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Chemical Reviews 10/2011; 112(3):1710-50. · 40.20 Impact Factor
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ABSTRACT: Binding of natural bile acids to human serum albumin (HSA) is an important step in enterohepatic circulation and provides a measure of liver function. In this article, we report on the use of four dansyl (Dns) derivatives of cholic acid (ChA) to demonstrate a regiodifferentiation in their relative affinity for the two binding sites of HSA. Using both steady-state and time-resolved fluorescence, formation of Dns-ChA@HSA complexes was confirmed; the corresponding binding constants were determined, and their distribution between bulk solution and HSA microenvironment was estimated. By means of energy transfer from Trp to the Dns moiety, donor-acceptor distances were estimated (21-25 Å) and found to be compatible with both site 1 and site 2 occupancies. Nevertheless, titration using warfarin and ibuprofen as specific displacement probes clearly indicated that 3α- and 3β-Dns-ChA bind to HSA at site 2, whereas their C-7 regioisomers bind to HSA at site 1. Furthermore, the C-3-labeled compounds are displaced by lithocholic acid, whereas they are insensitive to ChA, confirming the assumption that the former binds to HSA at site 2. Thus, Dns labeling provides a useful tool to modulate the relative affinity of ChA to the major binding sites of HSA and, in combination with other fluorescent ChA analogs, to mimic the binding behavior of natural bile acids.
The Journal of Physical Chemistry B 08/2011; 115(35):10518-24. · 3.70 Impact Factor
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ABSTRACT: Under radical reaction conditions, two different and competitive reaction pathways were observed for N-(α-methylbenzyl)trichloroacetamides with a N-3-cyclohexenyl substituent: 1,4-hydrogen translocation and radical addition to a double bond. However, for radicals with an acyclic alkenyl side chain, the direct cyclisation process was exclusively observed. The dichotomy between translocation and direct radical cyclisation in these substrates has been theoretically studied using density functional theory (DFT) methods at the B3LYP/6-31G** computational level.
Organic & Biomolecular Chemistry 03/2011; 9(9):3180-7. · 3.70 Impact Factor
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Krzysztof P Rembacz,
Jannes Woudenberg,
Mark Hoekstra,
Elles Z Jonkers,
Fiona A J van den Heuvel,
Manon Buist-Homan,
Titia E Woudenberg-Vrenken,
Jana Rohacova, M Luisa Marin,
Miguel A Miranda,
Han Moshage,
Frans Stellaard,
Klaas Nico Faber
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ABSTRACT: Bile acid-CoA:amino acid N-acyltransferase (BAAT) conjugates bile salts to glycine or taurine, which is the final step in bile salt biosynthesis. In addition, BAAT is required for reconjugation of bile salts in the enterohepatic circulation. Recently, we showed that BAAT is a peroxisomal protein, implying shuttling of bile salts through peroxisomes for reconjugation. However, the subcellular location of BAAT remains a topic of debate. The aim of this study was to obtain direct proof for reconjugation of bile salts in peroxisomes. Primary rat hepatocytes were incubated with deuterium-labeled cholic acid (D(4)CA). Over time, media and cells were collected and the levels of D(4)CA, D(4)-tauro-CA (D(4)TCA), and D(4)-glyco-CA (D(4)GCA) were quantified by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Subcellular accumulation of D(4)-labeled bile salts was analyzed by digitonin permeabilization assays and subcellular fractionation experiments. Within 24 hours, cultured rat hepatocytes efficiently (>90%) converted and secreted 100 μM D(4)CA to D(4)TCA and D(4)GCA. The relative amounts of D(4)TCA and D(4)GCA produced were dependent on the presence of glycine or taurine in the medium. Treatment of D(4)CA-exposed hepatocytes with 30-150 μg/mL digitonin led to the complete release of D(4)CA, D(4)GCA, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cytosolic marker). Full release of D(4)TCA, catalase, and BAAT was only observed at 500 μg/mL digitonin, indicating the presence of D(4)TCA in membrane-enclosed organelles. D(4)TCA was detected in fractions of purified peroxisomes, which did not contain D(4)CA and D(4)GCA. Conclusion: We established a novel assay to study conjugation and intra- and transcellular transport of bile salts. Using this assay, we show that cholic acid shuttles through peroxisomes for taurine-conjugation.
Hepatology 09/2010; 52(6):2167-76. · 11.66 Impact Factor
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ABSTRACT: Two cholic acid (ChA) analogues, containing a 4-nitrobenzo-2-oxa-1,3-diazole (NBD) moiety at C-3, were incorporated into ChA aggregates and submitted to fluorescence quenching by the two enantiomers of several tryptophan derivatives. In all cases, a significant stereodifferentiation was observed; the most remarkable effect was found when comparing the 3alpha and the 3beta diastereomers.
