Human Serum Amyloid a Impaired Structural Stability of High-Density Lipoproteins (HDL) and Apolipoprotein (Apo) A-I and Exacerbated Glycation Susceptibility of ApoA-I and HDL

Human serum amyloid A (SAA) is an exchangeable apolipoprotein (apo) in high-density lipoprotein (HDL) that influences HDL quality and functionality, particularly in the acute phase of inflammation. On the other hand, the structural and functional correlations of HDL containing SAA and apoA-I have not been reported. The current study was designed to compare the change in HDL quality with increasing SAA content in the lipid-free and lipid-bound states in reconstituted HDL (rHDL). The expressed recombinant human SAA1 (13 kDa) was purified to at least 98% and characterized in the lipid-free and lipid-bound states with apoA-I. The dimyristoyl phosphatidylcholine (DMPC) binding ability of apoA-I was impaired severely by the addition of SAA, while SAA alone could not bind with DMPC. The recombinant human SAA1 was incorporated into the rHDL (molar ratio 95:5:1, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC): cholesterol: apoA-I) with various apoA-I:SAA molar ratios from 1:0 to 1:0.5, 1:1 and 1:2. With increasing SAA1 content, the rHDL particle size was reduced from 98 Å to 93 Å, and the α-helicity of apoA-I:SAA was decreased from 73% to 40% for (1:0) and (1:2), respectively. The wavelength maximum fluorescence (WMF) of tryptophan in rHDL was red-shifted from 339 nm to 345 nm for (1:0) and (1:2) of apoA-I:SAA, respectively, indicating that the addition of SAA to rHDL destabilized the secondary structure of apoA-I. Upon denaturation by urea treatment from 0 M to 8 M, SAA showed only a 3 nm red-shift in WMF, while apoA-I showed a 16 nm red-shift in WMF, indicating that SAA is resistant to denaturation and apoA-I had higher conformational flexibility than SAA. The glycation reaction of apoA-I in the presence of fructose was accelerated up to 1.8-fold by adding SAA in a dose-dependent manner than that of apoA-I alone. In conclusion, the incorporation of SAA in rHDL impaired the structural stability of apoA-I and exacerbated glycation of HDL and apoA-I.     

高效亲和色谱法测定丹皮酚与固定化人血清白蛋白结合域

利用亲和色谱,在模拟人体生理环境下(37 ℃、pH 7.4),采用竞争置换法研究了丹皮酚(PAE)与固定化人血清白蛋白(HSA)的相互作用。通过对PAE的自我竞争分析及PAE与HSA上结合位点的标记物间的竞争置换分析,得到了PAE和HSA间的结合常数、结合位点数和结合域。结果表明: PAE在HSA分子中仅存在一类结合位点,结合常数为4.84×103 L/mol,该结合位点为HSA上的Sudlow siteII;通过对PAE与HSA相互作用的热力学研究,推断出二者间的作用力类型为氢键或范德华力。     

Comparative DSC study of human and bovine serum albumin

The thermal denaturation process of bovine and human both fatty acid containing and fatty acid free albumins in aqueous solution was studied by use of differential scanning calorimetry. Human serum albumins were found to be more stable than their bovine counterparts. Fatty acid free albumins were characterized as generally less stable, more susceptible to aggregation, their unfolding endothermic transition was less cooperative and with the smaller degree of reversibility. Deconvolution analysis with using a non-two-state model with two component transitions showed essential differences in the thermodynamic parameters between all studied albumins, particularly regarding the high-temperature component transition.     

