Sudlow's Site Research Articles

Design, synthesis and protein-binding character of an acylhydrazone anticancer candidate

Acylhydrazone derivatives have potential antitumor activities due to their metabolic modulation effect in cancer cells. Herein, an acylhydrazone compound, p-nitrobenzaldehyde-p-methylbenzoylhydrazone (MNB), was designed, prepared and characterized. The interaction of MNB with carrier protein is systematically deciphered using spectroscopic, electrochemical and modeling techniques. Ultraviolet (UV) and electrochemical impedance spectroscopy (EIS) indicate that MNB can couple with albumin through hydrophobic interaction. Molecular probe experiments reveal MNB is located at Sudlow's site I in albumin. Stern-Volmer equation, Langmuir adsorption isotherm, van’t Hoff equation and Arrhenius equation are applied to analyze the thermodynamic and kinetic characteristics of the interaction. The results demonstrate that the interaction is a typical spontaneous process dominated by entropy, whose activationenergy is ca. 26.78 kJ·mol−1. Circular dichroism (CD) spectra show that MNB has a small impact on the conformation of albumin. Molecular docking investigation simulates the structural details of the new complex, which is consistent with experimental conclusions. This study provides comprehensive information on the interaction of MNB with carrier protein, which lays the basis for its application as a new anticancer candidate.

Computational View of β-Asarone–Human Serum Albumin Interaction

β-Asarone (BAS), a bioactive phytochemical from the medicinal herb, Acorus calamus Linn., has shown many pharmacological activities. Computational docking studies unveiled the interaction site of BAS on the human plasma carrier, albumin. The primary binding arrangement of BAS was placed at Sudlow's Site I of HSA, which is pinpointed in subdomain IIA of albumin. Hydrophobic and van der Waals forces together with hydrogen bonds appear to secure the BAS-albumin complex. The BAS at Site I was surrounded by more hydrophobic and polar residues than those seen at Site II, as evidenced by LigPlot+. Therefore, the interaction between BAS and albumin at Site I seems to be comparatively more stable owing to more vital interactions.

Esterase Activity of Serum Albumin Studied by 1H NMR Spectroscopy and Molecular Modelling.

Serum albumin possesses esterase and pseudo-esterase activities towards a number of endogenous and exogenous substrates, but the mechanism of interaction of various esters and other compounds with albumin is still unclear. In the present study, proton nuclear magnetic resonance (1H NMR) has been applied to the study of true esterase activity of albumin, using the example of bovine serum albumin (BSA) and p-nitrophenyl acetate (NPA). The site of BSA esterase activity was then determined using molecular modelling methods. According to the data obtained, the accumulation of acetate in the presence of BSA in the reaction mixture is much more intense as compared with the spontaneous hydrolysis of NPA, which indicates true esterase activity of albumin towards NPA. Similar results were obtained for p-nitophenyl propionate (NPP) as substrate. The rate of acetate and propionate release confirms the assumption that there is a site of true esterase activity in the albumin molecule, which is different from the site of the pseudo-esterase activity Sudlow II. The results of molecular modelling of BSA and NPA interaction make it possible to postulate that Sudlow site I is the site of true esterase activity of albumin.

A simple method for obtaining human albumin and its use for in vitro interaction assays with indole-thiazole and indole-thiazolidinone derivatives

This work aimed to develop a simple and low-cost method to obtain human serum albumin (HSA) and its consequent application for in vitro drug interaction assays. The HSA was purified by classic principles of plasma precipitation and thermocoagulation, using a multiple-stage fractionation. The quality of the final product was assessed by electrophoresis, protein dosage by the Lowry method and the pharmacopeial thermal stability. At the end, an isotonic solution of HSA with a total protein concentration of 2.7 mg·mL−1 was obtained, which was visualized as a single band corresponding to the molecular weight of 66 kDa. After the thermal stability test, there was no indication of turbidity or color change of the solution. Finally, the HSA was useful for interaction assays with indole-thiazole and indole-thiazolidinone derivatives through UV–vis absorption and fluorescence spectroscopic studies, as well as by docking molecular analysis. Derivatives quenched the intrinsic fluorescence of HSA, disrupted the tryptophan residues microenvironment, and probably bind at Sudlow's site I. Therefore, the simplified methodology developed in this work proved to be effective in obtaining HSA that can be applied to research goals including drug interaction assays.

