Determination of Paraquat in Human Serum by HPLC

目的：建立HPLC法测定人血清中百草枯含量。方法：色谱柱：Kromasil C18柱（200mm×4．6mm，5μm），流动相：乙腈-水（含0．03mol·L-1庚烷磺酸钠，0．24mol·L-1磷酸）=3：97（用三乙胺调pH至2．0）；检测波长258nm；柱温25℃；进样体积20μl；流速0．8ml·min-1。结果：百草枯在0．106—10．6mg·L-1浓度范围内线性关系良好（r=0．9993），最低检出浓度为0．065mg·L-1。高、中、低3种浓度样品绝对回收率〉89．4％，方法回收率〉94．4％，日内精密度RSD在0．12％～1．74％之间，日间精密度RSD在0．44％～2．89％之间。结论：该方法操作简便易行、灵敏度高、专属性强，适用于百草枯的人体血清浓度测定。

Preliminary Studies on High-performance Liquid Chro-matography Chemiluminescence Determination of theSaturated Fatty Acids(C_(16) C_(18))in Human Serum

The peroxyoxalate chemiluminescence(CL)detection of fatty acids in human se- rum combined with high-performance liquid chromatography (HPLC)is described.Some fatty acids in serum were extracted with a 1 :1(v/v)mixture of chloroform-n-heptane.2-(4-Hydrazinocarbonyl- phenyl)-4,5-diphenylimidazole (HCPI) was used as a fluorescent labelling reagent of the fatty acids. The labelling reaction was carried out at 30℃ for 1 h at pH 6.5 and the resulting reaction mixture was sudjected to HPLC. The labelled fatty acid C_(17)(P-C_(17))was used as the internal standard. The la- belled fatty acids C_(16) and C_(18) were separated within 18 min on an ODS-8OTM column (150 mm× 6 mm ID,5μm,Tosoh Japan).The calibrlation curves of fatty acids from the spiked control serum were Y_1=-0.003 7 + 0.0028X_1,r=0.994 for FA C_( 16) and Y_2=0.00 1 2 + 0.00098X_2,r=0.999 for FA C_( 18),respectively.The average recoveries of facids from the spiked contrl serum were 107.2%(n=8,RSD=4.3%)for FA C_(16) and 97.35%(n=8, RSD=4.0%)for FA C_(18),respectively.The lower detection limits of fatty acids after reaction were 12μmol per 20μl injection for FA C_(16) and 18 μmol per 20μl injection for FA C_(18),respectively(signal to noise ratio, S/N=2).The HPLC/CL method was applied to the determination of FA C_(16) and FA C_(18) in normal human serum and the results showed that the concentrations of fatty acids in normal human serum were 0.134 ± 0.009 μ mol/ml serum(n=5) for FA C_(16) and 0.052±0.028 μmol/ml serum(n=5)for FA C_(18),respectively.

Effect of human autologous serum and fetal bovine serum on human corneal epithelial cell viability,migration and proliferation in vitro

AIM： To analyze the concentration-dependent effects of autologous serum （AS） and fetal bovine serum （FBS） on human corneal epithelial cell （HCEC） viability, migration and proliferation. METHODS： AS was prepared from 13 patients with non- healing epithelial defects Dulbecco＇s modified eagle medium/ Ham＇s F12 （DMEM/F12） with 5% FBS, 0.5% dimethyl sulphoxide （DMSO）, 10 ng/mL human epidermal growth factor, 1% insulin-transferrin-selenium, then were incubated in serum media： DMEM/F12 supplemented by 5%, 10%, 15% or 30% AS or FBS. HCEC viability was analyzed using cell proliferation kit XTI＇, migration using a wound healing assay, proliferation by the cell proliferation enzyme-linked immunosorbent assay （ELISA） BrdU kit. Statistical analysis was performed using the generalized linear model, the values at 30% AS or 30% FBS were used as the baselines. RESULTS： HCEC viability was the highest at 30% AS or 15% FBS and the lowest at 10% AS or 30% FBS application. HCEC migration was the quickest through 30% AS or 30% FBS and the slowest through 5% AS or 5% FBS concentrations. Proliferation was the most increased through 15% AS or 5% FBS and the least increased through 30% AS or 30% FBS concentrations. HCEC viability at 10% and 15% AS was significantly worse （P=0.001, P=0.023） compared to baseline and significantly better at 15% FBS （P=0.003） concentrations. HCEC migration was significantly worse （P〈0.007） and HCEC proliferation significantly better （P〈0.001） in all concentration groups compared to baseline. CONCLUSION： For the best viability of HCEC 30% AS or 15% FBS, for HCEC migration 30% AS or 30% FBS, for proliferation 15% AS or 5% FBS should be used. Therefore, we suggest the use of 30% AS in clinical practice.

