Fasting Gastric Juice Melatonin Levels in Relation to Gastroduodenal Pathologies Among Zambian Patients: A Cross Sectional Study
ABSTRACT
Background and Aims
Melatonin is a hormone released from the pineal gland and enterochromaffin cells of the gastrointestinal tract. The influence of melatonin on gastric pathologies is not fully investigated. Our aim was to evaluate the relationship between fasting gastric juice melatonin levels and gastroduodenal pathologies among Zambian patients.
Methods
This cross-sectional study was conducted in Lusaka, Zambia. We collected morning fasting gastric juice samples from patients presenting for oesophagogastroduodenoscopy (OGD) to quantify levels of melatonin using commercial ELISA kits. Interviewer administered questionnaires were used to collect baseline characteristics. Data were analyzed in Stata version 15.
Results
We collected gastric juice from 175 patients with a median age of 49 years, IQR (40–65). The number of females was 95 (54%) and males 80 (46%). Of these, 100 (57%) had normal OGD examinations, 49 (28%) peptic ulcers, 25 (14%) gastric cancer and 1 (1%) gastric polyp. The median level of melatonin in normal patients was 141.8 pg/mL, IQR (46.2–500.1). In those with gastroduodenal pathologies, the median was 339.4 pg/mL, IQR (101.8–500.1) and the difference was statistically significant, p = 0.02. Results also revealed significantly higher levels of melatonin among patients with history of cigarette smoking, p = 0.003.
Conclusion
There is an association between gastroduodenal pathology and morning gastric juice melatonin levels, a finding whose biological significance warrants further research.
1 Background
Melatonin is a circulating hormone mainly produced and released by cells in the pineal gland and enterochromaffin cells of the gastrointestinal tract (GIT) [1]. The hormone is a close derivative of serotonin, involved in regulation of circadian and seasonal rhythms [2, 3]. Melatonin has multiple other functions that influence different systems in the human body. In the GIT, it is synthesized in serotonin-rich enterochromaffin cells and is a major source of extra-pineal melatonin with high tissue concentrations that significantly contribute to circulating levels in peripheral blood, particularly during the day [1, 4].
Concentrations of melatonin in the GIT are much higher than in plasma or the pineal gland [4]. GIT melatonin has multiple functions exhibiting endocrine, paracrine, autocrine and luminal activity. Studies have shown involvement of melatonin in different anticancer mechanisms such as cell proliferation inhibition, reduction in tumor growth and metastases with a potential for future therapeutic approaches [5, 6]. The importance of melatonin has also been suggested in ulceration of the gastrointestinal mucosa, which could induce a reduction in the secretion of pepsinogen and hydrochloric acid and stimulation of the immune system [7].
The exact role of melatonin in development of gastric cancer or peptic ulceration is still under investigation as the interaction with other etiological drivers such as Helicobacter pylori (H. pylori) is yet to be established. With a potential of melatonin being used as adjuvant to standard therapy for benign and malignant gastroduodenal pathologies, it is imperative that its role be clearly understood.
In this study, we investigated the association between fasting gastric juice melatonin levels and gastroduodenal pathologies (peptic ulceration and gastric cancer). In addition, we evaluated for possible associations with risk factors such as H. pylori infection and exposure to biomass smoke.
2 Methods
This cross-sectional study was conducted at the University Teaching Hospital (UTH) endoscopy unit in Lusaka, Zambia. UTH is the biggest referral hospital in Zambia.
2.1 Study Procedure
Patients presenting to the UTH endoscopy unit for oesophagogastroduodenoscopy (OGD) were considered for enrollment. Only those above the age of 18 years, who gave informed written consent were included. Interviewer administered questionnaires were used to collect information on basic characteristics. Excluded from enrollment were patients with history of prior gastrointestinal cancer diagnosis or treatment and those with incomplete OGD examination due to luminal obstruction.
OGD procedures were conducted according to standard of care. Upon entry into the stomach, the water channel was flushed and gastric juice aspirated into sterile cryovials. It was then kept frozen at −80°C in readiness for testing. All OGD procedures were conducted in the morning between 08 and 11 am, and the patients had fasted for at least 8 h. Blood was collected to test for H. pylori antibodies using commercial ELISA kits (Biohit Oyj, Helsinki, Finland).