Determination of free iodide in human serum: Separation from other I-species and quantification in serum pools and individual samples

A method for the determination of free iodide in human serum was developed. For this purpose iodide from pooled serum samples was separated from the organic manner by SEC. The iodide fraction subsequently was freezedried and analyzed by ion chromatography for quantification. Investigations for recovery and precision were carried out and were found to show sufficient results. For quality assurance ICP-MS was taken additionally as an total I-detector [1], using native and iodide-spiked serum samples. The iodide results of ICP-MS as well as those of IC were well corresponding. Iodine containing SEC-fractions from iodide-spiked samples showed no increased I-values except that in the iodide fractions, proving that there was no iodide conversion into other I-species (and vice versa) during the whole procedure.Free iodide from two serum pools of different healthy persons was determined as 2.25 and 2.43 g I^–/L, respectively. The values are related to total iodine levels determined by ICP-MS. For comparative reasons a table of individual iodine and iodide values is presented.Abbreviations IC ion chromatography - ICP-MS inductively coupled plasma mass spectrometry - LPLC low pressure liquid chromatography - PED pulsed electrochemical detector - SEC size exclusion chromatography - RT retention time     

Comparison of mobility shift affinity capillary electrophoresis and capillary electrophoresis frontal analysis for binding constant determination between human serum albumin and small drugs

In this study, two capillary electrophoresis–based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l -tryptophan (l -TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%. Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l -TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.     

The purification and identification of human blood serum proteins with affinity to the antitumor active RL2 lactaptin using magnetic microparticles

The cytopoxic effect of RL2 lactaptin (the recombinant analog of proteolytic fragment of human kappa‐casein) toward tumor cells in vitro and in vivo presents it as a novel promising antitumor drug. The binding of any drug with serum proteins can affect their activity, distribution, rate of excretion and toxicity in the human body. Here, we studied the ability of RL2 to bind to various blood serum proteins. Using magnetic microparticles bearing by RL2 as an affinity matrix, in combination with mass spectrometry and western blot analysis, we found a number of blood serum proteins possessing affinity for RL2. Among them IgA, IgM and IgG subclasses of immunoglobulins, apolipoprotein A1 and various cortactin isoforms were identified. This data suggests that in the bloodstream RL2 lactaptin takes part in complicate protein–protein interactions, which can affect its activity.     

秋水仙碱与人血清白蛋白相互作用的光谱学和电化学研究

用荧光光谱、紫外吸收光谱和电化学方法研究了在近似生理条件下秋水仙碱与人血清白蛋白的相互作用.研究表明,秋水仙碱对人血清白蛋白的荧光猝灭主要是静态猝灭过程,秋水仙碱使人血清白蛋白的构象发生变化.与此同时,在pH=7.4、含0.05%吐温-20的磷酸盐缓冲液中,秋水仙碱在玻碳电极上出现一不可逆的氧化峰,加入人血清白蛋白后秋水仙碱的氧化峰电位正移,峰电流下降.此外,利用光谱学方法和电化学方法测定的秋水仙碱与人血清白蛋白相互作用的结合常数和结合位点数吻合.     

Separation and determination of selected psychotropic drugs in human serum by SPE/HPLC/DAD on C18 and Polar-RP columns

A simple and sensitive high-performance liquid chromatographic method with diode array detection is described for the quantification of some psychotropic drugs: fluoxetine, sertraline, alprazolam, perphenazine, zolpidem, and hydroxyzine in fortified human serum samples. The test compounds were extracted from human serum by solid-phase extraction using C18 extraction column and injected into C18 or Polar-RP analytical columns for separation. A mobile phase was composed of methanol, water, acetate buffer at pH 3.5, and diethylamine. The method was validated for the concentration range 0.4–5?µg?mL^?1 for zolpidem and 0.5–6?µg?mL^?1 for other drugs. Mean recoveries were from 87.79% to 107.94% with adequate precision (% RSD ≤2.1%). The full separation of all investigated drugs, good peaks’ symmetry, and simultaneously high system efficiency were obtained on Polar-RP column, which was used for the first time to analyze these drugs. System efficiency obtained on the column was significantly higher compared to that obtained on commonly used C18 column. The method seems to be suitable for the analysis of investigated drugs in human plasma for psychiatric patients in multiple drug overdoses as well as for control of pharmacotherapy, particularly in combination therapy.     