Exploration of the binding between cuminol and bovine serum albumin through spectroscopic, molecular docking and molecular dynamics methods

Cuminol (4-Isopropylbenzyl alcohol), found in the essential oils of several plant sources, is an important constituent of several cosmetics formulations. The interaction of cuminol with model plasma protein bovine serum albumin was studied in this paper. The experimental studies were mainly carried out using fluorescence spectrophotometry aided with UV visible and CD spectroscopies. Intrinsic fluorescence measurements showed that there was a weak binding between cuminol and BSA. The mechanism of binding involved static quenching with around 1:1 binding. The binding was chiefly supported by hydrophobic forces although a little contribution of hydrogen bonding was also found in the interaction and the values of enthalpy change were negative with positive entropy change. The secondary structure of BSA didn’t change significantly in presence of low concentrations of cuminol, however, partial unfolding of the former taken place when the concentration of the latter increased. Molecular docking analyses showed cuminol binds at the intersection of subdomains IIA and IIIA, i.e. its binding site is in between Sudlow sites I and II. Molecular dynamics simulations results have shown that BSA forms a stable complex with cuminol and the structure of the former didn’t change much in presence of later. Communicated by Ramaswamy H. Sarma

Spectroscopic Studies of Quinobenzothiazine Derivative in Terms of the In Vitro Interaction with Selected Human Plasma Proteins. Part 1.

Plasma proteins play a fundamental role in living organisms. They participate in the transport of endogenous and exogenous substances, especially drugs. 5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazinium salts, have been synthesized as potential anticancer substances used for cancer treatment. Most anticancer substances generate a toxic effect on the human body. In order to check the toxicity and therapeutic dosage of these chemicals, the study of ligand binding to plasma proteins is very relevant. The present work presents the first comparative analysis of the binding of one of the 5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazinium derivatives (Salt1) with human serum albumin (HSA), α-1-acid glycoprotein (AGP) and human gamma globulin (HGG), assessed using fluorescence, UV-Vis and CD spectroscopy. In order to mimic in vivo ligand–protein binding, control normal serum (CNS) was used. Based on the obtained data, the Salt1 binding sites in the tertiary structure of all plasma proteins and control normal serum were identified. Both the association constants (Ka) and the number of binding site classes (n) were calculated using the Klotz method. The strongest complex formed was Salt1–AGPcomplex (Ka = 7.35·104 and 7.86·104 mol·L−1 at excitation wavelengths λex of 275 and 295 nm, respectively). Lower values were obtained for Salt1–HSAcomplex (Ka = 2.45·104 and 2.71·104 mol·L−1) and Salt1–HGGcomplex (Ka = 1.41·104 and 1.33·104 mol·L−1) at excitation wavelengths λex of 275 and 295 nm, respectively, which is a positive phenomenon and contributes to the prolonged action of the drug. Salt1 probably binds to the HSA molecule in Sudlow sites I and II; for the remaining plasma proteins studied, only one binding site was observed. Moreover, using circular dichroism (CD), fluorescence and UV-Vis spectroscopy, no effect on the secondary and tertiary structures of proteins in the absence or presence of Salt1 has been demonstrated. Despite the fact that the conducted studies are basic, from the scientific point of view they are novel and encourage further in vitro and in vivo investigations. As a next part of the study (Part 2), the second new synthetized quinobenzothiazine derivative (Salt2) will be analyzed and published.

Elucidation of interaction between serum albumin and ginsenoside CK along with cytotoxic study

As a main metabolite of ginsenosides, compound K (CK) has a vast array of pharmacological effects. However, due to its low polarity and insoluble in water, its oral application has been greatly limited. In this work, the interaction between serum albumin and ginsenoside CK was elucidated by multi-spectroscopic studies. The result of ultraviolet/visible absorption spectroscopy showed that the conformation of serum albumin could be changed via binding with CK. The result of fluorescence spectroscopy suggested that CK could form complex with serum albumin. CK could quench the fluorescence and the fluorescence residues of serum albumin were located in or near the binding position. Molecular docking indicated that CK bound at Sudlow's site II of serum albumin and formed hydrogen-bonding interactions with three residues. Furthermore, the flexible side chain of CK was difficult to be stabilized at the binding site, resulting in its serious perturbation during dynamics simulation. This work also performed the cytotoxic study and the result showed that serum albumin enhanced the inhibitory effect of CK on the proliferation of both Caco-2 and HCT-116 cells. To sum up, this work revealed that serum albumin might be an appropriate carrier of hydrophobic compounds, with the advantage of improving their biocompatibility.

