Effect Of Human Serum Albumin Research Articles

Protective effect of albumin on VEGF and brain edema in acute ischemia in rats

Vascular endothelial growth factor (VEGF) is known to be an important stroke-related pathogenic factor for the formation of brain edema. We examined the therapeutic effect of human serum albumin on VEGF expression in acute ischemic stroke. Adult male Sprague–Dawley (SD) rats were subjected to Middle Cerebral Artery Occlusion (MCAO), the suture was withdrawn 2 h later, and 25% albumin (1.25 g/kg) or saline (5 ml/kg) was administered intravenously after reperfusion. The model was evaluated by 2,3,5-triphenyl-tetrazolium chloride (TTC) staining, neurological deficits and brain water content. Serum albumin level was determined. VEGF expression was studied by enzyme linked immunosorbent assay (ELISA), quantitative real-time PCR and immunohistochemistry. We demonstrated that albumin administration maintained the serum albumin at a higher level than the sham group at 6 h, 1 d, 2 d and 3 d after MCAO, and significantly improved the neurological deficits and decreased the brain water content. In addition, the strong up-regulation of VEGF expression at 6 h and 1 d after MCAO can be attenuated by albumin administration. However, albumin administration had no significant depressing effect on VEGF expression at 2 d, 3 d and 5 d after MCAO in the cortex and hippocampus. Strong up-regulation of VEGF immunoreactivity was noted in the saline group in the blood–brain barrier (BBB), and in neurons surrounding the peri-infarct area and periventricular area at 24 h after MCAO. The expression of VEGF in the albumin group was much weaker. Furthermore, there were high correlations between the brain water content with the serum albumin level, with serum VEGF protein level, and with brain VEGF mRNA expression at 24 h after MCAO. In conclusion, maintaining the serum albumin at a higher level, and attenuating endogenous VEGF expression at 6 h and 1 d, but not 2 d, 3 d, or 5 d after MCAO, may partially contribute to the protective effects of albumin on reduction of brain edema in the early stage of ischemia.

In vivo tumour and metastasis reduction and in vitro effects on invasion assays of the ruthenium RM175 and osmium AFAP51 organometallics in the mammary cancer model

We have compared the organometallic arene complexes [(eta(6)-biphenyl)M(ethylenediamine)Cl](+) RM175 (M=Ru(II)) and its isostructural osmium(II) analogue AFAP51 (M=Os(II)) for their ability to induce cell detachment resistance from fibronectin, collagen IV and poly-l-lysine, and cell re-adhesion after treatment, their effects on cell migration and cell viability, on matrix metalloproteinases production, and on primary tumour growth of MCa mammary carcinoma, the effect of human serum albumin on their cytotoxicity. There are differences between ruthenium and osmium. The Os complex is up to 6x more potent than RM175 towards highly-invasive breast MDA-MB-231, human breast MCF-7 and human epithelial HBL-100 cancer cells, but whereas RM175 was active against MCa mammary carcinoma in vivo and caused metastasis reduction, AFAP51 was not. Intriguingly the presence of human serum albumin in the growth medium enhanced the cytotoxicity of both compounds. RM175 increased the resistance of MDA-MB-231 cells to detachment from substrates and both compounds inhibited the production of MMP-2. These data confirm the key role of ruthenium itself in anti-metastatic activity. It will be interesting to explore the activity of osmium arene complexes in other tumour models and the possibility of changing the non-arene ligands to tune the anticancer activity of osmium in vivo.

Effect of plasma proteins on buprenorphine transfer across dually perfused placental lobule

Objective. The aim of this investigation is to determine the effect of human serum albumin (HSA) and α-acid glycoprotein (AAG) on buprenorphine (BUP) transplacental transfer and distribution.Methods. The technique of dual perfusion of placental lobule (DPPL) was utilised. BUP was co-perfused with the marker compound antipyrine (AP). In each experiment, the radiolabelled isotopes [3H]-BUP and [14C]-AP were added to enhance their detection limits. Human plasma proteins, HSA and AAG, were added to both the maternal and fetal circuits separately and in combination at their physiological concentrations in maternal and fetal circulations close to term.Results. Transplacental transfer of BUP, in absence of plasma proteins, is a two-step process: the first is its uptake by the syncytiotrophoblast from the maternal circuit, and the second is its transfer/release from the tissue to the fetal circuit. The addition of HSA to the perfusion medium affected only the second step of BUP transfer, but AAG affected both steps. The combined effect of HSA and AAG was not different from that observed in presence of the latter alone.Conclusions. Binding of BUP to circulating AAG could have an important role in the transfer of the drug from the maternal to fetal circulation.

