A pilot study to evaluate the serum Alpha-1 acid glycoprotein response in cats suffering from feline chronic gingivostomatitis.

BACKGROUND: Feline chronic gingivostomatitis (FCGS) is a multifactorial immune-mediated disease that can lead to chronic pain, anorexia, and weight loss and has substantial health and welfare effects. Currently, the recommended treatment includes dental extractions to decrease the inflammatory stimulation associated with dental plaque. However, complete remission is observed in less than half of the cases, and the majority need comprehensive medical management. This study aimed to evaluate the serum levels of the acute phase protein alpha-1 acid glycoprotein (AGP) in cats with FCGS and to examine whether dental extractions contribute to a significant decrease in the systemic inflammatory response at two postoperative time points. RESULTS: AGP serum concentrations in the cats with FCGS were significantly higher at all time points than that in the control groups and were significantly correlated with the global caudal stomatitis score at day 0 but not at day 30 or 60. A significant improvement of some clinical scores, such as perceived comfort and global caudal stomatitis, was observed 60 days after the dental extraction. However, the levels of AGP did not significantly change over time. CONCLUSIONS: Cats with FCGS were more likely to have a systemic inflammatory response compared with age- and dental disease-matched controls. Dental extractions, in most cases, did not contribute to a significant decrease of AGP both at 30 and 60 days. Therefore, this study reinforces the need to pursue comprehensive medical management after dental extractions to attenuate the systemic inflammatory response as a result of this disease.

Periapical infection may affect birth outcomes via systemic inflammation.

OBJECTIVES: Maternal dental periapical infections are associated with preterm birth and intrauterine growth restriction. This study investigates whether the association is mediated through bacterial spread from periapical lesions to placenta (direct pathway) or systemic inflammatory reaction (indirect pathway). MATERIALS AND METHODS: We compared birth outcomes in Malawian mothers with and without periapical infection. As markers of a direct pathway, we identified placental bacteria using a 16S rDNA approach and assessed histological evidence of inflammation in the placenta and amniotic membranes. We measured C-reactive protein, alpha-1-acid glycoprotein, and salivary cortisol as markers of an indirect pathway. We used regression models to associate the predictor variables with duration of pregnancy and newborn size. RESULTS: Of 1,024 women, 23.5% had periapical infection. There was no association of periapical infection with either bacterial DNA or histological inflammation in placenta or membranes. Periapical infection was associated with C-reactive protein, alpha-1-acid glycoprotein, and cortisol concentrations in a dose-dependent manner at 36 weeks. Addition of alpha-1-acid glycoprotein or cortisol concentration into regression models attenuated the association between periapical infection and pregnancy outcomes. CONCLUSION: There was no evidence of direct spread of periapical bacteria to the placenta. Periapical infections and adverse pregnancy outcomes are in part mediated through systemic inflammation.

Effects by periodontitis on pristane-induced arthritis in rats.

BACKGROUND: An infection-immune association of periodontal disease with rheumatoid arthritis has been suggested. This study aimed to investigate the effect of pre-existing periodontitis on the development and the immune/inflammatory response of pristane-induced arthritis. METHODS: We investigated the effect of periodontitis induced by ligature placement and Porphyromonas gingivalis (P. gingivalis) infection, in combination with Fusobacterium nucleatum to promote its colonization, on the development of pristane-induced arthritis (PIA) in rats (Dark Agouti). Disease progression and severity of periodontitis and arthritis was monitored using clinical assessment, micro-computed tomography (micro-CT)/intraoral radiographs, antibody response, the inflammatory markers such as α-1-acid glycoprotein (α-1-AGP) and c-reactive protein (CRP) as well as cytokine multiplex profiling at different time intervals after induction. RESULTS: Experimentally induced periodontitis manifested clinically (P < 0.05) prior to pristane injection and progressed steadily until the end of experiments (15 weeks), as compared to the non-ligated arthritis group. Injection of pristane 8 weeks after periodontitis-induction led to severe arthritis in all rats demonstrating that the severity of arthritis was not affected by the pre-existence of periodontitis. Endpoint analysis showed that 89% of the periodontitis-affected animals were positive for antibodies against arginine gingipain B and furthermore, the plasma antibody levels to a citrullinated P. gingivalis peptidylarginine deiminase (PPAD) peptide (denoted CPP3) were significantly (P < 0.05) higher in periodontitis rats with PIA. Additionally, there was a trend towards increased pro-inflammatory and anti-inflammatory cytokine levels, and increased α-1-AGP levels in plasma from periodontitis-challenged PIA rats. CONCLUSIONS: Pre-existence of periodontitis induced antibodies against citrullinated peptide derived from PPAD in rats with PIA. However, there were no differences in the development or severity of PIA between periodontitis challenged and periodontitis free rats.

