A GC-TOF-MS method was developed and validated for a metabolic fingerprinting in saliva of smokers and nonsmokers. We validated the method by spiking 37 different metabolites and 6 internal standards to saliva between 0.1 μM and 2 mM. Intraday coefficients of variation (CVs) (accuracies) were on average, 11.9% (85.8%), 8.2% (88.9%), and 10.0% (106.7%) for the spiked levels 25, 50, and 200 μM, respectively (N = 5). Interday CVs (accuracies) were 12.4% (97%), 18.8% (95.5%), and 17.2% (105.9%) for the respective levels of 25, 50, and 200 μM (N = 5). The method was applied to saliva of smokers and nonsmokers, obtained from a 24 h diet-controlled clinical study, in order to identify biomarkers of endogenous origin, which could be linked to smoking related diseases. Automated peak picking, integration, and statistical analysis were conducted by the software tools MZmine, Metaboanalyst, and PSPP. We could identify 13 significantly altered metabolites in smokers (p < 0.05) by matching them against MS libraries and authentic standard compounds. Most of the identified metabolites, including tyramine, adenosine, and glucose-6-phosphate, could be linked to smoking-related perturbations and may be associated with established detrimental effects of smoking.
Phosphatidylcholine (PC) is the major phospholipid of the hydrophobic gastric mucosal barrier and is chiefly released from mucous cells into the gastric mucus. Whereas the mucosa contains highly unsaturated PC, gastric mucus predominantly contains palmitoyl‐oleoyl‐PC and palmitoyl‐linoleoyl‐PC, indicating a selective release of these PC species into the gastric lumen. In order to understand gastric PC metabolism, we investigated synthesis and release of PC in cultivated porcine gastric mucous cells, using dual labelling with [methyl‐3H]‐choline and [1‐14C]‐palmitate, in the presence of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), indomethacin and prostaglandin E2 (PGE2). Linear incorporation of [methyl‐3H]‐choline and [1‐14C]‐palmitate into PC was achieved for at least 8 h. In contrast to type II pneumocytes TPA increased PC synthesis in gastric mucous cells but not its release. Indomethacin did not influence PC synthesis, but it decreased the release of newly synthesized PC. PGE2 antagonized the effect of indomethacin on PC release. We conclude that PC release by isolated porcine gastric mucous cells is regulated in a manner different from type II pneumocytes. PC release is impaired by indomethacin and this impairment is restored by PGE2.
The purpose of this study was to characterize time-dependent changes in pepsinogen (PG) synthesis of porcine gastric chief cells during long-term monolayer culture. Porcine chief cells were isolated by pronase/collagenase treatment of fundic mucosa and enriched by density gradient and counterflow centrifugation. PG isoenzymes were identified in [L-35S]methionine-labelled cultured chief cells by native polyacrylamide gel electrophoresis followed by phosphor imager analysis, protease detection and immunoblots with specific PG A and C antibodies. The obtained results suggest that porcine chief cell cultures, after an initial settling period, reached an approximate steady state in total protein content and synthesis as well as in PG content and isoenzyme pattern from days 3 to 9 of culture. The latter was characterized by the presence of at least two PG A and two PG C isoenzymes. During the supposed steady-state total PG synthesis averaged out at 34 +/- 2% of total protein synthesis, as detected by [L-35S]methionine incorporation, due to the synthesis of, mainly, PG A2 and, to a much lesser extent, PG C and A1. In line with an active secretion, PG A2 proportion was on average significantly higher in released (44 +/- 3%) than in intracellular labelled proteins (19 +/- 2%). In addition, PG release from chief cells cultured for 6 and 9 days could be stimulated by cholecystokinin-octapeptide. These data suggest that porcine chief cells in monolayer culture are a model well suited for the quantitative and qualitative characterization of PG isoenzyme synthesis and release during long-term investigations, for which an establishment of a culture steady state appears to be a useful prerequisite.
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