaged conditions. When we undertook clinical pharmacological studies, we had to face different hard-to-understand medical facts:

We suggested an answer to this question by applying different and precise biochemical methods in the study of human gastrointestinal tract (with and without the presence of the classical ulcer). Unfortunately, we had no methodology to answer this question.
These observations maintained the main trends of clinical pharmacology (e.g., time period with drugs, to keep the time period after cessation of treatment); however, we introduced the biochemical methodology to the pharmacology.
We tried to approach the biochemical events in the whole tissue by simultaneously using more parallel biochemical measurements (using the same tissue samples, with the measurements carried out at the same time).
Before the human biochemical examinations, we learned the biochemical methodology in animal experiments.

Methodologies of experimental models and clinical studies
The observations were carried out in CFY (Sprague-Dawley) (Gödöllő, Hungary) strain rats, weighing 180-210 g, and on the resecates of stomach and small intestine of patients who underwent gastric surgery because of unhealed ulcer disease (during 1970-1980). The patients suffered from classical peptic ulcer diseases (PUD) with clinical symptoms (decreased appetite, feeling of dullness and pain in the epigastric region of the abdomen, pyrosis, impaired gastric emptying and retention syndrome). These patients presented one month before the surgical intervention. The presence of gastroduodenal ulcers was endoscopically diagnosed, and thereafter these patients received medical treatments (anticholinergic agents, late H 2 receptor antagonist and antacids for one month). A possibility of surgical interventions was evaluated for those patients who were not healed after the treatment.
The indication of gastric surgery was done by physicians [consultations between internists (gastroenterologists) and surgeons] independently from us]. The resecates of stomach and small intestine (according to the method of Billroth II). A small group of patients underwent classical partial gastrectomy (according to the method of Billroth II), and jejunal ulcer was developed. These patients were also medically (pharmacologically) treated during 1970-1980. During surgical intervention, the stomach and small intestine were removed immediately and these were cut into two parts. One part was given for histological evaluation of resected tissues and the other part was immersed (after separation of mucosa and muscular layer) in liquid nitrogen and used for biochemical examinations. The mucosa specimens were also separated from each other (depending on the distance of ulcer edge). Both the biochemical measurements from the mucosa specimens and muscular layers (independently from the number of tissue specimens), obtained from one patient, and the surgical intervention were carried out at the same time.
The animal observations were carried out in both sexes of CFY-strain rats.
The following experimental models were used: 6. Gastric mucosal preventive effects of atropine, cimetidine, vitamin A and β-carotene in 4hour indomethacin-treated rats; 7. Stress ulcer in rats was caused by 4 hours of immobilization (Nagy et al., 1982;1983); 8. The stress ulcer in rats was caused due to 5 hours of swimming. In some animals, the stress ulcer provocation (swimming) was combined with pyloric ligation during the beginning of stress (Nagy et al., 1982;1983);

12.
All biochemical examinations were carried out in the control (non-ulcerated) in the ulcerated (mucosa up to 2 cm around the ulcer) antral, duodenal, jejunal mucosa or from the corpus (fundic), antral, duodenal and jejunal mucosa and from the tissues located below the mucosa (muscular layer). All patients with jejunal ulcers previously underwent a gastric partial resection because of duodenal ulcer. No direct provocative agents such as drugs or primary diseases (renal, endocrine, hematological, liver, pulmonary diseases) could be detected in the background of peptic ulceration ("genuine ulcer"). The tissue specimens were obtained during the surgery.
The following measurements were carried out in different observations: 1. Determination of gastric acid output. The gastric basal acid output (BAO) and maximal acid output (MAO) were determined in patients; 2. The extent of experimental gastric ulcer (except in Shay rats) was scored in the following way: score 0: no ulceration; score 1: the erosions were less than 1 mm; score 2: the erosions were between 2 and 4 mm; score 4: the erosions were greater than 4 mm; and score 5: represents the mucosal damage in all part of fundus. The values of scores were summarized for every stomach, and the average ± SEM was given (Mózsik et al., 1983 a, b);

