Evaluation on Intestinal Permeability of Phlorotannins Using Caco-2 Cell Monolayers

Among the phlorotannins of seaweed polyphenols, eckols which have a dibenzodioxin linkage are known to have various physiological functions. The purpose of this study was to investigate the intestinal epithelial absorption of eckols using Caco-2 cell monolayers of the small intestinal membrane model. Each compound permeated from the apical (AP) side to the basolateral (BL) side in the monolayers was identified and quantitated by liquid chromatography-mass spectrometry with electrospray ionization. In the transport assays using five types of eckols (eckol, fucofuroeckol A, phlorofucofuroeckol A, dieckol, and 8,8'-bieckol), only the monomeric eckol showed limited transepithelial absorption with relatively small apparent permeability values (0.30 ± 0.04 × 10−8 cm/s). Analyzing the Hanks’ balanced salt solution in the receiver on the BL side showed that phloroglucinol was detected in all experimental sections using eckols, and it's concentration increased with time over the course of the incubation. The other molecules corresponding to the unconjugated and conjugated metabolites of eckols were not detected in the AP and BL sides through the assays. These results suggest that eckols, including monomeric eckol, may be decomposed into phloroglucinol in the intestinal epithelium and the resulting phloroglucinol permeates to the BL side.

Conjugated Metabolites of Hydroxytyrosol and Tyrosol Contribute to the Maintenance of Nitric Oxide Balance in Human Aortic Endothelial Cells at Physiologically Relevant Concentrations

Nitric oxide (NO) is an important signaling molecule involved in many pathophysiological processes. NO mediates vasodilation and blood flow in the arteries, and its action contributes to maintaining vascular homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium. Dietary antioxidants and their metabolites have been found to be directly and/or indirectly involved in the modulation of the intracellular signals that lead to the production of NO. The purpose of this study was to investigate the contribution of conjugated metabolites of hydroxytyrosol (HT) and tyrosol (TYR) to the release of NO at the vascular level, and the related mechanism of action, in comparison to their parental forms. Experiments were performed in human aortic endothelial cells (HAEC) to evaluate the superoxide production, the release of NO and production of cyclic guanosine monophosphate (cGMP), the activation of serine/threonine-protein kinase 1 (Akt1), and the activation state of endothelial nitric oxide synthase (eNOS). It was observed that the tested phenolic compounds enhanced NO and cGMP concentration, inhibiting its depletion caused by superoxide overproduction. Moreover, some of them enhanced the activation of Akt (TYR, HT metabolites) and eNOS (HT, HVA, TYR-S, HT-3S). Overall, the obtained data showed that these compounds promote NO production and availability, suggesting that HT and TYR conjugated metabolites may contribute to the effects of parental extra virgin olive oil (EVOO) phenolics in the prevention of cardiovascular diseases.

Overcoming challenges associated with the bioanalysis of cysteine-conjugated metabolites in the presence of antibody–drug conjugates

Aim: Investigations have shown that for the antibody–drug conjugate (ADC) belantamab mafodotin, concentrations of the cysteine-conjugated metabolite, Cys-mcMMAF, were overestimated in the presence of the ADC during sample processing when utilizing a historical SPE method. Results: A new assay was developed utilizing an acidic protein precipitation to remove the ADC early in the extraction process, thus eliminating the risk of overestimating Cys-mcMMAF in the presence of belantamab mafodotin. In vitro experiments demonstrated a linear relationship between the concentration of belantamab mafodotin and the release of Cys-mcMMAF. Extensive stability assessments were performed to cover storage of study samples. Conclusion: This work emphasized the critical importance of understanding the performance of a bioanalytical method for free toxic payload in the presence of the ADC.

Resveratrol stereoselectively affected (±)warfarin pharmacokinetics and enhanced the anticoagulation effect

Resveratrol (RVT) has various beneficial bioactivities and popularly used as a dietary supplement. RVT showed inhibitions on CYP1A2/2C9/3A4, breast cancer resistance protein (BCRP), and some conjugated metabolites of RVT also inhibited BCRP. (±)Warfarin, an anticoagulant for cardiovascular disease but with narrow therapeutic window, were substrates of CYP1A2/3A4(R-form), 2C9(S-form) and BCRP. We hypothesized that the concurrent use of RVT might affect the metabolism and excretion of warfarin. This study investigated the effect of RVT on the pharmacokinetics and anticoagulation effect of (±)warfarin. Rats were orally given (±)warfarin (0.2 mg/kg) without and with RVT (100 mg/kg) in a parallel design. The results showed that RVT significantly increased the AUC0−t of S-warfarin and international normalized ratio. Mechanism studies showed that both RVT and its serum metabolites (RSM) inhibited BCRP-mediated efflux of R- and S-warfarin. Moreover, RSM activated CYP1A2/3A4, but inhibited CYP2C9. In conclusion, concomitant intake of RVT increased the systemic exposure of warfarin and enhanced the anticoagulation effect mainly via inhibitions on BCRP and CYP2C9.