The effect of sea buckthorn supplement on oral health, inflammation, and DNA damage in hemodialysis patients: a double-blinded, randomized crossover study.

OBJECTIVE: Chronic kidney disease is associated with inflammation, oxidative stress, malnutrition, poor oral health, and mouth dryness. The objective of this study was to evaluate effects of sea buckthorn oil (SBO) extract, which is rich in vitamins, phytochemicals, and polyunsaturated fatty acids, on oxidative stress, saliva production, and inflammation in hemodialysis patients. DESIGN SETTING AND SUBJECTS: This was a randomized, double-blinded, and placebo-controlled crossover study (2 × 8 weeks, 4-week washout). The study subjects were hemodialysis patients (n = 45) recruited from the Department of Renal Medicine at Karolinska University Hospital in Stockholm. INTERVENTION AND MAIN OUTCOME MEASURES: The patients received 4 capsules per day, each containing 500 mg of SBO or placebo, for 8 weeks. They were then crossed over to the other treatment after a 4-week washout period. Salivary gland biopsies, saliva, and blood samples were collected before and after each treatment period. Main outcomes were DNA breaks and oxidative DNA lesions in minor accessory salivary glands, salivary flow rates, and inflammation markers in blood (high-sensitivity C-reactive protein, antitrypsin, orosomucoid in plasma, leukocytes in blood). Blood markers including creatinine, urea in plasma, and hemoglobin in blood were investigated. RESULTS: The results showed no significant changes in DNA breaks, oxidative DNA lesions, salivary flow rates, or inflammation after SBO supplementation. However, plasma levels of phosphate and sodium increased and plasma levels of iron decreased. CONCLUSION: In conclusion, SBO supplementation as performed in this study did not protect against oxidative stress, nor improve oral health or inflammation status in hemodialysis patients.

[The investigation of alpha1-antitripsin, orozomukoid and ceruloplasmin in peripheral blood and oral fluid. Paradontitis is one of more risk factors of cardiovascular diseases].

This article presence the connection between paradontitis and cardiovascular diseases, and definition of the maintenance of acute phase proteins in an oral fluid at patients with acute myocardial infarction is obviously important for clinic. Results of own researches of change of the maintenance of three acute phase proteins: ceruloplasmin, alpha1-antitripsin and orosomucoid in an oral fluid and blood plasma at paradontitis and myocardial infarction allow to consider the paradontitis as one more risk factor of a cardiovascular pathology, except well-known hypertensions, smoking, a diabetes.

Interaction of silver nanoparticles with plasma transport proteins: A systematic study on impacts of particle size, shape and surface functionalization.

