Ratio Of Human Serum Albumin Research Articles

Human Serum Albumin Mediated Controllable Synthesis of Defect-Rich Copper Hydroxide Nanowire for Cuproptosis-Based Anti-Tumor Therapy.

Cuproptosis, as a newly identified form of programmed cell death, shows great promise in cancer treatment. Efficient Cu+ delivery while avoiding systemic toxicity and elimination of the resistance from over-expressed intracellular copper chelator glutathione (GSH) are critical for cuproptosis. Herein, this work innovatively constructs a biocompatible and defect-rich copper hydroxide nanowire (HCu nanowire) through a human serum albumin (HSA) mediated biomineralization method. This work finds that the morphology and size of HCu nanowires can be controlled adjusted by the feed ratio of HSA and Cu2+. Remarkably, except for outstanding biocompatibility, HSA coordination endows HCu nanowires abundant oxygen vacancies (OVs), and the defect-rich HCu nanowire possesses excellent GSH consumption efficiency. Density functional theory studies indicate that OVs change GSH absorption energy on defective HCu nanowires. In cancer cells, HCu nanowires deplete GSH and simultaneously produce sufficient free Cu+ for enhanced cuproptosis. Meanwhile, Cu+ can catalyze endogenous H2O2 into hydroxyl radicals (·OH) via a Fenton-like reaction. Thus, synergetic cuproptosis and ROS mediated apoptosis against tumor are achieved. The experimental results show that HCu nanowires have a better performance in both antitumor efficiency and safety compared with chemotherapeutic drug Dox at the same dose, demonstrating its great potential in clinical applications.

Considerations for efficient surface functionalization of nanoparticles with a high molecular weight protein as targeting ligand

Functionalization of nanoparticles with ligands is a powerful tool to achieve efficient targeting of receptors expressed on specific cell types. For optimal ligand-receptor interactions, the ligands should be attached on the nanoparticle surface in a predictable manner with specific orientations and density that preserve their bioactivity. While there are many publications on nanoparticles functionalized with small ligands that meet these requirements, achieving these conditions is particularly challenging for protein-based ligands of higher molecular weight. Proteins have complex and often fragile structures with numerous reactive residues, and they generally do not withstand harsh reaction conditions well. They are also prone to non-specific adsorption. Thus, conjugation strategies have to be considered carefully and optimized for each individual protein-based ligand as well as for the particle platform. In this study, we present a comprehensive approach for site-selective conjugation between aminated silica nanoparticles (SiNPs) and the single accessible thiol in human serum albumin (HSA) (66.5 kDa). We varied several reaction parameters including the density of amino groups on the particle surface, protein to amino group molar ratios, and linker length and evaluated their effect on colloidal stability, mode of protein attachment, protein density, and binding capacity of the tethered protein. We demonstrated that particle surface properties strongly impact covalent conjugation. For SiNPs with low amino group density (5,000 NH2/particle), only 25% of the available surface was covered with protein, and up to 90% of HSA was non-specifically adsorbed. Adjusting the molar ratio of HSA and lengthening the linker did not substantially increase the amount of covalently-attached ligand. In contrast, SiNPs with high amino group density (20,000 NH2/particle) showed high protein loading accompanied by low levels of non-specific adsorption. Using a short linker and 1:1 HSA to NH2 molar ratio resulted in 70% surface coverage with HSA molecules. The mode of attachment and protein density strongly impacted the functionality of the immobilized HSA. High non-specific adsorption resulted in the loss of its binding capacity, whereas predominately covalently-conjugated HSA showed binding affinities higher than that of soluble HSA and had a Kd value in the range of about 6 to 12 nM. Our findings indicate that reaction parameters should be carefully assessed to obtain site-selective and specifically oriented conjugation that maintains the protein's binding capacity. The approach presented here may serve as general instruction for the immobilization of high molecular weight targeting proteins to the surfaces of nanoparticles.