Simultaneous determination of phthalates and adipates in human serum using gas chromatography–mass spectrometry with solid‐phase extraction

A gas chromatography–mass spectrometry assay was developed and validated for the simultaneous determination of phthalates and adipates in human serum. The phthalates and adipates studied were dimethyl phthalate, diethyl phthalate, dibutyl phthalate, benzylbutyl phthalate, di‐2‐ethylhexyl phthalate, di‐n‐octyl phthalate, diethyl adipate, dibutyl adipate, diisobutyl adipate, bis(2‐butoxyethyl) adipate and di‐2‐ethylhexyl adipate, with diisooctyl phthalate as internal standard. The extraction and cleaning up procedure was carried out with solid‐phase extraction cartridges containing dimethyl butylamine groups, which showed extraction efficiencies over 88% for each analyte and the internal standard. The calibration curves obtained were linear with correlation coefficients greater than 0.98. For all analytes, the assay gave CV% values for intra‐day precision from 4.9 to 13.3% and mean accuracy values from 91.4 to 108.4%, while inter‐day precision was 5.2–13.4% and mean accuracy 91.0–110.2%. The limits of detection for the assay of phthalates and adipates were in the range 0.7–4.5 ng/mL. The method is simple, sensitive and accurate, and allows for simultaneous determination of nanogram levels of phthalates and adipates in human serum. It was successfully applied to an investigation on the level of phthalates and adipates in a non‐occupationally exposed population.     

Study of the interactions between fluoroquinolones and human serum albumin by affinity capillary electrophoresis and fluorescence method

The interactions between fluoroquinolones and human serum albumin (HSA) were investigated by affinity capillary electrophoresis (ACE) and fluorescence quenching technique. Based on the efficient separation of several fluoroquinolones using a simple phosphate buffer, the binding constants of fluoroquinolones with HSA were determined simultaneously during one set of electrophoresis by ACE method. The thermodynamic parameters were obtained from data at different temperatures, and the negative ΔH and ΔS values showed that both hydrogen bonds and van der Waals interaction played major roles in the binding of fluoroquinolones to HSA. The interactions were also studied by fluorescence quenching technique. The results of fluorescence titration revealed that fluoroquinolones had the strong ability to quenching the intrinsic fluorescence of HSA through the static quenching procedure. The binding site number n, apparent binding constant K_b and the Stern-Volmer quenching constant K_sv were determined. The thermodynamic parameters were also studied by fluorescence method, and the results were consonant with that of ACE.     

Atomic force microscopy of human serum albumin (HSA) on poly(styrene/acrolein) microspheres

Atomic Force Microscopy (AFM) in the tapping mode was used for the observation of bare poly (styrene/acrolein) P(SA) microspheres and microspheres with attached HSA. Prior to the AFM observations the P(SA) microspheres were immobilized covalently on the surface of quartz slides modified with -aminopropyltriethoxysilane. Atomic Force Microscopy pictures were registered for the dry samples. The partial coalescence of the P(SA) microspheres connected to the quartz surface with amino groups has been observed. The AFM pictures of the single P(SA) microspheres revealed that the surface of these particles is smooth and that any irregularities, if present, do not exceed 1 nm. The surface of microspheres with attached HSA has very clearly different morphology with regular pattern of HSA macromolecules. Cracks on the surfaces of some microspheres with HSA revealed that protein macromolecules are attached to these particles in several layers. In the case of some other microspheres the defects in protein attachment allowed the observation of the border between the bare surface of the P(SA) microspheres and the surface covered with protein macromolecules. Comparison of the thickness of the HSA layers on the P(SA) microspheres with the dimensions of HSA macromolecules, determined earlier from the x-ray studies, suggests that the first layer, 3.0±0.2 nm thick, is formed of the HSA macromolecules arranged flatly on the surface whereas protein macromolecules in the subsequent layers, each 8.6±1 nm thick, are adsorbed protruding from the surface.     

Quantification of acyclovir in dermal interstitial fluid and human serum by ultra‐high‐performance liquid–high‐resolution tandem mass spectrometry for topical bioequivalence evaluation

Time–concentration curves for the topical anti‐viral drug acyclovir can provide valuable information for drug development. Open flow microperfusion is used for continuous sampling of dermal interstitial fluid but it requires validated methods for subsequent sample analysis. Therefore, we developed a sensitive, selective and high‐throughput ultra‐high‐performance liquid chromatography–high‐resolution tandem mass spectrometry method to determine acyclovir in human dermal interstitial fluid and serum. We validated the method over a concentration range of 0.1–25 ng/mL for a sample volume of just 20 μL and employed cation‐exchange solid‐phase extraction in a fully automated sample treatment procedure. Short‐ and long‐term sample stability data and the analysis of 5000 samples from a clinical trial demonstrate the successful application of our method.     