Nutrient supplemented serum-free medium increases cardiomyogenesis efficiency of human pluripotent stem cells

AIM: To development of an improved p38 MAPK inhibitor-based serum-free medium for embryoid body cardiomyocyte differentiation of human pluripotent stem cells. METHODS: Human embryonic stem cells (hESC) differentiated to cardiomyocytes (CM) using a p38 MAPK inhibitor (SB203580) based serum-free medium (SB media). Nutrient supplements known to increase cell viability were added to SB medium. The ability of these supplements to improve cardiomyogenesis was evaluated by measurements of cell viability, total cell count, and the expression of cardiac markers via flow cytometry. An improved medium containing Soy hydrolysate (HySoy) and bovine serum albumin (BSA) (SupSB media) was developed and tested on 2 additional cell lines (H1 and Siu-hiPSC). Characterization of the cardiomyocytes was done by immunohistochemistry, electrophysiology and quantitative real-time reverse transcriptionpolymerase chain reaction. RESULTS: hESC cell line, HES-3, differentiating in SB medium for 16 d resulted in a cardiomyocyte yield of 0.07 ± 0.03 CM/hESC. A new medium (SupSB media) was developed with the addition of HySoy and BSA to SB medium. This medium resulted in 2.6 fold increase in cardiomyocyte yield (0.21 ± 0.08 CM/hESC). The robustness of SupSB medium was further demonstrated using two additional pluripotent cell lines (H1, hESC and Siu1, hiPSC), showing a 15 and 9 fold increase in cardiomyocyte yield respectively. The age (passage number) of the pluripotent cells did not affect the cardiomyocyte yields. Embryoid body (EB) cardiomyocytes formed in SupSB medium expressed canonical cardiac markers (sarcomeric α-actinin, myosin heavy chain and troponin-T) and demonstrated all three major phenotypes: nodal-, atrial- and ventricular-like. Electrophysiological characteristics (maximum diastolic potentials and action potential durations) of cardiomyocytes derived from SB and SupSB media were similar. CONCLUSION: The nutrient supplementation (HySoy and BSA) leads to increase in cell viability, cell yield and cardiac marker expression during cardiomyocyte differentiation, translating to an overall increase in cardiomyocyte yield.

Antioxidant Effects of Oats Avenanthramides on Human Serum

The antioxidant ability of capsules containing oats avenanthramides on human body was evaluated in present study.Healthy people were randomized to supplementation with oats-derived avenanthramides capsules or placebo for 1 mon.Plasma lipid peroxides and antioxidant status were measured.For 8 capsules （containing 3.12 mg avenanthramides） groups,the levels of serum superoxide dismutase （SOD） and reduced glutathione hormone （GSH） were significantly increased by 8.4 and 17.9%,respectively （P0.05）,and malondialdehyde （MDA） level significantly decreased by 28.1%.The total cholesterol （TC）,triglyceride （TG）,and low density lipoprotein cholesterol （LDL-C） levels were lowered by 11.1,28.1,and 15.1%,respectively （P0.05）.The high density blood lipoprotein cholesterol （HDL-C） levels in the same treat was increased by 13.2%.Based on our research,it can be concluded that oats extract containing avenanthramides possessed a high antioxidative activity on humans.It indicated that oat avenanthramides could be used to prevent hyperlipemic and angiocardiopathy.

