Plasma protein binding of ketoprofen enantiomers in man: method development and its application.

The protein binding of ketoprofen enantiomers was investigated in human plasma at physiological pH and temperature by ultrafiltration. 14C-labelled (RS)-ketoprofen was synthesized and purified by high-performance liquid chromatography and utilized as a means of quantifying the unbound species. In vitro studies were conducted with plasma obtained from six healthy volunteers. The plasma was spiked with (R)-ketoprofen alone, (S)-ketoprofen alone, and (RS)-ketoprofen in the enantiomeric concentration range of 1.0 to 19.0 micrograms/ml. The plasma protein binding of ketoprofen was nonenantioselective. At a racemic drug concentration of 2.0 micrograms/ml the mean (+/- SD) percentage unbound of (R)-ketoprofen was 0.80 (+/- 0.15)%. The corresponding value for (S)-ketoprofen, 0.78 (+/- 0.18)%, was not statistically different (P greater than 0.05). At this racemic drug concentration (2.0 micrograms/ml) the percentage unbound of each enantiomer was unaffected (P greater than 0.05) by the presence of the glucuronoconjugates of ketoprofen (10 micrograms/ml) in plasma. At clinically relevant concentrations, the plasma binding of ketoprofen did not exhibit enantioselectivity or concentration dependence nor was the binding of either enantiomer influenced by its optical antipode (P greater than 0.05).     

Inhibition of tubulin assembly and covalent binding to microtubular protein by valproic acid glucuronide in vitro

Acyl glucuronides are reactive metabolites of carboxylate drugs, able to undergo a number of reactions in vitro and in vivo, including isomerization via intramolecular rearrangement and covalent adduct formation with proteins. The intrinsic reactivity of a particular acyl glucuronide depends upon the chemical makeup of the drug moiety. The least reactive acyl glucuronide yet reported is valproic acid acyl glucuronide (VPA-G), which is the major metabolite of the antiepileptic agent valproic acid (VPA). In this study, we showed that both VPA-G and its rearrangement isomers (iso-VPA-G) interacted with bovine brain microtubular protein (MTP, comprised of 85% tubulin and 15% microtubule associated proteins {MAPs}). MTP was incubated with VPA, VPA-G and iso-VPA-G for 2 h at room temperature and pH 7.5 at various concentrations up to 4 mM. VPA-G and iso-VPA-G caused dose-dependent inhibition of assembly of MTP into microtubules, with 50% inhibition (IC₅₀) values of 1.0 and 0.2 mM respectively, suggesting that iso-VPA-G has five times more inhibitory potential than VPA-G. VPA itself did not inhibit microtubule formation except at very high concentrations (≥2 mM). Dialysis to remove unbound VPA-G and iso-VPA-G (prior to the assembly assay) diminished inhibition while not removing it. Comparison of covalent binding of VPA-G and iso-VPA-G (using [¹⁴C]-labelled species) showed that adduct formation was much greater for iso-VPA-G. When [¹⁴C]-iso-VPA-G was reacted with MTP in the presence of sodium cyanide (to stabilize glycation adducts), subsequent separation into tubulin and MAPs fractions by ion exchange chromatography revealed that 78 and 22% of the covalent binding occurred with the MAPs and tubulin fractions respectively. These experiments support the notion of both covalent and reversible binding playing parts in the inhibition of microtubule formation from MTP (though the acyl glucuronide of VPA is less important than its rearrangement isomers in this regard), and that both tubulin and (perhaps more importantly) MAPs form adducts with acyl glucuronides.     

Molecular dynamics simulations of apo and holo forms of fatty acid binding protein 5 and cellular retinoic acid binding protein II reveal highly mobile protein,retinoic acid ligand,and water molecules

