A peptic ulcer results from an imbalance between damaging forces of gastric acid-pepsin and gastro-duodenal mucosal defense barriers (
Metabolomics refers to the study of endogenous and exogenous metabolites in biological systems to provide comparative semi-quantitative information about all metabolites in the system ranging from cell cultures to human biological fluids such as urine, saliva, tissue, and blood. Metabolomics is an emerging and potentially powerful tool in discovering biomarkers and identifying perturbed pathways due to disease, which can clarify the mechanism of action (
In the present study, we hypothesized that the GC/MS-based inclusive analysis of tissue metabolites symbolizes the markers that can discriminate the PUD group from the normal mucosal group. Our study demonstrated major metabolic perturbations in the antral gastric regions of
Institutional Ethics Committee at Punjabi University, Patiala, has approved the protocol of this study (vide IEC approval no. IEC/05/09/15). As per protocol approved by the review board, written informed consent was taken from all the subjects prior to sample collection. The study using human subjects was performed in accordance with the approved ICMR guidelines and regulations.
Antral biopsies from peptic ulcer patients (N = 72) and healthy volunteers with normal mucosa (N = 20) were collected from freshly isolated resections using Upper Gastrointestinal Endoscopy (UGIE) carried out by trained professionals at Government Medical College & Hospital, Sector-32, Chandigarh (Punjab, India).
The tissue biopsies were collected from the margin of the ulcer, cleaned using saline solution, and finally using liquid nitrogen were snapped-frozen within 30 min of collection. The collected biopsies were then investigated for
Fifteen milligrams of antral biopsy samples were transferred to 2.0 mL Eppendorf tube and extracted with 500 μL methanol/chloroform solvent in a ratio of 3:1 (v/v) and vortexed for 30 s. The samples were then ultrasonicated (30% amplitude, with pulses 30 s ON, 10 s OFF) for 15 min and vortex-mixed for 30 s. Samples were stored for 10 min at −20°C and then centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was collected and transferred to a fresh vial and stored at −20°C until analysis. For GC/MS analysis, samples were prepared by adding 100 μL of ethyl acetate (EA) to each tissue extract and vortex-mixed for 1 min. Then, 100 μL of derivatization reagent, consisting of a mixture (3:1:1, v/v/v) of N, O-bis(trimethylsilyl) trifluoroacetamide (BSTFA), pyridine, and EA, was added, and samples were incubated for 16 h at room temperature. The resulting solution was then vortexed for 1 min, and GC/MS analysis was performed.
Using Perkin Elmer Clarus 500 Gas Chromatography system outfitted with DB-5MS capillary column (25 × 0.2 mm ID, 0.25 μm film thickness) having Perkin Elmer Clarus 500 annual injector analysis was performed. Helium at a constant flow rate of 1.0 mL/min was used as the carrier gas. An aliquot (1 μL) of each of the derivatized solutions was injected in splitless mode. The temperature of the inlet (270°C), transfer line (260°C), ion source (200°C) and quadrupole (150°C) were maintained, respectively. The chromatogram temperature program was run at 80°C (isothermal heating) for 2 min, followed by the sequential rise in oven temperature at the rate of 10 °C/min to 180°C, 5 °C/min to 240°C, 10 °C/min to 260°C and finally maintained at 280°C for 5 min. Data acquisition in MS was achieved in an electron impact mode at 60 eV and scanned for m/z values ranging from 5 to 400 with an acquisition rate of 20 spectra/s with 6 min of solvent delay time (
After analysis, each sample was symbolized by a GC/MS total ion current (TIC) chromatogram constituting various peaks. For further study, peaks detected in at least 80% of the samples were considered and analyzed by comparing their respective MS spectra and retention indices with reference data in the Mass Spectral Library of the National Institute of Standards and Technology (NIST) (Wiley Registry, 9th edition, 2008). The mass spectra of each metabolite were vigilantly investigated, and compounds with matching probability >80% were considered for further study. The data in context to ion intensities of respective detected peaks were normalized and then exported to PAST (PAleontological Statistics, Version 3.24) to perform principal component analysis (PCA) with 95% confidence ellipses, where distinctly separate alignment trend was observed in the data. Further, using statistically significant metabolites only (
For the present study, 20 healthy volunteers were enrolled, keeping in view the exclusion criteria viz a viz no previous medical history related to metabolic disorders or diet and stress-related illness and no intake of NSAIDs, antibiotics, or any kind of drug for at least six months prior to the current investigation. On the other hand, a total of 45
The presence of
Under optimal GC/MS conditions as mentioned earlier, typical representative total ion chromatograms (TIC) of tissue samples were derived for peptic ulcer patients and the normal controls as presented in
(A) Chromatogram of normal mucosa, and (B) chromatogram of peptic ulcer tissue.