2.2 ELISA Assay to Determine Melatonin Levels
We used commercially obtained ELISA kits (Wuhan Fine Biotech Co. Ltd, China), for melatonin quantification. The assays were conducted with strict adherence to the manufacturer's instructions. The upper limit for melatonin quantification was 500.1 pg/mL.
Frozen gastric juice samples were brought to room temperature about 20 min before testing. To prepare the standards, 1 mL of sample dilution buffer was added to each, mixed thoroughly and kept at room temperature for 10 min. Samples were similarly diluted and mixed thoroughly. The standards, test samples and controls were then placed in pre-coated wells, carefully recording their positions. Biotin-labeled antibody (50 μL) was added in each well and incubated for 45 min at 37°C. Washing was then done three times with wash buffer allowing it to stay for 1 min in the well. The wells were emptied by aspiration. HRP-Streptavidin Conjugate (SABC) 100 μL, was then added to each well and covered with a plate sealer, incubating for 30 min at 37°C. Washing was then done 5 times allowing the buffer to rest 1–2 min in the wells each time. TMB Substrate (90 μL) was then added into each well, covered and incubated at 37°C in the dark for 10–20 min. The stop solution, (50 μL) was then added into each well. An immediate color change to yellow was then observed and the OD absorbance read at 450 nm in Microplate Reader immediately after that. The results were therefore determined in accordance with the manufacturer's guidance.
Permission to conduct the study was obtained from the University of Zambia-Biomedical Research Ethics Committee, ref 4539 2023.
2.3 Data Entry and Analysis
Data were analyzed in Stata Statistical Software: Release 15. College Station, TX: StataCorp LLC, using standard statistical methods. The analysis that was conducted was pre-specified when designing the data collection tools. Continuous variables which included age and melatonin levels were summarized using medians and interquartile range (IQR). Normality was tested using the Shapiro–Wilk test. Associations were computed using Fisher's exact (for categorical variables such as comparison of sex against peptic ulceration) and Kruskal- Wallis tests (for continuous variables such as comparison of melatonin levels against gastroduodenal pathologies). These specifications are outlined as foot note on tables. Statistically, two-sided p values less than 0.05 were considered significant.
3 Results
We enrolled 175 patients, of which 100 (57%) had normal gastric mucosa, 49 (28%) peptic ulcers, 25 (14%) gastric cancer and 1 (1%) gastric polyp. Overall, 95 (54%) were females and 80 (46%) were males. The median age was 49 years; IQR 40–65 years.
There was no statistically significant difference between patients with normal gastric mucosa and those with pathologies, save the age for gastric cancer patients; they were significantly older, Table 1.
| Variable | All | Normal | Peptic ulceration | OR; 95% CI | p value | Gastric cancer | OR; 95% CI | p value |
|---|---|---|---|---|---|---|---|---|
| Total number of samples | n = 175 | n = 100 | n = 49 | n = 25 | ||||
| Age in years: | ||||||||