Metallic nanoparticles are an important and widely used materials in development of nano-enabled medicine. For that reason, their interaction with biological molecules has to be systematically examined, as use of nanoparticles can lead to altered biological functions. In this study, we evaluated the interaction between silver nanoparticles (AgNPs) and two important plasma transport proteins - albumin and α-1-acid glycoprotein. To investigate comprehensively how different physico-chemical properties impact interaction of proteins with nanosurface, AgNPs of different size, shape and surface coating was prepared. The study was conducted using UV-Vis absorption, fluorescence, inductively coupled plasma mass spectrometry, circular dichroism spectroscopy, transmission electron microscopy, dynamic and electrophoretic light scattering techniques. The results showed significant complexities of the nano-bio interface and binding affinities of proteins onto surface of different AgNPs, which were affected by both AgNPs and protein properties. The most significant role on AgNPs-protein interaction had the coating agents used for AgNPs surface stabilization. Our findings should improve safe-by-design approach to development of the metallic nanomaterials for medical use.

Membrane-induced conformational change of alpha1-acid glycoprotein characterized by vacuum-ultraviolet circular dichroism spectroscopy.

The tertiary structure of alpha1-acid glycoprotein (AGP) remains unresolved despite its novel function because AGP is a hard target in X-ray and NMR analyses. To elucidate the membrane-induced conformational change of AGP, the vacuum-ultraviolet circular dichroism (VUVCD) spectra of AGP and its constituent sugars were measured down to 160 nm in the presence or absence of phosphoglyceride liposome using a synchrotron-radiation VUVCD spectrophotometer. The secondary-structure contents and numbers of segments of AGP were estimated from the VUVCD spectra of the protein moiety obtained by subtracting the contributions of the glycan moiety. Further, the positions of secondary structures on the amino acid sequence were predicted by combining the VUVCD data with a neural network algorithm. These comprehensive secondary-structure analyses revealed that AGP consists of 11.4% alpha-helices (3 segments) and 39.9% beta-strands (12 segments) in the absence of liposome (pH 4.5), which are close to the proportions in the secondary structure of native AGP (pH 7.4) predicted by homology modeling, and that it consists of 47.5% alpha-helices (7 segments) and 2.7% beta-strands (2 segments) in the presence of liposome (pH 4.5). Detailed characterization of these alpha-helices of AGP bound to liposome suggested that two alpha-helices (residues 15-27 and 161-175) in the N- and C-terminal regions strongly interact with liposome. Most of the progesterone-binding residues of AGP were involved in the sequences transferring from beta-strands to alpha-helices or unordered structures, which coincided with the large decrease in progesterone-binding capacity of liposome-bound AGP. These results provide the first sequence-level information on the membrane-binding mechanism and structure-function relationship of AGP.

Controlled release of a protein kinase inhibitor UCN-01 from liposomes influenced by the particle size.

A protein kinase inhibitor UCN-01 binds with high affinity to human alpha 1-acid glycoprotein (hAGP) which may compromise the drugs therapeutic effectiveness. Liposomal formulations of UCN-01 have been evaluated as a means of reducing the impact of binding to hAGP. However, in an initial study, UCN-01 was released rapidly from liposomes added to rat plasma containing hAGP. The purpose of this study was to develop a liposomal formulation of UCN-01 that only slowly released drug. Liposomes composed of lipids with a high phase transition temperature and having an average particle size of 120 nm and above reduced leaking of UCN-01 when the formulations were evaluated by adding to rat plasma containing hAGP. Furthermore, formulations composed of larger liposomes were also more effective in vivo; in tests in which liposomal preparations were injected together with hAGP into rats, more UCN-01 was retained in liposomes for 24h after administration of 155 nm liposomes as compared to 112 nm liposomes.

The influence of a multifunctional, polymeric biomaterial on the concentration of acute phase proteins in an animal model.