No. | RT | Compound | Observed m/z values | Match | FC | ||
---|---|---|---|---|---|---|---|
Amino acids, peptides, and analogues | |||||||
1 | 6.13 | β-alanine | 81.1 | 99 | 3.14 | 0.035 | 0.061 |
2 | 7.49 | l-Aspartic acid | 70.4 | 82 | −0.096 | 0.046 | 0.065 |
3 | 8.06 | l-Arginine | 48.1 | 99 | NA | NA | NA |
4 | 9.32 | l-Valine | 120.0 | 97 | 0.662 | ** | *** |
5 | 10.12 | Glycine | 67.0 | 86 | −0.111 | 0.001 | 0.011 |
6 | 11.21 | l-Isoleucine | 132.1 | 98 | 13.353 | 0.0001 | 0.002 |
7 | 13.35 | l-Leucine | 73.1 | 96 | 0.336 | ** | *** |
8 | 14.05 | l-Alanine | 90.0 | 80 | −0.613 | 0.003 | 0.018 |
9 | 15.90 | l-Threonine | 72.1 | 80 | −0.080 | 0.04 | 0.058 |
10 | 19.80 | l-Proline | 116.07 | 80 | −0.059 | 0.001 | 0.009 |
11 | 21.01 | l-Cysteine | 168.2 | 85 | 0.498 | 0.001 | 0.008 |
12 | 22.96 | l-Glutamine | 129.0 | 80 | 1.831 | 0.03 | 0.054 |
Carbohydrates and carbohydrate conjugates | |||||||
13 | 6.35 | Arabinose | 204.0 | 83 | 0.028 | 0.025 | 0.046 |
14 | 7.64 | β-D-Galactopyranoside | 217.06 | 96 | 1.128 | 0.024 | 0.046 |
15 | 16.14 | Xylonic acid | 144.1 | 94 | −0.1 | 0.02 | 0.039 |
16 | 26.77 | Meglumine | 41.1 | 83 | −0.907 | ** | *** |
17 | 28.90 | D-Mannose | 58.2 | 98 | NA | NA | NA |
18 | 31.85 | D-Glucose | 207.0 | 82 | 1.498 | 0.04 | 0.065 |
19 | 32.09 | Arabinopyranose | 206.1 | 80 | 0.995 | 0.01 | 0.034 |
Fatty acids and conjugates | |||||||
20 | 13.98 | Valeric acid | 71.0 | 88 | 3.857 | 0.001 | 0.007 |
21 | 14.77 | Dodecanamide | 147.0 | 94 | 0.068 | 0.039 | 0.065 |
22 | 15.50 | 2-Dodecenol | 43.1 | 91 | NA | NA | NA |
23 | 16.54 | Palmitic acid | 78.1 | 94 | 0.021 | 0.02 | 0.050 |
24 | 17.18 | 1-Hexadecen-3-ol | 62.0 | 93 | 2.686 | 0.002 | 0.013 |
25 | 18.65 | Nonahexacontanoic acid | 47.1 | 83 | 1.309 | 0.003 | 0.016 |
26 | 19.11 | Stearaldehyde | 48.3 | 92 | −0.059 | ** | *** |
27 | 20.66 | Stearic acid | 101.2 | 80 | 6.042 | 0.01 | 0.033 |
28 | 22.07 | Arachidonic acid | 254.1 | 90 | −0.645 | ** | *** |
29 | 23.00 | Arachidic acid | 68.1 | 89 | NA | NA | NA |
30 | 27.31 | Caprylic acid | 189.1 | 96 | NA | NA | NA |
31 | 30.60 | Propionamide | 73.0 | 80 | 0.674 | 0.04 | 0.063 |
32 | 31.13 | 1-Heptadecanol | 62.0 | 80 | 0.155 | 0.003 | 0.013 |
Organic acids and derivatives | |||||||
33 | 11.73 | Butyric acid | 69.1 | 85 | NA | NA | NA |
34 | 6.62 | Propionic acid | 73.1 | 84 | 3.024 | 0.058 | 0.077 |
35 | 7.29 | Methylmalonic acid | 68.1 | 97 | NA | NA | NA |
36 | 7.79 | Acrylic acid | 79.0 | 80 | −0.015 | 0.01 | 0.026 |
37 | 7.86 | β-hydroxy pyruvic acid | 132.1 | 91 | 9.961 | 0.0001 | 0.002 |
38 | 10.68 | Maleamic acid | 152.4 | 93 | 0.416 | 0.01 | 0.031 |
39 | 13.83 | l-(+)-Lactic acid | 187.1 | 99 | NA | NA | NA |
40 | 14.59 | α-ketoglutaric acid | 176.0 | 90 | −0.072 | 0.004 | 0.016 |
41 | 14.94 | Eicosyl acetate | 88.0 | 93 | 15.922 | 0.0002 | 0.003 |
42 | 19.56 | Hydrocinnamic acid | 198.5 | 86 | −0.582 | ** | *** |
43 | 28.36 | 1,2-benzenedicarboxylic acid | 201.4 | 88 | 0.047 | 0.03 | 0.054 |
44 | 29.15 | Thioglycolic acid | 145.1 | 88 | 2.136 | 0.05 | 0.068 |