Characterization of Isoflavone Conjugated Metabolites in Human Plasma and Urine by Dietary Soybean Meals

Objectives Soy isoflavones are one of the best studied secondary plant metabolites for the last 30 years and associated with potential health benefits such as prevention of cancer, cardiovascular diseases, and menopausal symptoms. The study aimed to identify isoflavone conjugated metabolites from human plasma and urine after dietary soybean meals using a targeted metabolomic approach and to investigate the effect of fermentation on bioavailability of soy isoflavones. Methods In the cross-over intervention design with 10 candidates (6 women and 4 men), standardized meals with fermented soybean (FS, Cheonggukjang, by Bacillus subtilis) and non-fermented soybean (NFS) were served over two weeks in two randomized groups. Flavonoid derivatives in each freeze-dried tested meal and isoflavone conjugated metabolites in plasma and urine collected before and after the consumption of each meal were evaluated by UPLC-DAD-QToF/MS. Results Twenty-two isoflavones, including genistein, daidzein, glycitein, and its glycosides, were identified in both tested meals. In particular, 6”-O-succinoyldadizin and 6”-O-succinoylgenistin produced newly as acylated forms with succinic acid in fermented process. Also, in levels of simple glycosidic forms (e.g., daidzin and genistin), the FS meal showed higher than NFS meal, which might be explained by the degradation of malonylglucosides during the fermentation. Fourteen metabolites conjugated with glucuronic acid and sulfuric acid were identified in plasma and urine, among them, genistein 4’,7-di-O-glucuronide and daidzein 7-O-glucuronide were confirmed as major metabolites in plasma and urine, respectively. The bioavailability was differentiated by personal characteristics after intake soy isoflavones, and it was considered that genistein class was more excellent as well as FS meal was more effective when compared to NFS meal on bioavailability. Conclusions The results may contribute to not only understanding the potential health effects of fermented foods by modifying the bioavailability of soybean bioactive components but also application of other traditional foods related with fermentation. Funding Sources This work was supported by the National Institute of Agricultural Science of Rural Development Administration, Republic of Korea.

Microbiota Facilitates the Formation of the Aminated Metabolite of Tea Polyphenols Which Trap Deleterious Reactive Endogenous Metabolites (P06-022-19)

Objectives The in vivo mechanism of tea polyphenol-mediated prevention of many chronic diseases is still largely unknown. Studies have shown that accumulation of toxic reactive cellular metabolites, such as ammonia and reactive carbonyl species (RCS), is one of the causing factors to the development of many chronic diseases. The objective of this study is to investigated the in vivo interaction between tea polyphenols and ammonia and RCS. Methods In mice, we gave 200 mg/kg tea polyphenol ((-)-epigallocatechin-3-gallate (EGCG) or theaflavin) to CD-1 mice, 129/SvEv specific-pathogen-free (SPF) mice, or germ-free (GF) mice. Urinary and fecal samples were collected in metabolic cages for 24 h. In humans, two healthy volunteers drank 4 cups of Lipton green tea every day for four days. On the fourth day, 24 h urinary and fecal samples were collected after consuming the first cup of tea. Using LC tandem mass, we searched the formation of the aminated and RCS conjugated metabolites of tea polyphenols. Chemical standards were synthesized to confirm the structures of these metabolites. In order to study the impact of gut microbiota on the formation of these metabolites, we also quantified the concentrations of these metabolites in SPF and GF mice. Results We found that both EGCG and theaflavin could rapidly react with ammonia to generate the aminated metabolites. Both tea polyphenols and their aminated metabolites could further scavenge RCS, such as methylglyoxal (MGO), malondialdehyde (MDA), and trans-4-hydroxy-2-nonenal (4-HNE), to produce the RCS conjugates of tea polyphenols and the aminated tea polyphenols. Both the aminated and the RCS conjugated metabolites of EGCG were detected in human after drinking four cups of green tea per day. By comparing the levels of the aminated and the RCS conjugated metabolites in EGCG or theaflavin exposed germ-free (GF) mice and specific-pathogen-free (SPF) mice, we demonstrated that gut microbiota facilitate the formation of the aminated metabolites of tea polyphenols, the RCS conjugates of tea polyphenols, and the RCS conjugates of the aminated tea polyphenols. Conclusions Altogether, this study provides in vivo evidences that tea polyphenols have the capacity to scavenge toxic reactive metabolic wastes. This finding opens a new window to understand the underlying mechanisms by which drinking tea could prevent the development of chronic diseases. Funding Sources We gratefully acknowledge financial support from NIH R01 grant AT008623 to this work.