The effect of decontamination procedures on the pharmacokinetics of venlafaxine in overdose.

The aim of this work was to investigate the pharmacokinetics (PK) of venlafaxine in overdose and the effects of single-dose activated charcoal (SDAC) and whole-bowel irrigation (WBI), alone or in combination, as methods of decontamination. The data included 339 concentration-time points from 76 venlafaxine overdose events (median dose 2,625 (150-13,500 mg)); 69 were slow-release doses. SDAC, WBI, a combination of both, or no decontamination were administered to patients as decided by the treating clinician. The data were modeled using WinBUGS (Windows Bayesian Inference Using Gibbs Sampling). A one-compartment model with first-order input and elimination provided an adequate description of the data. SDAC increased clearance (CL) of venlafaxine by 35%, and SDAC and WBI combined reduced the fraction absorbed by 29%. However, the latter produced a greater reduction in maximum plasma concentration (C(max)) for a similar drop in area under the plasma concentration-time curve (AUC). Both SDAC alone, and a combination of SDAC and WBI, decreased the AUC after venlafaxine overdose, but the combination may be more beneficial because it reduces peak concentrations to a greater extent.

Influence of fluorapatite minor additions on behavior of hydroxyapatite ceramics.

Fluorinated hydroxyapatite is known to be less soluble by body fluids, resulting in enhanced resistance to biodegradation in vivo conditions, as compared to the pure hydroxyapatite ceramics. The present work was aimed at the investigation of the effect of minor additions of ultrafine fluorapatite (up to 10 wt%) on the sintering behavior and mechanical properties of hydroxyapatite ceramics. In vitro testing for the osteoblast-like cells viability and proliferation was performed with the samples of varying fluorapatite content. It was found that the fluorapatite addition hinders the sintering shrinkage and lowers the strength, but does not generally affect negatively the viability of the cells.

Adding functional entities to plasmids.

Non-viral gene therapy constitutes an alternative to the more common use of viral-mediated gene transfer. Most gene transfer methods using naked DNA are based upon non-sequence-specific interactions between the nucleic acid and cationic lipids (lipoplex) or polymers (polyplex). We have developed a technology in which functional entities hybridize in a sequence-specific manner to the nucleic acid (bioplex). This technology is still in its infancy, but has the potential to become a useful tool, since it allows the construction of highly defined complexes containing a variety of functional entities. In its present form the bioplex technology is based upon the use of peptide/nucleic acids (PNA) as anchors. Single, or multiple, functional entities are directly coupled to the anchors. By designing plasmids, or oligonucleotides, with the corresponding anchor target sequence, complexes with desired composition can easily be generated. The long-term aim is to combine functional entities in order to achieve optimal, synergistic interactions allowing enhanced gene transfer in vivo.

Protein adsorption onto two bioactive glass-ceramics.

Recent research suggests that the biocompatibility of an implant is to a large extent determined by selective adsorption of proteins from surrounding body fluids. Protein adsorption from human plasma onto two bioactive glass-ceramics (RKKP and AP40) which differ in La and Ta content, was studied by means of chromatography and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The quantitative analysis showed that the glass-ceramics have good protein binding capacities indicating multilayer formation. A correlation between chemical composition and the amount of adsorbed proteins was observed. The presence of La and Ta decreased the protein adsorption, so AP40 bound significantly more protein per surface unit then did RKKP. Preferential adsorption of apolipoprotein J, fibrinogen and fibronectin was observed.

Plasma protein adsorption pattern on characterized ceramic biomaterials.

The protein/biomaterial interactions of three biomaterials used in hard tissue surgery were studied in vitro. A dynamic flow system and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) were used to investigate the adsorption of proteins from diluted human plasma on hydroxyapatite, alumina and zirconia, with regard to total protein binding capacity, relative binding capacity for specific proteins and flow-through and desorption patterns. The ceramics were characterized regarding physicochemical properties; namely, chemical composition by elementary analyses and specific surface, pore volume and pore size distribution using the BET-method and Hg-porosimetry. The materials were found to adsorb a surprisingly low amount of plasma proteins, leaving more than 70% of the surface free. The cellular response will therefore be highly affected by the physico-chemical properties of the material, in contrast to a surface fully covered with proteins. Regarding the adsorption of proteins, most proteins exhibited similar flow-through patterns on the three adsorbents. The exceptions with different flow-through patterns were apolipoprotein D (Apo D), apolipoprotein J (Apo J), complement factor C1s (C1s), complement factor C3 (C3), ceruloplasmin, fibrinogen, alpha1 B glycoprotein and alpha2 HS glycoprotein and serum retinal-binding protein (SRBP). The role of these proteins on acceptance or rejection of implants has to be investigated.

Inter- and intra-molecular migration of peptide amide hydrogens during electrospray ionization.