Indocyanine green-loaded injectable alginate hydrogel as a marker for precision cancer surgery.

Accurate identification of tumor sites and boundaries is of paramount importance during minimally invasive surgery. Although laparoscopic resection is being increasingly and widely performed for early gastric and colorectal cancers, the detection of tumors located inside the stomach and intestine is difficult owing to the lack of tactile sensation. Here, we propose the application of an indocyanine green (ICG)-loaded alginate hydrogel system as a fluorescence surgical marker for precise laparoscopic operations. A physical complex of ICG and human serum albumin (HSA) was mixed with sodium alginate to form an injectable hydrogel system. Calcium carbonate and D-gluconic acid (GA) were added to the gel to control its strength and gelation time, respectively. The optimal conditions for the preparation of injectable hydrogels were determined by analyzing the fluorescence spectra and sol-gel transition time of the prepared samples at various concentrations and compositions. Next, the aqueous solutions of ICG, ICG-HSA, and ICG-HSA-loaded alginate were subcutaneously injected into nude mice (three mice per group), and near-infrared (NIR) fluorescence images of the mice (λex. =780 nm, λem. =845 nm) were obtained at different points in time for 8 days. Then, fluorescence intensities at the injection sites, target-to-background ratio, and areas of ICG fluorescence were analyzed. Finally, the potential utility of ICG-HSA-loaded alginate hydrogel as a surgical marker was evaluated in a porcine model. The ICG-HSA-loaded alginate solution was injected into three sites in the submucosal space of the porcine stomach via a catheter. A fluorescent laparoscopic system was installed on the abdomen of the pig 3 days post-injection, and the fluorescence signal generated from the fluorescence surgical marker located inside the stomach was evaluated using the fluorescence laparoscope system (λex. =785 nm, λem. =805 nm). The optimal concentration of ICG-HSA complex was determined to be 30 µM, and maximum fluorescence intensity of the complex was obtained at a 1:1 mole ratio of HSA to ICG. The subcutaneous injection of ICG or ICG-HSA solution in mice resulted in the rapid spread of the fluorescence signal around the injection site in 3 h, and a weak fluorescence was detected at the injection site 24 h post-injection. In contrast, the fluorescence detection time was effectively prolonged up to 96 h post-injection in the case of ICG-HSA-loaded alginate gel, while diffusion of the injected ICG from the injection site was effectively prevented. In the laparoscopic operation, injection sites of the hydrogel in porcine stomach could be accurately detected in real time even after 3 days. This alginate hydrogel system may be potentially useful as an effective surgical marker in terms of accuracy and persistence for laparoscopic operation.

Albumin Based Nanomedicine for Enhancing Tacrolimus Safety and Lymphatic Targeting Efficiency.

The increasing demand of organ transplantation and the frequent occurrence of transplant rejection necessitate the development of new therapies. Immunosuppressant marks an effective therapeutic strategy but is usually impeded by its toxicity, untargeted delivery to draining lymph node. The emergence of nanoformulations provides a possibly way to address these challenges. In this work, we prepared a novel human serum albumin (HSA) nanoformulation loaded with a well-known immunosuppressant tacrolimus (TAC) (termed TAC-HSA-NPs), via a simple self-assembly procedure of albumin in aqueous solution to enhance the solubility of TAC. By adjusting the molar ratio of HSA to TAC, we were able to take control of several key parameters of the obtained nanoparticles, such as loading capacity and particle size. Under the molar ratio of HSA:TAC = 1:10, TAC-HSA-NPs showed a mean size of 164 ± 51 nm with reliable pharmacokinetic stability, prolonged blood circulation time as well as decreased renal toxicity. More importantly, in vivo mice allo-heart transplantation verified the efficient lymphatic targeting of TAC-HSA-NPs, which is of crucial significance for immunosuppression in heart transplant recipients. Overall, our study proposed a new nanoformulation of TAC-HSA-NPs and confirmed the potential application in organ transplantation.