| < 45 | 62 (35%) | 45 (45%) | 15 (31%) | 0.11 | 2 (8%) | |||
| 45 or more | 113 (65%) | 55 (55%) | 34 (69%) | 0.5 (0.2–1.1) | 23 (92%) | 0.1 (0.01–0.5) | < 0.001 | |
| Sex: | ||||||||
| Female | 95 (54%) | 59 (59%) | 21 (43%) | 15 (60%) | ||||
| Male | 80 (46%) | 41 (41%) | 28 (57%) | 0.5 (0.2–1.1) | 0.08 | 10 (40%) | 1.1 (0.4–2.9) | > 0.99 |
| Hypochlorhydria (pH> 4): | ||||||||
| Yes | 93 (53%) | 52 (52%) | 25 (51%) | 15 (60%) | ||||
| No | 77 (44%) | 45 (45%) | 24 (49%) | 8 (32%) | ||||
| Missing | 5 (3%) | 3 (3%) | — | 0.9 (0.4–1.9) | 0.35 | 2 (8%) | 1.7 (0.6–4.9) | 0.35 |
| HIV infection: | ||||||||
| Yes | 24 (14%) | 14 (14%) | 7 (14%) | 3 (12%) | ||||
| No | 131 (75%) | 75 (75%) | 37 (76%) | 19 (76%) | ||||
| Missing | 20 (11%) | 11 (11%) | 5 (10%) | 1.1 (0.3–3.0) | 1.00 | 3 (12%) | 0.8 (0.1–3.5) | > 0.99 |
| H. pylori infection: | ||||||||
| Yes | 145 (83%) | 81 (81%) | 40 (82%) | 0.56 | 24 (96%) | |||
| No | 3 (2%) | 3 (3%) | 0 (0%) | 0 (0%) | ||||
| Missing | 27 (15%) | 16 (16%) | 9 (18%) | — | 1 (4%) | — | — | |
| Exposure to biomass smoke: | ||||||||
| Yes | 53 (30%) | 24 (24%) | 19 (39%) | 0.08 | 10 (40%) | |||
| No | 121 (69%) | 76 (76%) | 30 (61%) | 14 (56%) | ||||
| Missing | 1 (1%) | — | 2.0 (0.9–4.40) | 1 (4%) | 2.2 (0.8–6.2) | 0.13 | ||
| Cigarette smoking: | ||||||||
| Yes | 16 (10%) | 6 (6%) | 8 (16%) | 2 (8%) | ||||
| No | 146 (83%) | 86 (86%) | 38 (78%) | 3.0 (0.8–11.1) | 0.07 | 22 (88%) | ||
| Missing | 13 (7%) | 8 (8%) | 3 (6%) | 1 (4%) | 1.3 (0.1–7.9) | 0.67 | ||
| Alcohol: | ||||||||
| Yes | 37 (21%) | 19 (19%) | 14 (29%) | 4 (16%) | ||||
| No | 130 (74%) | 76 (76%) | 33 (67%) | 20 (80%) | ||||
| Missing | 8 (5%) | 5 (5%) | 2 (4%) | 1.7 (0.7–4.0) | 0.21 | 1 (4%) | 0.7 (0.2–2.8) | > 0.99 |
- Note: p values computed using the Fishers exact test.
3.1 Analysis of Fasting Gastric Juice Melatonin Levels
The median level of melatonin in gastric juice was 275.6 pg/mL (IQR, 56.5–500.1 pg/mL). Melatonin levels beyond the upper limit was observed in 58 (33%) of the patients, while in 7 (4%), no levels were detected.
We compared the values for peptic ulcer and gastric cancer patients against those with normal mucosa. The sole patient with a gastric polyp was left out of this part of the analysis. The median melatonin levels for patients with normal gastric mucosa was 141.8 pg/mL (IQR, 46.2–5001), which was significantly lower than that of peptic ulceration patients at 349.5 pg/mL (IQR, 116.6–500.1), p = 0.02. The difference with gastric cancer patients was not statistically significant 255.5 pg/mL (76.6–500.1), p = 0.30. However, comparison between all patients with gastroduodenal pathologies and those without yielded a statistically significant difference, p = 0.02, Table 2.
| n | Median melatonin level pg/mL | Interquartile range pg/mL | p value (compared to normal) | |
|---|---|---|---|---|
| Normal | 100 | 141.8 | 46.2–500.1 | |
| Peptic ulcer (PU) | 49 | 349.5 | 116.6–500.1 | 0.02 |
| Gastric tumor (GU) | 25 | 255.5 | 76.6–500.1 | 0.30 |
| Gastroduodenal pathologies (combined PU and GU) | 74 | 339.4 | 101.8–500.1 | 0.02 |
- Note: The sole patient with a gastric polyp with excluded from this analysis. p values computed using the Kruskal-Wallis test.
