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Nuclear Magnetic Resonance Analysis of Indomethacin-Induced Gastric Ulcers Saadallah Ramadan, Antonio M. Bonin, Brendan J. Kennedy, Trevor W. Hambley, and Peter A. Lay* Centre for Heavy Metals Research, School of Chemistry, University of Sydney, NSW, 2006, Australia Received July 21, 2004
Acetonitrile extracts of ulcerated and control rat stomachs were studied by various NMR techniques in an attempt to understand how indomethacin, a common and powerful nonsteroidal antiinflammatory drug (NSAID), induces ulcers in the stomach. One- (1D) and twodimensional (2D) NMR spectra of extracts of ulcerated and control stomachs revealed that glycolytic and Krebs cycle enzymes were partially inhibited in the ulcerated stomach as shown by the lactate/glucose ratio. The (total choline)/lactate ratio was also higher in the extract from the control stomach than in the ulcerated stomach. Glycerophosphoethanolamine and glycerophosphocholine concentrations were higher in the ulcerated stomach extract as compared with the control stomach extract. These results explain the gastrointestinal protective effect of D-glucose and Krebs cycle intermediates on NSAID-induced ulceration.
Introduction Two of the main causes of gastric ulcers are Helicobacter pylori bacterial infection and prolonged use of nonsteroidal antiinflammatory drugs (NSAIDs).1 The present work is concerned with NSAID-related ulcers since H. pylori has never been detected in rats (1). Some of the biochemical changes that are associated with NSAID-induced ulcers are briefly discussed below. It has been proven that uncoupling of mitochondrial oxidative phosphorylation (or inhibition of electron transfer) underlies the damage resulting from micromolar indomethacin (IndoH) dosing (2). Alkaline phophatase (brush border marker) is also deactivated after IndoH dosing. The activities of certain mitochondrial marker enzymes [succinate (Suc) dehydrogenase, monoamine oxidase, glutamate dehydrogenase, D-β-hydroxybutyrate dehydrogenase, and citrate synthase] were increased, which is yet to be explained. The glutamate/malate ratio was also inhibited in uncoupled mitochondria (2). The carboxylic acid group also seems to be associated with the uncoupling activity, which is associated with acidic NSAIDs, since the damage is minimized in the ester derivatives of the acidic NSAIDs or in nonacidic NSAIDs. Acidic NSAIDs also tend to inhibit mitochondrial respiration after uncoupling mitochondrial oxidative phosphorylation (3). Esterified or nonacidic NSAIDs are ineffective as mechanical proton translocators, which explains their improved gastrointestinal tolerability (4). Uncoupling of oxidative phosphorylation usually results in diminished cellular ATP production, cellular calcium toxicity, and the production of reactive oxygen species resulting in increased mucosal permeability. * To whom correspondence should be addressed. Tel: 612 9351 4269. Fax: 612 9351 3329. E-mail:
[email protected]. 1 Abbreviations: Cho, choline; CMC, carboxymethyl cellulose; Cr, creatine; For, formate; Fum, fumarate; Glyn, glycogen; GPE, glycerophosphoethanolamine; GPC, glycerophosphocholine; IndoH, indomethacin; Lac, lactate; Nico, nicotinamide; NSAID, nonsteroidal antiinflammatory drug; PCh, phosphocholine; Pi, inorganic phosphate; Sar, sarcosine; Suc, succinate; Tau, taurine; TMP, trimethyl phosphate.