The concentrations of the acute phase proteins alpha1-Acid Glycoprotein (AAG) and haptoglobin were determined in Sprague-Dawley-rats after implantation of a novel biodegradable multifunctional polymeric biomaterial for the reconstruction of a gastric wall defect (polymer group; n=42). For comparison, the concentrations of AAG and haptoglobin were measured as well after primary wound closure of the gastric wall defect without biomaterial implantation (control group; n=21) and in rats without any surgical procedure (baseline group; n=21). The implantation periods were 1 week, 4 weeks and 6 months. The concentrations of AAG and haptoglobin were measured by an ELISA assay. Gastrointestinal complications like fistula, perforation or peritonitis did not occur in any of the animals. No statistically significant differences in the concentrations of AAG and haptoglobin were detected between the polymer and the control group. An adequate mechanical stability of the polymeric biomaterial was detectable under the extreme pathophysiological conditions of the stomach milieu. In further examinations the correlation between the intraperitoneal cytokine levels of the animals and the following systemic inflammatory markers should be analysed. Further investigations are needed to analyse the mechanisms of the tissue integration of a biomaterial as well as the process of the tissue remodeling and the influence of the immune system on these mechanisms. The knowledge of these processes is necessary to adapt the multifunctional biomaterial and prepare it thus for the use and implantation in different body locations and to develop novel therapeutical options in medicine.

Determination of drug-protein binding using supported liquid membrane extraction under equilibrium conditions.

A technique for determination of drug-protein binding based on a membrane extraction technique termed "equilibrium sampling through membrane (ESTM)" is presented. It involves the establishment of an equilibrium between an aqueous buffer and either a blood plasma sample or a matched buffer, both containing the drug. Analysis of the aqueous buffer in the two cases gives the drug-protein binding. The principle bypasses some sources of systematic error found with common techniques for this measurement based on e.g. ultrafiltration, as it senses the equilibrium conditions without disturbing the sample. The technique is applied to some local anesthetic drugs as model substances and two alternative ways for the evaluation are presented. Results with these evaluation methods are compared with literature values for the drug-protein binding of these compounds. It is found that the drug-protein binding values obtained are lower than literature values, which is attributed to reduced systematic error.

Equilibrium sampling through membrane based on a single hollow fibre for determination of drug-protein binding and free drug concentration in plasma.

The determination of drug-protein binding and free drug concentration in plasma applying the equilibrium sampling through membrane (ESTM) technique has been studied using supported liquid membrane extraction in a single hollow fibre without any membrane carrier. In the extraction setup, the donor phase (plasma or buffer) was placed in the vial, into which was immersed the hollow fibre with the acceptor phase situated in the lumen. This proposed technique was applied to study the drug-protein binding of five local anaesthetics and two antidepressants as model substances, and the influence of the total drug concentration on the drug-protein binding was investigated. The brief theoretical background for determination of the drug-protein binding under equilibrium conditions is described. The developed method shows a new, improved and simple procedure for determination of free drug concentration in plasma and extent of drug-protein binding.

Proteome demonstration of alpha-1-acid glycoprotein and alpha-1-antichymotrypsin candidate biomarkers for diagnosis of enterovirus 71 infection.

BACKGROUND: Human enterovirus 71 (EV71) is the major causative agents of hand-foot-and-mouth disease and frequently associated with severe complications such as encephalitis and death. Understanding the host response following enteroviral infection may facilitate the development of biomarkers for EV71 infections. METHODS: We implemented two-dimensional gel electrophoresis technology on proteins prepared from serum obtained from 4 mild and 4 severe cases of EV71 infections and 4 healthy control children, to investigate the differentially expressed proteins. The differential expressed proteins were further identified with liquid chromatography-mass spectrometry/mass spectrometry analysis and western blotting validation. RESULTS: A total of 27 differentially expressed proteins were picked and identified with liquid chromatography-mass spectrometry/mass spectrometry. Of the 27 identified proteins, 6 proteins were up-regulated in the mild-infected and severe EV71-infected patients in comparison to the healthy control group. Two proteins, alpha-1-acid-glycoprotein (AGP1) and alpha-antichymotrypsin (AACT), were not detected in the EV71-infected patients, but appeared in the control patient. Western blotting analysis demonstrated that AGP1 and AACT proteins were negatively associated with the clinical severity of EV71 infection. Similarly, both of the proteins were not detected in the secretion medium from the EV71-infected neuroblastoma cells, but detected in the mock-infected cells, suggesting that differentially expressed AGP1/AACT protein levels are in response to EV71 infections. CONCLUSIONS: Two candidate proteins AGP1 and AACT, whose expression levels were reduced under the EV71 infection pathological condition, provide useful source of information for potential diagnostic biomarkers of EV71 infection in children.

