Metabolomic analysis of the effects of omeprazole and famotidine on aspirin-induced gastric injury
Gastric mucosal ulceration and gastric hemorrhage are frequently associated with treatment by non-steroid anti-inflammatory drugs (NSAIDs); however, no convenient biomarker-based diagnostic methods for these adverse reactions are currently available, requiring the use of endoscopic evaluation. We recently reported five biomarker candidates in serum which predict gastric injury induced by NSAIDs in rats, but were unable to clarify the mechanism of change in the levels of these biomarker candidates. In this study, we performed capillary electrophoresis–mass spectrometry-based metabolomic profiling in stomach and serum from rats in which gastric ulcer was induced by aspirin and prevented by co-administration of omeprazole and famotidine. Results showed drug-induced decreases in the levels of citrate, cis-aconitate, succinate, 3-hydroxy butanoic acid, and O-acetyl carnitine in all animals administered aspirin. In contrast, aspirin-induced decreases in the level of 4-hydroxyproline were suppressed by co-administration of omeprazole and famotidine. We consider that these changes were due to the prevention of gastric ulcer and decrease in the amount of collagen in stomach tissue by omeprazole and famotidine, without prevention of the NSAID-induced depression of mitochondrial function. In addition, the decreases in 4-hydroxyproline in the stomach was also detectable as changes in the serum. While further study is needed to clarify limitations of indications and extrapolation to humans, this new serum biomarker candidate of gastric injury may be useful in the monitoring of NSAID-induced tissue damage.
KeywordsMetabolomics Capillary electrophoresis–mass spectrometry (CE–MS) Gastric injury Non-steroid anti-inflammatory drugs (NSAIDs) Omeprazole Famotidine
Non-steroid anti-inflammatory drugs (NSAIDs) are widely prescribed (Steinmeyer 2000) for rheumatoid arthritis, osteoarthritis, acute pain, and fever (Gabriel and Fehring 1992). Among the adverse effects of these drugs, the most frequent are gastric ulceration and gastric hemorrhage. In particular, the reported relative risk of these events is three times greater in NSAID users than nonusers (Gabriel et al. 1991). Their use is particularly complicated by the fact that their effects in relieving pain may mask these side effects, which can make patients delay seeking medical attention and in turn delay ulcer healing (Hawkey 2000). The only way to detect NSAID-induced gastric ulceration and hemorrhage at present is endoscopy (Steinmeyer 2000). The identification of sensitive serum biomarkers would improve their safe usage.
In our previous paper (Takeuchi et al. 2013), we reported that the metabolic profiles of serum and stomach tissue extract obtained from rats treated with NSAIDs using capillary electrophoresis–mass spectrometry (CE–MS) showed drug-induced decreases in the levels of citrate, cis-aconitate, succinate, O-acetyl carnitine, 3-hydroxy butanoic acid, proline and 4-hydroxyproline. In addition, decreases in the levels of cis-aconitate, O-acetyl carnitine, 3-hydroxybutanoic acid, proline and 4-hydroxyproline in stomach tissue extracts were significantly correlated with similar changes in the serum levels of these compounds. These decreases can be assessed non-invasively in serum, suggesting that these new biomarkers of gastric injury may be useful for monitoring NSAID-induced tissue damage in place of the current invasive practice of endoscopy. With regard to the mechanism of change, we speculated that decreases in levels of cis-aconitate, O-acetyl carnitine, and 3-hydroxy butanoic acid might reflect the NSAID-induced dysfunction of these mitochondrial pathways, which might increase the risk of gastric ulceration. In contrast, we considered that the decreases in proline and 4-hydroxyproline reflected increased collagenase activity and the subsequent decrease in the amount of collagen in stomach tissue.
Metabolomics is a method which quantifies the concentrations of endogenous metabolites in biological fluids using sensitive analytical tools such as MS, and then compares differences between them among samples. Its use in identifying biomarkers is increasing (Xia et al. 2013). In details, the concentration of metabolites is first determined using any of several analytical tools, including MS with gas chromatography (Fiehn et al. 2000; Schauer et al. 2006), liquid chromatography (Plumb et al. 2003) and CE (Monton and Soga 2007; Ban et al. 2012; Ramautar et al. 2013), and nuclear magnetic resonance spectrometry (Nicholson et al. 2002). Second, concentrations of endogenous metabolites are then compared between patients and controls to identify metabolites or metabolic pathways which were changed by the disease or injury. In the present study, we selected CE–MS for metabolite analysis. This approach is currently used to identify biomarkers for various diseases (Soga et al. 2006, 2011; Hirayama et al. 2012), and its efficacy has been clarified.
