Proteomics of human liver membrane transporters: a focus on fetuses and newborn infants

Background: Hepatic membrane transporters are involved in the transport of many endogenous and exogenous compounds, including drugs. We aimed to study the relation of age with absolute transporter protein expression in a cohort of 62 mainly fetus and newborn samples. Methods: Protein expressions of BCRP, BSEP, GLUT1, MCT1, MDR1, MRP1, MRP2, MRP3, NTCP, OCT1, OATP1B1, OATP1B3, OATP2B1 and ATP1A1 were quantified with LC-MS/MS in isolated crude membrane fractions of snap-frozen post-mortem fetal and pediatric, and surgical adult liver samples. mRNA expression was quantified using RNA sequencing, and genetic variants with TaqMan assays. We explored relationships between protein expression and age (gestational age [GA], postnatal age [PNA], and postmenstrual age); between protein and mRNA expression; and between protein expression and genotype. Results: We analyzed 36 fetal (median GA 23.4 weeks [range 15.3–41.3]), 12 premature newborn (GA 30.2 weeks [24.9–36.7], PNA 1.0 weeks [0.14–11.4]), 10 term newborn (GA 40.0 weeks [39.7–41.3], PNA 3.9 weeks [0.3–18.1]), 4 pediatric (PNA 4.1 years [1.1–7.4]) and 8 adult liver samples. A relationship with age was found for BCRP, BSEP, GLUT1, MDR1, MRP1, MRP2, MRP3, NTCP, OATP1B1 and OCT1, with the strongest relationship for postmenstrual age. For most transporters mRNA and protein expression were not correlated. No genotype-protein expression relationship was detected. Discussion and conclusion: Various developmental patterns of protein expression of hepatic transporters emerged in fetuses and newborns up to four months of age. Postmenstrual age was the most robust factor predicting transporter expression in this cohort. Our data fill an important gap in current pediatric transporter ontogeny knowledge

Liquid chromatography–tandem mass spectrometry analysis of free and esterified fatty acid N-acyl ethanolamines in plasma and blood cells

The origin of N-acyl ethanolamides (NAEs) in plasma is not well understood, and it is possible that NAEs are present in plasma in esterified form. To test this hypothesis, a new and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of arachidonoyl ethanolamide, 2-arachidonoyl glycerol, docosahexaenoyl ethanolamide, dihomo-¿-linolenoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, and stearoyl ethanolamide in 100 µl of human plasma using a simple acetonitrile extraction step. Using this method, we determined (i) free and esterified NAE levels in human plasma, (ii) free and esterified NAE levels in plasma of mice fed with diets with different amounts of n-3 fatty acids, and (iii) esterified NAE levels in blood cells. Murine and human plasma extracts contained 20- to 60-fold higher levels of esterified NAEs than free NAEs. Moreover, the effect of dietary n-3 fatty acids on murine free plasma NAE profiles was similar for esterified NAEs. Finally, esterified NAEs were also present in murine blood cells, and their pattern followed the same diet effect as observed for free and esterified NAEs in plasma. Together, these data point to the presence of previously ignored pools of esterified NAEs in plasma and blood cells that correlated well with free NAE levels in plasma

Effects of Long- and Short-Chain Fatty Acids on the Release of Gastrointestinal Hormones using an ex Vivo Porcine Intestinal Tissue Model

Gastrointestinal (GI) peptide hormones play an important role in short-term regulation of food intake and blood glucose levels. Modulating their release is of potential relevance for weight management and possibly diabetes. As currently available models are hard to extrapolate to the human situation, the use of porcine intestinal tissue, collected from slaughter pigs, was investigated for this purpose. Intestinal tissue disks showed a predicted regional release pattern of GI peptides. Various long-chain fatty acids differentially stimulated release of glucagon-like peptide 1 (GLP-1) (up to 500%) and glucagon-like peptide 2 (GLP-2) (up to 200%) from ileal tissue disks, but effects on peptide YY (PYY) did not reach significance. Short-chain fatty acids had no effects on the release of GLP-1, GLP-2, and PYY in either the ileum or colon. In conclusion, this porcine tissue model shows to be of advantageous use in a tiered approach to study the potential of satiety-inducing compounds to be selected for studies in humans

Measurement of palmitoylethanolamide and other N-acylethanolamines during physiological and pathological conditions

Palmitoylethanolamide (PEA) belongs to the N-acyl ethanolamines (NAEs), a group of endogenous compounds involved in a variety of physiological processes, including energy homeostasis and inflammation. This review focuses on the analysis of PEA in plasma and tissues and discusses effects of diet and some pathological processes on PEA levels. Originally isolated from egg yolk, PEA has been detected in a variety of tissues and plasma of different species. The compound is present at relatively high levels compared to other NAEs and now mostly analysed using liquid chromatography coupled to mass spectrometry. PEA plasma concentrations show marked fluctuations during the day. However, concentrations in tissues are likely to be more relevant than those in plasma. Most studies suggest that compared to other NAEs, tissue PEA tissue levels are not influenced by changes in dietary fatty acid composition. Effects of inflammation and disease on PEA tissue levels show differences between different models and studies. Therefore, more research is needed on the endogenous role and tissue kinetics of PEA during disease. The rediscovery of the therapeutic potential of PEA has fuelled research and the development of new pharmaceutical formulations. With regard to this there is a need for better kinetic data and models, preferably also on its tissue disposition. Moreover, it is important to learn more about effects of exogenous PEA on the kinetics of other NAEs (and endocannabinoids) and effects of inhibiting its breakdown using inhibitors of the degrading enzymes fatty acid amide hydrolase or N-acylethanolamine-hydrolyzing acid amidase