The isotopic exchange of amide hydrogens in proteins in solution strongly depends on the surrounding protein structure, thereby allowing structural studies of proteins by mass spectrometry. However, during electrospray ionization (ESI), gas phase processes may scramble or deplete the isotopic information. These processes have been investigated by on-line monitoring of the exchange of labile deuterium atoms in homopeptides with hydrogens from a solvent suitable for ESI. The relative contribution of intra- and inter-molecular exchange in the gas phase could be studied from their distinct influence on the well-characterized exchange processes in the spraying solution. The deuterium content of individual labile hydrogens was assessed from the isotopic patterns of two consecutive collision-induced dissociation fragments, as observed with a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Results demonstrate that gas phase exchange in the high-pressure region between the capillary and the skimmer cause substantial depletion of the isotopic information of penta-phenylalanine and penta-aspartic acid. For penta-alanine and hexa-tyrosine, the amide hydrogens located close to the N-terminus are depleted from deuterium during mass analysis. Amide hydrogens located close to the C-terminus still retain the information of the isotopic state in solution, but they are redistributed by intra-molecular exchange of the amide hydrogens with the C-terminal hydroxyl group.

A new method for the accurate determination of the isotopic state of single amide hydrogens within peptides using Fourier transform ion cyclotron resonance mass spectrometry.

A new method is presented to accurately determine the probability of having a deuterium or hydrogen atom on a specific amide position within a peptide after deuterium/hydrogen (D/H) exchange in solution. Amide hydrogen exchange has been proven to be a sensitive probe for studying protein structures and structural dynamics. At the same time, mass spectrometry in combination with physical fragmentation methods is commonly used to sequence proteins based on an amino acid residue specific mass analysis. In the present study it is demonstrated that the isotopic patterns of a series of peptide fragment ions obtained with capillary-skimmer dissociation, as observed with a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, can be used to calculate the isotopic state of specific amide hydrogens. This calculation is based on the experimentally observed isotopic patterns of two consecutive fragments and on the isotopic binomial distributions of the atoms in the residue constituting the difference between these two consecutive fragments. The applicability of the method is demonstrated by following the sequence-specific D/H exchange rate in solution of single amide hydrogens within some peptides.

Conformational stability of adsorbed insulin studied with mass spectrometry and hydrogen exchange.

A new method is described for direct monitoring of the conformational stability of proteins that are physically adsorbed from solution onto a solid substrate. The adsorption-induced conformational changes of insulin are studied using a combination of hydrogen/deuterium (H/D) exchange and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The effect of the surface hydrophobicity on the adsorption-induced conformational changes in the insulin structure is probed by adsorbing insulin on a hydrophilic silica and a hydrophobic methylated silica surface before subjecting the insulin molecules to the isotopic exchange process. The present study describes the experimental procedure of this new application of MALDI. Results show that insulin is more highly and more irreversibly adsorbed to a hydrophobic methylated silica surface than to a hydrophilic silica surface. Hydrogen-exchange experiments clearly demonstrate that the strong interaction of insulin with the hydrophobic surface is accompanied by a strong increase in the H/D-exchange rates and thus in a reduction in the insulin native structural stability. In contrast, H/D-exchange rates of insulin are somewhat reduced upon adsorption on silica from solution.

TM-AFM Threshold Analysis of Macromolecular Orientation: A Study of the Orientation of IgG and IgE on Mica Surfaces.

Adsorption and orientation properties of two different types of immunoglobulin molecules on derivatized and native mica surfaces were investigated using TM-AFM. The analyses included height measurements at two different pH values and a new technique, presented here as threshold analysis, which displays the outer mantle shape of an adsorbed protein. A major difference in preferential orientation is observed upon comparing the adsorption of the two proteins onto the different surfaces. The characteristics of both the adsorbed immunoglobulin and the surface are important for any preferential orientation of the adsorbed protein. Copyright 1998 Academic Press.

Salt-promoted adsorption of proteins onto amphiphilic agarose-based adsorbents. II. Effects of salt and salt concentration.

The effects of different types of salts and salt concentrations on the selectivity in the adsorption of serum proteins have been compared for the amphiphilic agarose-based adsorbents Phenyl-Sepharose, Octyl-Sepharose, butyl-agarose and mercaptopyridine-derivatized agarose. By use of multivariate analysis, the complex interrelationships for the different combined effects were evaluated. From these analyses conclusions about similarities and/or dissimilarities in the mechanisms involved in adsorption of proteins on respective adsorbent were made.

Comparison of the protein adsorption selectivity of salt-promoted agarose-based adsorbents. Hydrophobic, thiophilic and electron donor-acceptor adsorbents.