Here, we applied a shotgun metabolomics approach based on CE–time of flight (TOF)-MS profiles of endogenous metabolites to analyze the effects of omeprazole and famotidine on aspirin-induced gastric injury. Omeprazole and famotidine are known to prevent aspirin-induced gastric ulcer by inhibiting gastric acid secretion (Takeda et al. 1982). We therefore classified the biomarker candidates according to whether levels changed on administration of aspirin or by gastric injury.
2 Materials and methods
2.1 Study design
Male Sprague–Dawley rats were purchased from Charles River Japan (Yokohama, Japan) and housed in plastic cages. They were fed a pellet diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and filtered tap water (containing 2 ± 1 ppm free chlorine adjusted with sodium hypochlorite) and kept under controlled environmental conditions throughout the experimental period. The animals were used when 6 weeks old and weighing 140–180 g, and acclimatized for 3 days prior to entry into the study.
Sixteen animals were randomly divided into four groups, a control group (0.5 % methylcellulose, p.o.) and one of three treatment groups, in which gastric ulcer was induced by single intragastric administration of aspirin (Nacalai Tesque, Kyoto, Japan) at 100 mg/kg. Treatment group rats also received an intragastric administration of famotidine (Wako Pure Chemical Industries, Ltd., Kyoto, Japan) at 5 mg/kg, omeprazole (Wako Pure Chemical Industries, Ltd., Kyoto, Japan) at 60 mg/kg, or vehicle (0.5 % methylcellulose) 30 min before the administration of aspirin.
All studies were conducted at the Kashima Facilities of Astellas Pharma, Inc., which have been awarded Accreditation Status by the AAALAC International. This study was approved by the Animal Ethical Committee of Astellas Pharma, Inc. (Tokyo, Japan).
2.2 Sample collection
Animals were sacrificed at 5 h after aspirin or vehicle administration by exsanguination via the abdominal aorta under anesthetization with isoflurane. Serum was collected in the same way as in our previous paper (Takeuchi et al. 2013). The stomach were quickly removed after exsanguination, incised along the greater curvature, assessed macroscopically, and photographed to determine the dimension of observed gastric ulcers using the WinROOF image analysis software (Mitani Corporation, Tokyo, Japan). The stomachs were weighed, and stored at −70 °C until sample preparation.
2.3 Sample preparation for CE–MS
Sample preparation was conducted in the same way as in our previous paper (Takeuchi et al. 2013). Briefly, to inactivate the enzymes, frozen stomach tissue was homogenized in methanol (1 mL) containing internal standards [10 μM each of methionine sulfone (Wako, Osaka, Japan), d-camphor-10-sulfonic acid (Wako) and 2-(n-morpholino)ethanesulfonic acid (Dojindo, Kumamoto, Japan)]. A 50-μL aliquot of serum was added to 450 μL of methanol containing the internal standards to inactivate the enzymes. Deionized water (200 μL) and 500 μL chloroform were then added and the mixture was centrifuged at 4,600×g for 20 min at 4 °C. An aliquot of the upper aqueous layer was then centrifugally filtered through a Millipore 5-kDa cutoff filter to remove proteins. The lyophilized filtrate was dissolved in 50 μL of Milli-Q water containing reference compounds (200 μM 3-aminopyrrolidine and 200 μM trimesate).
The chloroform used in the extraction is a volatile grade II carcinogen, and all operations involving its use were conducted under a fume hood.
2.4 Metabolite standards
As metabolite standards, we used the same chemicals as those described in our previous paper (Takeuchi et al. 2013). All chemical standards were obtained from common commercial sources. Standards were dissolved in Milli-Q water, 0.1 M HCl, or 0.1 M NaOH to obtain 10 or 100 mM stock solutions. Working standard mixtures were prepared by diluting stock solutions with Milli-Q water just prior to injection into the CE–TOF-MS system. All chemicals used as standards were of analytical or reagent grade.
All CE–TOF-MS experiments were performed using an Agilent 1600 CE system (Agilent Technologies, Waldbronn, Germany), an Agilent G3250AA LC/MSD TOFMS system (Agilent Technologies, Santa Clara, CA, USA), an Agilent1100 isocratic HPLC pump, an Agilent G1603A CE–MS adapter kit, and an Agilent G1607A CE–electro spray ionization (ESI)–MS sprayer kit. For anion analysis, Agilent G7100-60041 platinum needle was replaced with the original Agilent stainless steel ESI needle (Soga et al. 2009). System control and data acquisition were done using the Agilent G2201AA ChemStation software for CE and Analyst QS for TOF-MS.