Evaluation of the binding effect of human serum albumin on the properties of granules

The main objective of this study was the application of a solution of human serum albumin as a granulating fluid. The properties of the granules formed were evaluated and compared with those when a conventional binder was applied in the same concentration. The powder mixture contained a soluble (mannitol) and an insoluble component (different types of cellulose). The protein solution applied exerted an appropriate aggregating effect if the system contained microcrystalline celluloses. Powdered cellulose was not suitable for the granulation with human serum albumin solution. As compared with the same concentration of the conventionally applied cellulose ethers as binder, the prepared granules exhibited a larger particle size, a significantly better compressibility, a higher breaking hardness and a favourable deformation process. These findings mainly reflect the good adhesive properties of the protein. The best compressibility and mechanical behaviour were attained on the application of the microcrystalline cellulose Vivapur type 105. This favourable behaviour may be connected with the wettability of cellulose. These results suggest that the formulation of tablets may be easier from an active agent in the serum that binds to albumin (e.g. interferon) since the amount of additives (binder) can be reduced.

Effect of albumin on transplacental transfer and distribution of rosiglitazone and glyburide

Objective. The aims of this investigation were (i) to determine the rate and extent of rosiglitazone transfer across term human placenta, and (ii) to determine the effect of human serum albumin (HSA) on rosiglitazone and glyburide transfer and distribution.Methods. These aims were achieved by utilizing the technique of dual perfusion of placental lobule (DPPL). Each hypoglycemic drug was coperfused with the marker compound antipyrine (AP). In each experiment, the [3H]-isotope of the hypoglycemic drug and the [14C]-isotope of AP were added to enhance the detection limits of each drug. Human serum albumin (HSA) was added to both the maternal and fetal circuits in the experiments in which it was investigated.Results. Transplacental transfer of rosiglitazone and glyburide from the maternal to fetal circuits in media devoid of HSA was similar. However, the addition of HSA to the maternal and fetal circuits had different effects on the transfer and distribution of the two drugs, though their binding to HSA (99.8%) was almost identical. HSA increased the maternal (M) to fetal (F) transfer of rosiglitazone, as revealed by an increase in its F/M concentration ratio from 0.17 ± 0.01 (in the absence of albumin) to 0.33 ± 0.07 (p < 0.001). Moreover, the addition of albumin decreased the amount of rosiglitazone retained by placental tissue from 539 ± 148 to 60 ± 8 ng/g (p < 0.001). Conversely, the addition of HSA to the perfusion media resulted in a decrease in glyburide transfer, as revealed by the change of its F/M concentration ratio from 0.09 ± 0.02 (in the absence of albumin) to 0.03 ± 0.01 (p < 0.01). However, similar to rosiglitazone, glyburide retention by the tissue decreased from 103 ± 26 to 19 ± 6 ng/g (p < 0.001).Conclusions. These data indicate that the binding of the two drugs to albumin, though similar, is only one of the factors that could affect their placental transfer and distribution.

Albumin overload induces adaptive responses in human proximal tubular cells through oxidative stress but not via angiotensin II type 1 receptor

Proteinuria is pathogenic to proximal tubular cells (PTC) and linked with progression to renal failure. The aim of this study was to determine the effects of human serum albumin (HSA) overload on the changes in gene and protein expression stimulated by oxidative stress in PTC and any interaction with ANG II that is pivotal in disease pathogenesis. Markers of oxidative stress, antioxidant defences, transcription factor activation, and the expression of stress-related genes were measured in human PTC (HK-2 cells) overloaded with either globulin-free fatty acid free (GF/FAF) HSA or globulin-free (GF) HSA. The effects of ANG II were also determined. HSA overload in HK-2 cells caused PTC hyperfunction, increased oxidative stress, and an upregulation of adaptive responses and stress-related genes. Some responses were common to both HSAs but others were unique to either HSA and unaffected by addition of ANG II or candesartan (a specific ANG II type 1 receptor blocker). ANG II also independently induced oxidative stress and upregulated other stress-related genes. HSA overload in HK-2 cells stimulated increased oxidative stress and upregulated changes in stress-related gene expression indicating new pathways of PTC injury that are not mediated via ANG II type 1 receptors.