IndoH is a commonly used and powerful NSAID that is believed to act by inhibiting cyclooxygenase and, consequently, prostaglandin synthesis, but it also causes severe gastric ulceration in a certain percentage of the population (5). Recently, selective cyclooxygenase 2 (COX-2) inhibitors have made a significant impact on the antiinflammatory market for NSAIDs because they have much lower GI toxicities than drugs such as IndoH; however, the early promise held for this class of drugs has been diminished by findings that they induce ulcers as well as exacerbate preexisting ulcers and also cause renal damage, elevated blood pressure, and platelet aggregation (5). It is widely believed that IndoH gastrointestinal toxicity is a consequence of inhibition of cyclooxygenase 1 (COX-1), while the analgesic and antiinflammatory therapeutic effects are due to COX-2 inhibition. Biochemical effects that result from NSAID damage include uncoupling of oxidative phosphorylation (6) and COX inhibition (leading to a decrease in prostaglandin production, as illustrated in Figure 1) (4). Prostaglandin PGE2 has been proven to stimulate glucose metabolism (7). NSAIDs act principally on the intestinal absorption and secretion of sodium and chloride ions, which, in turn, relate to the decrease in prostaglandins. Other possible NSAID effects include inhibition of cAMP-dependent protein kinase and increase in levels of lipoxygenase products produced by the diversion of arachidonate as a consequence of prostaglandin COX inhibition (8). Production of the energy transfer compound, ATP, declines as a result of treatment with NSAIDs, which can be explained by the NSAID inhibition of enzymes involved in glycolysis and tricarboxylate (Krebs) cycle metabolism (9). The first in-vivo one-dimensional (1D) 31P NMR spectrum was acquired by Nakada et al. (10) by means of a custom-built zigzag surface coil. Control and IndoHinduced ulcerated stomachs were studied, and it was observed that the relative amounts of high energy phosphate compounds (PCr and ATP) were reduced in
10.1021/tx049806t CCC: $30.25 © 2005 American Chemical Society Published on Web 01/19/2005
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Figure 1. COX-1 catalyzes the conversion of arachidonic acid to prostaglandin.
Figure 2. Control (left) and IndoH-induced ulcerated stomachs (right).
the ulcerated stomach relative to the control stomach (10). In this work, we shed some light on the biochemical markers that result from IndoH-induced gastric ulcers in rats. An understanding of these changes may assist in the improved design of drugs aimed at reducing the adverse side effects that are usually associated with IndoH usage.
Experimental Procedures Animals. Male pathogen-free Sprague-Dawley rats, 6-8 weeks old, weighing 200-250 g, were the source of stomach tissue used in the present study (University of Sydney Animal Ethics Approval no: L07/6-02/2/3575). Rats were fasted overnight before being dosed with IndoH (10 mg/kg) in 2% CMC by gastric gavage (without sedation). Three hours after dosing, rats were sacrificed by cervical dislocation and their stomachs were removed, opened, and washed with water. Ulcers were abundant (in the range of 25-35 mm2 out of ∼4.5 cm2 of total stomach area) and macroscopically identifiable in the rats that received an IndoH dose, whereas control stomachs were free of lesions (Figure 2). Stomachs were then cut into small pieces with a pair of scissors and mechanically homogenized in an acetonitrile suspension. One advantage of acetonitrile extraction over other types of aqueous extraction methods (e.g., ice-cold perchloric acid extraction, among others) is the improved solubility of metabolites that are less soluble in aqueous media, as well as the enhanced spectral resolution due to the absence of any paramagnetic compounds (11, 12). No precipitates were detected in the final NMR sample solutions. The final suspension was centrifuged, and the supernatant was lyophilized. The residue was reconstituted in D2O to provide field frequency lock in the NMR experiments. The pH value was adjusted to 7.4 at 25 °C in all NMR measurements. NMR Experiments. All spectra were obtained with a Bruker DRX-400 MHz narrow bore NMR spectrometer, using a CHP z-gradient probe, with the temperature unit set to 300 K. Chemical shifts are expressed relative to the anomeric resonance of R-glucose (1H, δ 5.233; 13C, δ 92.9) (13). For 1H-13C correlation experiments in D2O solutions, standard Bruker (Xwin-NMR version 3.5) pulse programs were used without modification. Excellent water suppression was achieved by means of excitation sculpting with magnetic field gradients (14). Spectral widths were routinely 6 kHz for 1H and 28 kHz for one-bond and for long-range C-H correlation experiments. All two-dimensional