Impact of Ethylene Oxide Sterilization of Polymer-Based Prefilled Syringes on Chemical Degradation of a Model Therapeutic Protein During Storage

Materials from prefilled syringe systems—such as silicone oil, tungsten, glass, and rubber—may enhance therapeutic protein aggregation and particle formation. Also, the sterilization method used for syringes may impact aggregation and chemical degradation of biologics during storage. Syringes are generally sterilized by radiation, ethylene oxide gas (EO), or steam. Among the sterilization methods, EO has the potential to cause chemical degradation by the formation of adducts with susceptible amino acid residues in the protein. In this study, EO- and steam-sterilized syringes were compared to determine the influence of residual EO on human serum albumin (HSA) degradation. Although the amount of residual EO in the EO-sterilized syringes was less than 220 μg/syringe, well below the International Organization for Standardization limit, EO adduction to cysteine (Cys) and methionine (Met) in HSA was observed by liquid chromatography and mass spectrometry analysis. The EO adduct ratio of HSA stored for 2 weeks in EO-sterilized syringes was about 45%. In contrast, no chemical degradation was observed in HSA formulation stored in steam-sterilized syringes. Because of the propensity of EO to readily form adducts with proteins, an alternative to EO sterilization should be used for prefilled syringes that will be used for therapeutic protein products.

Bioanalytical verification of V-type nerve agent exposure: simultaneous detection of phosphonylated tyrosines and cysteine-containing disulfide-adducts derived from human albumin

Nerve agents still represent a serious threat to civilian and military personnel as demonstrated by the violent conflict in the Middle East. For verification of poisoning, covalent adducts with endogenous proteins (e.g., human serum albumin, HSA) are valuable long-term biomarkers. Accordingly, we developed a microbore liquid chromatography-electrospray ionization mass spectrometry/high-resolution mass spectrometry (μLC-ESI MS/HR MS) method for simultaneous detection of HSA-adducts with the V-type nerve agents VX, Chinese VX (CVX), and Russian VX (RVX). Following Pronase-catalyzed proteolysis, novel disulfide-adducts were detected in addition to phosphonylated tyrosine residues. Dipeptide disulfide-adducts were formed between the thiol-containing leaving group of the V-type nerve agents (2-(diisopropylamino)ethanethiol, DPAET, for VX and 2-(diethylamino)ethanethiol, DEAET, for CVX and RVX) and the free thiol group of Cys34 in HSA (DPAET-CysPro, DEAET-CysPro). We also identified tripeptide disulfide-adducts containing Cys448 (MetProCys-DPAET, MetProCys-DEAET) and to a lesser extent Cys514 (AspIleCys-DPAET, AspIleCys-DEAET). Synthetic tripeptide references were used for confirmation of the postulated structures by μLC-ESI MS/HR MS. Lower limits of detection were determined in human plasma, being nearly identical for the three V-type nerve agents, and corresponded to 1-6 μM nerve agent for tyrosine-adducts, 1-3 μM nerve agent for CysPro-adducts, and 6 μM nerve agent for MetProCys-adducts, thus covering concentrations of toxicological relevance. Characterization of proteolysis kinetics revealed stable plateaus for all adducts being reached between 60 and 90 min at 37 °C. Adduct formation kinetics were characterized by simultaneously monitoring the V-type nerve agent, its leaving group, and the corresponding disulfide dimer. Furthermore, adduct formation patterns were investigated as a function of the molar ratio of HSA to V-type nerve agent. Graphical abstract Modification of human serum albumin (HSA) by V-type nerve agents Chinese VX (CVX) and RussianVX (RVX). Various tyrosine residues (Tyr???)n (e.g. most reactive Tyr411) were phosphonylated and disulfide-adducts were formed between the thiol-containing leaving group 2-(diethylamino)ethanethiol (DEAET) and at least three cysteine residues (Cys34, Cys448 and Cys514). Pronase-mediated proteolysis produced low-molecular cleavage products including phosphonylated tyrosines, dipeptide (Cys34Pro) and tripeptide (MetProCys448, AspIleCys514) disulfide-adducts that were detected by microbore liquid chromatography-electrospray ionization mass spectrometry/high-resolution mass spectrometry (μLC-ESI MS/HR MS).