We further evaluated associations between fasting melatonin levels and risk factors linked to gastroduodenal pathology in Zambia. We found that the median levels in men, 377.4 pg/mL (IQR, 95.9–500.1) was significant higher levels than in females, 148.9 pg/mL (IQR, 51.0–500.1), p = 0.03. We also found that the median melatonin levels in patients with a history of cigarette smoking 500.1 pg/mL (IQR, 327.9–500.1) were significantly higher than in non-smokers 226.2 pg/mL (IQR, 54.5–500.1), p = 0.003. Table 3.
| Variable | Melatonin level Median (interquartile range) | p value |
|---|---|---|
| Age: | ||
| Less than 45 years | 315.6 (45.6–500.1) | 0.75 |
| 45 years or more | 266.1 (69.4–500.1) | |
| Sex: | ||
| Female | 148.9 (51.0–500.1) | 0.03 |
| Male | 377.4 (95.9–500.1) | |
| Hypochlorhydria: | ||
| Yes | 193.8 (62.1–500.1) | 0.79 |
| No | 319.3 (49.8–500.1) | |
| HIV infection: | ||
| Positive | 272.4 (100.0–500.1) | 0.75 |
| Negative | 300.7 (54.7–500.1) | |
| H. pylori infection: | ||
| Positive | 266.1 (56.5–500.1) | 0.34 |
| Negative | 69.3 (35.3–407.4) | |
| Exposure to biomass smoke: | ||
| Yes | 349.5 (90.1–500.1) | 0.28 |
| No | 203.7 (54.5–500.1) | |
| Smoking: | ||
| Yes | 500.1 (327.9–500.1) | 0.003 |
| No | 226.2 (54.5–500.1) | |
| Alcohol: | ||
| Yes | 379.2 (84.7–500.1) | 0.17 |
| No | 252.7 (54.8–500.1) |
- Note: p values computed using the Kruskal-Wallis test.
Using a multivariable regression analysis which included age, sex, alcohol intake, cigarette smoking and gastroduodenal pathologies, only cigarette smoking was independently associated with melatonin levels, p = 0.04.
4 Discussion
This study evaluated the relationship between levels of fasting melatonin in gastric juice and gastroduodenal pathologies. We found that high levels were significantly associated with these pathologies when using combined analysis. In addition, cigarette smoking was independently associated with higher melatonin levels.
Melatonin is one of the gastrointestinal hormones that have not been extensively studied, and therefore, our understanding of its role in gastrointestinal pathologies remains unclear. To our knowledge, this is the first study that has explored this question in an exclusively African population and we believe it will attract other gastrointestinal scientists on the continent to further investigate melatonin in the gut.
We found no association between high melatonin levels and age, but that females had lower levels than men. This is similar to a study among the Japanese population which found that older females had significantly lower levels of melatonin excretion than their male counterparts [8].
Our group has in the past investigated several risk factors for gastric cancer in Zambia. We previously reported that biomass smoke exposure was a significant risk factor for gastric cancer [9]. In addition, we reported a link to cigarette smoking and being of low socioeconomic status [9, 10]. We have also evaluated molecular characteristics for gastric cancer in Zambia [11, 12]. This however, was the first study in which we considered a gastric hormone as a possible risk factor or biomarker. We did not find a statistically significant association, although the level of melatonin was higher in gastric cancer patients than in those with normal mucosa. The effect could have been limited by the sample size, and therefore a larger study to evaluate this relationship is warranted. Some studies have documented that melatonin can inhibit different types of cancer both in vitro and in vivo [13]. Involvement of melatonin in different anticancer mechanisms including apoptosis induction, cell proliferation inhibition, reduction in tumor growth and metastases have also been reported [5, 6].
We found that individuals with peptic ulceration had higher levels of gastric melatonin than those without. This was despite the main risk factor H. pylori showing no association. Melatonin is a potent reactive oxygen metabolite scavenger and antioxidant that influences many physiological functions of the GIT. It is produced by enterochromaffin cells, playing a protective role on the gastric mucosa. Studies have shown that with mucosal ulceration is associated with elevated expression of melatonin receptors, with others demonstrating that melatonin has a gastroprotective effect against peptic ulceration [14].