Significance of functional hepatic resection rate calculated using 3D CT/^(99m)Tc-galactosyl human serum albumin singlephoton emission computed tomography fusion imaging

AIM: To evaluate the usefulness of the functional hepatic resection rate (FHRR) calculated using 3D computed tomography (CT)/99mTc-galactosyl-human serum albumin (GSA) single-photon emission computed tomography (SPECT) fusion imaging for surgical decision making.METHODS: We enrolled 57 patients who underwent bi- or trisectionectomy at our institution between October 2013 and March 2015. Of these, 26 patients presented with hepatocellular carcinoma, 12 with hilar cholangiocarcinoma, six with intrahepatic cholangiocarcinoma, four with liver metastasis, and nine with other diseases. All patients preoperatively underwent three-phase dynamic multidetector CT and 99mTc-GSA scintigraphy. We compared the parenchymal hepatic resection rate (PHRR) with the FHRR, which was defined as the resection volume counts per total liver volume counts on 3D CT/99mTc-GSA SPECT fusion images.RESULTS: In total, 50 patients underwent bisectionectomy and seven underwent trisectionectomy. Biliary reconstruction was performed in 15 patients, including hepatopancreatoduodenectomy in two. FHRR and PHRR were 38.6 ± 19.9 and 44.5 ± 16.0, respectively; FHRR was strongly correlated with PHRR. The regression coefficient for FHRR on PHRR was 1.16 (P < 0.0001). The ratio of FHRR to PHRR for patients with preoperative therapies (transcatheter arterial chemoembolization, radiation, radiofrequency ablation, etc.), large tumors with a volume of > 1000 mL, and/or macroscopic vascular invasion was significantly smaller than that for patients without these factors (0.73 ± 0.19 vs 0.82 ± 0.18, P < 0.05). Postoperative hyperbilirubinemia was observed in six patients. Major morbidities (Clavien-Dindo grade ≥ 3) occurred in 17 patients (29.8%). There was no case of surgery-related death.CONCLUSION: Our results suggest that FHRR is an important deciding factor for major hepatectomy, because FHRR and PHRR may be discrepant owing to insufficient hepatic inflow and congestion in patients with preoperative therapies, macroscopic vascular invasion, and/or a tumor volume of > 1000 mL.

Combined fluorescence and electrochemical investigation on the binding interaction between organic acid and human serum albumin

Human serum albumin （HSA） is a plasma protein responsible for the binding and transport of fatty acids and a variety of exogenous chemicals such as drugs and environmental pollutants. Such binding plays a crucial role in determining the ADME （absorption, distribution, metabolism, and excretion） and bioavailability of the pollutants. The binding interaction between HSA and acetic acid （C2）, octanoic acid （C8） and dodecanoic acid （C12） has been investigated by the combination of site-specific fluorescent probe, tryptophan intrinsic fluorescence and tyrosine electrochemistry. For the study of the fatty acid interaction with the two drug-binding sites on HSA, two fluorescent probes, dansylamide and dansyl-L-proline were employed in the displacement measurements. Intrinsic fluorescence of tryptophan in HSA was monitored upon addition of the fatty acids into HSA. Electrocatalyzed response of the tyrosine residues in HSA by a redox mediator was used to investigate the binding interaction. Qualitatively, observations from these three approaches were very similar. HSA did not show any change in the fluorescence and electrochemical experiments after mixing with C2, suggesting there is no significant interaction with the short-chain fatty acid. For C8, the measured signal dropped in a single-exponential mode, indicating an independent and non-cooperative binding. The calculated association constant and binding ratio were 3.1 × 10^6 L/mol and 1 with drug binding Site Ⅰ, 1.1 × 107 L/mol and 1 with Site Ⅱ, and 7.0× 0^4 L/mol and 4 with the tryptophan site, respectively. The measurements with C12 displayed multiple phases of fluorescence change, suggesting cooperativity and allosteric effect of the C12 binding. These results correlate well with those obtained by the established methods, and validate the new approach as a viable tool to study the interactions of environmental pollutants with biological molecules.