Structural and dynamic properties from a series of 300 ns molecular dynamics, MD, simulations of two intracellular lipid binding proteins, iLBPs, (Fatty Acid Binding Protein 5, FABP5, and Cellular Retinoic Acid Binding Protein II, CRABP-II) in both the apo form and when bound with retinoic acid reveal a high degree of protein and ligand flexibility. The ratio of FABP5 to CRABP-II in a cell may determine whether it undergoes natural apoptosis or unrestricted cell growth in the presence of retinoic acid. As a result, FABP5 is a promising target for cancer therapy. The MD simulations presented here reveal distinct differences in the two proteins and provide insight into the binding mechanism. CRABP-II is a much larger, more flexible protein that closes upon ligand binding, where FABP5 transitions to an open state in the holo form. The traditional understanding obtained from crystal structures of the gap between two β-sheets of the β-barrel common to iLBPs and the α-helix cap that forms the portal to the binding pocket is insufficient for describing protein conformation (open vs. closed) or ligand entry and exit. When the high degree of mobility between multiple conformations of both the ligand and protein are examined via MD simulation, a new mode of ligand motion that improves understanding of binding dynamics is revealed.     

Carnitine palmitoyltransferase activities: Effects of serum albumin,acyl-CoA binding protein and fatty acid binding protein

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low M concentrations of octanoyl-CoA. However, even a large excess (500 M) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low M concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above effect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolismin vivo.Abbreviations ACBP acyl-CoA binding protein - BSA bovine serum albumin - CPT carnitine palmitoyltransferase - CPT₀ malonyl-CoA sensitive CPT of the outer mitochondrial membrane - CPT malonyl-CoA insensitive CPT of the inner mitochondrial membrane - OG octylglucoside - OMV outer membrane vesicles - IMV inner membrane vesicles Affiliated to the Department of Experimental Medicine, University of Montreal     

肝型脂肪酸结合蛋白研究进展

肝型脂肪酸结合蛋白（liver fatty acid binding protein,L-FABP）是脂肪酸结合蛋白（fatty acid binding proteins,FABPs）家族重要的成员,在肝脏、小肠、肾脏等组织中均有表达。L-FABP在不饱和脂肪酸、饱和脂肪酸、胆固醇、胆汁酸等转运过程中扮演重要角色。目前研究显示L-FABP在脂肪肝、肝硬化以及肝癌发生发展中起到重要作用,并有望作为肝损伤的早期检测指标。此外,新近研究发现尿中L-FABP水平还可以用于预测1型糖尿病患者的临床结局。在2型糖尿病中,尿中L-FABP与糖尿病性肾病的病程有密切关系。主要就L-FABP的特性、结构及其与疾病的关系做一综述。     

In vivo and in vitro binding of 2,4-dichlorophenoxyacetic acid to a rat liver mitochondrial protein

2,4-dichlorophenoxyacetic acid (2,4-D) is a hormonal herbicide widely used in the world because of its efficacy in the control of broadleaf and woody plants. In this study we have demonstrated in vivo covalent binding of the phenoxyherbicide 2,4-D to a single protein of 52 kD (from rat liver mitochondrial preparation) detected through immunoblotting studies with the specific antiserum for 2,4-D. The direct involvement of 2,4-D in the formation of the adduct has also been demonstrated in vitro, using liver mitochondrial preparations exposed to 14C-UL-2,4-D. Radiolabeled protein separated by SDS-PAGE and afterwards electroeluted showed a single labeled protein of 52 kD. When mitochondria exposed to radiolabeled xenobiotic were devoid of their outer membrane, the specific activity observed suggest that protein involved in covalent interaction belongs to the inner mitochondrial membrane. We propose that covalent binding of the phenoxyherbicide 2,4-D to a very specific single protein of 52 kD observed in vitro and in vivo may be related to known alterations of the mitochondrial function.     

In vitro metabolism of 3-t-butyl-4-hydroxyanisole and its irreversible binding to proteins