7.
The separation and measurements of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) were completed according to the methods published earlier (Mózsik et al., 1967 b, c;1969 c;1976 a, b, c, d;1978 a, b;1979 a, b, c, d, e;Mózsik and Vizi, 1976 a, b); 8. The separation of membrane ATPase was carried out by the treatment with NaJ and differential centrifugation (Mózsik and Øye, 1969;Mózsik et al., 1974 a, b, c, d;1979 a, b, c, d, g, e;Schmidt and Tannhauser, 1945); 9. The ATPase activity was measured in vitro system by liberation of inorganic phosphorus, followed by the ATP transformation into ADP in the presence of Mg 2+ (Mg dependent) and Mg 2+ , Na + and K + (total) or Mg 2+ -, Na + -, K + -dependent ATPase. The Na + -and K +dependent ATPase were calculated by the difference between the ATPase activities obtained in the presence of Mg 2+ , Na + and K + and Mg 2+ (Mg 2+ -dependent part) (Mózsik and Øye, 1969;Mózsik, 1969 a, b;Mózsik et al., 1974 a, b, c, d); The enzyme activity was expressed as micromoles of Pi/mg membrane protein/hour. The results were given as means ± SEM (Mozsik and Øye, 1969;Mózsik, 1969 a, b).The Student "t" test was used for the statistical analysis of the parametric results and by Mann and Whitney's method for the severity of erosions.
We used the rats as experimental animals in these observations to approach the changes in the cellular energy systems and their regulation in different experimental conditions.
The rat's stomach is divided into two parts, namely glandular (fundic) and membranous (rumen). These parts can be separated well and clearly.
The following biochemical measurements were carried out from both parts of the rat's stomach: acid-soluble inorganic phosphates, acid-soluble organic phosphates, lipids, ribonucleic acids (RNA) and deoxyribonucleic acid (DNA) (see the scheme of these measurements in Table 6).
The measurements of these biochemical parameters generally represented the main components of the cells: lipids (as cell membrane), acid-soluble inorganic and organic phosphates (mitochondrion), RNA (partly the cytoplasm as well as nucleus) and DNA (nucleus). In other words, we tried to observe different compartments of cells. The measurements of amounts of acid-soluble inorganic phosphates in the different tissues are widely used to approach the dephosphorylation [i.e., these compounds originated from the splitting of adenosine triphosphate (ATP) independently from its different pathways]. The components of the acid-soluble organic phosphates were not known at that time; meanwhile, the presence of adenosine triphosphate (ATP), adenosine diphosphate (ADP), cyclic 3',5'-adenosine monophosphate (cAMP), adenosine monophosphate (AMP) and adenine and adenosine were incorporated in this tissue extract. Naturally, these methodologies were updated later by direct measurements of these compounds using thin-layer chromatographic and enzymatic methods.

General biochemistry of glandular (fundic) part and of forestomach (rumen) in 24-hour pylorusligated rats
The necessity of the biochemical analysis of the stomach (gastroduodenal) mucosa was suggested for a better understanding. The underline mechanisms involved in development of mucosal damage and prevention (1962)(1963)(1964) (Gheorghui, 1975;Mózsik et al., 1967 a, b;1969 a, b, c, d;Mózsik et al., 1970 a, b). These results stimulated us for doing further biochemical observations in the animal stomach on dependence of increased and decreased vagal activity.