45 | 33.67 | Glycinamide | 69.5 | 80 | −0.343 | 0.02 | 0.048 |
Others | |||||||
46 | 5.57 | Bicarbamamide (Biurea) | 41.1 | 87 | 4.34 | ** | *** |
47 | 6.86 | Phenylephrine | 68.0 | 80 | −0.997 | 0.006 | 0.022 |
48 | 7.17 | Phenanthrenol | 45.0 | 80 | −0.863 | 0.003 | 0.013 |
49 | 7.93 | 4-Hydroxy-2,6-dimethyl heptane | 40.1 | 81 | NA | NA | NA |
50 | 7.98 | β-mercaptoethylamine |
167.2 | 89 | NA | NA | NA |
51 | 8.49 | 3,5-dimethyl |
76.1 | 93 | 9.09 | 0.01 | 0.027 |
52 | 8.51 | 2-phenoxyethanol | 43.0 | 88 | −0.435 | ** | *** |
53 | 8.79 | Butanetriol | 63.1 | 81 | 1.254 | ** | *** |
54 | 9.02 | D-Sphingosine | 97.2 | 90 | −0.749 | ** | *** |
55 | 11.79 | Oxetane | 41.0 | 81 | −0.550 | 0.0001 | 0.003 |
56 | 11.80 | Anthrone | 63.0 | 84 | 0.842 | 0.047 | 0.065 |
57 | 12.09 | N-Docosane | 58.1 | 96 | −0.994 | 0.02 | 0.046 |
58 | 12.29 | Phenol | 87.0 | 84 | −0.185 | ** | *** |
59 | 13.53 | Phosphate | 52.1 | 80 | −0.696 | 0.005 | 0.019 |
60 | 14.56 | R-(ar)-Tumerone | 67.1 | 80 | NA | NA | NA |
61 | 15.48 | (R)-Pantolactone | 77.1 | 83 | 5.130 | ** | *** |
62 | 16.27 | Dodecane | 87.1 | 94 | 0.407 | 0.01 | 0.030 |
63 | 18.26 | Coumarin | 58.0 | 80 | 1.503 | 0.02 | 0.045 |
64 | 18.59 | Oxolane | 61.1 | 82 | −0.853 | 0.01 | 0.028 |
65 | 20.14 | Naphthalene | 127.0 | 84 | NA | NA | NA |
66 | 23.53 | Guanosine | 69.0 | 80 | 0.601 | 0.02 | 0.043 |
67 | 25.14 | Halostachine | 49.0 | 85 | −0.223 | 0.04 | 0.062 |
68 | 25.32 | Oleanitrile | 67.0 | 83 | NA | NA | NA |
69 | 27.86 | Oct-3-ene | 78.1 | 97 | 0.180 | 0.035 | 0.060 |
70 | 28.85 | Cholesterol | 378.1 | 94 | NA | NA | NA |
71 | 29.76 | dl-Amphetamine | 203.1 | 82 | −0.956 | ** | *** |
72 | 30.08 | Norephedrine | 143.0 | 91 | NA | NA | NA |
73 | 31.90 | 1-Decene | 71.1 | 89 | −0.871 | ** | *** |
74 | 32.58 | Peroxyergosterol | 219.1 | 89 | NA | NA | NA |
75 | 33.73 | β-phenyl ethylamine | 103.1 | 80 | 0.248 | 0.02 | 0.042 |
Note: **Insignificant
NA, Not Applicable (FC cannot be calculated because these metabolites are absent in normal gastric metabolic profile except cholesterol, which is absent in PUD group but present in normal one).
Contrastingly, 15 metabolites viz a viz 4-hydroxy-2,6-dimethyl heptane, arachidic acid (C20:0), butyric acid (C4:0), caprylic acid (C8:0), D-mannose, l-(+)-lactic acid, l-arginine, methylmalonic acid, naphthalene, norephedrine, oleanitrile, peroxyergosterol, R-(ar)-tumerone and cysteamine were generated in ulcer tissues but absent in normal mucosal tissue while cholesterol is the most depleted metabolite in ulcer tissues. However, four metabolites, i.e., naphthalene, oleanitrile, peroxyergosterol and R-(ar)-tumerone were not considered for further study as these are main constituents of different food sources and moreover not reported in human gastric metabolic profile yet. Further, the PCA scatter plot, as shown in
The figure illustrates the scatter plot of PCA analysis for tissue metabolomic data. The normal mucosa group tends to cluster to the left region, while the PUD tissue group generally clustered to the right area. The analysis demonstrated satisfactory modeling and achieved a fairly distinct separation between the metabolite profiles of the two groups.