2.6 CE–TOF-MS conditions for cationic metabolite analysis
Metabolite analysis was conducted in the almost same way as that reported in our previous paper (Takeuchi et al. 2013). Separations were carried out in a fused silica capillary (50-μm inner diameter × 100-cm total length) filled with 1 M formic acid as the electrolyte (Soga and Heiger 2000). Approximately 3 nL of sample solution was injected by applying 50 mbar for 3 s, followed by application of 30 kV. Capillary temperature was maintained at 20 °C, and the sample tray was cooled below 5 °C. Methanol/water [50 % (v/v)] containing 0.1 μM hexakis(2,2-difluoroethoxy)phosphazene was delivered as the sheath liquid at a flow rate of 10 μL/min. ESI–TOF-MS was operated in positive ion mode, and capillary voltage was set at 4 kV. A flow rate of heated dry nitrogen gas (heater temperature, 300 °C) was maintained at 10 psi. In TOF-MS, the fragmenter, skimmer, and Oct RFV voltage were set at 75, 50, and 500 V, respectively. Automatic recalibration of each acquired spectrum was achieved using the masses of reference standards ([13C isotopic ion of a protonated methanol dimer (2MeOH + H)]+, m/z 66.0632) and ([hexakis(2,2-difluoroethoxy)phosphazene + H]+, m/z 622.0290). Exact mass data were acquired at a rate of 1.5 spectra/s over a 50–1,000 m/z range.
2.7 CE–TOF-MS conditions for anionic metabolite analysis
Metabolite analysis was conducted in the almost same way as that mentioned in our previous paper (Takeuchi et al. 2013). For serum analysis, a commercially available COSMO (+) capillary (50-μm inner diameter × 111-cm total length; Nacalai Tesque, Kyoto, Japan) (Katayama et al. 1998), chemically coated with a cationic polymer, was used as the separation capillary. A platinum electrode was used as a substitute for the standard stainless spray needle to prevent galvanic reactions (Soga et al. 2009). A 50 mM ammonium acetate solution (pH 8.5) was used as electrolyte solution for CE separation. Approximately 30 nL of sample solution was injected by applying 50 mbar for 30 s, followed by application of −30 kV. Ammonium acetate (5 mM) in 50 % methanol/water (v/v) containing 0.1 μM hexakis(2,2-difluoroethoxy)phosphazene was delivered as the sheath liquid at a flow rate of 10 μL/min. ESI–TOF-MS was conducted in negative ion mode, with the capillary voltage set at 3.5 kV. For TOF-MS, the fragmenter, skimmer, and Oct RFV voltage were set at 100, 50, and 500 V, respectively. Automatic recalibration of each acquired spectrum was performed using reference masses of reference standards ([13C natural isotopic ion of deprotonated acetic acid dimer (2CH3COOH–H)]−, m/z 120.03834), and ([hexakis + deprotonated acetic acid (CH3COOH–H)]−, m/z 680.03554). Other conditions were identical to those previously reported (Soga et al. 2009). For stomach analysis, a COSMO (+) capillary (50-μm inner diameter × 107-cm total length was used and Oct RFV voltage was set at 200 V). Other conditions were identical to the serum analysis.
2.8 Data processing for generation of the metabolome differential display
Data processing was conducted in the same way as in our previous paper (Takeuchi et al. 2013) using Master Hands ver. 184.108.40.206 (in-house software developed by Keio University). To reduce data analysis time, raw datasets were preprocessed by binning the data along the m/z axis to 0.02 m/z resolution, subtracting the baseline from each electropherogram by robust nonlinear fitting of the data to a seventh order polynomial, and removing the noise from each electropherogram by leveling to 0 all signal intensity values that fell within 5 SD of the signal intensities from 1 to 4 min. The resulting data sets were then further binned to 1 m/z unit resolution along the m/z axis. A set of peaks was selected from each dataset using a modified Douglas–Peucker algorithm, with alignment of datasets along the migration time axis as described in the text. Annotation tables for both cation and anion modes were generated based on the results of CE–TOF-MS analysis of standard compounds. The annotation labels were aligned to the actual datasets in a similar fashion. Arithmetic operations were applied to whole datasets on a data point-by-data point basis to highlight differences of interest. Averaging the datasets within each group allowed visualization of absolute (difference between the corresponding intensities from the averaged datasets) and relative (absolute difference divided by the larger of the two corresponding intensities) differences.