Spectrometric study on the interaction of indocyanine green with human serum albumin

The interaction of indocyanine green(ICG) with human serum albumin(HSA) was investigated via various spectrometric(UV-visible, fluorescence and circular dichroism) techniques. The experimental results indicate that the interaction of ICG with HSA depends on the values of R(R is defined as the molar ratio of HSA to ICG). The interaction of ICG with HSA can form two complexes with intrinsic binding constants(Ka) of 2.97×105(R≤2) and 2.63×104(R>2), respectively. The fluorescence and induced CD(ICD) spectra of ICG demonstrate that binding the first mole of HSA to ICG can form a chiral ICG-HSA complex with strong fluorescence emission, and the chirality and fluorescence of ICG-HSA complex can be significantly reduced by adding another mole of HSA to ICG. Furthermore, although both ICG and ICG-HSA complexes followed an energy-dependent endocytosis process to enter living cells, the cellular uptaken dynamic mechanism of ICG was significantly affected by the HSA conjugation.

Synthesis, structure, and biological evaluation of a copper(ii) complex with fleroxacin and 1,10-phenanthroline.

A novel mixed-ligand Cu(ii) complex combined with the quinolone drug fleroxacin and 1,10-phenanthroline was synthesized in this work. The crystal structure of the complex was characterized via X-ray crystallography, which was the first reported single crystal complex of fleroxacin. Results showed that Cu(ii) was coordinated through pyridone oxygen and one carboxylate oxygen atom of fleroxacin, as well as two nitrogen atoms from 1,10-phenanthroline. Various characterization methods, including Fourier transform infrared, elementary analysis, thermogravimetry, and X-ray powder diffraction, were applied. The Cu(ii)-quinolone complex exhibited favorable biological activities, and was proved to be capable of transforming supercoiled PUC19 DNA into nicked form under hydrolytic conditions. The obtained pseudo-Michaelis-Menten kinetic parameter was 12.64 h(-1), which corresponded to a million-fold rate enhancement in DNA cleavage. In addition, the interaction capacity of the complex with human serum albumin (HSA) was investigated. The results demonstrated a moderately intense combination between HSA and the complex. The complex evidently quenched the fluorescence of HSA. Approximately 19.2% of the quenching was attributed to Förster resonance energy transfer (FRET), whereas the rest was caused by ground-state complex formation (molar ratio of HSA : complex = 1 : 2). The energy of the complex was excited during FRET, which increased the fluorescence of the complex by approximately 18%.

Modification of a PAMPA model to predict passive gastrointestinal absorption and plasma protein binding

The Parallel Artificial Membrane Permeability Assay (PAMPA) is a well-known high throughput screening (HTS) technique for predicting in vivo passive absorption. In this technique, two compartments are separated by an artificial membrane that mimics passive permeability through biological membranes such as the dermal layer, the gastrointestinal tract (GIT), and the blood brain barrier (BBB).In the present study, a hexadecane artificial membrane (HDM)-PAMPA was used to predict the binding of compounds towards the human plasma using a mixture of human serum albumin (HSA) and alpha-1-acid glycoprotein (AGP). The ratio of HSA and AGP was equivalent to that found in the human plasma for both proteins (∼20:1). A pH gradient (5.0–7.4) was performed to increase the screening capacity and overcome the issue of passive permeability for acidic and amphoteric compounds.With this assay, the prediction of passive GIT absorption was maintained and the compounds were discriminated according to their permeability (on a no-to-high scale). The plasma protein binding (PPB) was estimated via the correlation of the differences between the amount of compound crossing the artificial membrane in assays conducted with and without protein using only a two end-point measurement. The use of a mixture of HSA and AGP to modulate drug permeation was compared to the use of the same concentrations of HSA and AGP used separately. The addition of HSA alone in the acceptor compartment was sufficient for estimating PPB, while it was demonstrated that AGP alone could enable the estimation of AGP binding.

