Substance P, vasoactive intestinal peptide, and leu-enkephalin in plasma and gastric juice of patients with precancerous lesions and gastric cancer

Abstract

AIM: To investigate the changes of the brain-gut-peptide concentrations in the plasma and gastric juice and their relations to gastric diseases.

METHODS: A total of 83 subjects were part of the study. Of those, 28 had chronic atrophic gastritis with precancerous lesions, 22 had gastric cancer in an advanced stage, and 33 were healthy subjects for a control group. Samples of fasting blood and gastric juice were collected. Levels of substance P (SP), vasoactive intestinal peptide (VIP) and leu-enkephalin (LEK) in plasma and gastric juice were measured with radioimmunoassay kits expressed as ng/L.

RESULTS: In patients with gastric cancer, the SP levels (83.7 ± 11.0 vs 39.6 ± 4.5, P < 0.01; 24.0 ± 1.6 vs 17.8 ± 1.5, P < 0.05) and LEK in plasma and gastric juice (226.2 ± 15.4 vs 180.3 ± 13.1, P < 0.01; 55.0 ± 3.4 vs 30.7 ± 2.4, P < 0.05), and VIP of gastric juice (80.5 ± 2.9 vs 64.3 ± 4.1, P < 0.05) were higher than those in the controls. The SP and LEK levels of plasma correlated with those of gastric juice (r = 0.432 and 0.516, P < 0.05). In the post-surgical gastric cancer, plasma levels of SP and gastric juice LEK were lower than the pre-surgical levels (P < 0.05). In the precancerous lesions, plasma and gastric juice LEK levels and gastric juice VIP levels were increased (P < 0.05), and the plasma LEK level correlated with the gastric juice LEK level (r = 0.398, P < 0.05).

CONCLUSION: Measurement of concentrations of SP, VIP, and LEK in plasma and gastric juice is of clinical significance in detecting certain stomach diseases.

Key Words: Stomach neoplasms, Precancerous conditions, Substance P, Vasoactive intestinal peptide, Enkephalin, leucine

INTRODUCTION

Multifocal chronic atrophic gastritis with intestinal metaplasia (IM) of the gastric mucosa is considered a precancerous lesion of gastric cancer[1,2]. The increased prevalence of this lesion in high-risk population is of little use in identifying individuals at the highest risk. It has been proposed that some types of IM represent a more advanced stage in the precancerous process, as represented by the colonic type of morphology and the secretion of sulfated mucins[3]. The poor prognosis of advanced gastric cancer has not changed substantially over the years[4]. This dismal outcome is in sharp contrast to the excellent five-year survival rate of surgically treated early gastric cancer, reportedly to be 90%-95%[5]. Since most patients with early gastric cancer are probably asymptomatic or have only vague and/or nonspecific complaints, they are usually seen in the clinic at a rather advanced stage of the disease.

Various gut regulatory peptides, including substance P (SP), vasoactive intestinal peptide (VIP) and leu-enkephalin (LEK), may act as endocrine hormones influencing gastrointestinal functions. The alterations of levels of beta-endorphin, motilin,, and gastrin in plasma and gastric juice have been reported to occur in gastric cancer and precancerous lesions[6]. Abnormal plasma levels of SP, VIP and LEK have been reported to occur in stomach diseases[7,8]. The present study was undertaken to determine if the production of these peptides is altered in gastric cancer and precancerous lesions, and if the measurement of the levels of the peptides in gastric juice and plasma could assist in unraveling the pathogenesis and early diagnosis.