A molecular dynamics study on the binding of gemcitabine to human serum albumin

Human Serum Albumin (HSA) is introduced as one of the ideal protein-based nanoparticles for drug delivery due to some of its unique performances such as non-toxic, non-immunogenic, biocompatible, and biodegradable. Besides, the HSA has gained considerable attention owing to high compatibility without any severe side effects and extraordinary binding capacity of various drugs on its Sudlow sites (Ⅰ and Ⅱ). Hence, this study tries to explore and discuss the interactions between two Sudlow sites of HSA as the drug delivery agent and Gemcitabine (GEM), a drug used for treating cancerous tissues and cells, employing molecular dynamics. To investigate the adsorption phenomenon and the binding process of GEM on the Sudlow sites of HSA, the dynamics of GEM molecule, radial distribution function, radius of gyration, solvent-accessible surface area, hydrogen bonding, vdw energy, and free binding energy results are analyzed. The simulation results reveal that the binding of GEM molecules to the Sudlow sites of HSA induced slight conformational changes in the HSA structure. The results also illustrate that the GEM could strongly bind to the subdomain IIIA of HSA, and its stability on the site Ⅱ is higher than site Ⅰ. Hydrogen bonding as a non-bond interaction is the primary force in the stability of the GEM-HSA complex.

Investigating the binding affinities of fructose and galactose to human serum albumin: simulation studies

ABSTRACT Human serum albumin (HSA) is abundant in blood. HSA binds a wide range of drugs, metabolites, and nutrients. A glycated HSA is also a potential diabetes biomarker. Recently, crystal structures of glucose- and fructose-bound HSA have been reported. Both cyclic and acyclic sugar forms are trapped in Sudlow site I. Galactose can also bind HSA, but no atomic detail is available. Thus, molecular dynamics simulations were employed to study the structural and dynamic properties of fructose- and galactose-bound HSA in comparison to glucose-bound HSA from previous studies. Both bound sugars promote different degrees of domain motions which can affect a drug/solute binding affinity at Sudlow site I. A large and highly water-exposed Sudlow site I allows high mobility of bound sugars. Nonetheless, more protein contacts imply a tighter binding of fructose than galactose. Although galactose and glucose are epimers, galactose forms a different interaction network which disrupts a formation of interactions with K195 and K199 resulting in the escape of galactose. In contrast, fructose molecules are anchored inside by a number of protein interactions and sugar dimer structure. These highlights the importance of protein–sugar and sugar–sugar interactions for ligand binding in large and highly water-exposed cavity.

Synthesis and characterization of Pd(II) antitumor complex, DFT calculation and DNA/BSA binding insight through the combined experimental and theoretical aspects

A novel palladium(II) complex, [Pd(bpy)(acac)]NO3 (where bpy and acac are 2,2′-bipyridine and acetylacetonato), has been synthesized and fully characterized through 1H NMR, UV-Vis and FT-IR spectra, conductivity measurement and elemental analysis. Geometry optimization using DFT calculation demonstrated that a square-planar geometry is built around Pd(II) atom. The MEP and FMO analysis were also applied. Partition coefficient evaluation was conducted to determine the lipophilicity of the compound which follows the order complex > cisplatin. The antitumor activity of the complex has been evaluated on K562 cancer cells by MTT method, which exhibited an appropriate activity in compare to cisplatin. The interaction of [Pd(bpy)(acac)]NO3 towards biomolecules (CT-DNA and BSA) was investigated through in-detail fluorescence, UV–Vis and other techniques. The findings of fluorescence titration demonstrated that the Pd(II) complex binds to CT-DNA via hydrophobic and with BSA through van der Waals forces and hydrogen bond. UV–Vis experiment revealed a reduction in the absorption spectra of biomolecules in presence of the complex. The binding affinity was further endorsed for CT-DNA utilizing viscosity measurement and gel electrophoresis and for BSA via CD spectroscopy and FRET calculation. In addition, molecular docking supports the findings of experiments and indicates that the Pd(II) complex appears to be situated at DNA intercalation position and BSA Sudlow's site I. All results have shown that this compound may be a useful potential antitumor candidate for further medical purposes.