Figure 14.
The changes in the chemical composition of glandular stomach (fundus) and forestomach (rumen) in pylorus-ligated rats. The following parameters were measured: gastric secretory volume (mL), H + output, number of ulcers, wet tissue (g), acid-soluble inorganic (P i ) and organic (P i ) phosphates, lipid phosphates (μg), ribonucleic acid (RNA) (μg) and deoxyribonucleic (DNA) (μg) acids. The results were expressed as percentage values of sham-operated (=100%) animals. The statistical analysis was carried out between the sham-operated rats and 7-and 24-hour pylorusligated rats (means ± SEM There were many criticisms for this experimental ulcer model because the ulceration appeared in the forestomach (not in the glandular part of the animal stomach); however, different typical events of experimentally developed ulcer can be detected using this model: a. The gastric hypersecretion can be obtained before the ulcer development in accordance with the time (after surgical intervention); b. The peak of gastric acid hypersecretion can be obtained in this model in 7 hours after the surgical intervention (see Figure 2), and its value does not change from 7 to 24 hours after pyloric ligation; c. The time period between 4 and 7 hours offers excellent good possibility to study the stimulatory or inhibitory actions of different compounds on the gastric acid secretion in rats; d. We can very well study the possible correlations between gastric acid secretory responses and development of gastric ulcer.
Bearing these conclusions in mind, we started with the "general biochemical" approach -the main biochemical events during the development of gastric acid hypersecretion and ulcer (of course, respecting the actual level of international research). We have to emphasize that no general biochemical examinations were given in the gastrointestinal research for animals and patients earlier. So, these types of observations internationally opened a new avenue ("biochemistry") in the gastrointestinal research.
We tried to select the different biochemically measured parameters for providing nearest approach to the cell functions (e.g., membrane, mitochondrion, ribonucleic acid and deoxyribonucleic acid). The functions of the organs are specific events (gastric secretion, ulcer development); meanwhile, the biochemical mechanisms obtained in the target organs are extremely complicated. The biochemical extractums (e.g., acid-soluble inorganic and organic phosphates, lipids, ribonucleic acid and deoxyribonucleic acid) from the gastric tissues dominantly represent the cell membranes (lipids), mitochondrion (lipid-soluble organic and inorganic phosphates, partly ribonucleic acid) and nucleus (deoxyribonucleic acid).
At that time, the measurements of acid-soluble inorganic phosphate represent the cumulative effect of breakdown of adenosine triphosphate (ATP) (by different pathways) from the effector organs; meanwhile, the compounds of the acid-soluble organic phosphates (when these observations were carried out) were unknown. Now, we know that the acid-soluble organic phosphates contain the adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic 3',5'-adenosine monophosphate (cAMP) and adenosines and adenines.
We have to emphasize that we tried to approach only the main biochemical lines in the stomach during the development of gastric acid hypersecretion and ulcer after surgical intervention.
The obtained results of our present observations clearly indicated to us: a. All biochemical changes (in acid-soluble inorganic and organic phosphates, lipids, RNA and DNA) -depending on time -are similar to each other;  The critical evaluation of these experimental results called our attention to reconsider our previously created knowledge on the development of gastric acid hypersecretion and ulcer development in pylorus-ligated rats.
Our aims were (1) to study the effects of different parasympatholytics and surgical vagotomy on the biochemical parameters of stomach and (2) to compare the changes in the gastric mucosal biochemical parameters produced by "chemical" and "surgical" vagotomy.    Increasing inhibitory effects of parasympatholytics -depending on the extent of chemical diameters -did not run closely parallel with increasing diameter of tertiary and quaternary ammonium molecules, and we also perceived biochemical changes of gastric mucosal biochemistry. After bilateral surgical vagotomy, the quantities of acid-soluble inorganic phosphates decreased significantly in both glandular and membranaceous (forestomach and rumen) stomach wall. The alterations of acid-soluble inorganic and organic phosphates in the stomach wall showed contradictory trends of surgical vagotomy to those after administration of parasympatholytics ("chemical" vagotomy), the effects of surgical vagotomy on the nucleic acid metabolism being greater than the effects of different parasympatholytics.
A biochemical-cellular-morphological explanation of parasympatholytics and surgical vagotomy has been suggested ( Figure 18). According to this explanation, the nucleic acids are in the center of Figure 18, and phospholipids, acid-soluble inorganic and organic phosphates are in periphery of a "hypothetic cell." The products of cells (HCl secretion) are presented in an outer part of the figure. After a large alteration of periphery, the center will change to a small degree and vice versa. Table 17. Main steps of regulatory levels of cells in the living organs. The most stable regulatory steps are located at the level of DNA and the most instable regulatory steps are at the level of functions of organs.