MATERIALS AND METHODS

Subjects

Studies were performed on 22 patients with advanced gastric cancer (5 women and 17 men) with a mean age of 48.2 ± 2.6 years. A total of 28 patients had chronic atrophic gastritis with precancerous lesions (8 women and 20 men) with a mean age of 47.5 ± 3.1 years. A total of 33 healthy subjects (7 women and 26 men) with a mean age of 45.8 ± 2.5 years were used as controls. The diagnoses were based on the endoscopic, histologic or cytological data and the clinical and laboratory findings before therapy. Intestinal metaplasia of the gastric mucosa was considered precancerous lesions. Carcinoma was found in the antrum (n = 15), body (n = 3), and fundus (n = 4) of the stomach. Pathohistological diagnosis revealed adenocarcinoma (n = 16), squamous cancer (n = 1), and undetermined (n = 5). Patients with other diseases were not included in the study.

Methods

Patients under fasting conditions had 2 mL of a blood sample collected in prechilled heparinized tubes containing 1000 kIU aprotinin. The plasma was separated immediately by centrifugation, frozen and stored at -20 °C until analysis. The gastric juice samples (4 mL) were collected in tubes during gastroscopy at fasting and were immediately mixed with NaHCO₃ (0.1 N) to adjust the pH to 5-7, and stored at 20 °C until analysis. The concentrations of SP, VIP, and LEK were measured with radioimmunoassay kits (The General Hospital of Chinese PLA, Beijing, China)[9,10]. The coefficient variations were controlled to less than 9%. The smallest amount of peptides detectable with 95% confidence was 2 ng/L for SP, 0.5 ng/L for VIP, and 10 ng/L for LEK.

Statistical analysis

All values were given as x^-± s. The unpaired Student’s t test was used to compare the control group and patient groups. The paired Student’s t test was used to compare post- and pre-surgical gastric cancer patients. Correlations were calculated for the peptide values in both plasma and gastric juice with linear regression analysis. P < 0.05 was considered significant.

RESULTS

The levels of SP, VIP and LEK peptides in the control and patient groups are summarized in Table 1. In patients with gastric cancer, a statistically significant correlation was found between the levels of SP and LEK in plasma and gastric juice: r = 0.432, P < 0.05 for SP; and r = 0.516, P < 0.05 for LEK. In patients with precancerous lesions, r = 0.398, P < 0.05 for LEK.

Table 1 Comparison of plasma and gastric juice levels of substance P, vasoactive intestinal peptide and leu-enkephalin between patient and control groups (x^-± s, ng/L).

Diagnosis	n	Plasma			Gastric juice
		SP	VIP	LEK	SP	VIP	LEK
Gastric cancer	22	83.9 ± 11.0b	6.0 ± 1.0a	226.2 ± 15.4a	24.1 ± 1.6a	80.5 ± 2.9a	55.0 ± 3.4b
Precancerous lesion	28	42.0 ± 2.3a	7.0 ± 0.4a	213.9 ± 19.1a	20.0 ± 2.8a	78.4 ± 8.7a	41.7 ± 5.7a
Healthy control	33	39.6 ± 4.5	6.9 ± 1.0	180.3 ± 13.1	17.8 ± 1.5	64.3 ± 4.1	30.7 ± 2.4

^aP < 0.05,

^bP < 0.01, vs healthy controls. SP: Substance P; VIP: Vasoactive intestinal peptide; LEK: Leu-enkephalin.

In gastric cancer, the differences in the levels of plasma SP and LEK, and in the gastric juice peptides between adenocarcinoma and other cancers did not reach statistical significance (P > 0.05). In gastric cancer patients, post-surgery plasma levels of SP (47.2 ± 11.6 ng/L, P < 0.01) and gastric juice LEK (35.9 ± 3.8 ng/L, P < 0.01) were lower than pre-surgery, but changes in the other peptides did not reach statistical significance.

To test if the differences in peptide levels were due to the difference in sex distribution between the patient and control groups, comparisons were made for each sex. No statistically significant difference was found.

DISCUSSION

Neuropeptides form a part of the brain-gut-axis which may regulate gastrointestinal functions, including immune regulation and development of gastrointestinal cancer[7,8]. Our results showed that gastric juice peptide levels changed in the precancerous lesion, as well as in established gastric cancer. The levels of SP did not change significantly in the precancerous lesion. The plasma levels of SP and LEK changed in gastric cancer as compared with controls, as well as the level of LEK in the precancerous states.