High-Performance affinity chromatographic studies of repaglinide and nateglinide interactions with normal and glyoxal- or methylglyoxal-modified human albumin serum

During diabetes human serum albumin (HSA), an important drug transport protein, can be modified by agents such as glyoxal (Go) and methylglyoxal (MGo) to form advanced glycation end-products. High-performance affinity microcolumns and zonal elution competition studies were used to compare interactions by the anti-diabetic drugs repaglinide and nateglinide with normal and Go- or MGo-modified HSA at Sudlow sites I and II of this protein. Both drugs had their strongest binding at Sudlow site II for the normal and modified forms of HSA. The association equilibrium constants at this site for repaglinide and nateglinide with normal HSA were 6.1 (± 0.2) × 104 M−1 and 7.1 (± 0.8) × 105 M−1, respectively, at pH 7.4 and 37⁰C; these values increased by up to 3.6-fold for repaglinide and decreased by up to 45–55 % for nateglinide when HSA was modified by Go or MGo at levels seen in prediabetes or diabetes. Both drugs were also found to bind at Sudlow site I, with association equilibrium constants at this site on normal HSA of 4.2 (± 0.3) × 104 M−1 for repaglinide and 5.0 (± 0.1) × 104 M−1 for nateglinide. The binding strength for repaglinide at Sudlow site I increased by 1.3- to 1.7-fold with the Go-modified HSA and decreased slightly (i.e., up to 19 %) for the MGo-modified HSA, while nateglinide showed only a small or insignificant change in binding with the same modified HSA samples. These results indicated that binding by repaglinide and nateglinide with HSA can be altered significantly by modification of this protein with Go or MGo, making these modifications of potential interest in the treatment of patients with these drugs during diabetes.

Interaction and esterase activity of albumin serums with orphenadrine: A spectroscopic and computational approach

Herein, we have explored the comparative effect of antihistaminic drug, orphenadrine (ORP) on serum albumins - human serum albumin (HSA) and bovine serum albumin (BSA). The interaction between ORP and proteins was characterized using steady state fluorescence spectroscopy, UV-visible spectroscopy, circular dichroism (CD) and molecular docking methods. Binding constant for BSA-ORP and HSA-ORP systems were found to be 2.78 × 104 Lmol−1 and 3.09 ×104 Lmol−1, respectively. Far UV-CD results showed that the secondary structure of HSA becomes more compact in presence of ORP, however, the secondary structure of BSA decreases in presence of ORP. The functionality of both the proteins changes in presence of ORP which was confirmed by esterase like activity of BSA/HSA. Competitive binding results showed that ORP binds to Sudlow site I (subdomain II) of both BSA and HSA. The computational study was carried out to further our understanding of the experimental work.

Biomolecular interaction mechanism of an anticancer drug, pazopanib with human serum albumin: a multi-spectroscopic and computational approach

Pazopanib (PZP) is a multi-targeting tyrosine kinase inhibitor and is currently approved by FDA for the treatment of soft tissue sarcoma and renal cancer. Molecular interaction mechanism of PZP with human serum albumin (HSA) was explored under simulated physiological conditions (pH = 7.4), using fluorescence and UV absorption spectroscopy along with computational methods. Based on the inverse correlation between the Stern-Volmer constant (Ksv ) and temperature, it was concluded that PZP quenched the protein fluorescence through static quenching mechanism. This was also confirmed from the UV-vis absorption spectral results. Moderate binding affinity between PZP and HSA was evident from the Ka values (5.51 − 1.05 × 105 M−1) while PZP-HSA complex formation was driven by hydrophobic and van der Waals interactions as well as hydrogen bonds, as revealed by positive entropy change (ΔS = +98.37 J mol−1 K−1) and negative enthalpy change (ΔH = −60.31 kJ mol−1). Three-dimensional fluorescence spectral results disclosed microenvironmental perturbations around Trp and Tyr residues of the protein upon PZP binding. Interestingly, the addition of PZP to HSA significantly protected the protein against thermal stress. Competitive drug displacement results obtained with warfarin, phenylbutazone and diazepam elucidated Sudlow's Site I, positioned in subdomain IIA of HSA, as the preferred binding site of PZP which was well supported by molecular docking analysis, while molecular dynamics simulation results suggested the stability of the PZP-HSA complex. Communicated by Vsevolod Makeev