Protein adsorption of human serum onto six different agarose-based chromatographic gels that were representative of the salt-promoted adsorbent family [octyl- and phenyl-Sepharose, mercaptoethanol-divinyl sulfone agarose (T gel), mercaptomethylene pyridine-derivatized agarose gel (MP gel), tricyanoaminopropene-divinyl sulfone agarose (DVS-TCP gel), tricyanoamino-propene-bisoxirane agarose (bisoxirane-TCP gel)] was studied in the presence of moderate or high concentrations of the water structuring salt, sodium sulfate. Study of the protein adsorption selectivity by two-dimensional gel electrophoresis revealed an opposed selectivity for hydrophobic interaction adsorbents and electron donor-acceptor adsorbents. The T gel, MP gel and TCP gels belonged to the electron donor-acceptor adsorbents, displaying a main selectivity for immunoglobulins, whereas octyl-Sepharose belonged to the hydrophobic adsorbents, displaying a main selectivity for 'hydrophobic' proteins. Phenyl-Sepharose for its part was described as an example of a composite selectivity of both families. The conclusion of this work is two-fold: (1) hydrophobic interaction chromatography (HIC) and electron donor-acceptor chromatography (EDAC) have opposed protein selectivities and are both salt-promoted. As a main consequence, it means that high concentrations of a water-structuring salt can promote different types of weak molecular interactions, resulting in different protein adsorption selectivities: (2) thiophilic adsorption chromatography (TAC) should be renamed EDAC as similar protein selectivity is demonstrated for electron donor-acceptor ligand devoid of sulfur atoms.

Factors affecting protein interaction at sorbent interfaces.

Interactions between surfaces and macromolecules are the fundamentals in separation and detection of diverse solutes. In this very brief review the central aspects of protein-surface interactions are discussed with the intention of identifying the important factors influencing such processes and placing them in relation to the established knowledge in this field. Some perspectives of new techniques related to scanning probe microscopy for studying interactions at the nanometer level are also discussed.

Preparation and purification of an end to end coupled mEGF-dextran conjugate.

The amino terminus of mouse epidermal growth factor (mEGF) was coupled directly to the aldehyde end of dextran through a reductive amination procedure. The highest coupling efficiency was approximately 80% and could be reached after approximately 24 h of reaction time at pH 8. Gel filtration on Sephadex G-50 Fine removed free mEGF from the conjugate. Preparative polyacrylamide gel electrophoresis was used to separate the conjugate from excess noncharged dextran. The conjugate bound specifically to the EGF receptor on cultured glioma cells as shown in displacement tests with free mEGF. The conjugate was stable in the pH interval 4-9, in 2 M sodium chloride, in 7 M urea, and in human serum and could still bind to the EGF receptor after such treatments. The conjugates are candidates for targeted nuclide therapy.

Polyol-promoted adsorption of serum proteins to amphiphilic agarose-based adsorbents.

We tested the promotion of protein adsorption onto amphiphilic agarose-based adsorbents by addition of high concentrations of polyols during the adsorption phase. C3- to C5-polyols were inefficient in promoting protein adsorption, whereas some of the C6-polyols studied (sorbitol, dulcitol and mannitol) could promote serum protein adsorption onto mercaptomethylene pyridine-derivatized agarose, octyl- and phenyl-Sepharose. Sorbitol was the most potent protein adsorption promoter, with a direct relation between the amount of protein adsorbed and the concentration of sorbitol. For each chromatographic gel, the effects of increasing concentrations of sorbitol or sodium sulfate on protein adsorption were similar and two-dimensional electrophoresis revealed the preservation of the protein adsorption specificity whether sorbitol or sodium sulfate was used. These results show that a water-structuring salt or a polyol can promote protein adsorption in the same manner, presumably by a related mechanism.

Cosolvent-induced adsorption and desorption of serum proteins on an amphiphilic mercaptomethylene pyridine-derivatized agarose gel.

We studied the effects of the following cosolvents on the adsorption and desorption of serum proteins from an amphiphilic mercaptomethylene pyridine-derivatized agarose gel: glucose, sucrose, polyethylene glycol (PEG), 2-methyl-2,4-pentanediol (MFD), sorbitol, pentaerythritol, glycerol, and Na2SO4. The water-structuring salt 0.4 M Na2SO4 was the most potent promoter of protein adsorption, followed by 5 M sorbitol and, to a lesser extent, 0.2 M PEG 1000 and 2.25 M MPD. The other cosolvents (4 M glucose, 1.5 M sucrose, 0.3 M pentaerythritol, and 7.6 M glycerol) were unable to promote protein adsorption to the gel. Attempts to modulate the salt-promotion effect of Na2SO4 with different cosolvents demonstrated the occurrence of synergistic effects for pentaerythritol, sorbitol, and glucose and antagonistic effects for the other cosolvents. Sorbitol and glycerol were found to be the most interesting co-solvents studied, as the first promoted protein adsorption, whereas the other disrupted protein interaction. As a consequence of these novel findings we propose sorbitol and glycerol, both well-known protein stabilizers, as possible alternatives to water-structuring salts during the adsorption phase and to deleterious organic solvents during the desorption phase on amphiphilic gels.