Immunolocalization of VEGF-A and orosomucoid-1 in odontogenic myxoma.

OBJECTIVE: The aim of the present study was to determine and establish the immunohistochemical distribution of VEGF-A and ORM-1 protein in odontogenic myxomas to suggest a possible function in the biological behavior of odontogenic myxomas. MATERIALS AND METHODS: A total of 33 odontogenic myxoma cases and three tooth germs were included. Immunohistochemistry was performed to localize VEGF-A and ORM-1 proteins in tumor cells, endothelial cells and extracellular matrix in the odontogenic myxomas. The intratumoral microvessel density (MVD) was determined with CD34 and Factor VIII antibodies. RESULTS: Immunopositivity was strong in the endothelial cells, which compose various vessels, and in the randomly oriented stellate, spindle-shaped and round tumoral cells with long cytoplasmic processes. More than half of the extracellular matrix lacked expression of VEGF-A. ORM-1 expression was strong in both endothelial cells and tumor cells, and the myxoid extracellular matrix was positive, with moderate or strong immunoexpression in all cases. An important finding of this study was the statistically significant positive correlation between the expression of ORM-1 and VEGF-A in tumor cells (p=0.02). CONCLUSIONS: The results of this study suggest that the expression of VEGF-A and ORM-1 may be associated with two mechanisms (angiogenesis and tumor structural viscosity) that may influence tumor growth in odontogenic myxoma.

Kinetics and role of alpha 1-acid glycoprotein-dependent osmotic transport of water and ions in palmitoyl-L-oleoyl phosphatidylcholine liposomes.

alpha 1-Acid glycoprotein isolated from human blood plasma is known to influence cell permeability, although the mechanisms of this process are unclear. Here, the glycoprotein effects on the permeability of osmotically stressed phospholipid liposomes are studied as a model of membrane permeability. Liposomes containing glycoprotein were found to be osmotically sensitive to water and chloride salts of some monovalent (Na+, K+) and bivalent (Mg2+, Ca2+) ions. The permeations of these substances were determined by light-scattering measurements of the volume changes in liposomes after mixing with hyperosmotic solutions of chloride salts. The time courses of scattered light were recorded by means of stopped-flow spectrophotometry. Two processes were studied: the fast water outflow from liposomes and slower ion permeations through the lipid membrane. The second order permeation rate constants were determined at different glycoprotein concentrations for both processes. Values from 66 to 250 x 10(3) for water outflow and 2-500 M-1 sec-1 for the different ion permeations were obtained in order to characterize the permeations of solutes across the lipid membrane. The apparent activation energies also were calculated between 18 and 33 degrees C. The mercurial sulphydryl reagent pCMBS inhibited the ion permeations in the slow phase. When pCMBS was present in this phase, higher activation energies were obtained, indicating more difficult permeations. An interpretation of these results is that membrane permeability is mediated by aqueous pores. Membrane selectivity to monovalent metal ions also was demonstrated, but no correlation was observed between the ion radius of the corresponding metal cation and permeation rate constants. The discovery of non-specific pores in liposomes containing glycoprotein shows that they can serve as vehicles for the water and ions in the processes of passive transport through lipid membranes.

Clinical pharmacokinetics of unbound docetaxel: role of polysorbate 80 and serum proteins.