To investigate differences in the biomolecular composition of the peptic ulcer and normal mucosal samples, the Wilcoxon test was performed on statistically significant hits (
Metabolic profiling of gastric cancer using various analytical techniques has been reported earlier (
From the GC/MS analysis, several marker metabolites related to peptic ulcer disease have been discovered. Several intermediates of “Aerobic Glycolysis” (glucose, β-D-galactopyranoside, l-(+)-lactic and β-hydroxypyruvic acid) were found to be up-regulated in the peptic ulcer tissue, which is also supported by a previous study on gastric cancer (
Under excess glucose conditions, rapidly proliferating cancer cells tend to be highly glycolytic (Crabtree Effect). Due to disruption of oxidative phosphorylation, they tend to become more sensitive to mitochondrial dysfunctioning (
Oxidative stress is involved in natural aging and some human diseases like neurodegeneration, multiple sclerosis, cardiovascular disease, cancer, and rheumatoid arthritis. Elevated levels of reactive oxygen species (ROS), producing free radicals, results in molecular and tissue damage triggering a chain of destruction to promote oncogenic transformation; thus, free radicals have proven to be a useful index for depicting biological markers
PUD Biomarkers | |||
---|---|---|---|
Amino |
l-Isoleucine |
Organic Acids | Butyric acid |
Fatty Acids | Stearic acid (C18:0) |
Others | β-mercaptoethylamine |
Elevated amino acids are the major contributing factors in cancer cells; specifically, many cancer cell lines require l-glutamine, a well-known nutrient for their survival even under metabolic stress (
Glutamine basically tends to control the master regulator of protein translation mTORC1 required for anabolic growth of cells. In addition, it acts as a nitrogen donor for various metabolic enzymes and
The homeostasis of amino acids has been regulated by two protein kinases, namely mechanistic target of rapamycin complex 1 (mTORC1) and general control non-derepressable 2 (GCN2), required for cell growth and proliferation in response to numerous growth and stress signals. Increased endogenous levels of l-arginine and l-cysteine significantly activate mTORC1 (sensor of arginine and leucine) and GCN2 kinases. In response to amino acid-uncharged tRNA, kinases result in inhibition of protein translation that further induce the elevation of the cellular amino acid pool (
Organic acids such as l-(+)-lactic acid, methylmalonic acid and butyric acid (fatty acid intermediate) emerged as important biomarkers of PUD. Accumulation of lactic acid and disruption of TCA cycle as reported earlier in cases of colorectal, gastric, liver, and invasive ovarian cancers by