A method for the preparation of methyl-labelled 3-t-butyl-4-hydroxyanisole (BHA) is described. Metabolism of [14C]BHA using four different enzyme systems (liver microsomes + NADPH; liver microsomes + cumene hydroperoxide (CHP); sheep seminal vesicle (SSV) microsomes (as a source of prostaglandin synthetase) + arachidonic acid (AA); horseradish peroxidase (HRP) + hydrogen peroxide) was investigated. In all systems, BHA was oxidized to a variety of products including formaldehyde, a dimer di-BHA, polar and water soluble metabolites as well as a reactive intermediate(s) that binds irreversibly to proteins. With liver microsomes and NADPH, phenobarbital (PB) induction gave increased yields of all products while 3-methylcholanthrene (MC) induction specifically increased protein binding but decreased other metabolite formation. BHA addition effectively discharged the activated oxygen complex of cytochrome P-450 (liver microsomes) as well as Comp. I and Comp. II of HRP suggesting that it is a good one electron peroxidase donor. BHA addition also increased the net rate of NADPH oxidation in the presence of liver microsomes suggesting uncoupling. It is proposed that in all system investigated BHA is oxidized predominantly via a one electron oxidation process to yield first the BHA free radical which then dimerizes, forms more products or binds to proteins.     

Stereoselective plasma protein binding of amlodipine

The binding of the (R)‐ and (S)‐enantiomers of amlodipine to bovine serum albumin (BSA), human serum albumin (HSA), α₁‐acid glycoprotein (AGP), and human plasma (HP) was studied by equilibrium dialysis over the concentration range of 75–200 μM at a protein concentration of 150 μM. Unbound drug concentrations were determined by enantioselective capillary electrophoresis using 50 mM phosphate buffer, pH 2.5, containing 18 mM α‐cyclodextrin as background electrolyte. Saturation of the protein binding sites was not observed over the concentration range tested. Upon application of racemic amlodipine besylate, (S)‐amlodipine was bound to a higher extend by HSA and HP compared with (R)‐amlodipine, whereas the opposite binding of the enantiomers was observed for BSA and AGP. Scatchard analysis was used to illustrate the different binding affinities of amlodipine besylate enantiomers to BSA, HSA and AGP. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

The liver fatty acid binding protein - comparison of cavity properties of intracellular lipid-binding proteins

The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common -barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited portal region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound.     

In vitro plasma protein binding and aqueous aggregation behavior of astaxanthin dilysinate tetrahydrochloride

The tetrahydrochloride salt of astaxanthin di-L-lysinate (lys(2)AST) is a highly water-dispersible astaxanthin-amino acid conjugate, with an aqueous dispersibility of > or = 181.6 mg/mL. The statistical mixture of stereoisomers has been well characterized as an aqueous-phase superoxide anion scavenger, effective at micromolar (microM) concentrations. In the current study, the aqueous aggregation behavior and in vitro plasma protein binding [with fatty-acid-free human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP)] were investigated with a suite of techniques, including circular dichroism (CD) and UV-vis spectroscopy, ultrafiltration, competitive ligand displacement, and fluorescence quenching. Induced CD bands obtained in Ringer buffer solution of HSA demonstrated high affinity monomeric binding of the compound at low ligand per protein (L/P) ratios (in aqueous solution alone the carotenoid molecules formed card-pack aggregates). The binding constant ( approximately 10(6)M(-1)) and the binding stoichiometry (approximately 0.2 per albumin molecule) were calculated from CD titration data. CD displacement and ultrafiltration experiments performed with marker ligands of HSA indicated that the ligand binding occurred at a site distinct from the main drug binding sites of HSA (i.e., Sites I and II). At intermediate L/P ratios, both monomeric and aggregated ("chirally complexed") binding occurred simultaneously at distinct sites of the protein. At high L/P ratios, chiral complexation predominantly occurred on the asymmetric protein template. The tentative location of the chirally-complexed aggregation on the HSA template was identified as the large interdomain cleft of HSA, where carotenoid derivatives have been found to bind previously. Only weak binding to AGP was observed. These results suggest that parenteral use of this highly potent, water-dispersible astaxanthin-amino acid conjugate will result in plasma protein association, and plasma protein binding at sites unlikely to displace fatty acids and drugs bound at well-characterized binding sites on the albumin molecule.     