Data was analyzed by principal components analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) to obtain an overview of systematic variations among all observations. The results were analyzed using ANOVA and Dunnett’s test to compare compound levels between groups. PCA, PLS-DA, and analytical tests were performed using SIMCA-P+version 12.0 (Umetrics, AB, Umea, Sweden) and GraphPad Prism version 5.03 (GraphPad Software, San Diego, CA, USA), respectively.
3.1 Gastric ulceration
Severity of gastric ulceration evaluated as area of ulceration
Aspirin with omeprazole
Aspirin with famotidine
(Administered 30 min before test article administration)
Number of animals
Severity of gastric ulceration
5.70 ± 5.43
3.2 Metabolomic analysis of stomach tissue extracts
Statistical analysis of quantitative differences between groups showed decreases in the levels of intermediates of the TCA cycle (citrate and cis-aconitate) in the group administered only aspirin and aspirin with omeprazole. In contrast, no changes were observed in the group administered 100 mg/kg of aspirin with famotidine. The final product and marker of the inhibition of fatty acid beta-oxidation (O-acetyl carnitine and 3-hydroxy butanoic acid) were decreased or tended to be decreased in the all groups administered aspirin regardless of the co-administration of omeprazole or famotidine. The level of 4-hydroxyproline was decreased only in the group administered 100 mg/kg of aspirin, and no changes were observed in the groups administered 100 mg/kg of aspirin with either omeprazole or famotidine. In contrast, proline levels were not changed in any group.
3.3 Metabolomic analysis of serum
Here, to clarify the mechanism of change in biomarker candidates reported in our previous paper, we evaluated the effects of omeprazole and famotidine on aspirin-induced gastric injury using a metabolome differential display based on CE–TOF-MS profiles of stomach tissue extract and serum from rats (Takeuchi et al. 2013). Results showed that co-administration of omeprazole and famotidine had no effect on decreases in levels of citrate, cis-aconitate, succinate, O-acetyl carnitine, and 3-hydroxy butanoic acid in stomach. In contrast, the decrease in the level of 4-hydroxyproline with aspirin recovered to the control level on co-administration with these compounds. Further, the decrease in levels of 3-hydroxy butanoic acid and 4-hydroxyproline in stomach were also detectable as changes in serum. From these results, we speculate that the decrease in 3-hydroxy butanoic acid is thought to reflect the risk of aspirin-induced gastric ulcer and that the decrease in 4-hydroxyproline reflects the induction of aspirin-induced gastric ulcer in serum. If confirmed, monitoring of these biomarker candidates would be useful in identifying the risk and induction of NSAIDs-induced gastric ulcer, and could be expected to replace the current invasive practice of endoscopy.
Decreased prostaglandin production by suppression of cyclooxygenase and mitochondrial dysfunction by NSAIDs are known as causes of gastric ulcer (Lehmann and Beglinger 2005). NSAIDs were known to induce the uncoupling of oxidative phosphorylation, increases resting state respiration, and disrupts mitochondrial transmembrane potential (Petrescu and Tarba 1997; Masubuchi et al. 1999; Mahmud et al. 1996; Tomoda et al. 1998; Somasundaram et al. 1997; Moreno-Sanchez et al. 1999; Mingatto et al. 1996) by the opening of mitochondrial permeability transition pores (Szewczyk and Wojtczak 2002). The loss of ATP by this change results in a reduced ability to regulate normal cellular functions such as the maintenance of intracellular pH and the normal barrier function of mucosa (Lehmann and Beglinger 2005). These changes were observed as decreases in the levels of intermediates of the TCA cycle (citrate and cis-aconitate) and in the level of the final product of fatty acid beta-oxidation (O-acetyl carnitine and 3-hydroxy butanoic acid) in stomach by metabolomic analysis based on CE–MS.
In the present study, although no differences were observed between co-administration of omeprazole, famotidine, and vehicle, the control and other groups were clearly separated in the PCA score plot. These findings suggest that many metabolites were affected by the administration of aspirin, and that only a few metabolites are suitable as biomarkers of NSAID-induced stomach injury. On the other hand, clear separation related to the macroscopic changes in stomach was observed by PLS-DA of stomach and serum. The decrease in 4-hydroxyproline (written as “hydroxyproline” and indicated in red in Online Resources 2 and 5) in both stomach and serum in the animals with gastric ulcers was indicated by the PLS-DA loading plot of stomach tissue extracts and serum. A decrease in 4-hydroxyproline in stomach tissue extracts was seen only in the group administered 100 mg/kg of aspirin, and no changes were observed in the groups administered 100 mg/kg of aspirin with either omeprazole or famotidine. Hydroxyproline is a modified amino acid specifically found in collagen and it is known the amount of collagen in stomach tissue was decreased by NSAID-induced gastric ulcer (Hasebe et al. 1987). We therefore consider that these findings are indicative of decreased collagen levels in the stomach, in previous article (Takeuchi et al. 2013). Given that a decrease in 4-hydroxyproline level was observed only in those animals in which gastric ulcers were induced, 4-hydroxyproline appears to be a marker for the detection of gastric ulcer induction.