Correspondence of Fatty Acid and Drug Binding Sites on Human Serum Albumin: A Two-Dimensional Nuclear Magnetic Resonance Study

The ability of human serum albumin (HSA) to bind fatty acids (FA) in multiple sites has been revealed by many studies. Here we detect and characterize nine individual binding sites by two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy of 18-[(13)C]-oleic acid (OA) complexed with HSA. We characterize site-specific FA binding by addition of (i) different FA molar ratios (from 1:1 to 4:1 OA:HSA) to observe the order of filling and occupancy of binding sites; (ii) methyl-β-cyclodextrin, as a FA acceptor, to observe the dissociation of FA; and (iii) drugs (with known binding sites in the crystal structure) to reveal the correspondence of three NMR peaks with sites in the crystal structure. At 1:1 and 2:1 OA:HSA ratios, three sites were shown to fill sequentially. These high-affinity sites were well resolved from additional sites (one medium-affinity and five low-affinity) observed at 3:1 and 4:1 OA:HSA ratios. Methyl-β-cyclodextrin extracted OA from individual sites in the reverse order of filling. FA bound in three low-affinity sites were displaced by drugs shown to bind in crystalline HSA to FA sites 7 and 3 (Sudlow's drug sites I and II, respectively) and FA site 6. With this strategy, 2D NMR spectral analysis permits site-specific characterization of the binding of drugs and FA and provides a sensitive probe of the mutual effects of FA and ligand binding.

The reactivity of human serum albumin toward trans‐4‐hydroxy‐2‐nonenal

Mass spectrometry was used to probe the preferred locations of trans-4-hydroxy-2-nonenal (HNE) addition to the cysteine, histidine, and lysine residues of human serum albumin (HSA). Considering only those modified peptides supported by high mass accuracy Orbitrap precursor ion measurements (high confidence hits), with HNE:HSA ratios of 1:1 and 10:1, 3 and 15 addition sites, respectively, were identified. Using less stringent criteria, a total of 34 modifications were identified at the higher concentration. To gain quantitative data, iTRAQ labeling studies were completed. Previous work had identified Cys(34) , the only free cysteine, as the most reactive residue in HSA, and we have found that Lys(199) , His(242/7) , and His(288) are the next most reactive residues. Although the kinetic data indicate that the lysines and histidines can react at relatively similar rates, the results show that lysine addition is much less favorable thermodynamically; under our reaction conditions, lysine addition generally does not go to completion. This suggests that under physiological conditions, HNE addition to lysine is only relevant in situations where unusually high HNE concentrations or access to irreversible secondary reactions are found.

Elucidation of the human serum albumin (HSA) binding site for the Cu-PTSM and Cu-ATSM radiopharmaceuticals