Enhanced expression of adipocyte-type fatty acid binding protein in murine lymphocytes in response to dexamethasone treatment

Fatty acids have a great influence on the process of lymphocyte apoptosis which is considered as a modulating factor of immune response in both humans and animals. However the mechanism underlying the function of fatty acids in the process of lymphocyte apoptosis is not fully understood. In this study we show that the appearance of adipocyte-type fatty acid binding protein (A-FABP) is induced upon administration of dexamethasone (DEX) in both in vivo and cultured lymphocytes, and its distinct nuclear localization occurs in close relation to the DEX-induced apoptosis process. In immuunohistochemistry of mouse spleen, A-FABP-immunoreactivity starts to occur 3 h after DEX stimulation, and it massively localizes in the nucleus 8 h after the treatment, while no A-FABP-immunoreactivity is discerned in the lymphocytes of normal as well as 24 h post-injection spleen. In the murine T-cell leukemia CTLL-2 cells, A-FABP-immunoreactivity is also induced in both of the cytoplasm and nucleus when the apoptosis is induced by IL-2 retrieval together with DEX treatment, while in the presence of IL-2 A-FABP-immunoreactivity is confined to the cytoplasm with DEX trreatment. On the other hand, A-FABP-immunoreactivity is not detected by IL-2 retrieval alone. The present findings altogether suggest that A-FABP and its ligands, fatty acids, play an important role in the process of apoptosis and the immune modulation induced by DEX.     

Sialic acid binding protein of human endometrium: Its regulation by steroids

In the present study, we have observed that sialic acid binding protein (SABP – a 54 kDa glycoprotein which was isolated from human endometrial scrapings taken at various stages of the menstrual cycle from normal cycling females and purified to apparent homogeneity and was earlier reported from this laboratory) was found in sufficiently detectable amount in the endometrium of normal cycling women whereas it was found in lesser amount in tissue from women who have recently entered the post-menopause stage. SABP was observed in both follicular and luteal phase of menstrual cycle which was found by western blot analysis. In the de-novo synthesis experiment, synthesis and secretion of SABP was found to be stimulated by estradiol (E₂) whereas progesterone (P₄) was found to have no significant stimulatory effect on it which was also confirmed by HEC cell culture. In the HEC cell culture, priming of cells with E₂ was found to influence the effect of P₄ on SABP when it was added 2 h after E₂ administration. This was observed by doing immunoprecipitation followed by SDS-PAGE and autoradiography. Hence this report clearly indicates that E₂ regulates the synthesis and secretion of 54 kDa SABP from human endometrium. How E₂ priming of endometrium influences the effect of P₄ on SABP has been discussed.     

Urinary liver-type fatty acid binding protein as a useful biomarker in chronic kidney disease

Background: We reported that urinary L-FABP reflected the progression of chronic kidney disease (CKD). This study is aimed to evaluate the clinical significance of urinary liver type fatty acid binding protein (L-FABP) as a biomarker for monitoring CKD. Methods: Urinary L-FABP was measured using human L-FABP ELISA kit (CMIC.Co., Ltd., Tokyo, Japan). The relations between urinary L-FABP and clinical parameters were evaluated in non-diabetic CKD (n = 48) for a year. In order to evaluate the influence of serum L-FABP derived from liver upon urinary L-FABP, both serum and urinary L-FABP were simultaneously measured in patients with CKD (n = 73). Results: For monitoring CKD, the cut-off value in urinary L-FABP was determined as 17.4 μg/g.cr. by using a receiver operating characteristics (ROC) curve. Renal function deteriorated significantly more in patients with ‘high’ urinary L-FABP (n = 36) than in those with ‘low’ L-FABP (n = 12). The decrease in creatinine clearance was accompanied by an increase in urinary L-FABP, but not in urinary protein. Serum L-FABP in patients with CKD was not correlated with urinary L-FABP. Conclusion: Urinary excretion of L-FABP increases with the deterioration of renal function. Serum L-FABP did not influence on urinary L-FABP. Urinary L-FABP may be a useful clinical biomarker for monitoring CKD.     

