Quantitation of ^βN-Alkanoyl-5-hydroxytryptamides in Coffee by Means of LC-MS/MS-SIDA and Assessment of Their Gastric Acid Secretion Potential Using the HGT-1 Cell Assay

Abstract

A straightforward stable isotope dilution analysis (SIDA) for the reliable quantitative determination of (beta)N-C(18:0)- to (beta)N-C(24:0)-alkanoyl-5-hydroxytryptamides (C5HTs) in coffee powder and beverages by means of LC-MS/MS was developed. The developed SIDA showing accuracy values of 92.6-107% and precision between 0.5 and 7% relative standard deviation for the individual derivatives allowed the sensitive and selective quantification of the target compounds in coffee beverages. Depending on the type of coffee, quantitation revealed C5HT levels between 65 and 144 microg/L in filtered coffee and up to 3500 microg/L in a French press beverage, thus indicating that about 0.3 or 7.2% of the C5HTs were extracted from the coffee powder into the beverage when using the cellulose filter method or the French press, respectively. To estimate the potential contribution of the C5HTs to the phenomenon of stomach irritation after ingestion of coffee brew, in vitro cell studies were performed with pure individual 5-hydroxytryptamides and a mixture of the predominating derivatives in ratios matching those found in coffee. All substances tested induced a decrease in the intracellular proton index (IPX) coined as an indicator of stomach acid secretion. While the biomimetic C5HT mixture was highest in its inducing effect, the individual stearic acid, oleic acid, and linoleic acid 5-hydroxytryptamide did not differ significantly from each other, but showed a less pronounced effect compared to arachinic acid 5-hydroxytryptamide. In conclusion, not the grade of saturation seems to determine the C5HT's mode of action in driving the stomach acid secretion, rather than the fatty acid chain length.

Multi‐parametric approach to identify coffee components that regulate mechanisms of gastric acid secretion

“…Experiments with biomimetic combinations as well as omission experiments revealed that N-MP and CA partially compensated the pro-secretory effect of other pro-secretory compounds. These results are in accordance with the previous findings of our group [7,9,11,24], which also demonstrated that the pro-secretory activity of C5HT depends on the number of carbons in the C-chain of the corresponding fatty acid [10]. Furthermore, the effect of CAFF on gastric acid secretion has been often described as pro-secretory [3,8,25].…”

“…Our previous results have shown that one coffee component, N ‐methylpyridinium (N‐MP), down‐regulated proton secretion in human parietal gastric tumor cells (HGT‐1) , when applied in combination with a lyophilized regular coffee beverage. In contrast, we also found that other coffee components such as CAFF as well as C5HT stimulated proton secretion in HGT‐1 cells when applied in coffee representative concentrations .…”

A dark brown roast coffee blend is less effective at stimulating gastric acid secretion in healthy volunteers compared to a medium roast market blend

“…administration of a coffee beverage containing high amounts of C5HTs induced ulcer formation, whereas no ulcers were detected in rats that received a steam-treated, C5HTs-depleted coffee beverage [3]. The pivotal role of C5HTs in stimulating gastric acid secretion was confirmed in previous studies of our own group, using, first, an activity-guided approach [8,9] and, second, a multiparametric approach [10] in human gastric tumor cells (HGT-1 cells). Treatment of HGT-1 cells with C5HTs in coffee representative concentrations not only induced proton secretion, but also induced the expression of prosecretory genes, for example, those encoding for the H + ,K + -ATPase alpha-subunit or the acetylcholine receptor CHRM3, and prosecretory EGFr signaling pathways.…”

Activity-Guided Fractionation to Characterize a Coffee Beverage that Effectively Down-Regulates Mechanisms of Gastric Acid Secretion as Compared to Regular Coffee

“…However, in this first approach, we were not able to identify which of the coffee components affect mechanisms of proton secretion. In a following approach, we were able to identify different N-alkanoyl-5-hydroxytryptamides as potent stimulators of proton secretion in HGT-1 cells depending on their alkanoylic substitution (28). However, other coffee constituents may have an effect on stomach acid secretion as well.…”