(2D) experiments incorporated a relaxation delay of 2 s. The majority of resonance assignments were made with the data obtained from a z-gradient homonuclear correlation spectroscopy (COSY) experiment acquired with 1k increments of 4k data points and 32 scans per increment, a gradient-HSQC experiment (inverse correlation via double inept transfer) (15) for 1k increments of 8k data points and 40 scans per increment, and a gradient-assisted heteronuclear multiple-bond correlation (HMBC) experiment acquired with 1024 increments of 4k data points and 32 scans per increment. All experiments involved pulsed field gradients having a duration of 1 ms, were sinusoidal in shape, and were amplified by a Bruker Gradient Amplifier. The one-bond and long-range correlation experiments were optimized for 1JCH of 145 Hz and nJCH of 6.25 Hz, respectively. For the 1D carbon spectra, 27 000 scans were accumulated with a repetition time of 2 s. COSY spectra were routinely processed with zero-filling to 4k data points and multiplying with a sine bell window function in each dimension. Heteronuclear 2D experiments were also processed with zero-filling to 4k data points and multiplying with an exponential window function in each dimension. Evaluations of concentrations were obtained from fully relaxed 1H spectra (22 s intertransient delay) acquired over 64k data points over a spectral width of 6410 Hz. Phosphorus spectra were acquired in inverse-gated mode using a 90 ° pulse (21 µs), a recycle time of 50 s to allow for full magnetization recovery, 150 transients, and 6k data points. The spectral width was 3000 Hz. The free induction decay was zerofilled to 64k data points with a line broadening factor of 2 Hz applied before Fourier transforming the raw data. To allow for phosphorus quantification, 5 mM trimethyl phosphate [P(O)(OCH3)3, TMP, Aldrich] was added to ulcerated and control stomach extracts.
Results One-dimensional, fully relaxed, and water-suppressed H spectra of ulcerated and control stomach extracts are shown in Figure 3, whereas COSY and HSQC 2D spectra are shown in Figure 4. Detailed analysis of 1D and 2D NMR spectra (16-19) revealed the contents of the extracts that are listed in Table 1. It is worth noting that the 1H NMR spectra of control and ulcerated stomach extracts were very similar in content, but the relative amounts of some metabolites were significantly different. Table 2 shows that the lactate (Lac)/glucose ratio (0.016) present in the ulcerated stomach was less than that observed in the control stomach (0.028). The Suc/Lac ratio in ulcerated stomachs was also less than that in control stomach. The total choline (Cho)/Lac (Table 3) ratio was higher in the control stomach than in the ulcerated stomach. In control stomachs, the concentration of Cho was more than four times that of available glycerophosphocholine (GPC), whereas in the ulcerated stomachs, Cho was only 33% higher than the concentration of GPC. The Cho/Lac and total Cho/Lac ratios were higher in the control stomach than in the ulcerated stomach. 1
NMR Studies of Indomethacin-Induced Gastric Ulcers
Figure 3. Fully relaxed 400 MHz 1H NMR spectra of control (A) and ulcerated (B) acetonitrile extracts of rat stomachs in D2O. Metabolite assignments are also shown. The intensity comparison is only valid within each spectrum.
Figure 5 shows the 31P NMR spectra of the stomach extracts, and the corresponding spectral analysis is shown in Table 4. The concentration of glycerophosphoethanolamine (GPE) in the ulcerated stomach was more than double that found in the control stomach. A major difference between the 31P spectra of ulcerated and control stomachs was a higher concentration of inorganic phosphate (Pi) in the ulcerated stomach extract, which was more than three times the concentration of Pi found in the control stomach, as determined by 31P NMR spectra of the stomach extracts.