Fatty acid sensor for low-cost lifetime-assisted ratiometric sensing using a fluorescent fatty acid binding protein

Elevated free fatty acid (FA) levels lead to insulin resistance, hypertension, and microangiopathy, all of which are associated with type 2 diabetes. On the other hand, deficiencies of FA are indicative of certain neurodegenerative diseases, including autism. Thus, free FA levels are a diagnostic indicator for a variety of disorders. Here we describe the use of a commercially available FA binding protein labeled with acrylodan (ADIFAB), which we modified with a ruthenium metal-ligand complex with the intention of creating a low-cost FA sensor. The dual-labeled FA binding protein was used in lifetime-assisted ratiometric sensing (LARS) of oleic acid. For both steady-state and time-resolved luminescence decay experiments, the protein is responsive to oleic acid in the range of 0.02-4.7 microM. The emission at 432 nm, which is associated with the acrylodan occupying the FA binding site, decreases in intensity and red shifts to 505 nm on the addition of oleic acid. The intensities of the 505-nm peak due to the acrylodan displaced from the binding site by FA and of the 610-nm emission peak of ruthenium remained nearly unchanged. Fitting of the fluorescence decay data using the method of least squares revealed three emitting components with lifetimes of approximately 0.60, 4.00, and 370 ns. Fractional intensities of the emitting species indicate that changes in modulation between 2 and 10 MHz on binding of the protein with oleic acid are due mainly to the 4.00-ns component. The 0.60- and 370-ns components are assigned to acrylodan (505 nm) and ruthenium, respectively. Note that because ruthenium has a lifetime that is two orders of magnitude longer than that of acrylodan, the FA measurements were carried out at excitation frequencies lower than what can be done with acrylodan alone. Thus, low-cost instrumentation can be designed for a practical FA sensor without sacrificing the quality of measurements.     

Localization of epidermal-type fatty acid binding protein in macrophages in advanced atretic follicles of adult mice

Summary The localization of epidermal-type fatty acid binding protein (E-FABP) in the mature mouse ovary was examined by immuno-light and electron microscopy. Numerous macrophages immunopositive for both anti-E-FABP and F4/80 antibodies, together with immunonegative cells, were found in advanced atretic follicles that had eccentric lumens containing deformed ova. While some E-FABP-immunopositive macrophages were spider in shape and appeared singly, others, especially close to the lumen, were round and voluminous and tended to be aggregated. The voluminous macrophages contained phagosomes of various sizes and they were regarded as those actively involved in the phagocytosis of apoptotic granulosa cells. E-FABP-immunopositive macrophages and their processes were often apposed to adjacent immunonegative cells, and some of them lined the lumen containing deformed ova. On the other hand, E-FABP-immunonegative cells in the atretic follicles were classified into two types: the one, a minority, was characterized by small mitochondria containing non-tubular cristae and presumably represented residual granulosa cells, while the other dominant type was characterized by large mitochondria containing tubular cristae and presumably represented theca cells originally surrounding the follicles to be atretic. The present detection of E-FABP-immunopositivity selectively in macrophages of the atretic follicles suggests possible involvement of E-FABP and/or its ligand fatty acids in the process of follicular atresia, and it makes more reliable the identification of the advanced atretic follicles with the antral spaces obliterated, which could provide further details on the histology of the follicular atresia than before.     

Fatty acid binding protein 4 in human skeletal muscle

The mechanisms that regulate intramyocellular triglycerol (IMTG) storage and mobilization are largely unknown. However, during the last decades several intracellular fatty acid binding proteins (FABPs) have been identified. FABP3 is the dominating FABP in skeletal muscle. Expression of additional FABPs is suggested from findings in FABP3-null mutated mice. In the present study, our aims were to investigate if FABP4 is expressed within skeletal muscle fibers and if FABP3 and FABP4 are more abundant in skeletal muscle fibers in endurance-trained than in control subjects. We show that FABP4 protein is expressed within the skeletal muscle fibers and that FABP4 mRNA and protein are more abundant in the endurance trained subjects. Still, FABP4 is markedly less expressed than FABP3, which is the generally accepted dominating FABP in skeletal muscle tissue.     

Molecular dynamics simulations of palmitate entry into the hydrophobic pocket of the fatty acid binding protein

The entry of substrate into the active site is the first event in any enzymatic reaction. However, due to the short time interval between the encounter and the formation of the stable complex, the detailed steps are experimentally unobserved. In the present study, we report a molecular dynamics simulation of the encounter between palmitate molecule and the Toad Liver fatty acid binding protein, ending with the formation of a stable complex resemblance in structure of other proteins of this family. The forces operating on the system leading to the formation of the tight complex are discussed.