蟾毒配基磺酸化及基于自制串联固相萃取对磺酸化产物富集的研究

关于抗癌活性的蟾蜍二烯内酯的结构修饰化合物已达到100多个。甾体母核3位磺酸化的蟾蜍二烯内酯是从蟾蜍皮中分离得到的一类具有抗癌活性的微量成分,但目前尚无关于蟾蜍二烯内酯磺酸化修饰的报道。该文以沙蟾毒精为例,首次发展了一种高效的3位磺酸化沙蟾毒精的合成方法,对目标产物进行了结构鉴定。基于自制亲水性材料Click TE-Cys,发展了反相/亲水串联固相萃取的反应产物后处理方法,用于磺酸化沙蟾毒精的富集。依此法对总蟾毒配基进行了磺酸化和目标产物的富集及LC-MS定性分析,实现了大量具有潜在抗癌活性的新化合物的制备和富集,为进一步实现高效低毒化合物的筛选提供了物质基础。     

光谱-分子模拟法分析杨梅素与人血清白蛋白的分子作用机制

在模拟生理条件下,用多种光谱法结合分子对接法测定了杨梅素(MY)与人血清白蛋白(HSA)的相互作用.研究结果表明,MY能够明显猝灭HSA的荧光,MY与HSA的相互作用为复合式静态结合过程,结合强度较强.热力学和分子对接结果表明,MY与HSA是自发结合的,维持MY与HSA的相互作用力主要是氢键和范德华力.能量转移结果表明...     

Identification of modification sites on human serum albumin and human hemoglobin adducts with houttuynin using liquid chromatography coupled with mass spectrometry

Houttuynin, a β‐keto aldehyde compound, is the major active ingredient in herba houttuyniae injection. The injection was once used as an anti‐inflammatory drug associated with occasional serious hypersensitivity reactions in the clinic, which were proposed to be related to the formation of protein adducts. N^α‐Boc‐lysine, FEEM and IVTNTT were used as model amino acids or peptides containing one nucleophilic residue to investigate adduct types by liquid chromatography coupled with ion trap mass spectrometry (LC/MSⁿ) and high‐resolution quadrupole time‐of‐flight mass spectrometry (Q‐TOF MS). These adducts were respectively characterized as Schiff bases formed by 1:1 reaction of houttuynin with lysine or N‐terminal residue and pyridinium adducts by 2:1 reaction. LC/MSⁿ analysis of trypsin digests of HSA/Hb incubations with houttuynin revealed that houttuynin‐modified HSA adducts were formed mainly at N‐terminal amino acid and lysine residues, specifically at Lys‐212, Lys‐414 and Lys‐525 for Schiff base adducts, and at Lys‐414 and Lys‐432 for pyridinium adducts, and houttuynin adducted more readily with N‐terminal valine of the α‐ and β‐chains in Hb and lysine amine (Lys‐62) of the β‐chain for Schiff base adducts. The results showed the direct modification of houttuynin to HSA/Hb in vitro, which was speculated to be responsible for the adverse reactions induced by houttuyniae injection. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterization of interaction kinetics between chiral solutes and human serum albumin by using high-performance affinity chromatography and peak profiling

Peak profiling and high-performance columns containing immobilized human serum albumin (HSA) were used to study the interaction kinetics of chiral solutes with this protein. This approach was tested using the phenytoin metabolites 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) as model analytes. HSA columns provided some resolution of the enantiomers for each phenytoin metabolite, which made it possible to simultaneously conduct kinetic studies on each chiral form. The dissociation rate constants for these interactions were determined by using both the single flow rate and multiple flow rate peak profiling methods. Corrections for non-specific interactions with the support were also considered. The final estimates obtained at pH 7.4 and 37°C for the dissociation rate constants of these interactions were 8.2-9.6 s(-1) for the two enantiomers of m-HPPH and 3.2-4.1 s(-1) for the enantiomers of p-HPPH. These rate constants agreed with previous values that have been reported for other drugs and solutes that have similar affinities and binding regions on HSA. The approach used in this report was not limited to phenytoin metabolites or HSA but could be applied to a variety of other chiral solutes and proteins. This method could also be adopted for use in the rapid screening of drug-protein interactions.     