Discussion NMR Spectra. The high degree of signal overlap (Figure 3) makes the assignment of signals and their integration very difficult, if not impossible. Signal overlap can also be a significant problem in 1H-1H and 1H-13C correlation 2D spectra. 1H-13C HSQC can be very useful, however, since the 1H chemical shift in one dimension is
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correlated with the corresponding 13C chemical shift in another dimension. The HMBC experiment is the best approach to determine long-range connectivity in a given molecule. The heteronuclear inverse-detected two-dimensional NMR experiments greatly improved the signalto-noise ratio (20). Cross-peaks from HSQC were generally split by 1H-1H scalar coupling in the F2 dimension. When HSQC spectral data are supplemented by the corresponding cross-peaks in the COSY spectrum, they often provide convincing evidence for the identity of a metabolite. The splitting of HSQC cross-peaks by H-H couplings along F2 also helped to confirm the identity of a certain metabolite. The assignment process involved comparing the chemical shift obtained from the mixture to the chemical shift of a single metabolite obtained from the literature (16-19). Slight variation of the chemical shift as a function of pH was taken into account in the assignment process. The much less congested aromatic region in 2D COSY spectra proved to be very helpful in identifying various compounds present in the complex mixture of metabolites. Cross-peaks were detected between the β protons and H5,5′ protons of phenylalanine. For tyrosine, cross-peaks were also detected between the β protons and the aromatic protons that are closest to the side chain. Glycogen (Glyn) was not abundant in ulcerated or control extracts since most of the stored Glyn is depleted during the 24 h fasting period before the rats were dosed. This fasting was necessary and consistent with accepted protocols used to intensify the effect of IndoH (21). Metabolic Inhibition. Results in Table 2 are in accordance with IndoH inhibition of glycolytic enzymes (6, 9) of COX-1, which, in turn, inhibits prostaglandin biosynthesis that simulates glycolysis (7). The smaller Suc/Lac ratio in ulcerated stomachs (Table 2) is suggestive of a slower Krebs cycle in IndoH-treated rats. The finding that NSAID-related gastric damage is greatly reduced by glucose and Krebs cycle precursor intake supports the above observation (8). Levels of Metabolites. Lower levels of Cho (Table 3) are usually associated with biological malfunctioning (22)
Figure 4. Control COSY NMR spectrum (top right) and ulcerated COSY NMR spectrum (bottom right); control HSQC NMR spectrum (top left) and ulcerated HSQC NMR spectrum (bottom left).
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Table 1. Metabolic Profile of Acetonitrile Control and Ulcerated Stomach Extracts; Data Were Obtained from 1D, COSY, HSQC, and HMBC NMR Spectra 1H
molecule
δ 13C
assignment (mul, j)
71.70
3.94
tyrosine
CH
C2
57.14
63.50
6.91
tyrosine
C6
116.74
C1 54.74
7.20
tyrosine
C5
131.74
67.34
5.81
uracil
aromatic CH (j56 ) 9 Hz) aromatic CH (j56 ) 9 Hz) CH (d, j ) 7.9 Hz)
C5
101.64
71.44
7.53
uracil
CH (d, j ) 7.9 Hz)
C6
144.04
62.04
5.89
uridine
CH (d, j ) 8.2 Hz)
C5
103.41
62.54 40.94 30.54 56.97 117.84 136.54
7.87 0.99 1.04 2.29 3.62
uridine valine valine valine valine