Hormonal influences on IM and malignant cells remain an important area of research in the study of stomach neoplasms and precancerous conditions. SP[11], VIP[12] and LEK[13] have each been localized to mammalian gastric enteric nerves using immunohistochemical and radioimmunological techniques. SP-containing nerve fibers are present in the myenteric plexus and circular smooth muscle of gastric corpus of several species. VIP has been localized to enteric neurons of the same gastric smooth muscle layers as the other neuropeptides[14]. LEK has important clinical implications between opiates and human immune system.

SP, an 11-amino acid peptide, is a natural polypeptide produced by the cells of the intestinal mucosa and brain. The primary action of this hormone is, by endogenous means, to stimulate the smooth muscle of stomach to contract. SP has been shown to play a secondary role in the pathogenesis of gastric cancer[15]. In the present study, we have demonstrated that SP levels in patients with gastric cancer were significantly increased. This may suggest either a destruction of the function of gastrointestinal cells by the malignant process or a primary failure which facilitates carcinogenesis.

VIP, a 28-amino acid peptide, is found extensively throughout the central nervous system and the gastrointestinal tract and is considered a neurotransmitter. Like SP, VIP has been shown to induce the release of histamine from mast cells. The release of histamine is inhibited by SP antagonist, suggesting that SP and VIP act on a common site or via a common receptor[16]. There is evidence to suggest that VIP modulates the neurogenic inflammatory response induced by SP, an effect probably related to its vasodilator activity[17]. VIP has also been shown to induce pepsinogen secretion in gastric glands, probably by increasing cAMP levels[18]. In several studies, VIP has been found to decrease gastric acid secretion by releasing somatostatin[19], whereas, in others, VIP had an inhibitory or no effect on gastric somatosatin[14,20]. Increased levels of VIP in gastric juice have been reported in the gastric cancer patients[15]. Karmeli et al[14] have provided evidence that VIP is involved in the pathogenesis of acute ethanol-induced gastric mucosal damage. In patients with gastric cancer or precancerous lesions, the changes in levels of VIP in gastric juice suggest that VIP may stimulate contraction of the smooth muscle of the stomach. The VIP mechanism may be enhanced by gastroduodenal mucosa since gastric emptying is influenced in gastric cancer or intestinal metaplasia involving gastric mucosa.

LEK is a 5-amino acid peptide. The fluctuations in LEK in various other gastrointestinal disorders are unclear. It is possible that gastrointestinal dysmotility may play a role in the development of gastric cancer and precancerous lesions. The regulation of gastrointestinal motility is a complex process involving the inherent properties of the mural musculature, both intrinsic and extrinsic neural control mechanisms, hormonal influences, and other factors. A wide range of gastrointestinal peptides, such as SP, VIP and LEK may influence the motor function of the gut. LEK levels have been previously reported to increase in patients with gastric cancer[15]. In our study, we have demonstrated that LEK levels in patients with gastric cancer and precancerous lesions were significantly elevated.

A good correlation was found between the plasma and gastric juice concentrations of SP and LEK in gastric cancer patients, and of LEK in precancerous lesions, although in individual patients there was a clear difference. Many gut hormones, including SP, VIP and LEK, will change their levels in plasma or gastric juice as a result of the disease of the stomach[15,16,21]. Our results show that the difference in levels of SP, VIP, and LEK between the patient and control groups are significant. Therefore, a diagnostic laboratory should choose one peptide for routine screens. In practice the values of SP, VIP, and LEK are not likely to be useful in the diagnosis and the follow-up of patients with gastric cancer and precancerous lesions. However, given the established interactions between stress and opiates and the immune system, our findings might justify further consideration of these factors in relation to causation of gastric cancer.