OBJECTIVE: Our objectives were to study the extent of docetaxel binding to plasma in the presence and absence of its excipient, polysorbate 80 (Tween 80; Imperial Chemical Industries PLC, London, United Kingdom), in vitro and to evaluate the pharmacokinetics of unbound docetaxel in vivo. METHODS: Equilibrium dialysis was used for determination of the fraction unbound (f(u)) docetaxel and was applied to study the pharmacokinetic behavior of unbound docetaxel in 23 patients with cancer receiving an intravenous infusion of the drug formulated in polysorbate 80 (Taxotere; Aventis Pharma SA, Vitry-sur-Seine Cedex, France). RESULTS: Polysorbate 80, added at clinically relevant concentrations (up to 1.0 microL/mL), increased f(u) in vitro by 13% (7.84% +/- 0.0752% versus 6.95% +/- 0.0678%, P <.00001). Similarly, f(u) calculated on the basis of the observed area under the plasma concentration-time curve (AUC) values [f(u)(AUC)] in vivo was 12% higher than f(u) in pretreatment samples [f(u)(pre)] (6.00% +/- 1.03% versus 5.49% +/- 1.01%, P =.038). Of various serum proteins evaluated, only alpha(1)-acid glycoprotein was significantly related to f(u) (P <.0018), with higher f(u) in the presence of lower protein levels. Total docetaxel clearance was related to alpha(1)-acid glycoprotein (R(2) = 0.13, P =.058), f(u)(pre) (R(2) = 0.15, P =.039), and f(u)(AUC) (R(2) = 0.29, P =.0048). CONCLUSION: This study demonstrates that the plasma binding of docetaxel is influenced by both alpha(1)-acid glycoprotein and its formulation vehicle. Further investigation is required to resolve the potential clinical significance of these observations.

Selected aspects of the microencapsulation of mammalian cells in HEMA-MMA.

Microencapsulation of live mammalian cells is one means of creating hybrid artificial organs, like an artificial pancreas or an artificial liver. In addition to creating and developing the methodologies for enclosing cells within the appropriate semipermeable and biocompatible membranes, novel techniques are needed to assess the various features of the resulting capsules. The small size of a capsule or its heterogeneity can lead to additional complexities that go beyond the problem of examining cell behavior in the presence of biomaterials. These problems are illustrated here by comparison of protein release by microencapsulated HepG2 cells within large and small HEMA-MMA (hydroxyethyl methacrylate-methyl methacrylate) capsules, by assessment of the effect of processing conditions on HEMA-MMA microcapsule permeability to horseradish peroxidase at the individual capsule level, and by a confocal microscopy technique for assessing intracapsule cell viability.

Targeted gene delivery into HepG2 cells using complexes containing DNA, cationized asialoorosomucoid and activated cationic liposomes.

Unilamellar activated cationic liposomes containing 3beta[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol, dioleoyl phosphatidylethanolamine (DOPE) and the N-hydroxysuccinimide ester of cholesteryl hemisuccinate (4:5:1, molar ratio) have been prepared and their DNA-binding capacity has been assessed in a gel retardation assay. Ternary complexes composed of activated cationic liposomes, carbodiimide-cationized asialoorosomucoid (Me+AOM) and pRSVL plasmid DNA were assembled for receptor-mediated DNA delivery into cells expressing the asialoglycoprotein receptor (ASGP-R). Binding of complexes in which Me+AOM was replaced by fluoresceinated Me+AOM (FMe+AOM) to the human hepatocellular cell line HepG2 at 4 degrees C was severely reduced by co-incubation with asialoorosomucoid (AOM). Moreover, assemblies containing liposomes, pRSVL DNA and Me+AOM (8:1:4, w/w/w) promoted high levels of luciferase activity in this cell line (1.3 x 10(7) relative light units/mg soluble cell protein). Assays conducted in the presence of a hundred-fold excess of the ligand AOM afforded considerably lower levels of transfection (2.5 x 10(5) relative light units/mg soluble cell protein). In contrast, the highest level of luciferase activity achieved with liposome, pRSVL DNA, AOM complexes was only a quarter of the best levels obtained with liposome, pRSVL DNA, Me+AOM assemblies. These findings strongly support the notion that complexes gain entry into hepatocyte-derived cells by ASGP-R mediation and that they are potentially useful gene carriers to liver hepatocytes.