Effect of human serum albumin on transplacental transfer of glyburide

Glyburide is a second-generation sulfonylurea hypoglycemic drug used for the treatment of select women with pregestational and gestational diabetes mellitus (GDM). In vitro and in vivo investigations demonstrated its very low transplacental transfer to the fetal circulation. However, the factors influencing its low transfer across the human placenta remain unclear. Therefore, the goal of the current investigation was to determine the effect of human serum albumin (HSA) on the transfer and distribution of glyburide across the human placenta. To achieve this goal, the technique of dual perfusion of the placental lobule was utilized. The effect of HSA on the transfer of glyburide was determined at the range of glyburide to HSA molar ratios of 1:2–1:100. The transfer rate of free/unbound glyburide to the fetal circuit was 73 ± 10% of the freely diffusible marker compound antipyrine (AP). Data obtained indicates the dependence of glyburide transfer and its retention by the placental tissue on the concentration of HSA.

The effect of human serum albumin on the extended storage of human oral keratinocyte viability under mild hypothermia

Isolated oral keratinocytes in suspension provide a number of advantages for use in maxillofacial surgery, however, the poor stability of this cell preparation at physiological temperatures is an apparent barrier preventing their use. The purpose of the present study was to evaluate whether human serum albumin (HSA) could serve as an effective constituent of a storage medium to enhance human oral keratinocyte (HOK) viability under conditions of mild hypothermia. Primary human oral keratinocytes were isolated from small pieces of the non-inflamed gingival tissues obtained during the extraction of the third molars of patients. HOK were cultured on collagen type I-coated culture dishes in keratinocyte growth medium (KGM). After the trypsinization of a culture dish (passage 2 or 3), freshly isolated HOK were stored for 24, 48, and 72 h at 4 °C or at room temperature in KGM, saline, Dulbecco’s modified Eagle’s medium (DMEM), saline supplemented with 10% HSA or DMEM supplemented with 10% (v/v) HSA under one atmosphere pressure. After storage, HOK cell survival was determined by dye exclusion using trypan blue and colony-forming assay and cell cycle change was obtained by flow cytometry. Highest cell viability was obtained in saline supplemented with 10% HSA and DMEM supplemented with 10% (v/v) HSA at 4 °C and at room temperature. Under these conditions no significant decline in keratinocyte viability was observed for at least 48 h. The cell cycle profiles of these cells were also maintained for at least 48 h at room temperature. These observations demonstrate that HSA might be better at preserving the viability of HOK stored under hypothermic and mild hypothermic conditions up to 48 h.

Structural Changes of Phospholipid Monolayers Caused by Coupling of Human Serum Albumin: A GIXD Study at the Air/Water Interface

Phase behavior and structural changes were studied for phospholipid monolayers coupled with human serum albumin (HSA) at the air/buffer (pH 3.8) interface by film balance and grazing incidence X-ray diffraction (GIXD) measurements. For comparison, the lipid headgroup was varied from small ionic L-α-dipalmitoyl-phosphatidic acid (DPPA) to large ionic L-α-dipalmitoyl-phosphatidyl-L-serine (DPPS) and zwitterionic L-α-distearoyl-phosphatidylcholine (DSPC). The presence of HSA leads to a more or less pronounced plateau in the pressure/area isotherms of all phospholipids. GIXD diffraction patterns illustrate that the phase sequences of DPPS and DPPA on buffer (oblique−rectangular−hexagonal) are changed because of the binding of HSA. The oblique−rectangular phase transition disappears and the rectangular−hexagonal phase transition shifts to a lower surface pressure. However, there is no effect of HSA on the structure of the DSPC monolayer (NN tilted rectangular packing). Phase diagrams of DPPA/HSA, DPPS/HSA, and DSPC/HSA have been established. A tilt angle decrease in both DPPA/HSA and DPPS/HSA monolayers is observed, but no change of the tilt angle for DSPC molecules in the mixed DSPC/HSA monolayer is found. Thus, whereas HSA binding stabilizes the liquid-expanded phase at low pressures most probably via penetration, it condenses the ordered phases of anionic monolayers via reducing the headgroup repulsion. The positional correlation lengths ξi parallel and perpendicular to the tilt direction change because of the binding of HSA.