CH (d, j ) 8.2 Hz) CH3 (d, j ) 7 Hz) CH3 (d, j ) 7 Hz) CH (m) CH (d, j ) 5 Hz)
C6 Me Me C3 C2
142.34 18.85 18.85 29.87 61.23
C4 22.64
2.33/2.40 4.15 8.23 8.35 1.48
2-hydroxybutyrate 2-hydroxybutyrate adenine adenine alanine
CHH CHOH H2 H8 CH3 (d, j ) 7.5 Hz)
C3 47.34 C2 66.54 undeta undeta C3 16.82
3.78 4.78 3.21 3.52 4.06 3.08 3.93 4.14 4.36 5.91 6.06 7.84 8.46 6.52 3.41 3.53 3.72 5.23 3.24 3.41 3.45 3.50 4.64 2.09 2.34 3.77 2.14 2.40 3.76 3.55 3.67 3.78 3.56 4.79 5.39c 3.68 4.33 3.90/3.96
alanine alanine Cho Cho/GPC Cho Cr Cr cytidine cytidine cytidine cytidine cytidine For Fum glucose (R) glucose (R) glucose (R) glucose (R) glucose (β) glucose (β) glucose (β) glucose (β) glucose (β) glutamate glutamate glutamate glutamine glutamine glutamine glycerol glycerol glycerol glycine glycine Glyn GPC GPC GPC
C2 51.22 nab 54.74 68.34 56.44 38.00 54.80 85.11 74.65 90.16 C5 97.04 C6 142.44 172.70 136.04 C4 70.34 C2 71.99 C3 73.54 C1 92.90 C2 75.24 C4 70.44 C5 76.64 C3 76.59 C1 96.74 C3 27.79 C4 34.34 C2 55.54 C3 27.74 C4 34.34 C2 55.54 63.36 63.36 72.89 C2 42.24 nab C1 1D only 67.20 60.40 67.60
3.92
GPC
CH (q, j ) 7.5 Hz) NH2 (CH3)2 N-CH2 CH2-OH CH3 CH2 CHCH2 CHOHCN CHN CCH (d, j ) 7.8 Hz) NCH (d, j ) 7.8 Hz) CHOOCH CH CH CH CH CH CH CH CH CH CH2 CH2 CH CH2 CH2 CH CHH CHH CHOH CH2 NH2 CH N-CH2 N-CH2-CH2-OP PO-CH2-CH(OH)CH2-OH PO-CH2-CH(OH)CH2-OH PO-CH2-CH(OH)CH2-OH (CH3)3
GPC
3.87/3.98 GPE 3.87
GPE
4.12
GPE
4.12 3.29 3.02/3.24 3.99 7.14 7.97
GPE GPE histidine histidine histidine histidine
HO-CH2-CH(OH)CH2-OP HO-CH2-CH(OH)CH2-OP HO-CH2-CH(OH)CH2-OP CH2-CH2-NH2 CH2-NH2 CH2 CH CH CH
δ 13C
assignment (mul, j)
69.35 22.84 22.84 25.10 40.61 54.34 16.44 30.64 29.76 54.84 124.94 136.64 154.34 148.54 36.24 57.04 130.34 128.54 129.98 37.97 42.20 C2 57.25 C3 61.04 C2 34.35 35.95 48.06 C4 20.64 C2 60.90 C3 66.47 C3 27.12 C2 56.24 C10 116.96 C9 123.19 C5 126.16 C8 undeta C11 119.68 C3 37.74 C3 37.74
2-hydroxybutyrate CH3
3.23
molecule
CH3 (dd, j ) 7.5 Hz, j ) 7.5 Hz) 1.02 isoleucine CH3 (d, j ) 6.45 Hz) 1.27/1.47 isoleucine CH2 (m) 1.99 isoleucine CH(m) 3.68 isoleucine CH (d, j ) 4 Hz) 1.33 Lac CH3 (d, j ) 7 Hz, jC-H ) 128 Hz) 4.11 Lac CH (q, j ) 7 Hz) 0.95 leucine CH3 1.00 leucine CH3 1.71 leucine CH 1.72 leucine CH2 3.74 leucine CH 2.12 methionine S-CH3 2.16 methionine CH2 2.65 methionine CH2 3.87 methionine CH 7.59 Nico aromatic CH 7.89 Nico aromatic CH 8.59 Nico aromatic CH 8.95 Nico aromatic CH 3.27 phenylalanine CH2 3.92 phenylalanine CH 7.34 phenylalanine aromatic CH 7.39 phenylalanine aromatic CH 7.43 phenylalanine aromatic CH 3.00 Sar CH3 3.60 Sar CH2 3.85 serine CH 3.97 serine CH2 2.43 Suc CH2 3.26 Tau CH2-SO3H 3.43 Tau CH2-NH2 1.33 threonine CH3 (d, j ) 7 Hz) 3.61 threonine CH (d, j ) 10 Hz) 4.26 threonine CH (m) 3.31 tryptophan (Trp) CH2 4.06 Trp CH 7.20 Trp aromatic CH 7.29 Trp aromatic CH 7.33 Trp aromatic CH 7.53 Trp aromatic CH 7.73 Trp aromatic CH 3.07 tyrosine CHH 3.22 tyrosine CHH
1.20
3.64/3.65 GPC
1H
C3 C2 C8 C6
0.93
isoleucine
C5
11.85
C4 CH2 C3 C2 C3
15.54 24.69 36.81 60.34 20.67
C2 C5 C6 C4 C3 C2 C5 C3 C4 C2 C5 C4 C6 C2 C3 C2 C5 C7 C6
a Cross-peak was not detected in HSQC spectrum. b Not applicable. c Glyn signal only detected in 1D proton spectrum as a broad signal. The signal was not detected in COSY and HSQC, probably due to the relatively short relaxation times of the corresponding nuclei.