Quantitative structure-retention relationships in the examination of the topography of the binding site of antihistamine drugs on alpha 1-acid glycoprotein.

Quantitative relationships between the structure of antihistamine drugs (AHD) and their retention on an alpha 1-acid glycoprotein (AGP) HPLC column (QSRR) were studied in order to identify characteristic structural features of the binding site for AHD on AGP. The hydrophobicity of AHD was determined by HPLC on an immobilized artificial membrane (IAM) column. A highly significant QSRR equation was obtained which describes the retention of AHD on AGP in terms of the chromatographically determined hydrophobicity parameter, electron excess charge on the aliphatic nitrogen and a molecular size descriptor. The topography of the AHD-binding site on AGP was suggested to be a conical pocket with lipophilic regions at the mouth of the receptor and an anionic region close to the spike of the cone. Protonated aliphatic nitrogen is supposed to guide a drug molecule towards the anionic region of the binding site. Hydrophobic aryl moieties provide anchoring of the molecule in the lipophilic regions of the binding site. Steric hindrance prevents the molecule from plunging into the binding site.

Behavior of a cell line derived from normal human hepatocytes on non-physiological and physiological-type substrates: evidence for enhancement of secretion of liver-specific proteins by a three-dimensional growth pattern.

The behavior of a recently described cell line, HH25, derived from normal human hepatocytes, has been investigated on several different substrates--tissue-culture plastic, glass, a thin layer of rat-tail collagen I, and thin layers or thick gels of extracellular matrix derived from the Engelbreth-Holm-Swarm murine sarcoma (EHS matrix). Cellular morphology, proliferation, and secretion of three hepatocyte-specific proteins (albumin, alpha1 acid glycoprotein, and alpha1 antitrypsin) have been examined. There were no differences in morphology, proliferation, or differentiated function in the cells on either plastic, glass, collagen, I, or a thin layer of EHS matrix, but on a thick EHS matrix gel the cells altered their morphology (forming three-dimensional colonies with canalicular-like structures) and their production of albumin and alpha1 acid glycoprotein was enhanced. This suggests that the enhanced differentiated function is associated with the morphological change (occurring only on the thick EHS gel) rather than with receptor-mediated cell-matrix interactions (which can also occur on the thin layer of EHS matrix). This cell line is therefore a good in vitro cellular model for the investigation of the roles of morphological changes and of cell-cell and cell-matrix interactions in the control of human hepatocyte behavior without the need for an extensive source of primary tissue.

Assessment of the contribution of alpha 1-acid glycoprotein to the serum binding of basic drugs using serum treated with sulphosalicylic acid and DEAE-cellulose.

Treatment of human serum with DEAE-cellulose in acid conditions almost completely removed alpha 1-acid glycoprotein (alpha 1-AG) with little change in the concentration of albumin and beta-lipoprotein, while treatment with sulphosalicylic acid removed almost all the proteins except alpha 1-AG. The binding of various drugs to serum treated as above was measured by equilibrium dialysis and the contribution of alpha 1-AG to drug binding by human serum was assessed. Sulphosalicylic acid-treated serum exhibited a saturable binding for propranolol, which was considered to be due to the binding to alpha 1-AG while DEAE-cellulose-treated serum mostly exhibited non-saturable binding, for which albumin and beta-lipoprotein may be responsible. With this treated serum, alpha 1-AG was estimated to contribute approximately 40% to the binding of therapeutic concentrations of propranolol, 15% to that of imipramine and 15-20% to that of desipramine, respectively, in serum samples pooled from healthy adults. However, no contribution of alpha 1-AG was observed in the binding of salicylic acid to the serum. Dissociation constants of the propranolol binding to the high affinity site in control serum, sulphosalicylic acid-treated serum and purified alpha 1-AG showed similar values (3.7-6.7 microM). These results suggest that treatment of serum with sulphosalicylic acid and DEAE cellulose is useful in assessing the contribution of alpha 1-AG to the serum binding of basic drugs.