The Cu-PTSM (pyruvaldehyde bis(N(4)-methylthiosemicarbazonato)copper(II)) and Cu-ATSM (diacetyl bis(N(4)-methylthiosemicarbazonato)copper(II)) radiopharmaceuticals exhibit strong, species-dependent binding to human serum albumin (HSA), while Cu-ETS (ethylglyoxal bis(thiosemicarbazonato)copper(II)) appears to only exhibit nonspecific binding to human and animal serum albumins. This study examines the structural basis for HSA binding of Cu-PTSM and Cu-ATSM via competition with drugs having known albumin binding sites. Warfarin, furosemide, ibuprofen, phenylbutazone, benzylpenicillin, and cephmandole were added to HSA solutions at drug:HSA mole ratios from 0 to 8:1, followed by quantification of radiopharmaceutical binding to HSA by ultrafiltration. Warfarin, a site IIA drug, progressively displaced both [(64)Cu]Cu-PTSM and [(64)Cu]Cu-ATSM from HSA. At 8:1 warfarin:HSA mole ratios, free [(64)Cu]Cu-PTSM and [(64)Cu]Cu-ATSM levels increased 300-500%. This was in contrast to solutions containing ibuprofen, a site IIIA drug; no increase in free [(64)Cu]Cu-PTSM or [(64)Cu]Cu-ATSM was observed except at high ibuprofen:HSA ratios, where secondary ibuprofen binding to the IIA site may cause modest radiopharmaceutical displacement. By contrast, and consistent with earlier findings suggesting Cu-ETS exhibits only nonspecific associations, [(64)Cu]Cu-ETS binding to HSA was unaffected by the addition of drugs that bind in either site. We conclude that the species-dependence of Cu-PTSM and Cu-ATSM albumin binding arises from interaction(s) with the IIA site of HSA.

How do fatty acids cause allosteric binding of drugs to human serum albumin?

This study was undertaken to investigate how fatty acids cause the allosteric binding of drugs to human serum albumin (HSA). The influence of fatty acids on the binding of ketoprofen (KP), an NSAID, to HSA was examined by using a photoaffinity labeling technique. Ultrafiltration was performed to quantitate the concentration of free KP. HSA, photolabeled with KP in the presence of myristate (MYR), octanoate, and diazepam, was cleaved with cyanogen bromide, separated by Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently analyzed by autoradiography. The addition of MYR at molar ratios from 4 to 5, but not from 1 to 2, causes substantial increases in unbound KP for KP:HSA ratios of 0.5 and 1. The addition of two or more moles of MYR, octanoate, and diazepam per mole of HSA caused a pronounced decrease in the labeling of the 11.6- and 13.5-kDa peptides. However, only MYR showed an increase in labeling of the 20 kDa and, especially, the 9.4-kDa peptides. At MYR:HSA ratios in excess of 3, a decrease in the extent of labeling of the 9.4-kDa peptide was observed. Long-chain fatty acids regulate the binding properties of HSA in a complex manner, in which a simultaneous competitive and allosteric mechanism operates and which mainly involves domain I.

A Water-Soluble Synthetic Bilirubin with Carboxyl Groups Replaced by Sulfonyl Moieties

The first symmetrical bilirubin analog with CO2H groups replaced by SO3H, 8,12-bis-(2-sulfo-ethyl)-3,17-diethyl-2,7,13,18-tetramethyl-(10H,21H,23H,24H)-bilin-1,19-dione, was synthesized from methyl (2,4-dimethyl-5-ethoxycarbonyl-1H-pyrrol-3-yl) acetate in nine steps via the sulfonic acid analog of xanthobilirubic acid (XBR) and isolated as its disodium salt. The sulfonic acid group was introduced at an early stage of the synthesis by reaction of an intermediate, ethyl 4-(2-bromoethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate, with sodium sulfite. The disodium bilirubin disulfonate exhibits NMR spectroscopic properties rather similar to those of the parent carboxylic acid, mesobilirubin-XIIIα; however, its UV/Vis spectra are blue-shifted and broadened relative to those of the parent compound. Like mesobilirubin, the disulfonate displays a positive exciton chirality circular dichroism spectrum, albeit with weaker Cotton effects, in a buffered aqueous solution (pH = 7.4) containing a 2:1 molar ratio of human serum albumin.

Protein conjugates of synthetic saccharides elicit higher levels of serum IgG lipopolysaccharide antibodies in mice than do those of the O-specific polysaccharide from Shigella dysenteriae type 1.