Investigation of the binding behavior of human serum albumin and phosphorothioate oligodeoxynucleotide

Aim To study the binding behavior between human serum albumin （HSA） and phosphorothioate oligodeoxynucleotide （PS- ODN） and the effects of bivalent cations on the interaction. Methods Surface plasma resonance, circular dichroism and fluorescence experiments were conducted. Results （ 1 ） the binding ability was decreased along with the increase of pH; （2） Zn^2＋and Ni^2＋ enhanced the interaction between PS-ODN and HSA; （3） Upon PS-ODN binding, the conformation of HSA was changed with an increase of β - sheet. Conclusion The results provide experimental evidences to the hypothesis that PS-ODN binds with HSA in the positive potential region, and histidine residues located in the region play a crucial rule in the interaction.

Transferrin and Its Isoforms from Normal Human Serum Revealed by Several Analytical Techniques

Transferrin（TF） and its isoforms have been widely reported via various analytical techniques, including a noticeable increased number of isoforms with low content of sialic acid（asialo-, monosialo-, and disialo-transferrin） and asialo-TF as well as disialo-TF, with one or several oligosaccharides released in human serum transferrin（hTf）. Here, hTf has been purified by native gradient polyacrylamide gel electrophoresis（PAGEso） before use. The hTf extracted with the electron-transfer approach showed a single subunit band（77.1 Da） in the SDS-PAGE gel, but it exhibited two bands in the native and denatured isoelectric focusing（IEF） gels, namely, hTf-2Fe^3＋ and apo-hTf, without finding any other transferrin isoforms. A reversed phase HPLC（RP-HPLC） equipped with a C18 column effectively separated hTf and its polymers and combined off-line techniques, including peptide mass fingerprinting（PMF）, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF-MS） and database search, and identified the high homology among hTf, apo-hTf, and their isoforms. Moreover, the elution solution consisting of acetonitrile and formic acid could easily denature both hTf and apo-hTf to form various isoforms during separation with HPLC, indicating that chemical factors lead to the formation of various isoforms in transferrin, artificially, during extraction and separation. The authors claimed that only two transferrin isoforms existed in the NHS, namely, hTf-2Fe^3＋ and apo-hTf, which could be employed in biomarkers, to distinguish the healthy population from many disease sufferers, such as, carbohydrate-deficient transferrin（CDT）

High Glucose Promotes the CTGF Expression in Human Mesangial Cells via Serum and Glucocorticoid-induced Kinase 1 Pathway

The role of serum and glucocorticoid-induced kinase 1 （SGK1） pathway in the connective tissue growth factor （CTGF） expression was investigated in cultured human mesangial cells （HMCs） under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with PIRES2-EGFP- S422D hSGK1 （SD） could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with PIRES2-EGFP （FP） under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with PIRES2-EGFP- K127N hSGK1 （KN） could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.

Insight into the interaction of inhaled corticosteroids with human serum albumin: A spectroscopic-based study

It is well known that the safety and efficacy profile of an inhaled cortocosteroid(ICS) is influenced by the pharmacokinetic properties and associated pharmacodynamic effects of the drug. Freely circulating,protein unbound, and active ICS can cause systemic adverse effects. Therefore, a detailed investigation of drug-protein interaction could be of great interest to understand the pharmacokinetic behaviour of corticosteroids and for the design of new analogues with effective pharmacological properties. In the present work, the interaction between some corticosteroids and human serum albumin(HSA) has been studied by spectroscopic approaches. UV–Vis spectroscopy confirmed that all the investigated corticosteroids can bind to HSA forming a protein-drug complex. The intrinsic fluorescence of HSA was quenched by all the investigated drugs, which was rationalized in terms of a static quenching mechanism. The thermodynamic parameters determined by the Van't Hoff analysis of the binding constants(negative ΔH and ΔS values) clearly indicate thathydrogen bonds and van der Waals forces play a major role in the binding process between albumin and betamethasone, flunisolide and prednisolone, while hydrophobic forces may play a major role in stabilizing albumin-triamcinolone complexes.