The comparative interaction of human and bovine serum albumins with CYP2C9 in human liver microsomes

The effect of human serum albumin (HSA), in its endogenous, free fatty acid free (FAF) and globulin free (GF) form, on the activity of CYP2C9 was studied in human liver microsomes using tolbutamide as the substrate. The widely used BSA was included to assess the differential effect of BSA and HSA. CYP2C9 activity was expressed as CL int (Vmax/Km). HSA(FAF) and BSA showed a concentration-dependent and biphasic (activation and inhibition) interaction with CYP2C9 activity. HSA(GF) and HSA exhibited an inhibitory effect, with an inhibition constant, Ki, of 19.9 μM (0.13% albumin) and 42.2 μM (0.35% albumin), respectively. Enzyme-kinetics revealed that the activation is accompanied by a decrease in Km values, while with inhibition Km values increased. A simplified method to calculate clearance, utilizing a single slope (V/S) determination based on V over the lowest linear range of [S] (designated as CL one) was assessed. Virtually identical values were obtained for CL int and CL one. The free-drug hypothesis was tested by comparing ratios of relative CL int/unbound fraction (FDH Test ratio). The FDH Test ratio for HSA was about 1, indicating that HSA binding of tolbutamide reduced the CYP2C9 activity in accord with the free-drug hypothesis. The FDH Test ratios for BSA and HSA(FAF) were 3.7 and 3.0, revealing a monophasic activation of CYP2C9. For 2%HSA(GF) the ratio of 0.3 confirmed inhibition. As revealed by their removal, free fatty acids and globulins, significantly alter the interaction of HSA with CYP2C9. In addition, HSA and BSA showed different effects on the oxidation of tolbutamide by CYP2C9.

Albumin and hydroxyethyl starch modulate oxidative inflammatory injury to vascular endothelium.

Human serum albumin is used clinically to maintain colloid osmotic pressure and is viewed to serve an antioxidant role in the vascular compartment via binding of redox-active metal complexes, transport of nitric oxide, and the oxidant-scavenging reactions of the single thiol of human serum albumin, cys34. Because of these potentially desirable adjunctive actions, we evaluated the purity and thiol redox state and compared the relative effects of clinically available 25% human serum albumin preparations with a starch-derived colloid, 6% hydroxyethyl starch, in in vitro models of inflammatory vascular injury. Bovine aortic endothelial cell responses to chemical, enzymatic, and cell-derived reactive inflammatory mediators in the presence of human serum albumin or hydroxyethyl starch were assessed. The cys34 thiol of fresh human serum albumin preparations was 70-85% oxidized and contained a population of human serum albumin (approximately 25% of total) having the cys34 resistant to reduction by 2-mercaptoethanol and NaBH4. Treatment of bovine aortic endothelial cells with human serum albumin dose-dependently protected from HOCl-mediated 14C-adenine release, with this protective effect of human serum albumin not dependent on protein thiol status. Addition of human serum albumin to cell media provided no protection from the cytotoxic actions of peroxynitrite and xanthine oxidase-derived reactive species. Binding of activated polymorphonuclear leukocytes to bovine aortic endothelial cells was significantly amplified by hydroxyethyl starch and inhibited by human serum albumin administration. The binding of neutrophil-derived myeloperoxidase to bovine aortic endothelial cells, a mediator of multiple oxidative and nitric oxide-consuming reactions, was also inhibited by human serum albumin and enhanced by hydroxyethyl starch. Clinical human serum albumin preparations show modest intrinsic non-thiol-dependent antiinflammatory properties in vitro, a phenomenon that was not observed with hydroxyethyl starch.