Another prominent observation recorded in the present study was the perturbation of fatty acid metabolism in ulcer tissues. Metabolomic fingerprinting revealed significant enhancement in the levels of saturated fatty acids (arachidic acid, caprylic acid, nonahexacontanoic acid, stearic acid and valeric acid) significantly increases in PUD tissues; while the reduction in the endogenous level of unsaturated fatty acid (arachidonic acid) was reported as previously observed in case of lung cancer and non-Hodgkin lymphoma (
Elevated endogenous levels of methylmalonic acid (MMA) observed in PUD tissue specimens might be because of its inability to convert into succinyl CoA signifying vitamin B12 deficiency (
Cysteamine (also known as β-mercaptoethylamine) is a biological compound produced in the gastrointestinal tract and hypothalamus of all animals acting upon the somatotrophic axis. It helps in improving nutrient digestion and absorption by enhancing portal drained visceral blood flow and net portal absorption. Further, cysteamine also downregulates the secretion of gastroenteropancreatic plasma and an inhibitory hormone named somatostatin (
In summary, by using a combinational approach involving highly sensitive analytical techniques and multivariate and univariate statistical tests, significant metabolic shifts and perturbations of amino acids, carbohydrate, fatty acids, organic acids, and sterols were identified in the case of peptic ulcer tissues. The results obtained in the study revealed the potential of the MS-based robust metabolic profiling approach for early diagnosis of PUD and contributed substantial evidence to understand pathophysiological responses during the development of PUD in humans. However, the study could not provide a direct correlation of age, gender, diet, and smoking with the development of PUD, as the cohort size was very limited.
The results obtained in the study could be utilized to develop serosurvey kits for early diagnosis of PUD using marker metabolites excreted in blood or urine of the
Authors also acknowledge the Department of Medicine and Department of Pathology, Government Medical College & Hospital, Chandigarh for providing biopsy samples of concerned patients and histopathology investigation with informed consent for the research purpose; and Sophisticated Analytical Instrument Lab, Thapar University, Patiala (PB), India for providing GC/MS facility. The authors would like to extend their sincere appreciation to the Researchers Supporting Project No. (RSP-2020/19), King Saud University, Riyadh, Saudi Arabia.
Group | Total Patients enrolled | Cases under study | Gender | Age (Years) | BMI | Diet | Smoking | Alcohol | Psychological | |||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Median | IQR | Median | IQR | V | NV | Consumption stress | ||||||
20 | 20 |
Male (N = 8) | 36 | 21–56 | 24.75 | 17.2–28.3 | 7 | 1 | 0 | 0 | 0 | |
Female (N = 12) | 45 | 28–70 | 19.35 | 17.9–29.5 | 10 | 2 | 0 | 0 | 0 | |||
72 | 45 |
Male (N = 35) | 39 | 20–70 | 20.3 | 13.8–29.8 | 19 | 16 | 15 | 22 | 4 | |
Female (N = 10) | 42 | 28–70 | 20.9 | 17.2–28.3 | 2 | 0 | 2 | 0 | 3 |
Note: *BMI = Body Mass Index; V = Vegetarian; NV = Non-vegetarian; IQR = Interquartile range.