Imaging of single antigens, antibodies, and specific immunocomplex formation by scanning force microscopy.

The most sensitive analytical techniques available today for detecting immuno assay complexes are radio or enzyme immuno analytical techniques, by which quantities of 10(7)-10(8) analyte molecules can be detected. With the introduction of scanning force microscopy, a new method for detecting biological processes became available. Here, we examine the feasibility of using scanning force microscopy as a biosensitive tool. We demonstrate that single or multiple rabbit anti-human serum albumin molecules form complexes with preadsorbed single human serum albumin molecules on mica. However, no interaction is observed between human immunoglobulin G molecules and preadsorbed single albumin molecules; only separate antigens and antibodies are observed at random positions on the mica. This shows the ability of scanning force microscopy to act as a biosensor for detection of immunocomplexes, and to act as a very powerful tool to study molecule-surface interactions in general.

Influence of salts on protein interactions at interfaces of amphiphilic polymers and adsorbents.

The protein-binding capacity of two different amphiphilic adsorbents was investigated to determine the effect of solvent additives on the binding of proteins in hydrophobic-interaction chromatography. There was no simple correlation between binding capacity and the lyotropic series such as those suggested by the two different theories proposed by Arakawa and Narhi and Melander and Horváth. Proteins are known to be dynamic flexible objects which continuously undergo changes in conformation and which may well be influenced by chaotropic salts. Are conformational changes of proteins at interfaces an important parameter involved in protein interactions with amphiphilic polymers and adsorbents? In an attempt to answer this question, the reactivity of the thiol group in human serum albumin (HSA) toward N-ethyl-3-(2-pyridyldisulfanyl)propionamide dextran was used as a model system to evaluate its correlation with the lyotropic series. The results indicate that the thiol-disulfide exchange reaction at interfaces of amphiphilic polymers is influenced by the type of salt used.

Amphiphilic agarose-based adsorbents for chromatography. Comparative study of adsorption capacities and desorption efficiencies.

A number of hydrophobic derivatives attached to cross-linked agarose were studied as protein adsorbents. Differences in the adsorption and desorption behaviour were determined as functions of type and concentration of selected salts. Whereas octyl- and phenyl-Sepharose adsorb serum albumin preferentially, pyridyl-S-agarose shows a much stronger preferential affinity for IgG in the presence of high concentrations of lyotropic salts, such as sulphates. In contrast to pyridyl-S-agarose, a large portion of proteins remained fixed to octyl- and phenyl-Sepharose after extensive washing with 1 M NaOH.

Adsorption and activation of zymogens at solid-liquid interfaces. I. Chymotrypsinogen on alkylamino modified silica derivatives.

Silica beads were modified with alkylamino groups of different lengths (C2, C4, C6, C8, and C10) and hydrophobicity. The relationship between surface structure and adsorption of chymotrypsinogen followed by its activation with trypsin at the solid-liquid interface was studied. From the adsorption isotherms, it follows that underivatized silica adsorbed chymotrypsinogen (CTG) well. The adsorption of CTG on alkylamino modified silicas appeared to correlate with the hydrophobicity of the latter. The longer the alkyl chains were, the higher was the amount of adsorbed CTG. The activation of adsorbed CTG with trypsin at the solid-liquid interface was a slower process when compared with the activation conducted in solution. Parallel experiments were performed with chymotrypsin (CT). The adsorption behavior was similar to that of CTG. The activity of adsorbed CT was inversely proportional to the hydrophobicity of the beads. These results correlated well with the desorption of CT after repeated washings. Repeated addition of substrate (Gly-Gly-Phe-NAp) to the CT covered beads resulted in the CT desorption. The higher the hydrophobicity of the beads was, the lower was the desorption of CT.

Thiophilic adsorbents for RIA and ELISA procedures.

Three types of agarose derivatives have been prepared and investigated as adsorbents for radioimmunoassay and ELISA analysis. The analytical systems were evaluated using beta 2 microglobulin as a model. After a competitive reaction between the immunocomponents in solution, the formed immune complexes were adsorbed onto the adsorbent in the presence of 0.5 M potassium sulfate in 0.1 M Tris, pH 7.5. The binding constant between the interaction site on human IgG and the adsorbent 3-(2-pyridylthio)-2-hydroxypropylagarose (Py-S-gel) was determined to be 1.5 x 10(7) M-1 and the binding capacity was 20 mg/ml gel. The immune complex was desorbed by deleting potassium sulfate from the buffer, and only 0.5% of the total applied protein remained after washing the adsorbent with 0.5 M NaOH. The same adsorbent can be used repetitively with different systems.