Mechanistic insight into glycation inhibition of human serum albumin by vitamin B9: Multispectroscopic and molecular docking approach

Advanced glycation end products (AGEs) formation produces free radicals that play a role in diabetes mellitus; hence inhibition of glycation plays a part in minimizing diabetes-related complications. This study was intended to examine the AGEs formation of HSA upon prolonged incubation of 28 days at 37 °C and further investigate the antiglycation potential of folic acid (FA). FA shows a significant binding affinity to the HSA with a binding constant (K) of 104 M−1. The evaluation of enthalpy change (∆H0) and entropy change (∆So) implied that the HSA-FA complex is stabilized primarily by hydrophobic interaction and hydrogen bonding. Molecular docking analysis depicted that FA binds with HSA in subdomain IIA (Sudlow's site I) with a binding energy of −7.0 kcal mol−1. AGEs were characterized by free lysine and thiol groups, carbonyl content, and AGEs specific fluorescence. The presence of FA significantly decreased glycation from free lysine and carbonyl content estimation and AGEs specific fluorescence. Multispectroscopic observations and molecular docking and examination of various biomarkers demonstrate the antiglycation activity of FA and its capacity to prevent disease progression in diabetes.

Analyzing the Long Time-Scale Dynamics of Uremic Toxins Bound to Sudlow Site II in Human Serum Albumin.

Protein bound uremic toxins (PBUTs), a series of chemicals that remain a challenge for removal strategies used on patients suffering with chronic kidney disease, could be strong candidates for MD study in order to better understand the interactions and time scales associated with binding mode transitions. Currently, traditional dialysis methods cannot satisfactorily remove PBUTs from the bloodstream. This is at least partly due to these toxin's high level of affinity for protein binding sites, particularly the prominent human serum albumin (HSA) and two of its drug binding sites (Sudlow site I and II). We investigate the dynamics of binding site transitions and interactions by MD simulations targeting four well-known toxins: indoxyl sulfate (IS), p-cresyl sulfate (PCS), indole-3-acetic acid (IAA), and hippurate acid (HIP). Long-time scale dynamics are obtained by the use of time-structure independent component analysis (tICA) for dimensionality reduction followed by spectral analysis of a Markov state model (MSM) scored using the generalized matrix Rayleigh quotient (GMRQ). Our results add new insights to prior findings related to the key role of charge-pairing in governing toxin-protein interactions. We find that IAA, the bulkiest hydrophobic toxin studied, observes the slowest process of at least 3 times slower than the smaller, less hydrophobic toxins. In general, we find that the processes slower than 15 ns are correlated with a transition from dominantly hydrophobic interactions deep in the binding pocket to a gain in hydrogen bonding partners near the mouth of the pocket. Our results indicate that aromatic residues such as PHE play a part in a type of toxin stabilization akin to π-stacking. In conclusion, this work presents mechanistic descriptions of interactions/transitions for a set of important PBUTs that bind Sudlow site II on time scales relevant to the underlying binding kinetics of most interest.

Binding mechanism of 4−octylphenol with human serum albumin: Spectroscopic investigations, molecular docking and dynamics simulation

4−Octylphenol (OP) is an environmental estrogen that can enter organisms through the food chain and cause various toxic effects. Here, the interaction between OP and human serum albumin (HSA) was explored through multipectral, molecular docking and dynamics simulation. The results showed that OP and HSA formed a ground state complex through a static quenching mechanism, and the interaction was spontaneously driven by hydrogen bonds and hydrophobic interaction forces. The binding constant at different temperatures was measured to be on the order of 105 L mol−1. Site competition experiments suggested that OP interacted with amino acid residues Lys195, Cy245 and Cys246 located at the Sudlow site I of HSA, resulting in a more stretched protein peptide. The presence of OP increased the surface hydrophobicity of HSA, and reduced the content of α-helix in HSA by 3.4%. FT−IR spectra showed that OP interacted with the C=O and C-H groups of the polypeptide backbone. Molecular docking demonstrated that OP mainly bound to Site I of HSA and hydrogen bonds participated in the interaction. In addition, molecular dynamics simulations further explored the stability and dynamic behavior of the OP−HSA complex through RMSD, RMSF, SASA and Rg.