A different view on human albumin—authors' reply

We thank Drs Nohe and Dieterich for their comments on our recent article in this journal, in which we reported that physiological concentrations of bovine serum albumin (BSA) and recombinant human serum albumin (HSA) selectively inhibit TNFα-induced expression of vascular cell adhesion molecule-1 (VCAM-1) in human aortic endothelial cells [1]. In contrast, neither E-selectin nor intercellular adhesion molecule-1 (ICAM-1) expression were inhibited by BSA [1]. We also observed that BSA inhibits TNFα-induced VCAM-1 expression in human umbilical vein endothelial cells (HUVEC) [2]. This result is consistent with the inhibitory effect of HSA on TNFα-mediated VCAM-1 expression in HUVEC reported by Nohe et al. [3]. However, these authors also found that contaminants in two different preparations of HSA can upregulate expression of ICAM-1 and E-selectin in resting and TNFα-stimulated HUVEC, respectively [3]. Nohe …

Human serum albumin and its structural variants mediate cholesterol efflux from cultured endothelial cells

In the present study, we used the human EA.hy926 endothelial cell line as the model system to investigate the effect of human serum albumin (HSA) and its structural variants on cholesterol efflux. Initial studies showed that HSA promoted cholesterol efflux in a dose- and time-dependent manner, reaching a plateau at 10 mg/ml at 90 min. As a control, gelatin displayed no significant effect on efflux, while HSA was significantly more efficient than ovalbumin and bovine serum albumin (BSA) in promoting cholesterol efflux. Equal molar concentrations of HSA and apolipoprotein A-I (apoA-I) showed that apoA-I had considerably higher efficiency in efflux. However, the prevailing high plasma concentrations of HSA may compensate for its lower efflux rate compared to apoA-I. To characterize the mechanism of HSA-mediated cholesterol efflux, we studied the effects of cAMP and temperature on efflux using both EA.hy926 endothelial cells and murine RAW 264.7 macrophages. We found that HSA-mediated efflux occurred via a cAMP-independent and relatively temperature-insensitive pathway. We next examined the nature of HSA–cholesterol interaction by comparing the effects of various HSA mutants to wild-type HSA on cholesterol efflux. We found specific interactions between subdomains 2A and 3A and cholesterol, as indicated by the changes in the efflux rate of various HSA mutants. In conclusion, our study provides evidence for the role of HSA in cholesterol efflux, and shows that the substitution of specific amino acid residues in subdomains of 2A and 3A may be important structural determinants in its ability to bind to cholesterol and participate in cholesterol efflux.

Beneficial effects of human serum albumin on stability and functionality of alginate microcapsules fabricated in different ways.

A key engineering challenge in designing microcapsules made from biocompatible alginate is maintaining adequate exchange of nutrients and oxygen between the entrapped cells and the environment, while simultaneously avoiding swelling and subsequent failure of the microcapsule. Approval for the use of alginate in pharmaceutical and/or biomedical applications also strictly requires that the components of the microcapsule material must meet the safety criteria of the ASTM and FDA. Incorporation of foetal calf serum (FCS) into the microcapsules for stabilization is not in accordance with the guidelines affirmed by these organizations. FCS should be substituted by microcapsule-stabilizing additives that are medically approved. In this communication, it is shown that 10% FCS can be replaced by 1% human serum albumin (i.e. by an agent for which medical approval is granted) without compromising effects on long-term in vitro stability. Furthermore, it is demonstrated that human serum albumin (HSA) significantly enhances cell survival and, particularly, insulin secretion of encapsulated rat islets over a time period of 3 weeks when incubated in culture medium. Thus, HSA-stabilized microcapsules made from UHV(Lam) alginate are apparently a promising system for immunoisolation of cells, particularly when alginate is cross-linked by injection of BaCl(2) crystals into the alginate droplets. Slight adjustments of the alginate concentration can tailor the microcapsule permeability to the released therapeutic factor.

Beneficial effects of human serum albumin on stability and functionality of alginate microcapsules fabricated in different ways

A key engineering challenge in designing microcapsules made from biocompatible alginate is maintaining adequate exchange of nutrients and oxygen between the entrapped cells and the environment, while simultaneously avoiding swelling and subsequent failure of the microcapsule. Approval for the use of alginate in pharmaceutical and/or biomedical applications also strictly requires that the components of the microcapsule material must meet the safety criteria of the ASTM and FDA. Incorporation of foetal calf serum (FCS) into the microcapsules for stabilization is not in accordance with the guidelines affirmed by these organizations. FCS should be substituted by microcapsule-stabilizing additives that are medically approved. In this communication, it is shown that 10% FCS can be replaced by 1% human serum albumin (i.e. by an agent for which medical approval is granted) without compromising effects on long-term in vitro stability. Furthermore, it is demonstrated that human serum albumin (HSA) significantly enhances cell survival and, particularly, insulin secretion of encapsulated rat islets over a time period of 3 weeks when incubated in culture medium. Thus, HSA-stabilized microcapsules made from UHVLam alginate are apparently a promising system for immunoisolation of cells, particularly when alginate is cross-linked by injection of BaCl2 crystals into the alginate droplets. Slight adjustments of the alginate concentration can tailor the microcapsule permeability to the released therapeutic factor.