Chemical Communications 07/2010; 46(27):4965-7. · 6.17 Impact Factor
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ABSTRACT: Interaction between bile acids and plasma proteins has attracted considerable attention over past decades. In fact, binding of bile acids to human serum albumin (HSA) determines their level in plasma, a value that can be used as a test for liver function. However, very little is known about the role that bile acids-HSA complexes play in hepatic uptake. In the present paper, we report on the utility of the singlet excited state properties of 4-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorescent derivatives of cholic acid (ChA); namely, 3alpha-NBD-ChA, 3beta-NBD-ChA, 3beta-NBD-ChTau, 7alpha-NBD-ChA, and 7beta-NBD-ChA to clarify key aspects of bile acids-HSA interactions that remain poorly understood. On the basis of either absorption or emission measurements, formation of NBD-ChA@HSA complexes with 1:1 stoichiometry has been proven. Enhancement of the fluorescence emission upon addition of HSA has been used for determination of the binding constants, which are in the range of 10(4) M(-1). Energy transfer from tryptophan to NBD-ChA occurs by a FRET mechanism; the donor-acceptor distances have been determined according to Forster's theory. The estimated values (27-30 A) are compatible with both site I and site II occupancy and do not provide sufficient information for a safe assignment; however, fluorescence titration using warfarin (site I probe) and ibuprofen (site II probe) for displacement clearly indicates that the employed cholic acid derivatives bind to HSA at site I.
The Journal of Physical Chemistry B 03/2010; 114(13):4710-6. · 3.70 Impact Factor
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ABSTRACT: Four new fluorescent derivatives of cholic acid have been synthesized; they incorporate a dansyl moiety at 3alpha-, 3beta-, 7alpha- or 7beta- positions. These cholic acid analogs are UV photoactive and also exhibit green fluorescence. In addition, they have been demonstrated to be suitable for studying the kinetics of bile acid transport by flow cytometry.
Organic & Biomolecular Chemistry 12/2009; 7(23):4973-80. · 3.70 Impact Factor
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ABSTRACT: A comprehensive study of the ketone-photoinduced formation of gold nanoparticles (AuNPs) from gold ions in aqueous and micellar solution has been carried out. Ketones are good photosensitizers for nanoparticle synthesis not because of the energy they can absorb or deliver but rather because of the reducing free radicals they can generate; thus, efficient nanoparticle generation requires a careful selection of substrates and experimental conditions to ensure that free-radical generation occurs with high quantum efficiency and that gold ion precursors do not cause UV screening of the organic photosensitizers. A key consideration in achieving AuNP synthesis with short exposure times is minimizing excited-state quenching by gold ions; this can be achieved by temporal or spatial segregation or a combination of the two. Temporal segregation can be accomplished by using unimolecular precursors, such as benzoins, that yield ketyl radicals from triplet precursors with lifetimes of a few nanoseconds. Spatial segregation can be achieved by using self-assembled structures such as micelles. In this case, the process can be assisted by selecting ketones with n,pi* triplet states and by adding good hydrophobic hydrogen donors such as 1,4-cyclohexadiene. Systems involving bimolecular reactions of ketones are catalytic in that the ketone is recovered at the end of the reductive process. Rate constants have been determined for the quenching of excited triplets and for the scavenging of ketyl radicals by Au(I) and Au(III); in general, these values are within an order of magnitude of the rate constant for diffusion control. This article provides a paradigm for the photochemical production of nanoparticles of gold and other metal ions that highlights ten aspects that must be considered in order to design successful photochemical systems for nanoparticle generation.
Journal of the American Chemical Society 01/2009; 130(49):16572-84. · 9.91 Impact Factor
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ABSTRACT: Cholylamidofluorescein (CamF) has been selected as a fluorescent bile acid scaffold to perform a full characterization of its photophysical properties. In aqueous medium, under nitrogen, the absorption spectrum of CamF was expectedly dependent on pH. Under air, the presence of CO(2) resulted in a partial protonation of the photoactive form, reducing the absorbance of CamF. The fluorescence spectrum of CamF in ethanol (lambda(exc) = 481 nm) showed a broad band with maximum at 518 nm; the fluorescence quantum yield was 0.67, and the fluorescence lifetime was 4.8 ns. Laser flash photolysis of CamF showed the triplet state transient with a broad maximum at ca. 540 nm and a lifetime of 19 mus. Flow cytometric kinetic assay of CamF uptake in real time was performed in suspensions of rat hepatocytes, showing that living hepatocytes accumulated slowly but constantly CamF along the 5-minute experimental period. Besides, intracellular fluorescence of live cells was found to be clearly dependent on the extracellular concentration of CamF. Thus, flow cytometry has allowed us to demonstrate that CamF is specifically taken up by living rat hepatocytes in a concentration-dependent fashion, suggesting the suitability of this molecule for further studies on bile-acid transport in liver cells.
Photochemical and Photobiological Sciences 08/2008; 7(7):860-6. · 2.58 Impact Factor
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