Investigation on the differences of four flavonoids with similar structure binding to human serum albumin

Flavonoids are structurally diverse and the most ubiquitous groups of polyphenols distributed in the various plants,which possess intensive biological activities.In this study,the interaction mechanisms between four flavonoids containing one glucose unit with similar molecular weight isolated from the Tibetan medicinal herb Pyrethrum tatsienense,namely.apigenin-7-O-β-D-glucoside（1）,luteolin-7-O-β-D-glucoside（2）.quercetin-7-O-β-D-glucoside（3）.quercetin-3-O-β-D-glycoside（4）.and human serum albumin（HSA）,were investigated by fluorescence.UV-vis absorbance,circular dichroism,and molecular modeling.The effects of biological metal ions Mg2＋,Zn2＋,and Cu2＋ on the binding affinity between flavonoids and HSA were further examined.Structure-activity relationships of four flavonoids binding to HSA were discussed in depth and some meaningful conclusions have been drawn by the experiment data and theoretical simulation.In addition,an interesting phenomenon was observed that the microenvironment of the binding site I in HSA has hardly changed in the presence of 4 differentiating from the other three flavonoids on the basis of conformation investigations.

Thermodynamic study on the interaction between anti-tumor drug tegafur and human serum albumin

The changes of thermodynamic properties of the system on interaction between tegafur and human serum albumin （HSA） and the changes of secondary structure units of HSA in the system at 298.15 K have been investigated by the Nano-Watt-Scale isothermal titration calorimetry （ITC）, the Langmuirs binding model and the circular dichroism （CD） spectrometry.

Characterization of Gallic Acid Interaction with Human Serum Albumin by Spectral and Molecular Modeling Methods

The binding of drugs with human serum albumin（HSA） is a crucial factor influencing the distribution and bioactivity of drugs in the body.To understand the action mechanisms between gallic acid（GA,3,4,5-trihydroxybenzoic acid） and HSA,the binding of GA with HSA was investigated by a combined experimental and computational approach.The fluorescence properties of HSA and the binding parameters of GA collectively indicate that the binding is characterized by static quenching mechanism at one high affinity binding site.According to the estimated molecular distance between the donor（HSA） and the acceptor（GA）,the binding is related to the fluorescence resonance energy transfer.As indicated by the thermodynamic parameters,hydrophobic interaction plays a major role in the GA-HSA complex.Further,the experimental results reveal that GA is bound in the large hydrophobic cavity of subdomain IIA in the site I of HSA,which is well approved by molecular docking.

Determination of Tramadol in Human Serum by Capillary Electrophoresis with the End-Column Electrochemiluminescence Detection

A capillary electrophoresis (CE) coupled with end-column electrochemiluminescence (ECL) detection method for the analysis of tramadol (TMD) has been investigated. ECL detection was working electrode biased at 1.2 V in a 20mmol·L-1 sodium phosphate buffer (pH = 8.0) containing 5 mmol·L-1 Ru (where bpy = 2,2’-bipyridyl). Linear correlation (r ≥ 0.997) between ECL intensity and drug concentration was obtained in the range 3 × 10-4 - 6 × 10-6 mol·L-1. The limits of detection (LODs) for tramadol in water was 3.012 × 10-8 mol·L-1(S/N = 3). The relative standard deviation values on peak size (10-5 mol·L-1 level) and migration time for the tramadol were 4.58% and 1.39% (n = 10), respectively. Applicability of the CE-ECL method to the analysis of human serum spiked with tramadol was

On-line Sweeping Sample Concentration in Micellar Electrokinetic Chromatography with Enhanced Sensitivity for the Deter-mination of Carbamazepine in Human Serum

A sensitive and simple micellar electrokinetic chromatography （MEKC） method was developed for the determination of antiepileptic drug, carbamazepine （CBZ）, using sweeping on-line concentration method with photodiode array detection. Under the optimal conditions, the calibration curve was linear over a range of 0.5-40μg·mL^-1 for CBZ with a correlation coefficient of 0.998. The detection limit （S/N = 3：1） of CBZ was 0.10 μg·mL^-1. The sweeping-MEKC method has been successfully applied to the analysis of CBZ in human serum.