including ulceration. The Cho-GPC reversible conversion is catalyzed by GPC phosphodiesterase, and it appears that this higher GPC/Cho ratio in the case of ulcerated stomachs could be induced by a disturbance of the ChoGPC equilibrium via modification of the enzyme kinetics. It is worth noting that phosphocholine (PCh), GPC, and
CDP-Cho are formed from Cho and can be hydrolyzed to form Cho (23). The Cho/Lac ratio was higher in the control stomach than in the ulcerated stomach. Cho is involved in the synthesis of platelet activating factor, a glycerolipid that is released by phagocytic blood cells in response to
NMR Studies of Indomethacin-Induced Gastric Ulcers Table 2. Metabolite Integrals and Ratios Found in Control and Ulcerated Stomachsa source of data
control stomach
Lac (integral) glucose (integral) Lac/glucose
glycolysis COSY 41.5 COSY 1475 COSY 0.028
Suc Lac Suc/Lac
Krebs cycle 1D 1D 1D
0.02 1 0.02
ulcerated stomach 30 1848 0.016 0.006 1 0.006
a Integrals are ∼95% accurate. The 1D spectra were fully relaxed, and integrated peaks were not close to either end of the spectrum. Signal-to-noise ratios for 1D spectra of control and ulcerated stomach extracts were 8700 and 7400, respectively. Spectral baselines were sufficiently flat, and peaks were sufficiently digitized to minimize errors. For 2D spectra, relaxation parameters for a particular metabolite are considered to be the same in both samples, and the same spectral areas were integrated in both spectra. Enough digitization was employed to allow suitable integration.
Table 3. Comparison of Key Metabolites in Control and Ulcerated Stomachs Obtained from 1H NMR Spectraa metabolite Lac Cr total Cho Cho GPC
chemical shift (ppm)
control integral
ulcerated integral
1.33 3.04
1 0.12 1.79 1.44 0.35
1 0.13 1.30 0.78 0.52
3.21 3.23
a Comparisons are only valid within control stomach data or ulcerated stomach data. Data were obtained from fully relaxed 1H 1D spectra. One-dimensional integrals are ∼95% accurate. The 1D spectra were fully relaxed, and integrated peaks were not close to either end of the spectrum. Signal-to-noise ratios for 1D spectra of control and ulcerated stomach extracts were 8700 and 7400, respectively. Spectral baselines were sufficiently flat, and peaks were sufficiently digitized to minimize errors. For 2D spectra, relaxation parameters for a particular metabolite are considered to be the same in both samples, and the same spectral areas were integrated in both spectra. Enough digitization was employed to allow suitable integration.
Figure 5. Fully relaxed 31P NMR spectra of control (A) and ulcerated (B) stomach extracts.
various inflammatory stimuli (24). Phagocytes emigrate out of the blood stream and into tissues where an infection has developed. The decrease in Cho-containing compounds detected above could also be partially due to permanent alterations in the level of water soluble Chocontaining compounds in the ulcerated stomach. The
Chem. Res. Toxicol., Vol. 18, No. 2, 2005 127 Table 4. Comparison of Key Metabolites in Control and Ulcerated Stomach Extracts Obtained from 31P NMR Spectraa metabolite
chemical shift (ppm)
control concn (mM)
ulcerated concn (mM)
TMP Pi unassigned GPE GPC
3.93 2.66 1.84 1.31 0.78
5.0 1.49 0.14 0.36 0.19
5.0 4.81 0.14 0.87 1.31
a One-dimensional integrals are ∼95% accurate. The 1D spectra were fully relaxed, and integrated peaks were not close to either end of the spectrum. Signal-to-noise ratios for 1D spectra of control and ulcerated stomach extracts were 146 and 131, respectively. The spectral baselines were sufficiently flat, and peaks were sufficiently digitized to minimize errors.