Multi-spectroscopic and molecular modeling studies on the interactions of serum albumin with 20(S, R)-protopanaxadiol and 20(S, R)-protopanaxatriol that inhibit HCT-116 cells proliferation

Serum albumin (SA) acts as the carrier protein to transport drugs and plays a crucial role in the pharmacokinetics of distribution. In this work, the cytotoxicities of 20(S, R)-protopanaxadiol [20(S, R)-PPD] and 20(S, R)-protopanaxatriol [20(S, R)-PPT] against HCT-116 cell lines were assessed. 20(S)-PPD, 20(R)-PPD, 20(S)-PPT, and 20(R)-PPT inhibited the cells growth in a concentration-dependent manner with the IC50 values of 30.27, 18.80, 68.28, and 14.38 μM, respectively. The result of ABTS assay showed that 20(S, R)-PPD-BSA exhibited a higher antioxidant activity than that of 20(S, R)-PPD alone. Subsequently, the binding affinity and interaction mechanism of ginsenosides with SA were studied by the ultraviolet–visible absorption and fluorescence spectroscopy, which indicated that ginsenosides interfered with the microenvironment around amide bonds in SA. Through the calculation of Stern-Volmer plot, the quenching rate kq values of 20(S)-PPD, 20(R)-PPD, 20(S)-PPT, and 20(R)-PPT were 6.75, 4.30, 2.37, and 3.31 ( × 1011 M−1 s−1), respectively. These results suggested that the quenching was not caused by dynamic collision, but the formation of ginsenoside-SA complexes. The results of molecular docking suggested that these compounds tended to bind more strongly to Sudlow's site II of HSA rather than Sudlow's site I. In addition, 20(S)-ginsenosides showed more stable binding to HSA in comparison with 20(R)-ginsenosides, which may help elucidating the mechanisms that account for the stronger bioactivities of 20(S)-ginsenosides.

Delivery Mechanism of the Pharmaceutical Complex of Genistein-Adenine Based on Spectroscopic and Molecular Modelling at Atomic Scale.

Genistein (GS) exhibits various biological activities, but its clinical application is limited because of the low bioavailability. In this study, a GS-adenine pharmaceutical complex was prepared through solvent evaporation to improve the bioavailability of GS, and a molecular model of a two-component supramolecular pharmacological transport mechanism was established. The structure of GS-adenine was characterized, in addition, interaction patterns between GS and adenine were investigated using density functional theory. The results showed that the solubility of GS-adenine was five times higher than that of GS, and the cumulative release rate of GS-adenine was 86 %. The results of fluorescence spectroscopy and molecular dynamic simulations showed that GS-adenine bound to the Sudlow's site I of HSA mainly through hydrophobic interactions. This study provides a useful reference for synthesizing pharmaceutical complexes to improve solubility and for exploring the mechanism of multiple pharmaceutical components in vivo.

Probing the interactions of novel europium coordination complexes with serum albumin.

Molecular interactions between novel europium coordination complexes (EC) possessing superior cytotoxic activity and bovine serum albumin (BSA), the most prominent representative of plasma proteins, were assessed using fluorescence spectroscopy and molecular docking techniques. Cumulative results from fluorescent probe binding, fluorescence quenching and Förster resonance energy transfer studies revealed that the europium complexes V4 and V8 do not perturb the BSA structure, while V3, V5, and V7 induce partial unfolding of the polypeptide chain. Molecular docking studies coupled with analysis of the three-dimensional structure of the BSA-EC complexes showed that V4 and V8 reside in the vicinity of the protein IIA subdomain (Sudlow's site I), while V3, V5 and V5 were localized predominantly in the BSA IIIA subdomain (Sudlow's site II). Due to the intactness of the protein structure upon association with V4 and V8, these compounds may be recommended for further evaluation as potential antineoplastic agents.

Recreational drugs 25I-NBOH and 25I-NBOMe bind to both Sudlow's sites I and II of human serum albumin (HSA): biophysical and molecular modeling studies

25I-NBOH and 25I-NBOMe simultaneously bind to sites I and II of HSA, which may affect their distribution and effects.