Pharmacological and biochemical studies in rats after chronic "chemical" and "surgical" vagotomy and cholinesterase inhibitor treatment
Biochemical observations were carried out to study the changes in these biochemical parameters of rat's stomach after chronic "chemical vagotomy" (2 × 1.0 mg i.p. atropine for 25 days) and cholinesterase inhibitor treatment (2 × 0.25 mg of neostigmine i.p for three weeks).
The biochemical examinations in the drug-treated groups of animals were also divided into two different groups. The biochemical observations of the first group were carried out immediately after the end of the drug treatment, whereas the observations of the second group were carried out after cessation of drug treatments (10 days after the cessation of atropine treatment and three weeks after cessation of cholinesterase inhibitor treatment).
To compare the changes in the stomach after a chronic "chemical" vagotomy (atropine treatment), the surgical vagotomy was carried out in a group of animals (without any other treatment), and the biochemical measurements were done one month after surgical vagotomy.
The control animals were treated with saline solution for 25 days. It has been suggested that the results of these animal observations will give a biochemical explanation for the effects of increased cholinergic activity (produced by cholinesterase inhibitor), for "chemical" vagotomy and "surgical" vagotomy ("use" vs. "disuse" of vagal nerve on the metabolism of gastric tissues). The results of the biochemical examinations are presented in cases of chronic atropine treatment and chronic neostigmine (see the forthcoming tables). The biochemical results after one month of surgical vagotomy are presented only in comparison with the changes in the biochemical parameters obtained in rats treated chronically with atropine and neostigmine.  The treatment's effects on the body weight and weight of the glandular and membranaceous (forestomach) parts are shown in Table 18: there was a significant decrease in body weight (0.01 > P > 0.001), but no significant change in the weight of the parts of the stomach. After prolonged atropine, there was a decrease in acid-soluble inorganic phosphate (P = 0.02), acid-soluble organic phosphates (P < 0.001), phospholipids phosphates (P < 0.001), ribonucleic acid (P = 0.03) and deoxyribonucleic acid (P < 0.05) in the glandular part. Ten days after cessation of atropine treatment, levels of acid-soluble organic phosphates (P = 0.02) and phospholipid phosphates (P< 0.001) improved but levels of ribonucleic acid and deoxyribonucleic acid did not. Prolonged atropine treatment did not alter the acid-soluble inorganic and organic, phospholipids phosphate and ribonucleic acid, but there was a decrease in deoxyribonucleic acid (0.01 > P > 0.001). Ten days after cessation of atropine treatment, there was a further reduction of ribonucleic acid (P = 0.02) and increase of deoxyribonucleic acid (P > 0.05). The results obtained from chronic neostigmine treatment provided the following conclusions: 1. The cholinergic dominance involves the decrease of the weight and biochemical constituents of the membranaceous (forestomach). This is the effect of cholinergic dominance on the membranaceous stomach wall.

2.
Examined biochemical constituents of the glandular stomach behave differently during the existence of the cholinergic dominance due to the decrease of acid-soluble inorganic phosphates, phospholipid phosphates and ribonucleic acid and the increase of acidsoluble organic phosphates during neostigmine treatment.

3.
We had observed "short-term" and "long-term" biochemical changes in the glandular stomach wall after neostigmine treatment. The "short-term" biochemical change (acidsoluble organic phosphates) was a reversible process lasting up to one month after cessation of neostigmine treatment. At the same time, the "long-term" biochemical changes (acid-soluble inorganic phosphate, phospholipids phosphate and ribonucleic acid) were observed as irreversible processes. It is interesting to note that the stomach can "remember" to the neostigmine treatment one month after cessation of treatment.
In the 1970s, there was a famous topic on physiology to approach the possible backgrounds of "use" and "disuse" of the neural regulation (especially after denervation of muscles) (Graff et al., 1965 a, b, c;Gregory, 1962). The surgical ablation of nerves was used extensively in these types of observations. There was a general note that the denervated organ became to b seniitive to mediators than that inavated organ. Emmelin and Rosenblueth (1951), Emmelin and Muren (1951 a, b;1952), and Elin (1952, 1961 observed that the efficenciees of drugs and mediators changes after a prolonged reatment (including the atropine). In these observations, no surgical manipulation was done with the nerves of different organs, however, they dichronic drunt was done to inhibit the neural functions at the levels of synapses or at the levels of tra to organs. This phenomenon was named as "pharmacological denervation" and was associated with the supersensitivity (Emmelin, 1952(Emmelin, , 1961. We were the first authors, who demonstrated the existence of supersensitivity of "pharmacologic denervation" phenomenon, together along with opment of tolerance to drugs used in the treatment and cross-tolerance to the pharmacologicallypharmarugs, but that are not used in the trea, under classical medical treatment with parasympatholytics in patients with peptic ulcer (see chapters of Sections 2.2-.2.3--2.4) There was an important note that the efficacy of atropine decreased during a chronic atropine treatment in patients with peptic ulcer, however, the decrease effect of atropine returned in time 0 days after cessation of atropine treatment.
These human observations called our attention to carry out different biochemical observations in the rat's stomach after cessation of atropine and neostigmine treatment.
We tried to approach the biochemical backgrounds of the "use" and "disuse" of the gastric tissues in rats (under experimental conditions). The changes in gastric mucosal constituents were presented in percentage values of "sham treated" (with physiological saline solution) (=100%) after chronic "chemical" and "surgical" vagotomy and neostigmine treatments. The