higher total Cho/Lac ratio in the control stomach relative to the ulcerated stomach may have been triggered by a series of energy-draining biochemical processes used to counteract the adverse effects of IndoH. For instance, the levels of Cho in the blood decrease during prolonged physical exercise (25) and Cho is consumed in order to boost mitochondrial energy production (26). 1 H NMR signals from intermediates involved in eicosanoid metabolism (in which COX-1 plays a central role), as well as lipid peroxides (that result from COX-1 inhibition), were not detected since these intermediates are hydrophobic long chain fatty acids and are water insoluble. One would expect, however, to detect deactivation of the eicosanoid metabolism due to IndoH-induced COX-1 inhibition. 31 P NMR Spectra. The higher concentration of Pi in the ulcerated stomach extract, which is more than three times the concentration found in the control stomach (31P NMR spectra, Figure 5), agrees with in vivo magnetic resonance spectroscopy studies reported earlier (10). There was also a substantial difference in metabolite concentrations resonating at 1.31 (GPE) and 0.78 (GPC) ppm (Table 4). Some of the detected GPE and GPC may be products of the breakdown of cell membrane phospholipids. The higher concentration of GPE in the ulcerated stomach (more than double that in the control) may also be due to altered cell-membrane phospholipid metabolism, as postulated for the higher GPE concentrations observed in olivopontocerebellar atrophy patients (27). Concentrations of GPC and GPE were also found to be elevated in Alzheimer’s disease patients, an indication of a possible abnormality of phospholipid metabolism (28). The greater than 6-fold increase in the concentration of GPC in the ulcerated stomach extract as compared with that of the control may arise because GPC is a precursor of ACh (a neurotransmitter) and a parasympathomimetic agent (29), i.e., a compound that produces effects resembling those of stimulation of the parasympathetic nerve supply to an organ. The parasympathetic nervous system is involved in keeping tissue inflammation under control by releasing ACh. Thus, the inflammation associated with the gastric administration of IndoH is likely to be responsible for the higher concentrations of GPC. The unassigned and weak signal (1.84 ppm) observed at equal relative intensity in the 31P NMR spectra of the control and ulcerated stomach extracts may be an impurity introduced during the sample preparation or a metabolite that is present in both samples at similar levels. High energy phosphates such as ATP and 2,3-BPG
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were not detected in the spectra, probably due to storage and freeze-thaw treatment of the stomach before preparing the acetonitrile extracts. Fasting of the rats may have also contributed to the reduction of high energy phosphates. Conclusions. The detected differences in the profiles of metabolites are new findings. The reduction in glycolytic and Krebs cycle intermediates caused by IndoH indicates that some of the enzymes in these cycles were inhibited. A higher GPC/Cho ratio was also found in the ulcerated stomach. In the future, in vivo magnetic resonance spectroscopy, in which the NMR signal is selectively acquired from a particular tissue, may be invaluable to gain further insights, if the spectral component resolution is sufficient to determine the above metabolite ratios. The current findings, however, may be used to improve IndoH-based formulations or other NSAID formulations. The results are also consistent with the finding that NSAID-related gastric damage can be greatly reduced by glucose and Krebs cycle precursor intake (8) and provide the first definitive evidence for why such treatments work. Thus, the inclusion of glucose or Krebs cycles intermediates, or even phospholipid precursors, in NSAID formulations is worthy of further investigation in order to reduce NSAID-induced GI toxicity.
Acknowledgment. We acknowledge the support of this work by the University of Sydney Major Equipment fund for the NMR spectrometer, an Australian Research Council (ARC) SPIRT grant and ARC RIEF grants (NMR spectrometer), an ARC Professorial Fellowship (P.A.L.), and Nature Vet Pty. Ltd. for the funding of S.R. Dr. Ian Luck is thanked for useful discussions and proofreading of the manuscript.
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