Glutathione-dependent interaction of heavy metal compounds with multidrug resistance proteins MRP1 and MRP2

The interactions of three heavy metal-containing compounds, cisplatin (CDDP), arsenic trioxide (AS(2)O(3)), and mercury dichloride (HgCl2), with the multidrug resistance transporters MRP1 and MRP2 and the involvement of glutathione (GSH)-related processes herein were investigated. In Madin-Darby canine kidney cells stably expressing MRP1 or MRP2, viability, GSH content, calcein efflux and polarized CSH efflux were measured as a function of exposure to CDDP, As2O3 and HgCl2. In isolated S integral]9-MRP1 and S integral 9-MRP2 membrane vesicles, the interaction with MRP-associated ATPase activity was measured. In the latter model system adduct formation with GSH is not an issue. The data show that (1) CDDP interacts with both MRP1 and MRP2, and GSH appears to play no major role in this process, (2) As2O3 interacts with MRP1 and MRP2 in which process GSH seems to be essential, and (3) HgCl2 interacts with MRP1 and MRP2, either alone and/or as a metal-GSH complex

Docosahexaenoic acid and eicosapentaenoic acid are converted by 3T3-L1 adipocytes to N-acyl ethanolamines with anti-inflammatory properties

n-3 PUFAs have beneficial health effects which are believed to be partly related to their anti-inflammatory properties, however the exact mechanisms behind this are unknown. One possible explanation could be via their conversion to N-acyl ethanolamines (NAEs), which are known to possess anti-inflammatory properties. Using fatty acid precursors we showed that 3T3-L1 adipocytes are indeed able to convert docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to their NAE derivatives docosahexaenoyl ethanolamine (DHEA) and eicosapentaenoyl ethanolamine (EPEA), respectively. This synthesis took place on top of an apparent background formation of these NAEs in standard culture medium. In addition we were able to demonstrate the presence of DHEA, but not of EPEA, in human plasma. DHEA and EPEA were found to decrease LPS induced adipocyte IL-6 and MCP-1 levels. Results of combined incubations with PPAR-¿ and CB2 antagonists suggest a role of these receptors in mediating the reduction of IL-6 by DHEA. Our results are in line with the hypothesis that in addition to other pathways, formation of N-acyl ethanolamines may contribute to the biological activity of n-3 PUFAs. Different targets, including the endocannabinoid system, may be involved in the immune-modulating activity of these “fish-oil-derived NAEs.

Presence, formation and putative biological activities of N-acyl serotonins, a novel class of fatty-acid derived mediators in the intestinal tract

Following the discovery of the endocannabinoid arachidonoyl ethanolamide (anandamide) and other N-acyl-ethanolamines, several other compounds have been found in which amino acids or neurotransmitters rather than ethanolamide are linked to fatty acids. Studies have shown that the local availability of fatty acid precursors, which in turn is modulated by dietary intake of lipids, determines the pattern of conjugates formed. Less information is available whether the same might be true for the amines or neurotransmitters involved. We hypothesized that N-arachidonoyl-serotonin (AA-5-HT) and its analogs could be endogenously present in those tissues that have high contents of serotonin. We investigated the endogenous presence of N-acyl serotonins in different parts of the gastro-intestinal tract of pigs and mice. We discovered that AA-5-HT, oleoyl-serotonin, palmitoyl-serotonin, and stearoyl-serotonin were endogenously present, particularly in the jejunum and ileum. Their formation in vitro was stimulated by the addition of serotonin to intestinal tissue incubations. Furthermore, in a mouse study we showed that the pattern of formation is dependent on the relative amount of fatty acids in the diet. The formation of docosahexaenoyl-serotonin and eicosapentaenoyl-serotonin was elevated in mice fed with a diet containing fish oil. Preliminary data showed that several of the serotonin conjugates are able to inhibit glucagon-like peptide-1 secretion and FAAH activity in vitro. Taken together, our data suggest that N-acyl serotonins are a novel class of lipid mediators present in the gut with highly promising biological propertie

Metabolism of ATP-binding cassette drug transporter inhibitors: complicating factor for multidrug resistance

Membrane transport proteins belonging to the ATP-binding cassette (ABC) family of transport proteins play a central role in the defence of organisms against toxic compounds, including anticancer drugs. However, for compounds that are designed to display a toxic effect, this defence system diminishes their effectiveness. This is typically the case in the development of cellular resistance to anticancer drugs. Inhibitors of these transporters are thus potentially useful tools to reverse this transporter-mediated cellular resistance to anticancer drugs and, eventually, to enhance the effectiveness of the treatment of patients with drug-resistant cancer. This review highlights the various types of inhibitors of several multidrug resistance-related ABC proteins, and demonstrates that the metabolism of inhibitors, as illustrated by recent data obtained for various natural compound inhibitors, may have considerable implications for their effect on drug transport and their potential for treatment of drug resistance