Effect of albumin and cytosol on enzyme kinetics of tolbutamide hydroxylation and on inhibition of CYP2C9 by gemfibrozil in human liver microsomes.

The effect of human serum albumin (Hsa) and human liver cytosol (Hlc) on the in vitro enzyme kinetics of the formation of hydroxytolbutamide (CYP2C9 marker reaction) and the inhibitory effect of gemfibrozil on tolbutamide hydroxylation were examined using human liver microsomes. The addition of Hsa greatly decreased the unbound concentrations of tolbutamide and gemfibrozil in the incubation medium, whereas Hlc only slightly decreased them. The unbound K(m) value for tolbutamide hydroxylation was 123 microM without Hsa and Hlc, and 73, 88, and 64 microM in the presence of Hsa (5 mg/ml), Hlc (0.5 mg/ml), and Hsa plus Hlc, respectively. The predicted in vivo hepatic clearance (CL(h)) of tolbutamide based on enzyme kinetics without Hsa and Hlc (0.06 ml/min/kg) was 40% of its in vivo clearance (0.15 ml/min/kg) based on published data. Addition of 5 mg/ml Hsa and 0.5 mg/ml Hlc to the incubation medium distinctly improved the prediction, with the coaddition of Hsa and Hlc yielding the most accurate value (0.14 ml/min/kg). The K(i) (6 microM) of gemfibrozil for CYP2C9, calculated using total drug concentrations, was increased by Hlc (8 microM), Hsa (40 microM), or both (72 microM). However, when the unbound substrate and inhibitor concentrations were considered, the K(i) (6 microM without Hsa and Hlc) was not markedly altered by Hsa (4 microM), Hlc (8 microM), or both Hsa and Hlc (9 microM). The present findings suggest that the addition of Hsa and Hlc to microsomal incubation media may yield enzyme kinetic estimates more comparable with in vivo results.

Interaction of plasma proteins with cytochromes P450 mediated metabolic reactions: inhibition by human serum albumin and α-globulins of the debrisoquine 4-hydroxylation (CYP2D) in liver microsomes of human, hamster and rat

The effect of human serum albumin (HSA), α1-acid glycoprotein (α1-AGP), and α- and γ-globulins on the in vitro metabolism of debrisoquine in human, hamster and rat liver microsomes was studied. Interaction of albumin with cytochrome P450 mediated phenytoin metabolism has been reported. Since plasma protein binding of phenytoin is high, in the present study a weakly protein bound drug, debrisoquine, was studied. Debrisoquine is a substrate of CYP2D6. The debrisoquine 4-hydroxylation was measured using a radio-TLC method. Among the four plasma proteins, α-globulins had the strongest inhibitory effect on the debrisoquine 4-hydroxylase activity. The inhibition with 2% α-globulins was 42±18% for human and higher for hamster and rat liver microsomes (65–71%). HSA had less effect than α-globulins. In the presence of HSA, the decrease in activity was between 18 and 35% for all liver microsomes studied. The debrisoquine 4-hydroxylase activity was not significantly changed by α1-AGP or γ-globulins. Using an ultra-filtration method, the protein binding of debrisoquine to 4% HSA, 0.5% α1-AGP, 2% α-globulins and 2% γ-globulins was found to be 22, 20, 22 and 5%, respectively. Since the observed inhibition is inconsistent with level of protein binding, it appears, particularly in the case of α-globulins, that the plasma proteins interact with CYP2D directly.

The effect of serum albumin on amphotericin B aggregate structure and activity.