Our development of vaccines to prevent shigellosis is based on the hypothesis that a critical (protective) level of serum IgG to the O-specific polysaccharide (O-SP) domain of Shigella lipopolysaccharide (LPS) confers immunity. The O-SP is a hapten and must be conjugated to a protein to induce serum antibodies. The O-SP of Shigella dysenteriae type 1 (approximately 27 tetrasaccharide repeat units), prepared by acid hydrolysis of the LPS, was bound to human serum albumin (HSA) by multiple point attachment (O-SP-HSA): The molar ratio of HSA to O-SP was 1.0. Synthetic saccharides, composed of one or multiples of the O-SP tetrasaccharide, equipped with a spacer at their reducing end, were bound to HSA by a single point attachment: The average molar ratios of the saccharides to HSA ranged from 4 to 24. Serum IgG anti-LPS, elicited in mice by O-SP-HSA or synthetic tetra-, octa-, dodeca-, and hexadecasaccharide fragments, was measured by ELISA. Outbred 6-week-old female mice were injected s.c. three times at biweekly intervals with 2.5 micrograms of saccharide as a conjugate and were bled 7 days after the second and third injections. Excepting the tetramer, conjugates of the octamer, dodecamer and hexadecamer elicited IgG LPS antibodies after the second injection, a statistically significant rise (booster) after the third injection, and higher levels than those vaccinated with O-SP-HSA (P = 0.0001). The highest geometric mean levels of IgG anti-LPS were elicited by the hexadecamer with 9 chains or 9 moles of saccharide/HSA (15.5 ELISA units) followed by the octamer with 20 chains (11.1 ELISA units) and the dodecamer with 10 chains (9.52 ELISA units). Clinical evaluation of these synthetic saccharides bound to a medically useful carrier is planned.

NMR spectroscopic diffusion, chemical shift and linewidth measurements of low-affinity binding of ibuprofen enantiomers to human serum albumin

The binding of racemic, (R)(-) and (S)(+)-ibuprofen (IBP) to human serum albumin (HSA) was studied using NMR spectroscopy. Having saturated the tight binding sites, the extent of weak binding was then investigated. The molecular diffusion coefficients of IBP in HSA solution at different concentration ratios indicate that there is no significant difference in binding capacity of the two enantiomers of IBP to HSA at high IBP:HSA ratios. However, there are chemical shift and linewidth changes in the (NMR)-N-1 spectrum of IBP in the presence of HSA induced by the binding and this suggests that the major binding interaction involves the sec-butyl chain of the IBP molecule binding to HSA in hydrophobic pockets. Copyright (C) 1999 John Wiley & Sons, Ltd.

The extracellular control of intracellular aspirin hydrolysis.

We have previously presented evidence that an intra-erythrocyte arylesterase is primarily responsible for the deacetylation of aspirin to salicylate. We now report studies on extracellular control factors. The fractional acetylsalicylic acid (ASA) hydrolysis at the completion of 20-minute incubations in suspensions of washed red blood cells (RBC) with human serum albumin (HSA) decreased sharply, from 0.6 at 0 gm/dl HSA to 0.2 at 5.0 gm/dl HSA. In RBC suspensions with oleic, linoleic, and lauric acid, ASA hydrolysis increased as fatty acid:HSA ratios increased. The addition of salicylate (SA) also resulted in an increase in the fraction hydrolyzed. In a separate, more direct, ultracentrifugation assay, unbound ASA levels were similarly affected by HSA and the other ligands. When SA and oleic acid were incubated with ASA in RBC, additive competition for binding sites was apparent at relatively low levels of the fatty acid. In the presence of increasing concentrations of sodium SA, 5-amino SA, and 4-amino SA, ASA hydrolysis in RBC suspensions was increased by an average of 137%, 75%, and 54%, respectively. The most reasonable explanation for these findings is that the intact ASA molecule binds to albumin and that the binding of competing ligands can modulate the levels of free ASA in the circulation. This, in turn, affects the amount of drug available as substrate for penetration into the RBC and reaction with the intracellular hydrolase which catalyzes ASA deacetylation. Such factors controlling drug availability, if validated in vivo, could be important with regard to the antiinflammatory and anti-platelet properties of aspirin.