Electrochemical determination of muscle relaxant drug tetrazepam in bulk form,pharmaceutical formulation,and human serum

Tetrazepam dissolved in the Britton-Robinson universal buffer of various pH values (2.5–11.5) containing 10 vol. % of ethanol was reduced at the mercury electrode in a single 2-electron irreversible step due to reduction of the 4,5 C=N double bond of the seven-membered ring. Differential pulse polarography (DPP) and adsorptive cathodic stripping voltammetry (AdCSV) techniques (Linear sweep LS, differential pulse DP and square-wave SW modes) for quantification of tetrazepam in bulk form and in myolastan tablets are presented. Moreover, the described linear sweep, differential pulse, and square-wave adsorptive cathodic stripping voltammetry was successfully applied in quantification of tetrazepam in spiked human serum without any prior extraction of the drug. The obtained results showed an increased sensitivity of the described electro-analytical procedures for the quantification of tetrazepam in the following order DPP, DP-AdCSV, LS-AdCSV, and SW-AdCSV, since the observed limits of tetrazepam quantitation by these electroanalytical techniques were 5 × 10⁻⁶ mol L⁻¹, 3 × 10⁻⁷ mol L⁻¹, 1 × 10⁻⁸ mol L⁻¹, and 3 × 10⁻⁹ mol L⁻¹, respectively.     

GC‐FID determination and pharmacokinetic studies of oleanolic acid in human serum

Analytical interest of OA determination in human serum has increased owing to the increasing interest in pharmaceutical research by pharmaceutical properties. A simple, specific, precise and accurate GC method with flame ionization detector (FID) developed and validated for the determination of oleanolic acid (OA) in human serum (HS). To an aliquot of HS, internal standard was added and a combination of liquid–liquid extraction with a mixture of diethyl ether‐isopropyl alcohol, filtration and consecutive GC resulted in separation and quantification of OA. The organic phase was analyzed using a GC system equipped with a 30 × 0.25 mm i.d. Rtx‐65TG capillary column and FID detection. Total chromatographic time was 10 min and no interfering peaks from endogenous components in blank serum were observed. The OA/internal standard peak area ratio was linearly fitted to the OA concentration (r = 0.992) over the range 10–1500 ng/mL. The mean serum extraction recovery of OA was 96.7 ± 1.0% and the lower limit of quantification based on 5 mL of serum was 10.7 ng/mL. The intra‐day coefficient of variation ranged from 1.3 to 3.6% and inter‐day varied from 1.4 to 4.5%. The developed method was used to study the pharmacokinetics of OA after oral administration in humans. The assay was simple, sensitive, precise and accurate for the use in the study of the mechanisms of absorption and distribution of OA in humans. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of blood glucose parameter from human blood serum by using a quartz crystal microbalance sensor coated with phthalocyanines compounds

Determining the blood glucose level is important for the prevention and treatment of diabetes mellitus. We developed a sensor system using Quartz Crystal Microbalance (QCM) to determine the blood glucose level from human blood serum. This study consists of two experimental stages: artificial glucose/pure water solution tests and human blood serum tests. In the first stage of the study, the QCM sensor with the highest performance was identified using artificial glucose solution concentrations. In the second stage of the study, human blood serum measurements were performed using QCM to determine blood glucose levels. QCM sensors were coated with phthalocyanines (Pcs) by jet spray method. The blood glucose values of 96 volunteers, which ranged from 71 mg/dL to 329 mg/dL, were recorded. As a result of the study, human glucose values were determined with an average error of 3.25%.