Mild heat treatment of Fungizone (FZ, an amphotericin B:deoxycholate preparation) leads to a new self-associated form (HFZ) that demonstrates improved therapeutic index in vivo. The origin of the improvement may lie in the differential stability in the presence of serum proteins. The purpose of this study is to assess the effect of human serum albumin (HSA) on the structure and stability and in vitro channel forming ability of these two preparations against model fungal and mammalian membrane vesicles. Kinetic absorption and CD spectroscopy were used to assess the kinetic and equilibrium stability of the characteristic amphotericin B complexes in the presence of HSA. Kinetic fluorescence spectroscopy of pyranine entrapped in model fungal and mammalian membrane vesicles was used to measure the cation-selective channel forming ability of HZ and HFZ delivered from HSA. It is shown that FZ is rapidly converted from its aggregated form to a protein-bound monomer in the presence of HSA, whereas HFZ demonstrates greater stability by persisting as a stable inactive aggregate. Fluorescence measurements of ion currents show that HSA attenuates the membrane-activity of both preparations. However, the activity of both HFZ and FZ remains significant against ergosterol-containing membranes. This is the first direct measurement of the intrinsic channel forming abilities of these amphotericin B preparations in the presence of serum proteins. These data provide a mechanistic rationale for the similar efficacy and lower toxicity of HFZ.

Human Serum Amyloid P Component is a Single Uncomplexed Pentamer in Whole Serum

Serum amyloid P component (SAP) is a universal constituent of amyloid deposits and contributes to their pathogenesis. SAP also has important normal functions in the handling of chromatin in vivo and resistance to bacterial infection. The atomic resolution crystal structure of SAP is known, but its physiological oligomeric assembly remains controversial. In the absence of calcium, isolated human SAP forms stable decamers composed of two cyclic disk-like pentamers interacting face to face. However, in the presence of its specific low molecular weight ligands and calcium, SAP forms stable pentamers. In the presence of calcium, but without any ligand, isolated human SAP aggressively autoaggregates and precipitates, imposing severe constraints on methods for molecular mass determination. Gel filtration chromatography and density gradient ultracentrifugation were used to compare SAP with the closely related molecule, C-reactive protein (CRP; which is known to be a single pentamer) and the effect of human serum albumin on SAP autoaggregation was investigated. In most physiological buffers and with the necessary absence of calcium, SAP, whether isolated or from whole serum samples, eluted from gel filtration columns clearly ahead of CRP. This is consistent with the existence of a monodisperse population of SAP decamers, as previously reported. However, in Tris/phosphate buffer, SAP was pentameric, suggesting that decamerization involved ionic interactions. On density gradients formed in undiluted normal human serum, SAP sedimented as single pentamers not complexed with any macromolecular ligand, regardless of the presence or absence of calcium. The calcium-dependent autoaggregation of isolated SAP was completely inhibited by physiological concentrations of albumin and the SAP remained pentameric. Human SAP exists within serum as single uncomplexed pentamers in the presence or absence of calcium. This oligomeric assembly, thus, does not require a calcium-dependent small molecule interaction. The usual >2000-fold molar excess of albumin over SAP in plasma is apparently sufficient to keep SAP in its physiological conformation.

Effect of laser welding with human serum albumin on the expression of P-selectin on platelets.

Artery repair by means of laser energy induces activation of platelets with a risk of thrombosis and local inflammatory reactions. The aim of this study was to investigate the effect of human serum albumin, the most common solder in laser surgery, on platelet activation. Platelet activation was evaluated in canine blood by using two-color flow cytometry with a phycoerythrin-labeled antibody to a common platelet marker, glycoprotein IIb/IIIa and fluorescein isothiocyanate-labeled antibody to a platelet activation molecule, P-selectin. Human serum albumin was applied in vitro and in vivo, as a solder during laser reconstruction of canine arteries. In vitro, albumin significantly (P < 0.01) reduces the expression of P-selectin on platelets. This is most likely related to the blockage of P-selectin by albumin, which binds to the platelet surface, as confirmed by flow cytometry with fluorescein isothiocyanate-labeled albumin. In vivo, application of albumin solder tended to result in a lower percentage of P-selectin-expressing platelets in laser-repaired arteries compared to suture-repaired arteries. Albumin decreases the percentage of P-selectin-expressing platelets in vitro. Further research may allow the platelet activation inhibiting properties of albumin to be further optimized in vivo.