Preparation and evaluation of mixed-mode resins with tryptophan analogues as functional ligands for human serum albumin separation

Five tryptophan analogues with a hydrophobic indole ring and an amino group on each molecule were used as functional ligands of mixed-mode resins for human serum albumin(HSA) purification. Their adsorption performance was evaluated and the effects of p H and salt addition on HSA adsorption were studied. The resins prepared showed typical p H-dependent adsorption and the highest adsorption capacity and affinity were found at pH 5.0for all the resins tested. The saturated adsorption capacity was 138.02 mg·g^(-1)with the tryptaminefunctionalized resin, which significantly decreased at p H below 4.0 due to electrostatic repulsion between ligands and HSA. Moreover, the addition of Na Cl or(NH_4)_2SO_4in media reduced HSA adsorption capacity, although the two salts showed different affecting profiles. The tryptamine-functionalized resin showed the best salt-tolerant performance, and its high adsorption capacity was maintained under high salt concentrations. In addition, the five resins prepared showed good adsorption selectivity for recombinant HSA from Pichia pastoris broth. Molecular docking results between tryptamine and HSA indicated that tryptamine was favorable to bind on Site II(indole-binding site) of HSA.

Assay of Cysteine in Human Serum with Quinine-Ce^(4+) Chemiluminescence System

A sensitive and selective chemiluminescence (CL) method was developed for the determination of cysteine. This method is based on that the weak CL of cysteine oxidized with cerium (IV) can be greatly enhanced by quinine, and the total cysteine in human serum can be detected through simply diluting with water, showing a simpler analytical characteristic.

Non-covalent binding analysis of sulfamethoxazole to human serum albumin:Fluorescence spectroscopy,UV-vis,FT-IR,voltammetric and molecular modeling

This study was designed to examine the interaction of sulfamethoxazole （SMZ） with human serum albumin（HSA）. Spectroscopic analysis of the emission quenching at different temperatures revealed that the quenching mechanism of human serum albumin by SMZ was static mechanism. The binding constant values for the SMZ-HSA system were obtained to be 22,500 L/mol at 288 K, 15,600 L/mol at 298 K, and 8500 L/mol at 308 K. The distance r between donor and acceptor was evaluated according to the theory of Foster energy transfer. The results of spectroscopic analysis and molecular modeling techniques showed that the conformation of human serum albumin had been changed in the presence of SMZ. The thermodynamic parameters, namely enthalpy change （ΔH^0） - 36.0 kJ/mol, entropy change （ΔS^0） - 41.3 Jim01 K and free energy change （ΔG^0） - 23.7 kJ/ mol, were calculated by using van＇t Hoff equation. The effect of common ions on the binding of SMZ to HSA was tested.

Electrochemical Determination of Alkaline Phosphatase in Human Serum by Differential Pulse Voltammetry

Differential pulse voltammetry(DPV) was applied to the determination of alkaline phosphatase(ALP) activity in human serum with phenyl phosphate as the substrate. Phenyl phosphate can enzymatically be hydrolyzed to produce phenol which is quantified by DPV at +660 mV(vs.Ag/AgCl) in the concentration range of 2.0_100 μmol/L. The standard curve for ALP is linear over the range from 0.06 to 1000 U/L with a relative standard deviation of 3.0%. The conditions for the enzymatic reaction and voltammetric detection were optimized and the kinetic constants were also examined.The human serum samples were tested by this method and the results were in good agreement with those obtained by the routine p-nitrophenyl phosphate spectrophotometric method.