We also performed targeted analysis of the biomarker candidates reported in our previous paper (Takeuchi et al. 2013). The final products and markers of fatty acid beta-oxidation (O-acetyl carnitine and 3-hydroxy butanoic acid) were decreased in all groups administered aspirin regardless of the co-administration of omeprazole or famotidine. This implies that coadministration with these drugs did not inhibit the suppression of fatty acid beta-oxidation by aspirin.
In addition to this, the levels of intermediates of the TCA cycle (citrate and cis-aconitate) were also decreased in the group administered aspirin only, and aspirin with omeprazole. In contrast, no changes were observed in the group administered 100 mg/kg of aspirin with famotidine. Although the effects of famotidine on the TCA cycle have not been reported, given that indomethacin, another NSAID which induces gastric ulcer, is reported to decrease levels of 2-oxoglutarate, another intermediates of the TCA cycle in urine, and that this effect was blocked by co-administration with cimetidine, another H2 blocker which can ameliorate NSAID-induced gastric ulcer (Um et al. 2012), we consider that H2 blockers, including famotidine, have potential in preventing the NSAID-induced suppression of the TCA cycle.
Proline is another amino acid found in collagen, but no decrease in the levels of this amino acid was observed. In a previous study, in contrast, a decrease in proline levels in the stomach was observed in rats given aspirin at 300 mg/kg. In the present study we used aspirin at the lower dose of 100 mg/kg because the gastric ulcer induced at 300 mg/kg is too severe to be prevented by omeprazole or famotidine. The difference in the results of these studies might therefore be due to the difference in aspirin dose.
In serum, while decreases were observed in the levels of 3-hydroxy butanoic acid and 4-hydroxyproline, those of citrate, cis-aconitate, O-acetyl carnitine, and proline showed no change. In our previous study, we reported that all these metabolites were decreased in serum with aspirin at 300 mg/kg. We therefore speculate that he difference in aspirin dose between these studies also explains the difference in the response of these metabolites in serum. Given that the serum levels of citrate, cis-aconitate, O-acetyl carnitine, and proline were not changed, these marker candidates are considered to lack sensitivity in the detection of NSAID-induced gastric ulcer. In contrast, levels of 4-hydroxyproline clearly decreased, indicating that this metabolite can be used as a new serum biomarker to monitor the induction of NSAID-induced tissue damage.
5 Concluding remarks
In this study, we evaluated the effects of omeprazole and famotidine on gastric injury induced by aspirin using a metabolomic analysis of stomach tissue extract from rats. The results showed that the decrease in levels of 4-hydroxyproline in stomach induced by aspirin recovered to the control level on co-administration with omeprazole and famotidine. In contrast, the levels of O-acetyl carnitine, 3-hydroxy butanoic acid, citrate, and cis-aconitate in stomach did not change on co-administration with these agents. Given that the changes in 4-hydroxyproline were considered due to a decrease in the amount of collagen in stomach tissue by lesions in the stomach, whereas the changes in O-acetyl carnitine, 3-hydroxy butanoic acid, citrate, and cis-aconitate were considered due to NSAID-induced depression of mitochondrial function, omeprazole and famotidine were able to prevent the decrease in the amount of collagen in stomach tissue by lesions in the stomach but not to prevent NSAID-induced depression of mitochondrial function. In addition, the change in 4-hydroxyproline levels was also observed in serum. We therefore conclude that decreases in the serum level of 4-hydroxyproline is biomarker which indicate the induction of gastric ulcer by NSAIDs.
We are grateful to Eisuke Kobayashi and Yutaka Nakahara for their technical assistance in caring for the rats, preparing samples, and estimating the dimensions of observed gastric ulcers.
All institutional and national guidelines for the care and use of laboratory animals were followed.
Conflict of interest
The authors have no conflict of interest of any kind related to the work presented in this publication. This study was carried out with support from a Grant from the Ministry of Health, Labour and Welfare, Drug Discovery Platform Research (H20-bio-ippan-011).
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