Melatonin-induced gastroprotection is characterized by an increase in blood flow, enhancement of mucosal PGE2 generation, luminal NO content and plasma gastrin levels [15]. Melatonin is thought to scavenge reactive oxygen metabolites, exerting anti-oxidizing and anti-inflammatory actions [15]. In addition, it inhibits the formation of metalloproteinases- 3 and -9 both of which are implicated in the pathogenesis of gastric ulcers [15]. One study reported that melatonin causes an 88% reduction of endogenous hydroxyl radicals [16].
It is therefore possible that the high levels we found in our ulcer patients were produced in response physiologically induced healing processes as melatonin plays a role in peptic ulcer healing [17]. Some authors have suggested that because of its unique properties, melatonin could be considered for prevention or treatment of other GIT conditions including colorectal cancer, ulcerative colitis, irritable bowel syndrome, and childhood colic but this required more investigation [1].
Biomass smoke is considered one of the leading environmental risk factors for several diseases [18] and recent studies have demonstrated that it is capable of penetrating the GIT, leading to alterations in gastric histology and physiology. Biomass smoke like cigarette smoke, contains a number of carcinogenic substances that have been linked to certain types of gastrointestinal cancers [19]. Analysis of our results revealed no association between biomass smoke and gastric melatonin levels. By contrast, cigarette smoking was significantly associated with melatonin with levels being higher among smokers than non-smokers. Similar to peptic ulceration, we postulate that the high melatonin among smokers was produced in response to the smoke, in an attempt to dampen its effects. Smoking is known to induce oxidative stress [20], and therefore this could partially explain the increased melatonin levels that we observed. Other researchers reported that melatonin alleviated cigarette-smoke induced endothelial cell pyroptosis through inhibiting the ROS/NLRP3 axis [21].
We found no association between melatonin levels and alcohol intake. However, our questionnaire was not designed to evaluate alcohol intake in great detail. We did not have information on the duration, type or quantities of alcohol being consumed. This limits the interpretation of this finding. The levels of gastric acid at the time of sample collection also showed no association with melatonin levels. However, as this was a cross-sectional study, we did not evaluate diurnal or long-term variations that might influence this relationship.
The main limitation of this study was the fact that we did not have repeated samples for each individual. It is known that hormonal production can be altered by many factors, and therefore, this being a cross sectional study, we were not able to analyze changes of melatonin levels. Secondly, the assay used had an upper limit of 500.1 pg/mL. We therefore, did not have exact values for the high readings, information that could have demonstrated further differences. However, our findings are still valid and helpful, as they provide baseline information that can be built on future. There is need to conduct prospective studies to comprehensively understand the role of melatonin in gastric diseases.
5 Conclusions
Peptic ulceration and a history of smoking are associated with fasting gastric juice melatonin levels. The biological significance of these findings is yet to be established.
Author Contributions
Jonathan Sibande: conceptualization, investigation, visualization, writing – review and editing, writing – original draft. Mutinta Nyama Hankolwe: methodology, validation, visualization, writing – review and editing. Paul Kelly: conceptualization, visualization, writing – review and editing, investigation, supervision, resources. Violet Kayamba: conceptualization, investigation, funding acquisition, writing – original draft, methodology, validation, visualization, writing – review and editing, resources, supervision.
Acknowledgments
This study was produced with the financial assistance of the European Union (Grant no. DCI-PANAF/2020/420-028), through the African Research Initiative for Scientific Excellence (ARISE), pilot programme. ARISE is implemented by the African Academy of Sciences with support from the European Commission and the African Union Commission. The contents of this document are the sole responsibility of the authors and can under no circumstances be regarded as reflecting the position of the European Union, the African Academy of Sciences, or the African Union. No known conflict of interest was encountered during the entire project. The funders had no role in study design; collection, analysis, and interpretation of data; writing of the report; or the decision to submit the report for publication.
Conflicts of Interest
The authors declare no conflicts of interest.
Transparency Statement
The lead author Violet Kayamba affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.
Open Research
Data Availability Statement
Data used in the manuscript can be access upon reasonable request to the corresponding author.
